Intratumoral delivery of FLT3L with CXCR3/CCR5 ligands promotes XCR1^+ cDC1 infiltration and activates anti-tumor immunity

Domain: neuroscience
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
Tumor infiltration by XCR1+ conventional dendritic cells (cDC1) correlates strongly with favorable prognosis and improved responses to immunotherapy. Yet, tumor-driven immunosuppressive programs restrict efficient cDC1 recruitment, highlighting the need for strategies to increase cDC1 access to the tumor microenvironment. Here, we establish a proof-of-concept cell-based immunotherapy that enhances the infiltration of circulating cDC1 progenitors and supports their local expansion. Intratumoral engraftment of autologous mesenchymal stromal cells engineered to express membrane bound FLT3L promotes cDC1 recruitment when combined with poly(I:C). We identify poly(I:C)-induced CXCL9 and CCL5 as essential chemokines controlling intratumoral cDC1 infiltration. Stromal cell–mediated local delivery of FLT3L together with CXCL9 and CCL5 is sufficient to enhance cDC1 infiltration in mice or humanized mice settings. Finally, this approach activates anti-tumor immunity and partially overcomes resistance to immune checkpoint blockade. Collectively, our data support the therapeutic potential of expanding intratumoral cDC1 through local and sustained delivery of FLT3L, CXCL9, and CCL5.

Introduction
An important factor limiting the development of anti-tumor immunity lies in the extent of tumor infiltration by immune cells, including dendritic cells (DCs). DCs are sentinel cells of the immune system initiating the tumor-immunity cycle1,2.

Tumor-associated DCs (TADCs) up-take antigens by phagocytosis and migrate to tumor draining lymph nodes (tdLNs) after up-regulating CCR7. If engaged in immunogenic maturation by the activation of proper innate signalling pathways, DCs can prime anti-tumor CD4+ and CD8+ T cells in tdLNs2,3. Once activated, effector T lymphocytes can migrate back to tumors via efferent lymph and then blood2. All these activities define DCs as initiators of the cancer-immunity cycle.

In addition to this virtuous cycle of T cell activation in tdLN, intratumoral DCs also have local roles. Indeed, they participate to the recruitment of CXCR3+ effector T cells via the release of CXCL9 chemokines4–6. Also, intratumoral DCs shape niches supporting the survival of tumor-infiltrating T cells (TILs) by delivering specific trophic cues. For instance, CXCL16+ TADCs attract CXCR6+ TCF1− effector and promote their survival by trans-presenting IL157. DCs establish local interactions with CD28+ T cells and preserve their fitness8. Finally, CCR7+ LAMP3+ DCs also populate Tertiary Lymphoid Structures9, some “stem immunity hubs” related-lymphoid aggregates where they colocalize with TCF1+ CD8+ TILs6,10 and establish some tripartite interactions with CXCL13+ CD4+ T cells and TCF1+ CD8+ TILs11.

DCs are classified into two major subsets: XCR1+ type 1 conventional DCs (cDC1s), which express IRF8 and an heterogenous population of CD11b+ DCs expressing IRF4 and encompassing cDC2s and DC3s. cDC1s play a non-redundant role in the rejection of immunogenic tumors12 and are essential for the efficacy of immune checkpoint blockade (ICB) therapies, as demonstrated in pre-clinical models13,14. Furthermore, cDC1 infiltration in human tumors correlates with favorable clinical outcomes and response to ICB15,16. Mechanistically, cDC1 would fine tune the CD8+ T cell response by maintaining the pool of TCF1+ TILs along tumor development thereby fueling a reservoir of T cells potentially promoting effector anti-tumor responses17. However, cDC1 infiltration within tumors remains generally limited18–20. COX enzymes in tumor cells limit the activation of NK-dependent DCs recruitment16,21. Also, oncogenic pathways such as β-catenin limit the recruitment of cDC1s by limiting CCL4 production19. Therefore, enhancing the recruitment and activation of intratumoral cDC1s remains an unmet therapeutic need to overcome cancer-induced immune suppression.

Mechanistically, recruitment of circulating DCs have been investigated in the context of pre-clinical models of immunogenic tumors15,19,21. Böttcher et al. have identified that cDC1 recruitment depends on NK-cell derived CCR5 ligands (CCL4, CCL5), as well as XCR1 ligands (XCL1, XCL2)21. It is unclear if infection activated CCR2-dependent pathways22 also control cDC1 recruitment.

The FLT3L-FLT3 signaling pathway is essential for the homeostasis of conventional DCs, including cDC1s, in both mice23,24 and humans25. Intratumoral FLT3L is an important biomarker associated to the density of TADCs, particularly cDC1s, in human melanoma15. FLT3L-FLT3 pathway supports DC development and maintenance in both lymphoid organs and peripheral tissues24. Tissue FLT3L supports the proliferation of DC precursors (pre-DCs) in bone marrow and peripheral sites24,26. Of note, radio-resistant cells control homeostatic levels of FLT3L27. Within secondary lymphoid organs, stromal cells contribute to DC pool maintenance. For example, a CCL19+ GREMLIN+ fibroblastic niche support DC survival at homeostasis28 via FLT3L production29. However, the cellular sources of FLT3L within tumors are not very well known. Barry et al. have shown that NK cells represent a substantial source of FLT3L15 but others sources like T cells30 or mast cells27 might participate to local FLT3L production.

Administration of recombinant soluble FLT3L is an effective approach to expand circulating DCs31 in humans but it faces questionable safety issues with general modification of haematopoiesis and expansion of tolerogenic DCs32. Furthermore, systemic recombinant FLT3L might promote immune tolerance, as it leads to the expansion of regulatory T cells33,34. Lastly, combination therapies of FLT3L with innate agonists such as anti-CD4020 or poly(I:C)14 raise some toxicity concerns associated to systemic inflammation.

In this work, we have reasoned that targeted, local interventions in the tumor microenvironment (TME) could optimize local generation of immunogenic DCs35, while avoiding most systemic effects underpinning immune regulation or toxicity. Here, we explore the therapeutic potential of delivering engineered autologous stromal cells within the TME. Engrafted stromal cells are expressing chemotactic factors supporting DC infiltration, differentiation, proliferation, and maintenance. We identify that stromal membrane bound form of FLT3L combined with poly(I:C) or CXCL9 and CCL5 is sufficient to activate the local recruitment and accrual of cDC1s, T and NK/ILC1 cells within tumors. Furthermore, we show that these immunotherapies stimulate anti-tumor immunity and partially overcome resistance to ICB. Altogether, these findings open an avenue to rewire the tumor microenvironment by local cell-based therapeutic intervention.

Results
Intratumoral delivery of engineered mesenchymal stromal cells expressing membrane bound FLT3L does not alter intratumoral and systemic cDC1 populations

We have previously demonstrated that engineered stromal cells expressing human FLT3L act as “synthetic niches” able to support the differentiation of human tissue DCs in immunodeficient mice36. In a first attempt to stimulate intratumoral cDC accumulation, we generated stable, autologous, fibroblastic cell lines from embryonic fibroblasts of C57BL/6 mice. These cells are characterized as CD45−CD31−CD44+CD140a+gp38/PDPN+ (Supplementary Fig. 1A) and termed as engineered mesenchymal stromal cells thereafter (eMSC). eMSC-FLT3L express homogeneously the membrane form of human FLT3L, as detected by flow cytometry (Fig. 1A). We found that eMSC-FLT3L but not eMSC-control (eMSC-ctrl) stimulated the differentiation of cDC1s and cDC2s, when co-injected with CD45.1 bone-marrow hematopoietic stem cell progenitors (HSPCs) in basal membrane extract plugs in the dermis of tumor free mice, showing that FLT3L expressed in eMSC-FLT3L was functional (Fig. 1B, C and Supplementary Fig. 1B). On the contrary, recombinant FLT3L (rec-FLT3L) did not stimulate cDC differentiation within the plugs. We next decided to inject eMSC-FLT3L within subcutaneous B16 melanoma tumors expressing ovalbumin (B16-OVA), which have a primary resistance to immune checkpoint blockade (ICB). First, we found that gp38+GFP+ eMSC-FLT3L were still detectable on site 14 days after intratumoral injection, validating their persistence within the TME (Fig. 1D, E). To further evaluate the long-term biodistribution and persistence of eMSCs, we engineered nano-luciferase-expressing eMSCs and monitored their persistence using in vivo imaging for 8 days post engraftment in the B16-OVA model (Fig. 1F). We observed that eMSCs remained within the tumor for at least 8 days (Fig. 1G), with their activity confined to the tumor site (Fig. 1H). These observations indicate that, once injected, eMSCs stay localized within the tumor and do not disseminate systemically. We next compared intratumoral engraftment of eMSC-FLT3L to B16F10 cells overexpressing human FLT3L (B16-huFLT3L) and assessed their impact on cDC differentiation. In contrast with B16-huFLT3L, B16F10 tumor bearing mice that had received intratumoral engraftment of eMSC-FLT3L at day 7 did not display an elevation of systemic FLT3L serum levels at day 12 (Fig. 1I, J). Similar results were obtained using the B16-OVA model (Supplementary Fig. 1C). However, we did not observe an increase of cDC1s in the tumor (Fig. 1K), the tumor draining lymph node (tdLN) (Fig. 1L) nor in the spleen (Fig. 1M).

We conclude that intratumoral injection of eMSC-FLT3L alone does not alter intratumoral and systemic cDC1 populations.

Intratumoral delivery of eMSC-FLT3L synergizes with poly(I:C) to activate anti-tumor immunity

We next wondered if innate agonist administration would circumvent the inability of eMSC-FLT3L to expand intratumoral DCs. We chose to use poly(I:C) because it had previously been shown to stimulate anti-tumor immunity in combination with recombinant, soluble FLT3L14. We designed our intratumoral cell-based therapy as two injections of eMSC-FLT3L and one injection of poly(I:C) within B16-OVA tumors (Fig. 2A). We found that only eMSC-FLT3L in combination with poly(I:C) induced tumor regression as assessed by the tumor growth (Fig. 2B) and the tumor weight (Fig. 2C). In addition, the combination therapy extended the survival of mice. Importantly, 10% of the mice treated with the combination therapy eliminated the tumor without relapses until day 90 (Fig. 2D). Interestingly, intratumoral administration of recombinant FLT3L in combination with poly(I:C) also delayed tumor growth (Supplementary Fig. 2A) and improved survival (Supplementary Fig. 2B) but less efficiently than eMSC-FLT3L. Furthermore, mice treated with the therapy did not exhibit weight loss, unlike those receiving anti-CD40 treatment as reported elsewhere37–39 (Fig. 2E, F). In addition, serum levels of ALT (Fig. 2G) and AST (Supplementary Fig. 2C), markers of hepatocellular injury, remained unchanged in mice treated with eMSC-FLT3L+poly(I:C) compared to the anti-CD40 group. Furthermore, mice having received the anti-CD40 antibody exhibited necrotic lesions in the liver (Supplementary Fig. 2D). These results demonstrate the lack of noticeable toxicity associated to immunotherapy by eMSC-FLT3L+poly(I:C) engraftment. In a clinical translational point of view, we asked if adult, autologous fibroblasts would represent a suitable option for eMSC-based immunotherapy. To test this, we generated a CD45−CD31− stromal fraction from adult murine visceral and inguinal adipose tissues. We transduced them to express membrane bound human FLT3L (eAMSC-FLT3L) (Fig. 2H). Following the same experimental plan as previously described (Fig. 2A), we found that eAMSC-FLT3L displayed similar efficiency as eMSCs-FLT3L, when combined with poly(I:C), as showed by the reduced tumor growth (Fig. 2I) and tumor weight (Fig. 2J). We conclude that adult fibroblasts are amenable to engineering for local immunotherapy. Lastly, to further validate the therapeutic efficacy of the eMSC-FLT3L+poly(I:C) strategy, we tested it across multiple tumor models, including MC38 colon carcinoma (Fig. 2K–N), TC-1 lung carcinoma transformed with HPV16 E6/E7 (Fig. 2O–R), and the orthotopic E0771 breast carcinoma (Fig. 2S–V). In each model, treatment delayed tumor growth (Fig. 2L, P, T), reduced individual tumor size variation relative to the day of the first intratumoral injection (Fig. 2M, Q, U), and improved overall survival (Fig. 2N, R, V).

Altogether, these experiments show that local cell-based delivery of FLT3L within the TME in combination with poly(I:C) is a potent therapy to control tumor growth in multiple tumor contexts.

Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the infiltration of cDC1s, that are required for immunotherapy efficiency induction

Having established the immunotherapeutic potential of eMSC-FLT3L+poly(I:C) combination, we next sought to address if this intervention would modify cDC infiltration in the TME. To do so, we analysed intratumoral cDCs 24h and 6 days after a poly(I:C) injection (Fig. 3A). We observed that 24 hours post poly(I:C) injection (day 10), tumor cDC1s underwent phenotypic maturation, as probed by CD40 and CD86 up-regulation within both eMSC-Ctrl and eMSC-FLT3L deliveries (Fig. 3B, C). Poly(I:C) injection also induced a decrease in the number of cDC1s in both eMSC-Ctrl and eMSC-FLT3L treated tumors (Fig. 3C). In contrast, 6 days post poly(I:C) injection (day 15) cDC1s underwent expansion in mice treated with both poly(I:C) and eMSC-FLT3L but not in mice treated with poly(I:C) or eMSC-FLT3L alone (Fig. 3C). This shows that sustained cDC1 expansion at the tumor site, uniquely achieved by the synergy of eMSC-FLT3L and poly(I:C), correlates with the efficiency of the therapy. Of note, similar results were found for cDC2s (Supplementary Fig. 3A). In order to test the role of cDC1s in the efficiency of the therapy, we injected B16-OVA in WT and in cDC1-deficient mice (Xcr1crexRosalsl-DTA (Xcr1DTA)40) and treated the tumors with eMSC-FLT3L and poly(I:C) immunotherapy. We found that the tumor control (Fig. 3D) and the survival extension (Fig. 3E) provided by the therapy, were totally abrogated in the absence of cDC1s.

We conclude that the synergy between eMSC-FLT3L and poly(I:C) expands intratumoral cDC1s, that are required for the therapeutic benefits of this intervention.

Intratumoral delivery of eMSC-FLT3L and poly(I:C) increase mature migratory cDC1s in tumor-draining lymph nodes

Having identified cDC1s anti-tumor role in our therapeutic approach, we next wondered if this effect relied on tumor-draining lymph nodes (tdLNs). As previously described, we analysed the tdLNs 24h (day 10) and 6 days (day 15) after the poly(I:C) administration (Fig. 4A). At 24h post poly(I:C) injection, we observed an increase in the migration of mature (CD40high, CD86high) cDC1s in the tdLNs of tumors treated with eMSC-FLT3L and poly(I:C) (Fig. 4B, C). However, 6 days after poly(I:C) injection, no significant differences were observed between groups in either the number of migratory cDC1s or their maturation status, suggesting that the effect of poly(I:C) is transient (Fig. 4C). Therefore, and in correlation with the previous results (Fig. 3C), we conclude that eMSC-FLT3L combined with poly(I:C) initially promotes the migration of mature cDC1s to the tdLN, followed by the maintenance of a pool of immature cDC1s within the tumor.

Intratumoral delivery of eMSC-FLT3L and poly(I:C) enables cross-priming of tumor-specific CD8+ T cells

Since cDC1s at day 10 in the tdLNs are mature and that these cells play a crucial role in the therapy, we investigated whether there was an induction of the adaptive immune response in the tdLN. At day 15, we found that the absolute number of activated CD8+CD44+ T cells (TACT), exhausted CD8+CD44+ Tim-3+PD-1+ T cells (TEX) and stem-like CD8+CD44+ Tim-3-PD-1+Slamf6+ T cells (TSL) significantly increased upon eMSC-FLT3L and poly(I:C) therapy (Fig. 4D, E). This effect was associated with an increase in proliferation, as probed by Ki67 staining, in both TCF1+CD44+ stem-like CD8+ and more differentiated TCF1−TIM3+ T cells (Supplementary Fig. 4A, B). Functionally, CD8+ T cells increased their capacity to secrete IFN-γ and effector to Treg-ulatory cells (Tregs) ratio increased (Fig. 4F, G and Supplementary Fig. 4C, D). Interestingly, we observed a significant increase in the absolute number of CD4+Foxp3− cells but not in Tregs (Supplementary Fig. 4D, E). Furthermore, CD4+Foxp3- cells of tumors treated with eMSC-FLT3L and poly(I:C) increased their secretion of IFN-γ (Supplementary Fig. 4F). Finally, the eMSC-FLT3L and poly(I:C) immunotherapy induced the expansion of tumor specific CD8+ T cells that recognize the OVA 257–264 immunodominant peptide, as shown with the tetramer staining (Fig. 4H, I). Altogether, we conclude that the therapeutic efficiency of eMSC-FLT3L and poly(I:C) associates with increased cross-priming of anti-tumor CD8+ T cells.

Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the infiltration of T and NK cells within tumor beds, that are required for immunotherapy efficiency

We next wondered if eMSC-FLT3L and poly(I:C) immunotherapy had an impact on the infiltration of intratumoral lymphocytes. First, we characterized lymphoid infiltrates at day 15 within B16-OVA tumors, treated with eMSC-FLT3L and poly(I:C) (Fig. 5A). We found that eMSC-FLT3L and poly(I:C) synergized to expand intra-tumoral NK1.1+ conventional NK cells (cNK) and CD62L−CD49a+ type 1 innate lymphoid cells (ILC1) (Fig. 5B, C). We also observed an increase in the proliferation of these two cell types, as probed by Ki67 staining (Fig. 5B, C). Furthermore, we noticed an increase in intratumoral CD8+CD44+ activated T cells (TACT) (Fig. 5D, E). By looking closer to the phenotype of these cells, we found that eMSC-FLT3L+poly(I:C) immunotherapy increased both CD8+CD44+TIM3+PD-1+ exhausted T cells (TEX) and CD8+CD44+TIM3−TCF1+ stem-like T cells (TSL) (Fig. 5D, E). Of note, the intratumoral abundance of TSL is associated to responses to immunotherapy in multiple cancer types41–43. Interestingly, expansion of TSL cells was associated with a significant increase in cell cycling activity, as probed by Ki67 staining (Supplementary Fig. 5A, B). By contrast, TEX cells displayed a high cycling activity both in control and treated groups. Strikingly, while intratumoral CD4+ T cells were increased and showed enhanced proliferation (Supplementary Fig. 5C, D), the abundance and cycling activity of intratumoral Tregs were not up-regulated following eMSC-FLT3L+poly(I:C) immunotherapy (Supplementary Fig. 5C, D). We next wondered whether these changes would associate to increased tumor-antigen-specific T cell response. We found that eMSC-FLT3L+poly(I:C) immunotherapy induced a striking expansion of tumor specific CD8+ T cells for the OVA 257-264 peptide, as detected by the tetramer staining (Fig. 5F) and the IFNγ-secreting T cells upon peptide-specific (SIINFEKL) ex vivo restimulation (Fig. 5G). Having established the local infiltration of tumor-specific T cells during the onset of tumor growth, we next wondered if the few survivor mice that had undergone eMSC-FLT3L+poly(I:C) immunotherapy displayed evidence of tumor-specific T cell memory. To test this, we injected the SIINFEKL synthetic peptide in tumor-free or B16-OVA survivor mice (d90 post primary tumor injection) (Fig. 5H). We found that this re-challenge induced the expansion of CD8+ T cells specific for the OVA 257–264 -H2Kb complex (Fig. 5I) in inguinal lymph nodes from survivor mice but not from tumor free naïve mice. Furthermore, those CD8+ T cells displayed a circulating effector memory phenotype (Fig. 5J). We conclude that eMSC-FLT3L+poly(I:C) immunotherapy induces a sustained memory CD8+ T cell response. Finally, in order to probe the requirement for conventional CD4 and CD8 T cells as well as NK cells, we performed eMSC-FLT3L+poly(I:C) immunotherapy in the presence of control, NK1.1 or CD4 and CD8 depleting antibodies, alone or in combinations (Fig. 5K). We found that both NK and CD4/CD8 contributed to the therapeutic effect of eMSC-FLT3L+ poly(I:C), as probed by the reversed inhibition of the tumor growth (Fig. 5L) and of the extension of survival (Fig. 5N). Importantly, inhibition of both NK and CD4/CD8 T cells achieved the maximal inhibition of eMSC-FLT3L+poly(I:C) immunotherapy. To address the involvement of CD8⁺ T cells and to differentiate total CD4⁺ T cells from Tregs, a second experiment was conducted in which CD8⁺, total CD4⁺, and Treg cells were individually depleted. CD8⁺ T-cell depletion confirmed their critical role, as evidenced by the loss of tumor control (Supplementary Fig. 5E) and reduced survival (Supplementary Fig. 5F). In contrast, depletion of CD4⁺ T cells or Tregs did not affect tumor growth or survival, indicating that CD8⁺ T cells are the primary mediators of the anti-tumor response in this context. Altogether, we conclude that eMSC-FLT3L+poly(I:C) immunotherapy activates and relies on both innate and adaptive anti-tumor immune response.

Intratumoral delivery of eMSC-FLT3L and poly(I:C) overcomes resistance to anti-PD-1/CTLA-4 immune checkpoint blockade

Lastly, we wondered if stimulation of T and NK-dependent anti-tumor immunity by eMSC-FLT3L and poly(I:C) immunotherapy would bypass resistance to immune checkpoint blockade (ICB). To test this, we submitted B16-OVA to a PD-1/aCTLA-4 ICB immunotherapy (Fig. 5N). We found that ICB slightly expanded the survival of mice as compared to isotype control-treated mice. However, mice treated with both ICB and eMSC-FLT3L+poly(I:C) immunotherapies exhibited a significant reduction of tumor growth (Fig. 5O) and had extended survival as compared to isotype-treated control groups (Fig. 5P). Of note, the combination of both ICB and eMSC-FLT3L+poly(I:C) improved eMSC-FLT3L+poly(I:C) alone in terms of survival, indicating a proper synergistic effect between both interventions.

Altogether, these data indicate that eMSC-FLT3L+poly(I:C) immunotherapy induce T and NK-dependent immunity against B16-OVA tumor and this defines a way to partially overcome resistance to ICB immunotherapy.

CXCR3 and CCR5 ligands are induced by poly(I:C) and control its impact on cDC1 and T cell infiltration

We next wondered which mechanism induced by poly(I:C) could explain the synergy between eMSC-FLT3L and poly(I:C). Since eMSC-FLT3L alone are able to recapitulate DC development from hematopoietic progenitors when engrafted in the dermis of tumor free mice (Fig. 1C and Supplementary Fig. 1B), we asked whether eMSC-FLT3L would also recapitulate DC development from DC progenitors within the TME. To test this, we engrafted eMSC-FLT3L together with bone marrow hematopoietic stem and progenitor cells (HSPCs), containing DC precursors and obtained from CD45.1 mice, within B16-OVA tumors (Fig. 6A). Three days after the engraftment of HSPCs, we found that CD45.1+ cDC1s had differentiated from progenitors co-engrafted with eMSC-FLT3L but not eMSC-ctrl (Fig. 6B). In the same tumors, eMSC-FLT3L did not increase cDC1s from the recipient mice, consistently with previous results (Fig. 6C). We conclude that availability of DC precursors and not specific features of the TME restrict the differentiation of pre-DCs into cDC1s within B16-OVA tumors. We next wondered if poly(I:C) would regulate the infiltration of pre-cDC1s via chemokine regulation. Using a chemokine multiplex array, we found that poly(I:C) up-regulated multiple chemokines within the TME of eMSC-FLT3L-treated tumors (Fig. 6D). This includes CXCL9 and CXCL11, which both act as ligands for the CXCR3 receptor, and CCL5, that acts as ligand for CCR544 (Fig. 6E). Notably, poly(I:C) monotherapy also elicited an upregulation of CXCL9, as evidenced by ELISA measurements performed on tumor homogenates (Supplementary Fig. 6A). CXCR3 and CCR5 are found on activated T and NK cells but also on circulating DC precursors4,45,46. To test the contribution of these chemokine signalling pathways to the eMSC-FLT3L+poly(I:C) therapy, we treated B16-OVA bearing mice receiving the therapy with CXCR3 and/or CCR5 antagonists (Fig. 6F). We found that CXCR3 and CCR5 inhibition prevented the anti-tumoral impact of eMSC-FLT3L+ poly(I:C) immunotherapy, as shown with the tumor growth (Fig. 6G) and the tumor weight (Fig. 6H). Furthermore, blockade of CXCR3 and/or CCR5 inhibited the accrual of intratumoral cDC1s (Fig. 6I) and the increase of activated CD8+ T cells within tdLN (Fig. 6J).

Altogether, these data highlight the crucial role of CXCR3 and CCR5 signalling pathways in mediating the immunotherapeutic effect of eMSC-FLT3L+poly(I:C) immunotherapy.

Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5 stimulates the infiltration of immature pre-cDC1s and cDC1s in WT and BRGSF human immune system (HIS) mice

We next reasoned that stromal cell-based delivery of CXCL9 and/or CCL5 could substitute the effect of poly(I:C) on eMSC-FLT3L therapy. To test this, we produce eMSCs engineered to express CXCL9 and CCL5, either alone or in combination with FLT3L (Fig. 7A). As confirmed by ELISA on culture supernatants, these cells secrete high concentrations of both chemokines when expressed alone or in combination (Fig. 7B, C). Then, we asked whether eMSC-FLT3L-CXCL9-CCL5 would allow the entry of circulating pre-cDC1s within B16-OVA tumors. To test this, we injected IRF8-GFP reporter mice with two tumors on either flank and injected eMSC-FLT3L on one side (left) and eMSC-FLT3L-CXCL9-CCL5 on the other side (right) (Fig. 7D). IRF8-GFP mice are useful to identify IRF8-expressing pre-cDC1s and cDC1s. Twenty four hours after the injection, we found that CCL5 and CXCL9 enriched tumors had an increased infiltration of pre-cDC1s, as defined by Lin-CD45RB+FLT3+MHCII+CD26+Ly6C−CD172a−XCR1−IRF8highSIRPa− cells (Fig. 7E). We next wondered if this infiltration of pre-cDC1s would translate into local cDC1 expansion at a later time point and following the same experimental design as previously but substituting poly(I:C) with the engineered cells (Fig. 7F). We found that tumors treated with eMSC-FLT3L-CXCL9-CCL5 had a significant increase in both the absolute number (Fig. 7G) and the frequency (Fig. 7H) of cDC1s compared with untreated tumors or those treated with eMSC-FLT3L alone. Furthermore, we noticed that upon the therapy, tumor cDC1s tend to be more activated, as shown by CD40 and CD86 up-regulation (Supplementary Fig. 7A). Altogether, these data show that stromal cells expressing CXCL9 and CCL5 can substitute the effect of poly(I:C) on cDC recruitment and maturation.

We then wondered whether eMSC-FLT3L-CXCL9-CCL5 co-localized with cDC1s within the tumors and whether they were more effective than eMSC-FLT3L in recruiting cDC1s. We injected eMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5 in Xcr1crexRosaLsL-tdTomato mice bearing melanoma tumors. In these mice, cDC1s are genetically labeled with tdTomato fluorescence. First, we found that cDC1s established contact with eMSCs at the border of tumor sites (Supplementary Fig. 7B). Next, we observed that cDC1s were more efficiently attracted by eMSC-FLT3L-CXCL9-CCL5 than by eMSC-FLT3L (Fig. 7I, J), underscoring the critical role of these chemokines in supporting interaction between FLT3+ cDC1s and membrane bound FLT3L-expressing eMSCs.

Since CCR5 and CXCR3 are expressed by activated T and NK cells4, we wondered if the infiltration of pre-cDC1s/cDC1s was indirect, due to the recruitment, at first, of activated lymphoid cells. To test this, we used BRGS mice that are deficient in murine T and B cells (Rag2−/−), murine NK cells (IL2rγ−/−)—and macrophage phagocytosis of human cells (SirpαNOD)47. BRGS mice were injected subcutaneously with basal membrane extract plugs containing either eMSC-ctrl (left flank), or eMSC-FLT3L-CXCL9-CCL5 (right flank). (Fig. 7K). First, we found that eMSC-FLT3L-CXCL9-CCL5 triggered a local accumulation of cDC1s in the absence of lymphocytes (Fig. 7L). We next wondered if this response was actually dependent on the FLT3 receptor tyrosine kinase that is required for the homeostasis of tissue cDC1s in non-lymphoid tissues24. To test this, we used BRGSF mice deficient in FLT3 (BRGSF Flt3−/−)48. We observed no cDC1 recruitment in mice deficient in tissue cDCs, both in control and treated plugs (Fig. 7L). From these results, we conclude that the recruitment of cDC1 induced by eMSC-FLT3L-CXCL9-CCL5 is independent on B, T and NK/ILCs cells but dependent on FLT3 signalling. Finally, we wondered whether our findings would be transferable to human settings. To test this in controlled experimental settings, BRGSF mice were engrafted with CD34+ HSPCs from human cord blood to create HIS mice. Seventeen-week-old BRGSF HIS mice showed multi-lineage human immune reconstitution comprising B, T, NK and DC. Reconstituted BRGSF HIS mice were injected subcutaneously with basal membrane extract plugs containing either eMSC-Ctrl (left flank) or eMSC-FLT3L-CXCL9-CCL5 (right flank) (Fig. 7M). After 10 days, mice were sacrificed, and the cellular infiltrates populating the plugs in the dermis of the mice were analysed by flow cytometry (Fig. 7N). We found that plugs containing eMSC-FLT3L-CXCL9-CCL5 had a significant increase in human cDC1s, as compared to plugs containing eMSC-ctrl, both in absolute number (Fig. 7O) and frequency (Fig. 7P). Of note, similar trends were observed for human cDC2s (Supplementary Fig. 7C, D).

Altogether, we conclude that the engraftment of eMSC expressing FLT3L, CXCL9 and CCL5 represent an efficient platform to promote the recruitment and expansion of cDC1s within tissues and that the system is amenable to human cDC1s.

Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5 stimulates the infiltration of activated CD4 and CD8 T cells in the tumor

We next wondered if the secretion of CXCL9 and CCL5 would also impact the attraction of activated T cells. To test this, we first set-up an in vitro migration assay in Boyden chambers, to study the chemo-attraction of activated polyclonal CD4+ and CD8+ T cells towards eMSCs expressing CCL5 and/or CXCL9 (Fig. 8A). As expected, after 3 hours of migration, we observed a synergistic effect of eMSC-CCL5 and eMSC-CXCL9 in their ability to attract activated CD4+ (Fig. 8B) and CD8+ T cells (Fig. 8C). We then wanted to know if this attraction could allow the recruitment of activated T cells in B16-OVA tumors. To test this, we injected eMSC expressing CCL5 and/or CXCL9 in the tumors and adoptively transferred ex vivo activated polyclonal CD45.1 T cells intravenously (Fig. 8D). A synergistic effect between eMSC-CCL5 and eMSC-CXCL9 was again observed in their ability to recruit activated CD4+ (Fig. 8E) and CD8+ (Fig. 8F) CD45.1 T cells in the tumor. In addition, we observed that eMSC-CCL5 and eMSC-CXCL9, either individually or in combination, tended to promote the infiltration of endogenous activated CD44+ T cells, particularly the CD44+ CD8+ T-cell subset (Fig. 8G, H). We conclude that eMSC-CXCL9 and eMSC-CCL5 represent a workable platform to deliver locally bioactive chemokines.

Lastly, we wanted to assess if the eMSC-FLT3L-CXCL9-CCL5 immunotherapy would have an impact on the infiltration of intratumoral lymphocytes. To test this, we injected eMSC-FLT3L-CXCL9-CCL5 in B16-OVA tumors and looked at a later time point the immune infiltrate (Fig. 8I). We observed that tumors treated with eMSC-FLT3L-CXCL9-CCL5 were more infiltrated with NK, ILC1, CD4+ and CD8+ T cells (Fig. 8J). Of note, absolute number of activated and exhausted CD8+ T cells were also increased in treated tumors.

Altogether, these data show that eMSC-FLT3L-CXCL9-CCL5 immunotherapy induce the recruitment of lymphoid cells in B16-OVA tumors.

Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5 recapitulates anti-tumor immunity

Having shown that eMSC-FLT3L-CXCL9-CCL5 immunotherapy had an impact on the infiltration of immune cells in the tumors, we wondered if it would also confer anti-tumor immunity. To test this, we did three injections of eMSC-FLT3L-CXCL9-CCL5 in B16-OVA tumors (Fig. 9A). We observed a significant reduction in tumor growth (Fig. 9B), tumor size (Fig. 9C) and tumor weight (Fig. 9D) at a later time point.

Finally, we wanted to assess if the eMSC-FLT3L-CXCL9-CCL5 immunotherapy would also overcome resistance to ICB, by treating the mice with or without aPD-1/aCTLA-4 immunotherapy (Fig. 9E). We found that even if ICB alone reduced the tumor growth (Fig. 9F) and expand the survival of mice (Fig. 9G), the synergy of both eMSC-FLT3L-CXCL9-CCL5 and ICB was needed to eradicate the tumor in a fraction of treated mice (Fig. 9G).

Altogether, these data indicate that eMSC-FLT3L-CXCL9-CCL5 immunotherapy activates anti-tumor immunity and partially overcomes resistance to ICB.

Methods
Mice
Mice were housed at the INSERM U1149 CRI, Medicine school site Bichat or at the Paris Institut Pasteur animal house facilities. Wild type C57BL/6J and CD45.1 (CByJ.SJL(B6)Ptprca/J) mice were purchased from Janvier and kept in our facility. Irf8-GFP (B6.Cg Irf8tm2.1Hm/J), Xcr1cre (B6 Xcr1tm1Ciphe), ROSA26-LSL-RFP (B6.Cg Gt(ROSA)26Sortm1Hjf/J) and ROSA26-LSL-DTA (B6.129P2 Gt(ROSA)26Sortm1(DTA)Lky/J)77 mice were kindly provided by Marc Dalod ROSA36-LSL-tdTomato (B6.Cg Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) were kindly provided by Dr. Tessa Bergsbaken. Xcr1DTA, Xcr1RFP, Xcr1tdTomato mice were generated as described40. OT-I Rag2−/− CD45.1 mice were kindly provided by Dr. Sebastian Amigorena. BALB/c Rag2−/− IL2rγ−/− SirpαNOD (BRGS), BALB/c Rag2−/− IL2rγ−/− SirpαNOD Flt3−/− (BRGSF) mice and BRGSF-HIS mice were provided by the Human Disease Model Core facility (Institut Pasteur, Paris). The study was approved by the local ethics committee (Comité d’éthique Paris Nord n°121 or CEEA89) and the French Ministry of Education and Research under the authorization number APAFIS#15373 and APAFIS#48865. Animal care and treatment were conducted with national and international laws and policies (European Economic Community Council Directive 86/609; OJL 358; December 12, 1987). All experiments were performed in specific pathogen-free animal facilities, under controlled environmental conditions with ambient temperature maintained at 22±2°C, a relative humidity between 40 and 60% and on a 12h light/dark cycle, in accordance with the Federation of European Laboratory Animal Science Association (FELASA) guidelines, institutional guidelines and the French law.

BRGSF humanized immune system mice (BRGSF-HIS)
Briefly, cord blood CD34+ cells (Biological Resource Center (CRB)-Banque de Sang de Cordon, AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, Paris, France) were isolated with affinity matrices according to the manufacturer’s instructions (Miltenyi Biotec) and subsequently phenotyped for CD34 and CD38 expression. Newborn (5–9 days old) BRGSF mouse pups received sublethal irradiation (3 Gy) and were injected intra-hepatically with 3×105 CD34+ human cord blood cells. Twelve and 16 weeks after graft, reconstitution of the human immune system was confirmed by analysis of human CD45, CD19, CD3, CD4, and CD8 expression on live leukocytes in the blood using flow cytometry. All animal studies were approved by the Pasteur Institute Safety Committee in accordance with French and European Guidelines (Committee for Ethics in Animal Experimentation 240006) and validated by the French Ministry of Education and Research (APAFIS#49211-2024042612197924v3). The use of human CD34⁺ cells was ethically and scientifically approved, as the CRB providing these samples is AFNOR-certified under the ISO 20387 standard. Furthermore, the scientific project was approved by the CRB management and a scientific board. Lastly, sample transfer for research purposes was authorized by the French Ministry of Higher Education and Research and the Île-de-France Regional Health Agency (authorization AC-2024-6640). Written informed consent was obtained from all patients, and a formal agreement between AP-HP and the Pasteur Institute governed the transfer and use of these samples.

Tumor cell lines
B16-OVA and YUMM-OVA cell lines were generated by lentiviral transduction using a pCDH_CMV7-OVA-EFI-G418 plasmid. TC-1-Luc cells were kindly provided by Dr. Alexandre Boissonnas, E0771 cells were kindly provided by Stéphanie Hugues, and MC38 cells were kindly provided by Dr. Philippe Bousso. B16F10, B16-FLT3L78, B16-OVA and TC-1-Luc cell lines were cultured in RPMI Medium 1640 (ThermoFisher Scientific) supplemented with GlutaMAX, 10% heat-inactivated fetal bovine serum (FBS) (ThermoFisher Scientific), 50μM β-mercaptoethanol (ThermoFisher Scientific) and 100U/mL penicillin-streptomycin (ThermoFisher Scientific). YUMM-OVA, MC38, and E0771 cell lines were cultured in DMEM (ThermoFisher Scientific) supplemented with GlutaMAX, 10% heat-inactivated FBS (ThermoFisher Scientific), 50μM β-mercaptoethanol (ThermoFisher Scientific) and 100U/mL penicillin-streptomycin (ThermoFisher Scientific). Cells were maintained at 37°C and 5% CO2.

Tumor models
For the grafted tumor model, tumor cells were harvested during the exponential growth phase using 0.05% Trypsin-EDTA (Gibco), washed twice in PBS, and counted using trypan blue exclusion. 8-to 12-week-old male (B16-OVA, MC38, YUMM-OVA) and female (E0771, TC-1) mice were subcutaneously inoculated with 5×105–1×106 tumor cells suspended in 100μL of not supplemented RPMI or DMEM medium. Tumor dimensions were recorded three times per week using a digital caliper, and tumor volume (V) was calculated using the formula V=(L×W×W)/2, with volume expressed in mm³. Mice were monitored daily, and endpoints were established based on signs of distress, including pain, lethargy, dehydration, ulceration, or tumor necrosis. To generate survival curves, mice were sacrificed at ethical endpoints, the main one being the tumor volume (2000 mm3). In some cases, this limit has been exceeded the last day of measurement, and the mice were immediately euthanized.

Generation of engineered mesenchymal stromal cells
Generation of mesenchymal stromal cells. Mesenchymal stromal cells (MSC) were generated from mouse embryonic fibroblasts (MEFs) from individual C57/BL6 embryos at embryonic day 13 and were kindly provided by Loredana Saveanu. Practically, the embryo body was digested with 0.05% trypsin and 0.02% EDTA in Hanks balanced salt solution at 37°C for 30 min. The single cell suspension obtained after digestion was resuspended in high glucose DMEM medium containing 10% FCS, 2mM L-glutamine, 100U/ml penicillin and 100μg/ml streptomycin and seeded on 0.1% gelatin (Sigma G1890) coated tissue culture dishes at 5×104 cells/cm2. Cells growing on the gelatin-coated dishes were harvested, and cell passages were repeated in DMEM medium containing 10% FCS, 2mM L-glutamine, 100U/ml penicillin and 100μg/ml streptomycin.

Generation of adipose-derived mesenchymal stromal cells. Method was adapted from Dr. Geneviève Marcelin. Inguinal and visceral fat of C57BL/6 mice were cut in small pieces and digested with 6–8 mL of digestion mix, containing high glucose DMEM (ThermoFisher Scientific), 1mg/mL collagenase A (Roche), resuspended in HBSS (ThermoFisher Scientific), 100U/mL HEPES (ThermoFisher Scientific), 100U/mL penicillin/streptomycin (ThermoFisher Scientific) and 1mg/mL BSA. After 40 min digestion at 37°C, the digestion was mechanically ended with a syringe and a needle of 18G. Cell suspension was then filtered on a 100 µm cell strainer and centrifuged. Floating adipocytes and red blood cells (ACK lysis, ThermoFisher Scientific) were removed. Cells were enriched in CD45-Ter119- cells using a StemCell Magnet and biotin anti-CD45 and anti-Ter119 antibodies. Enriched cells were then cultured in an adherent flask with DMEM (Gibco®) supplemented with GlutaMAX, 20% heat-inactivated FBS (Gibco®), 50μM β-mercaptoethanol (ThermoFisher Scientific) and 100U/mL penicillin-streptomycin (ThermoFisher Scientific). Cells were maintained at 37°C and 5% CO2.

Engineering of mesenchymal stromal cells. Human FLT3L, CXCL9 and CCL5 were amplified by PCR from previously obtained or commercial cDNA (Horizon Discovery) and cloned into pMX retroviral vectors (detailed in the Supplementary Table 1). To generate viral particles, the Platinum-E (Plat-E) Retroviral Packaging Cell Line (kindly provided by Dr. Laurie Menger) was transfected with 1,2 µg plasmid, 21 mM CaCl2 and HEPES-Buffered Saline (HBS, homemade solution). MSCs were then transduced with the viral supernatant and 8µg/mL polybrene (Santa-Cruz Biotechnology). Transduced MSCs were selected using 2μg/mL puromycin (ThermoFisher Scientific) and sorted on their expression of membrane bound FLT3L (antibody details provided in the Supplementary Table 1) and/or their expression of eGFP or mCherry reporters. Engineered MSCs were cultured in DMEM (Gibco®) supplemented with GlutaMAX, 10% heat-inactivated FBS (Gibco®), 50μM β-mercaptoethanol (ThermoFisher Scientific), 2µg/mL puromycin (ThermoFisher Scientific) and 100U/mL penicillin-streptomycin (ThermoFisher Scientific), under standard culture conditions of 37°C and 5% CO2. MSCs expressing the nano-luciferase were obtained by transducing MSCs with a lentiviral vector kindly provided by Laleh Majlessi.

In vitro experiments
T cell isolation, activation and polarization. CD4⁺ and CD8⁺ T cells were isolated from the draining lymph nodes and spleens of 10–12-week-old CD45.1 mice. Purification was performed using negative selection with a biotin-conjugated antibody cocktail, followed by anti-biotin magnetic beads and separation with a Miltenyi magnet and LS columns (Miltenyi Biotec). For in vitro stimulation, purified T cells were cultured on plates coated with anti-CD3 (2μg/mL; clone 17A2) and anti-CD28 (2μg/mL; clone 3751; BioLegend) in the presence of recombinant IL-2 (5μg/mL; BioLegend). To promote Th1 differentiation, cultures were supplemented with recombinant IL-12 (10ng/mL; BioLegend) and blocking antibody anti-IL-4 (5µg/mL; clone MP4-25D2; BioLegend).

Migration assay. Cell migration assays were conducted using 24-well transwell inserts with 5-μm pore polycarbonate membranes (Corning). Engineered mesenchymal stromal cells (eMSCs) expressing CCL5 or CXCL9 were seeded at a density of 250,000 cells per well in the lower chamber. Once the eMSCs reached confluency, the culture medium was replaced with serum-free DMEM. A total of 100,000 CD45.1 Th1-polarized T cells were resuspended in serum-free DMEM, and 100μL of the cell suspension was carefully added to the upper chamber of the Transwell insert. The upper chamber was then gently placed into the lower chamber using sterile forceps, ensuring proper immersion in the culture medium. The plate was incubated at 37°C for 3h. Following incubation, the supernatant was collected to quantify T-cell migration by flow cytometry.

In vivo experiments
HSPC differentiation in membrane extract plugs. Immunomagnetic negative selection was used to isolate mouse stem and progenitor cells (HSPC) from CD45.1 mice using the Mouse Hematopoietic Progenitor Cell Isolation Kit (StemCell), following the manufacturer’s instructions. A total of 104 hematopoietic stem and progenitor cells (HSPCs) per plug were subcutaneously injected alongside engineered mesenchymal stromal cells (eMSCs) at a 1:50 ratio (HSPC/eMSC) in 200μL of ice-cold Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher). Mice were euthanized on day 12 of differentiation via cervical dislocation, and GelTrex plugs were excised for further analysis. The plugs were sectioned and enzymatically digested in HBSS (Life Technologies) containing 1% FBS, 0.37 U/mL Collagenase D (Roche), 10μg/mL DNase I (Roche), and 1mg/mL Dispase (Sigma Aldrich) at 37°C for 30 min. Following digestion, the tissue was smashed on a 100-μm strainer (Corning), and the resulting cell suspension was collected and resuspended in FACS buffer for flow cytometry analysis. For experiments involving recombinant FLT3L, human FLT3L was used at a concentration of 100ng/mL (Celldex).

In vivo treatment with engineered stromal cells. Intratumoral injections of eMSCs started when the tumors were between 50 and 100mm3. Mice were treated with 5×105–1×106 eMSC-FLT3L, in combination with 25µg/mouse of poly I:C LMW (InVivoGen). We designed our intratumoral cell-based therapy as two injections of eMSC-FLT3L (day 7 and day 12) and one injection of poly(I:C) (day 9). Mice that were later treated with 5×105–1×106 eMSC-FLT3L-CXCL9-CCL5 received three injections of eMSCs. In the latest experiments, mice developed tumors with the desired injectable size few days later than the initial schedule. Thus, the start of injections was postponed but the number and frequencies of injections was kept.

In vivo bioluminescence. A total of 1×106 eMSC-NanoLuc cells were administered intratumorally into palpable B16-OVA tumors. Mice were imaged using the IVIS Spectrum CT 2 In Vivo Imaging System 4h after injection and again at days 4 and 8. Prior to imaging, mice were anesthetized and received an intravenous injection of 0.44µmoles fluorofurimazine substrate (Promega). On day 8, mice were sacrificed, and various organs were collected and imaged ex vivo to assess bioluminescence. Photon flux was quantified using the Living Image 4.8.2 software. Regions of interest (ROIs) were quantified as average radiance (photons/s/cm2/sr) represented as color-scaled images superimposed on grayscale photos of mice using Living Image software.

In vivo treatment with recombinant FLT3L. Intratumoral injections of recombinant FLT3L started when the tumors were between 50 and 100mm3. Mice were treated 4 times (every 2 days) with 10µg/mouse of recombinant FLT3L (Celldex).

Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) assay and hematoxylin-eosin coloration. Serum was collected 48h after poly(I:C) intratumoral or anti-CD40 (100µg/mice, clone FGK 4.5) intraperitoneal injection. Blood was allowed to clot at room temperature for 30 min and centrifuged two times at 1000×g for 10 min. ALT and AST levels were measured using the ALT/GPT kit (ThermoFisher Diagnostics) following the manufacturer’s instructions. Samples were run on a Konelab analyzer (ThermoFisher Scientific). Liver of the same mice were harvested and put in 4% paraformaldehyde (ThermoFisher Scientific) overnight. The next day, livers were transferred in 70% ethanol, paraffin embedded, cut, and stained with hematoxylin and eosin (H&E) at the histology platform of the Pasteur Institute. Slides were scanned using the Axioscan 7 (Zeiss).

Immune checkpoint blockade (ICB) treatment. Mice received intraperitoneal (i.p.) injections of 200μg anti-PD-1 (clone 29F.1A12, BioXCell) and 200µg of anti-CTLA-4 (clone 9H10, BioXCell). An isotype antibody (clone 1–113, BioXCell) and IgG control (clone MPC11, BioXCell) was used as a control. All antibodies were administered intraperitoneally at treatment initiation and subsequently every 2 days until reaching 5 injections.

CD4, CD8, CD25 and NK cells depletion. For the depletion of CD4⁺ T cells, CD8⁺ T cells, Tregs and NK1.1⁺ cells, mice were treated with a first dose of 500μg of anti-CD4 (clone GK1.5, BioXCell), anti-CD8 (clone 2.43, BioXCell), anti-CD25 (clone PC-61.5.3, BioXCell), and anti-NK1.1 (clone PK136, BioXCell). As controls, 300μg of IgG2a isotype (clone C1.18.4, BioXCell) and 500μg of rat IgG2b isotype (clone LFT-2, BioXCell) were used. All antibodies were administered intraperitoneally at the initiation of treatment and subsequently every 3 days at a dose of 200µg, until reaching 4–6 injections.

CXCR3 and CCR5 antagonists treatments. Mice received intraperitoneal injections of either maraviroc (500µg/mouse, MedChemExpress), a CCR5 antagonist, or AMG 487 (200µg/mouse, MedChemExpress), a CXCR3 antagonist. Both agents were dissolved in a vehicle of dimethyl sulfoxide (DMSO). Administration started at the initiation of treatment and continued every two days until day 15, where samples were collected for flow cytometry analysis.

In vivo re-challenge OVA peptide in mice. Survivor mice that fully rejected B16-OVA tumors treated with eMSC-FLT3L+poly(I:C) underwent in vivo restimulation via subcutaneous injection of 1µg OVA 257–264 peptide (SIINFEKL, IBA Lifesciences) dissolved in 100µl phosphate-buffered saline (PBS). This experiment was conducted 90 days after primary tumor injection. The injection site was located in the dorsal flank region. Three days post-restimulation, mice were euthanized, and inguinal draining lymph nodes were excised for subsequent analysis.

Adoptive T cell transfer. 1×105 eMSCs were injected in B16-OVA tumors at day 7, 9 and 12. At day 9, 2×106 Th1-polarized CD45.1 T cells (previously described) were intravenously administered.

Synthetic niches (plugs). 1×106 eMSC-ctrl or eMSC-FLT3L-CXCL9-CCL5 were injected subcutaneously (s.c.) in 200µL solution, containing 150µL of Geltrex LDEV-free reduced growth factor basement membrane matrix and 50µL of PBS. The synthetic niches (plugs) were harvested and analyzed by flow cytometry 10 days after the injection.

Mouse tissues processing
Murine tumors, tumor-draining lymph nodes (LNs), spleens, or synthetic niches were harvested and subjected to enzymatic digestion. Tissues were incubated in 3mL of digestion buffer, comprising Hank’s Balanced Salt Solution (HBSS, ThermoFisher Scientific) supplemented with calcium and magnesium (ThermoFisher Scientific), 75µg/mL Collagenase D (Roche), and 0.02 mg/mL DNase I (Roche), at 37°C for 30 min. Single-cell suspensions were then generated by smashing the digested tissues through 70µm cell strainers (Corning). Spleen red blood cells were lysed using ACK lysis buffer (ThermoFisher Scientific). The absolute number of live immune cells in each tissue cell suspension was assessed using AccuCheck Counting Beads (ThermoFisher Scientific) along with anti-CD45 and DAPI stainings on BD LSR-Fortessa X20, BD Symphony A5 (BDBiosciences) or spectral SONY ID7000 (SONY).

Flow cytometry analysis and fluorescent-activated cell sorting
After tissue processing and cell counting, Fc-receptor were blocked using Fc Block for 20 min at 4°C. Viability was assessed using 7-AAD (Biolegend), Zombie dyes (BioLegend), or Live/dead fixable stains (ThermoFisher Scientific). Extracellular staining was performed in FACS buffer (PBS (Life Technologies), 3% FBS (ThermoFisher Scientific), and 2mM EDTA (Life Technologies)) during 30 min at 4°C. Labelling with H2Kb-SIINFEKL tetramers (MBL) was performed at room temperature for 30 min. For intra-cellular stainings, single cell suspensions were then fixed during 30 min and stained during 30 min using a fixation/permeabilization kit (eBiosciences, transcription factor staining buffer). For intra-cellular IFNγ staining, single-cell suspensions were first restimulated ex vivo with 2µM OVA257–264 peptide (SIINFEKL, IBA Lifesciences) for 5h at 37°C in 5% CO2, followed by a 4-h incubation with 5µg/mL Brefeldin A (Sigma Aldrich). Cells were subsequently labeled with surface antibodies, fixed either with the Cytofix/Cytoperm kit (BD, cytokines stainings) or the Transcription Factor Staining Buffer Set (ThermoFisher Scientific, nuclear intracellular stainings), and stained with an intracellular cytokine staining (ICS) antibody panel. A comprehensive list of antibodies used is provided in Supplementary Table 1. Flow cytometry data were acquired using BD LSR-Fortessa X20, BD Symphony A5 (BD Biosciences) or ID7000 Spectral (Sony). Cells sorting was performed using a BD FACSMelody™ Cell Sorter equipped with BD FACSDiva software. Data were analyzed with FlowJo software.

Multiplex chemokines analysis
Dissected tumor samples were weighted and homogenized in 1mL of 1× phosphate-buffered saline (PBS) supplemented with protease inhibitor, using a Fisherbrand™ 150 handheld homogenizer. Homogenates were then centrifuged at 14,000×g for 15 min, and the resulting supernatants were collected. The concentrations of pro-inflammatory chemokines within the supernatants were quantified using the LEGENDplex™ MU Proinflammatory Chemokine Panel 1 and 2 (BioLegend), following the manufacturer’s instructions. Samples were analyzed by flow cytometry on a BD FACS LSRFortessa. Data were analyzed using LEGENDplex™ software (BioLegend). Chemokine concentrations were normalized to total protein concentration, which was estimated using the initial tumor mass.

Immunofluorescence
1×106 YUMM-OVA tumor cells were injected in the flank of Xcr1RFP or Xcr1tdTomato mice. Intratumoral injection of eMSC-ctrl, eMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5 started when tumors were between 50 and 100mm3. Mice were sacrificed two days later. Protocol was adapted from Laforêts et al.79. Tumors were harvested and embedded in 5% agarose. 300–350µm thick slices of non-fixed fresh samples were obtained using a vibratome (LEICA, VT1200S), with blade speed between 0.5 and 0.75 mm/s and the amplitude at 1 mm. Samples were then collected in phenol-red free DMEM medium, containing 10% FBS, 1% penicillin/streptomycin and 4mM L-glutamine. Live tumor samples were then put in a 35mm glass bottom dish, filled with the before mentioned medium. Images were taken with a Nikon Ti2E, equipped with a 20× objective, a spinning disk and a temperature-controlled chamber at 37°C. Images were analyzed with Fiji software. To quantify the number of cDC1s surrounding each eMSC, a threshold was applied to each channel of every image, based on the maximum intensity projection, generating one channel containing cDC1s and another containing eMSCs. For eMSC-FLT3L-CXCL9-CCL5, which co-express Cherry and GFP, cDC1s were isolated by subtracting the green channel from the red, while eMSCs were obtained by merging the two channels. The number of cDC1s around each eMSC was then manually determined.

Statistical analysis
Statistical analysis was performed using Prism 10 (GraphPad Software Inc., USA). Statistical analysis and verification of normal distribution were performed using GraphPad Prism software. Outliers were detected using the ROUT method in GraphPad Prism (Q=0,5%) and subsequently removed from the dataset prior to analysis. For comparisons of two groups, two-tailed paired or unpaired Student’s t test was used. When three or more groups were compared, a one-way or two-way analysis of variance (ANOVA) test was used. Precise tests are described in the figure legends. A p-value of less than 0.05 was considered as significant. The number of biological as well as experimental replicates is indicated in figure legend. Data are presented as means±SD or SEM, box and whiskers, or violin plots as described in the figure legends. SD standard deviation; SEM standard error of the mean.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work by Gorline et al. investigates the use of a cell therapy-based approach to remodel the TME through the delivery of FLT3L as a means to enhance the presence of XCR1+DC1+ cells in the tumor vicinity. Well designed mechanistic studies were also conducted and identified CXCR3/CCR5 ligands as major players in the induction of an anti-tumoral effect. The paper is well laid-out overall.\nAlthough the study will have a great impact on the field, it does have some pitfalls that need to be addresses prior to publication. Here are the major points that need to be worked-out:\n1 - The authors refer to the used cells as mesenchymal stromal cells (MSCs) yet, there is no evidence provided to prove that these cells are indeed MSCs, which is surprising! For instance, the authors refer to these cells as \"fibroblastic cell lines\", which indicates that they are not MSCs. The authors should therefore clarify this point. If these are indeed MSCs, then they need to show their phenotypic analysis (CD31-, CD44+, CD45-, CD73+, CD90+ and CD105+).\n2 - What is the expression profile of PD-1 and PD-L1 on these modified cells? This is important as pro-inflammatory signals will trigger or enhance the expression of these ICs on the cell surface.\n3 - The authors gene-engineered their cells to express FTL3L and other chemokines. They should however provide evidence that there is no autocrine loop taking place whereby the gene-engineered cells express the cognate receptors and respond to the produced ligands.\n4 - In figure 1, the authors seem to compare human to murine FLt3L. Is that correct? If so, why is that?\n5 - The authors always show tumor growth up to day 15. This is not impressive at all. Why not showing the growth until day 30 or 40?\n6 - By looking at Fig. 1L and N, one can say that the tumors that seem to be controlled by the eMSC-FLT3L + Poly I:C have regrown, which goes with my previous concern.\n7 - Why choosing the B16F10 model? The authors should validate their therapeutic in vivo potency in at least 2 additional tumor models.\n8 - The authors used the term cross-priming to refer to CD8 T-cell activation. Usually this process occurs if a given antigen is cross-presented. Have the authors provided any evidence for that?\n9 - Why are TIM3-TCF1+CD8+ Tpex referred to as \"exhausted\". No convincing data are presented in that regard.\n10 - Why are surviving animals not rechallenged with tumors?\n11 - Are the eMSCs presented in Fig. 6A expressing FLT3L?\n12 - Fig. 6B/C are missing numbers on the Y axis. Plz fix it.\n13 - Figure 6M is confusing. We see percentages between 80-100% in all groups, which is high! The authors should better explain this figure and its relation to Fig. 6N and O.\n14 - Fig. 8F is disappointing especially if compared to the first set of in vivo data in Fig. 1. Once would have expected better tumor control at that point... In fact, the authors should combine the eMSC-FLt3L + Poly I:C with the anti-PD1 and anti-CTLA4 to see if this combo therapy induces stronger or more effective anti-tumoral responses.\n15 - The discussion is very thin and needs more development within the context of cancer immunotherapy. In this current format, it looks more like a summary of findings. Plz discuss the drawback, limits, pitfalls of this technology and put it in a clinical context or compare it to other experimental approaches.\n16 - Why was the use of MSCs in the context of cancer vaccination not discussed? There are series of papers describing the use of gene-engineered or pharmacologically-reprogrammed MSCs in the context of cancer vaccination. Such concept should be discussed as well.", "reviewer_2": "This manuscript by Dr. Guermonprez and colleagues presents an elegant and innovative strategy for enhancing anti-tumor immunity through intra-tumoral delivery of engineered mesenchymal stromal cells expressing FLT3L and the chemokines CXCL9 and CCL5. The experiments are well-designed, and the mechanistic insights into cDC1 recruitment and T cell priming are compelling. I particularly appreciated the use of both mouse and humanized models, which enhances the translational relevance of the findings.\nNevertheless, a few key points could be addressed to strengthen the manuscript:\n1. Cellular source of chemokines: The cellular source of CXCL9 and CCL5 following poly(I:C) treatment remains undefined. Further characterization of this axis would help clarify the underlying mechanism.\n2. Safety considerations: Although systemic cytokine levels appear unaffected, the long-term biodistribution, persistence, and potential immunogenicity of the eMSCs warrant additional safety evaluation.\n3. Model generalizability: The study would benefit from validation in additional tumor models to demonstrate generalizability beyond B16-OVA. This model is somewhat peculiar, as most OVA-expressing tumors are highly immunogenic and are often spontaneously rejected in immunocompetent mice. This limitation could be addressed in a follow-up study in which intra-tumoral delivery of FLT3L-expressing eMSCs is tested in other transplantable or spontaneous tumor models.\n4. Mechanistic synergy with checkpoint blockade: The authors should discuss more thoroughly the mechanism by which this therapy synergizes with checkpoint blockade.\n5. Clinical implications: The authors should discuss better the translational potential of eMSC therapy, as the intratumor route of delivery would not be amenable to most human cancers\nAddressing these points will enhance the impact of the study. However, given the novelty and important implications of the work, I recommend the manuscript undergo minor revision prior to acceptance.\nMinor specific points:\nIn the experiments shown in Figures 3B, S2, and S3, the observed effects on cDC1 maturation and T cell activation could be attributed to poly I:C treatment alone. The authors should include a comparison between eMSC + poly I:C and poly I:C alone to demonstrate that the presence of eMSCs is essential for enhancing cDC1 maturation and IFN-γ secretion by T cells.", "reviewer_3": "The manuscript by Gorline et al analyses the anti-cancer efficacy of engineered mesenchymal stem cells expressing factors that enhance DC and T/NK cell activation/infiltration in a mouse tumour model harbouring an ectopic immunodominant antigen. The use of humanized mice/human cells is highly appreciated and the authors perform a comprehensive analysis of the majority of relevant aspects of DC-mediated induction of anti-cancer T cell immunity. The authors basically report the effective repurposing of existing knowledge, the combination of a previously reported system (eMSCs) with known anti-cancer factors for the treatment of cancer in mice. Despite an eventual lack of novelty, the manuscript is clearly interesting from a translational point of view.\nIn light of this, the choice of mouse cancer models that are most closely resembling human disease to demonstrate therapeutic efficacy may be of high relevance. Unfortunately, the manuscript practically exclusively relies on the B16-OVA cancer model. Moreover, several treatment controls are missing, which are relevant to understand if the efficacy of the new treatment strategy developed by the authors outcompetes previously used therapies. If those points are strengthened (see my suggestions to do so below), the manuscript will be a valuable addition to current efforts to improve cancer immunotherapy.\nMain comment 1 – only one pre-clinical cancer model expressing an ectopic immuno-dominant antigen is used in the manuscript\nThe strongest suit of the present manuscript is its therapeutic relevance. The key findings of every Figure should, therefore, be demonstrated in one or more cancer model that closely resembles human disease. Ideally not a cell line (for example spontaneous models, if possible, or at least engraftment of primary cancer cells) and certainly not cancer cells expressing ectopic antigens.\nI believe the only Figure that didn’t use B16-OVA is Figure 1F-J with B16/F10 tumours, which is an experiment to “exclude” undesired effects. Probably in exactly such “excluding experiments”, the authors should be consistent in tumour model and use B16-OVA.\nMajor comment 2 – missing controls (especially known factors as mono-treatment) and no repetition of experiments throughout the manuscript\nIntratumoral injection of PolyI:C and it’s clinically approved derivatives is a frequently explored approach and known to be effective in pre-clinical mouse models – via a plethora of pathways (PMID: 31046839, 34824158, 34531246, 40079116). The authors only explore chemokine induction by polyI:C, which raises a few questions on the effects of polyI:C (dependent or not on eMSC-FLT3L combination treatment):\n(1) How do eMSC-FLT3L compare with intratumoral injection of rec-huFLT3L in combination with polyI:C?\n(2) Figure 4A-D and O-Q: the eMSC-FLT3L and, most importantly, the reference-control polyI:C mono-therapy groups are missing. That is important because many of those effects have previously been shown to be induced by polyI:C alone (see citations above)\n(3) Figure 5D – is the upregulation of CXCL9, CXCL11 and CCL5 unique to eMSC-FLT3L + poly(I:C) or also found be polyI:C mono-treatment?\nMany experiments using treatment with eMSC-FLT3L-CXCL9-CCL5 are missing the eMSC-FLT3L control, for example:\n(1) Figure 6D-I: are those untreated compared to treated tumors? The control of eMSC-FLT3L-treated tumours is missing, especially for Fig. 6H-I.\n(2) Figure 6L-O: highly appreciated, but probably eMSC-FLT3L would have been the appropriate control here? I understand this is very challenging to repeat, but could the authors explain their choice of control here?\nFinally, the majority of experiments have only been performed once. Every experiment (especially in vivo tumour-treatments) have to be performed 2 independent times to generate robust and reproducible data.\nMajor comment 3 – some conclusions should be revised and may require further testing\nThe unique induction of activated and non-exhausted CD8+ T cells in the eMSC-FLT3L+ PolyI:C-treated group is not clear, because a lot of data from the other groups are omitted/not shown. Also, it is not clear if the efficacy of eMSC-FLT3L+ PolyI:C treatment is CD8+ T cell dependent or Treg cell dependent. Please consider the following for a better understanding of the effects of the therapy:\n(1) Figure 3F – please add a quantification of all experimental groups – not only the untreated and combo\n(2) Figure 3G – please add a quantification of Tregs and IFNgamma-producing CD8+ T cells for all experimental groups – not only the ratio with CD8 T cells\n(3) In Figure 4K-N, CD8 blockade should be tested alone to confirm an involvement of CD8 T cells. Also, to distinguish total CD4 from Treg cells, please block Tregs specifically using Foxp3-ko mice or a blocking antibody (for example anti-CD25).\nAdditional comments\nIn their explanations on conventional DCs (cDCs) the authors should probably adhere to the currently established nomenclature as much as possible and use the abbreviations cDC1 and cDC2 (instead of DC1 and DC2) to prevent confusion for the reader.\nDefine what “rec-huFLT3L” is. I understand recombinant human Flt3L.\nThe authors assess “Intra-tumoral delivery of eMSC-FLT3L” in Figure 1A-J. However, the experimental setup is a co-injection of tumour cells and MSCs from the beginning. The caveat of this strategy should be stated.\nAs the treatment with eMSC-FLT3L is of a transient nature, how to the authors envisage clinical translation? Please discuss and explain.\nFigure 4 is missing panel M\nFigure 4D – add percentages to representative FACS plot please\nResults from Figure 4H-J should be reinterpreted: it is clear that tumour-experienced mice will develop a T cell memory and it is not clear how much the therapy contributed to that. For confirmation, tumour-resected mice would be necessary. And many more markers than displayed in Figure 4J would be necessary to confirm a resident-memory phenotype. I do not think this is a necessary message of the manuscript, but please omit any statements like “We conclude that eMSC-FLT3L + poly(I:C) immunotherapy induce local, systemic and long- term immunity against tumor antigens in the CD8+ T cell compartment” throughout the manuscript.\nFigure 7D-F: could the authors also show non-transferred, endogenous T cell infiltration into tumours of this experiment? That would be more relevant than quantifying adoptively transferred T cells.\nFigure 7G-H: at day 17, tumours already have a significantly different size (according to Figure 8). Hence, the differences in T cell infiltration could be a consequence rather than a cause. T cell infiltration into tumours should be analyzed at an earlier time-point, when tumours have equal sizes between groups.\nThe authors state: From these results, we conclude that the recruitment of DC1 induced by eMSC-FLT3L-CXCL9-CCL5 is independent on B, T and NK/ILCs cells but dependent on FLT3 signalling. Yet, FLT3L overexpression alone is not sufficient to induce cDC1 recruitment by itself. This is puzzling and the authors should comment on this. To decipher this and understand if Flt3L is actually required (but not sufficient), the authors could consider to show if eMSC-CXCL9-CCL5 (without FLT3L expression) are able to recruit cDC1s to tumours (but that is not vital for the manuscript).\nFigure 8E-F: a tumor model that is less susceptible to aPD1/aCTLA4 blockade should be used to evaluate synergy with eMSC-FLT3L-CXCL9-CCL5 treatment. I don’t think this is necessary for the manuscript and the data could be removed."}, "author_response": "Point-by-point response to the reviewers\nWe s inc ere ly t hank th e re vie wers for t heir th oro ugh an d c ons tructive eval ua tion of ou r\nman uscript. In this point-b y-po int re ply, we a ddress ea ch of the r evi ewer s’ comme nts\nin detail, wh ich have h elp ed us to substa nt ially improve the q uality of o ur work. The\\au thor s’ response s are writte n in blue , and t he rev ised or new ly added text in the\nman uscrip t i s hi ghligh ted in green . Figures (inc luding su pplem enta l fig ures) ar e\nnumbe red ac cordin g to th eir order of ap pearance in the revised manusc ript, with\nthe correspon ding num ber s of the ori ginal figure s i ndica ted aft er, where applic ab le.\nREVIEWER COMMENTS\nReviewer #1:\nThe work by Gorline et al. investigates the use of a cell therapy-based approach to\nremodel the TME through the delivery of FLT3L as a means to enhance the presence\nof XCR1+DC1+ cells in the tumor vicinity. Well-designed mechanistic studies were also\nconducted and identified CXCR3/CCR5 ligands as major players in the induction of an\nanti-tumoral effect. The paper is well laid-out overall.\nWe would like to sincerely thank the reviewer for their positive and encouraging\nassessment of our work. We greatly appreciate the recognition of our study’s design\nand the mechanistic insights into the role of CXCR3/CCR5 ligands in mediating the\nanti-tumoral effect.\nAlthough the study will have a great impact on the field, it does have some pitfalls that\nneed to be addresses prior to publication. Here are the major points that need to be\nworked-out:\n1- The authors refer to the used cells as mesenchymal stromal cells (MSCs) yet, there\nis no evidence provided to prove that these cells are indeed MSCs, which is surprising!\nFor instance, the authors refer to these cells as \"fibroblastic cell lines\", which indicates\nthat they are not MSCs. The authors should therefore clarify this point. If these are\nindeed MSCs, then they need to show their phenotypic analysis (CD31-, CD44+,\nCD45-, CD73+, CD90+ and CD105+).\nWe appreciate the reviewer’s insightful comment and the opportunity to clarify this\npoint. We fully acknowledge that the terminology surrounding mesenchymal stem cells\nand mesenchymal stromal cells (MSCs) can lead to confusion. In our study, we\nspecifically used mesenchymal stromal cells (MSCs) as a platform for cell-based\ndelivery. We chose this broader terminology because it encompasses a variety of cell\ntypes and origins.\nIn our work, we engineered primary mouse embryonic fibroblasts (MEFs) that were\npropagated from individual C57BL/6 embryos at embryonic day 13. These cells are\ncharacterized as CD45⁻, CD31⁻, CD44⁺, CD73⁻, CD90.2⁻, CD140a⁺, and\ngp38/PDPN⁺, as shown in the histograms provided in the supplementary figure S1A.\nStromal cells from inguinal fat pad were used as positive control in those experiments.\nNonetheless, it is difficult to ascertain negativity for CD73 and CD90, as the protein\ncould have been cleaved by trypsin during cell detachment prior to analysis.\nInguinal\nfat pad\neMSCs\nCD45 CD31 CD44 CD73 CD90.2 CD140a gp38/PDPN\nInguinal\nfat pad FMO\neMSCs eMSCs\nCCR5 CXCR3 FLT3 PD-L1 PD-1\nSup Figure 1: Phenotypic characterization and biological activity of eMSCs\n(A) Phenotypic analysis by flow cytometry of eMSCs using various classical and\nconventional markers for stromal and stem cells.\n2- What is the expression profile of PD-1 and PD-L1 on these modified cells? This is\nimportant as pro-inflammatory signals will trigger or enhance the expression of these\nICs on the cell surface.\nWe thank the reviewer for this comment. We fully agree that assessing PD-1 and PD-\nL1 expression is relevant to understand the immunomodulatory potential of our\nmodified cells. Additional flow cytometry analysis on cultured cells revealed that these\nmodified cells do not express PD-1 nor PD-L1, as shown by the histograms we added\nin the supplementary figure S1A (see above). We did not, however, assess PD-1 or\nPD-L1 expression on eMSCs after their injection into tumors. Since these cells rapidly\ndecline after injection (see figure 1D-H), their presence and effect are transient.\nTherefore, even if they were to express these markers, it is unlikely that this would lead\nto immunosuppression.\n3- The authors gene-engineered their cells to express FTL3L and other chemokines.\nThey should however provide evidence that there is no autocrine loop taking place\nwhereby the gene-engineered cells express the cognate receptors and respond to the\nproduced ligands.\nWe thank the reviewer for this comment. Additional flow cytometry analysis revealed\nthat the eMSCs do not express FLT3 (or CXCR3 and CCR5), as shown by the\nhistograms we added in the supplementary figure S1A (see above), indicating that no\nautocrine loop could take place. These findings are in line with the general notion that\nFLT3 is a receptor tyrosine kinase restricted to the hematopoietic lineage and that\nCXCR3 and CCR5 are important for lymphocyte traffic.\n4- In figure 1, the authors seem to compare human to murine FLT3L. Is that correct? If\nso, why is that?\nWe would like to clarify that only human FLT3L was used throughout the study. We\ndeliberately chose the human ligand because it displays high cross-reactivity between\nhuman and murine FLT3 receptors, making it suitable for use in our mouse model. In\naddition, using cells that already express human ligands facilitates subsequent human\nproof-of-concept studies in humanized mice. We acknowledge that the labeling in our\nfigure may have been misleading. To avoid any confusion, we have removed the “hu”\ndesignation from the first figure in the revised version of the manuscript.\n5- The authors always show tumor growth up to day 15. This is not impressive at all.\nWhy not showing the growth until day 30 or 40?\nMost untreated mice reached the ethical endpoint by day 15 (tumor volume of 1500 or\n2000mm3, depending on mouse facility and ethical agreements). Therefore, tumor\ngrowth curves are shown only up to the last time point when all groups contained the\nsame number of mice, ensuring fair and reliable comparison between groups. For a\nlonger follow-up, we included survival data, which provide complementary information\non treatment efficacy (see figure 2D, previously figure 1N). We show that eMSC-\nFLT3L immunotherapies actually increase survival alone (see figure 2D, previously\nfigure 1N), or in combination with immune checkpoint blockade (see figure 5P, 9G,\npreviously figure 4Q and 8G).\n6- By looking at Fig. 1L and N, one can say that the tumors that seem to be controlled\nby the eMSC-FLT3L + Poly I:C have regrown, which goes with my previous concern.\nWe agree with the reviewer’s observation. Indeed, only a small proportion of mice\nachieved complete tumor eradication following treatment. In most cases, the eMSC-\nFLT3L + poly(I:C) therapy led to a marked delay in tumor growth but did not result in\nfull tumor elimination. Consequently, tumors tended to regrow once the treatment was\ndiscontinued. These findings indicate that while the therapy exerts a strong anti-tumor\neffect, it may require combination with additional therapies, such as the immune\ncheckpoint blockades (see figure 5P, previously figure 4Q), to achieve more durable\ntumor control and partial complete response.\n7- Why choosing the B16F10 model? The authors should validate their therapeutic in\nvivo potency in at least 2 additional tumor models.\nWe thank the reviewer for this comment, and we agree that validating the therapeutic\nefficacy of the therapy in additional tumor models would strengthen the paper.\nTherefore, eMSC-FLT3L + poly(I:C) therapy was validated in three other tumor models:\nthe breast cancer E0771 (CRL-3461), the colon carcinoma MC38 (Corbett et al. 1975)\nand the lung cancer TC-1 cell line (Lin et al. 1996). In each of this model, eMSC_FLT3L\nand poly(I:C) immunotherapy delayed tumor growth (see figure 2L, P, T) and improved\nsurvival (see figure 2N, R, V). These data have been added to a new figure, now\nfigure 2.\n25 20\n15\n10\n5\nexpressing membrane bound FLT3L stimulates anti-tumor immunity in the\npresence of poly(I:C).\n(K, O, S) Experimental design to evaluate anti-tumoral effects of eMSC-FLT3L and\npoly(I:C) in MC38 (K), TC-1 (O) and E0771 (S) tumor models.\n(L, P, T) Tumor growth curves (n=6-8 mice per group, one experiment)\n(M, Q, U) Individual tumor size variation compared to the day of the first eMSC-FLT3L\ninjection. (n=6-8 mice per group, one experiment, *p < 0.05, **p < 0.01 two-way\nANOVA-test with Šídák’s multiple comparisons test).\n(N, R, V) Survival curves. (n=6-8 mice per group, one experiment, *p < 0.05, log-rank\n(Mantel-Cox) test).\n8- The authors used the term cross-priming to refer to CD8 T-cell activation. Usually\nthis process occurs if a given antigen is cross-presented. Have the authors provided\nany evidence for that?\n+\nWe agree that we did not formally demonstrate that CD8 T cells specific for tumor-\nassociated antigens would depend on cross priming. Indeed, it could be hypothesized\n+\nthat MHCI-peptide complexes formed on tumor cells could be transferred to XCR1\ncDC1s for the activation of tumor-specific T cells. This process is termed as cross\ndressing. To test this possibility, we attempted to develop a model of tumor devoid of\nnoitairav\nezis\nromuT\n8D ot\nderapmoc\n71D\nta\n30 0.1141 100\n20\n50 10\n0 0\n0 20 40 60\n**\nDays post TC-1 injection\n0 1020304050\nDays post E0771 injection\n)³mm(\nemulov\nromuT\nUntreated\neMSC-FLT3L + poly(I:C)\n2000\n100 *\n1500\n1000\n50\n500\n0 0\n10 20 30 40 50\nDays post E0771 injection\nnoitairav\nezis\nromuT\nnoitairav\nezis\nromuT\n6D ot\nderapmoc\n71D\nta\n21D\not derapmoc\n32D\ta\nL M N\n100\n50\n0\n0 10 20 30 40\nDays post MC38 injection\nP Q R\nT U V\nlavivrus\ntnecreP\n2000 *\n1500\n1000\n500\n0\n0 10 20 30\nDays post MC38 injection\n)³mm(\nemulov\nromuT\n2500 2000\n1500\n1000\n500\n0\n0 20 40 60 Untreated\nDays post TC-1 injection eMSC-FLT3L + poly(I:C)\n)³mm(\nemulov\nromuT\n15\n10\n5\n*\neMSC-FLT3L + poly(I:C)\nlavivrus\ntnecreP\nlavivrus\ntnecreP\nK\ns.c.MC38\nD8 D13 D17 D24\nC57BL/6 D10 D20\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nO\ns.c.TC-1\nD6 D11 D14 D20\nC57BL/6 D8 D17\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nS\nC57BL/6 D12 D17 D21 D29\ns.c.E0771D14 D25 i.t.eMSC-FLT3L\ni.t.Poly(I:C)\nof the OVA model antigen enabling to track the T cell response. However, unfortunately,\nthese tumor cell lines were spontaneously rejected, preventing the possibility to\nperform our experiment. Nonetheless, the studies that highlighted a role of cross-\ndressing identified that it was a cDC1-independent process taking place in interferon-\nactivated type 2 cDCs (Duong et al. 2022). By contrast, cDC1-associated genes like\n+\nWDFY4 control cross presentation by cDC1s and the cross priming of CD8 T cells\nmediating tumor control (Theisen et al. 2018). Altogether, we assume that cDC1-\ndependent tumor control (see figure 3D-E, previously figure 2D-E) is afforded by cross\npriming by cDC1s.\n9- Why are TIM3-TCF1+CD8+ Tpex referred to as \"exhausted\". No convincing data are\npresented in that regard.\nTpex stands for precursor of exhausted cells, as mentioned in the text, which refers to\n+\nCD8 T cells possibly giving rise to exhausted cells expressing multiple inhibitory\nreceptors (PD1, TIM3, LAG3) and loosing polyfunctional cytokine response upon TCR\nrecall. To avoid confusion, we have termed these cells as T for stem-like T cells in\nSL\nthe revised manuscript.\n10- Why are surviving animals not rechallenged with tumors?\nThis is an interesting suggestion. Given the limited number of mice that completely\ncleared their tumors, we were unable to assess both tumor-specific memory T-cell\nresponses and the functional efficacy of this memory. Therefore, we re-challenged the\nsurviving animals with the SIINFEKL peptide to determine whether long-term immune\nmemory had been established in protected mice. As shown by data in figure 5I\n(previously figure 4I), protected mice (and not control, naïve animals), displayed potent\n+\nmemory CD8 T cell responses against a tumor-associated antigen. We conclude that\n+\ntumor control is concomitant to the activation of tumor specific CD8 T cell memory\nresponses.\n11- Are the eMSCs presented in Fig. 6A expressing FLT3L?\nTo clarify this point, several engineered cell types were generated: eMSC-FLT3L,\neMSC-CXCL9, eMSC-CCL5, and eMSC-FLT3L-CXCL9-CCL5. In this figure,\nconstructs expressing either eMSC-CXCL9 or eMSC-CCL5 were initially shown. To\nprovide a more complete picture and avoid any misunderstanding, the results obtained\nwith all the different constructs were also included in the figure 7A-C (previously figure\n6A-C).\nA\nFigure 7: Intratumoral delivery of eMSC co-\nφ expressing FLT3L, CXCL9 and CCL5\n5'LTR CCL5 IRES GFP 3'LTR\nφ stimulates cDC1 infiltration in mice and\n5'LTR CXCL9 IRES GFP 3'LTR\nφ humanized mice.\n5'LTR FLT3L IRES mCherry 3'LTR\nφ (A) Constructions used to produce eMSC-\n5'LTR CXCL9 P2A CCL5 IRES GFP 3'LTR\nCCL5, eMSC-CXCL9 and eMSC-FLT3L-\nCXCL9-CCL5.\n(B-C) Quantification by ELISA of CCL5 (B)\nand CXCL9 (C) released by the eMSCs\ntransduced with the various ligands. (n=2-6 for\nthe quantification of CCL5 and n=3-8 for the\nquantification of CXCL9, one experiment).\n12– Fig. 6B/C are missing numbers on the Y axis. Plz fix it.\nMissing numbers were added.\n13-Figure 6M is confusing. We see percentages between 80-100% in all groups, which\nis high! The authors should better explain this figure and its relation to Fig. 6N and O.\nWe have used 3 antibodies to characterize human cDC1s: CD141, BTLA and XCR1.\nOf note, XCR1 can be upregulated during the terminal differentiation of cDC1s in\ntissues. As can be seen, the percentage of cDC1s expressing XCR1 within\n+ +\nCD141 BTLA is high in both eMSC-containing control (100%) vs eMSC-FLT3L-\nCXCL9-CCL5 containing plugs (92%). However, the % of cDC1s within total CD45+\nis highly increased in eMSC-FLT3L-CXCL9-CCL5 group as compared to eMSC-control\n(see figure 7P, previously figure 6O). Indeed, the treated eMSC-FLT3L-CXCL9-CCL5\ngroup consistently shows higher percentages throughout the gating steps (see figure\n7N, previously figure 6M), supporting the overall trend and the results shown in figure\n7O-P (previously figure 6N-O).\n14- Fig. 8F is disappointing especially if compared to the first set of in vivo data in Fig.\n1. Once would have expected better tumor control at that point... In fact, the authors\nshould combine the eMSC-FLt3L + Poly I:C with the anti-PD1 and anti-CTLA4 to see\nif this combo therapy induces stronger or more effective anti-tumoral responses.\nenikomehC\n)Lm/gp(\nB C\nCCL5 CXCL9\n800\n600\n400\n200\nCXCL9 - - + - + - - + - +\nCCL5 - - - + + - - - + +\nYes, indeed! And the result can be found in figure 5N-P (previously figure 4O-Q). The\neMSC-FLT3L + poly(I:C) immunotherapy indeed induces stronger and more effective\nanti-tumoral responses when combined with ICB, as compared with the eMSC-FLT3L-CXCL9-CCL5 treatment. This outcome is somewhat expected, as poly(I:C) not only\npromotes chemokine secretion but also activates multiple signaling pathways,\nincluding the induction of type I IFNs downstream TLR3 in cDC1s and MDA5 in other\ncells. Type I IFN signaling, in turn activates the immunogenic maturation of cDCs\n(Longhi et al. 2009) (see figure 3B-C, 4B-C, previously figure 2B-C, 3B-C).\n15- The discussion is very thin and needs more development within the context of\ncancer immunotherapy. In this current format, it looks more like a summary of findings.\nPlz discuss the drawback, limits, pitfalls of this technology and put it in a clinical context\nor compare it to other experimental approaches.\n16- Why was the use of MSCs in the context of cancer vaccination not discussed?\nThere are series of papers describing the use of gene-engineered or pharmacologically\nreprogrammed MSCs in the context of cancer vaccination. Such concept should be\ndiscussed as well.\nWe agree with the comment. The discussion has been completely rewritten,\nhighlighting: i) the specific advantage of eMSCs for the local delivery of FLT3L and\nother factors within tumors; ii) practical and technological bottleneck in the road to\ntranslate the system to an immunotherapeutic intervention in clinical settings. Please,\nfind below in green the revised paragraphs, taking these comments into account.\n“In this study, we developed a novel immunotherapeutic platform based on\nthe intratumoral engraftment of ex vivo–engineered, autologous mesenchymal\nstromal cells (eMSCs). In the first part, we demonstrate a potent therapeutic synergy\nbetween eMSCs expressing a membrane-bound form of FLT3 ligand (FLT3L) and\nthe adjuvant poly(I:C). These findings are consistent with previous reports showing\nthat combined administration of recombinant FLT3L and poly(I:C) enhances cDC1\nexpansion and maturation, promotes tumor regression, and improves\nresponses to immune checkpoint blockade (Salmon et al. 2016; Hammerich et al.\n2019; Oba et al. 2020). However, systemic delivery or secretion of FLT3L has\nbeen shown to perturb hematopoiesis and induce peripheral and intratumoral Treg\nexpansion (Darrasse-Jze et al. 2009; Swee et al. 2009). Moreover, the\nrequirement for repeated injections of poly(I:C) and/or FLT3L in these studies raises\nsafety and feasibility concerns for clinical translation. This calls for the development\nof delivery methods limiting FLT3L protein production to the TME.\nHere, we show that intratumoral delivery of eMSCs expressing membrane bound\nFLT3L elicits potent anti-tumor immunity while restricting FLT3L activity to the\ntumor site, thereby avoiding systemic toxicities. Treated mice exhibited no elevation\nin serum FLT3L levels or Treg frequencies, and no evidence of weight loss or liver\ninjury, suggesting that this localized, cell-based strategy mitigates systemic adverse\neffects commonly associated with DC-targeted immunotherapies (Siwicki et al.\n2021). Autologous eMSC-FLT3L proposes an alternative option to the intratumoral\ninjection of recombinant FLT3L (Salmon et al. 2016), viral vectors encoding FLT3L\n(Snchez-Paulete et al. 2018), CAR-T cell transduced FLT3L (Lai et al. 2020) and\nHerpes Virus oncolytic virus encoding FLT3L (Wan et al. 2025). Indeed, local retention\nof eMSC has the advantage to restrict expression of FLT3L to the TME, a feature not\nafforded by CAR T cell mediated systemic delivery (Lai et al. 2020). In addition, our\nsystem, in contrast to viral vectors, is not susceptible to immunity.\nThis study expands the therapeutic scope of engineered mesenchymal stromal\ncells (eMSCs) to include modulation of the dendritic cell (DC) lineage. Previous studies\nhave shown that IL-2–based, T cell–targeting eMSCs confer substantial benefits by\npromoting the expansion of non-exhausted PD-1⁺TIM-3⁻ CD8⁺ T cells (Stagg et al.\n2004; Bae et al. 2022). Likewise, eMSCs engineered to express type I interferons or\nIL-12 have been reported to enhance the infiltration and cytotoxic effector functions of\nintratumoral CD8⁺ T cells (Ryu et al. 2011; Choi et al. 2017; ułach et al. 2021; Zhang\net al. 2022) and NK cells (Ren et al. 2008). It remains to be determined whether eMSCs\nthat act directly on lymphocytes could synergize with DC-targeting eMSCs such as\nthose developed in this study. Exploring such combinatorial strategies may further\namplify both antigen presentation and effector responses, providing a versatile\nplatform for next-generation cell-based immunotherapies.\nAn important finding of this study is that elevation of FLT3L levels alone in the\nTME is not sufficient to induce DC recruitment and bring immunotherapeutic benefits.\nWe found that poly(I:C) addition synergizes with eMSC-FLT3L. Therefore, we next\ninvestigated the mechanisms underlying the enhanced efficacy of the eMSC-FLT3L +\npoly(I:C) therapy and identified a marked upregulation of multiple chemokines within\nthe tumor microenvironment (TME), notably CXCL9 and CCL5, which serve as ligands\nfor CXCR3 and CCR5, respectively. Pharmacological blockade experiments\ndemonstrated that both CXCR3 and CCR5 are essential for the intratumoral\naccumulation of cDC1s and for the establishment of effective antitumor immunity. To\nminimize the potential toxicities associated with poly(I:C), we replaced poly(I:C) by the\nco-expression of CXCL9 and CCL5 on eMSC-FLT3L. This substitution similarly\nenhanced the infiltration of cDC1s, T cells, and NK cells, while maintaining robust\nantitumor activity. These results align with previous studies highlighting the role of\nCXCR3- and CCR5-mediated recruitment in sustaining immune effector infiltration into\nthe TME (Chow et al. 2019; Dangaj et al. 2019). Importantly, although CXCR3 and\nCCR5 are expressed on circulating pre-DCs (Cook et al. 2018) as well as on activated\nT and NK cells, our findings indicate that T and NK cells are dispensable for the\nCXCL9/CCL5-driven recruitment of DCs. It is known that intratumoral chemokines may\nderive from multiple sources, including NK cells and cDC1s themselves (Böttcher et\nal. 2018; Chow et al. 2019; Meiser et al. 2023) and are essential to support the\nsustained infiltration of cDC1 within the TME. Here, we show that this pathway of cDC1\nrecruitment associated to immunogenic tumors is activatable in the context of poorly\nimmunogenic tumors by eMSC-FLT3L-CCL5-CXCL9 engraftment. We demonstrate\nthat local chemokine release by eMSC increase the interaction of cDC1s with eMSC-\nFLT3L. Translatability of the approach to human cDC1s is demonstrated because\neMSC-FLT3L-CCL5-CXCL9 treatment supports the local recruitment of human cDC1s\nin the dermis of BRGSF immunodeficient mice reconstituted with human CD34⁺\nhematopoietic stem cells. However, the identity of the circulating progenitor population\nresponding to this therapy remains to be elucidated, as several candidate precursors\nhave been proposed as human equivalents of murine pre-DCs (Lee et al. 2015; Villani\net al. 2017; See et al. 2017).”\n“The prospect of locally implanted, eMSCs acting as versatile, sustained\nfactories for therapeutic molecule delivery is highly attractive for cancer\nimmunotherapy. This is particularly relevant for chemokines, which generate spatially\norganized gradients governing leukocyte trafficking and tissue compartmentalization.\nNonetheless, several technological and translational challenges must be addressed to\nrealize the clinical potential of eMSCs.\nFirst, standardized and GMP-compatible protocols for autologous eMSCs\nisolation, engineering, and production must be established. Such methods should\nensure reproducible recovery of stromal populations of controlled complexity that are\namenable to ex vivo expansion, viral transduction, and post-transduction enrichment.\nAlthough the present study primarily employed murine embryonic fibroblasts as an\nMSC source, we demonstrate that adipose-derived stromal cells can be expanded,\ntransduced, and used to generate immunologically competent eMSCs. Other groups\nhave successfully implemented bone marrow–derived eMSCs for immunotherapy\n(Stagg et al. 2004; Ren et al. 2008; ułach et al. 2021; Zhang et al. 2022). Validation\nin human systems will be critical for clinical translation. Furthermore, the phenotypic\nheterogeneity resulting from ex vivo expansion and transduction remains to be fully\ncharacterized. Despite homogeneous transgene expression (e.g., via IRES-linked\nfluorescent reporters), we can not exclude that this cellular preparation is functionally\nheterogeneous and that only a subset of eMSCs may be functionally active in vivo.\nIntegration of single-cell RNA sequencing and spatial transcriptomics in eMSC-treated\ntumors could provide essential insight into stromal diversity, fate, and functional states.\nSecond, optimization of eMSC delivery methods could improve therapeutic\npersistence and efficacy. In our current model, intratumoral injection of cell\nsuspensions resulted in transient eMSC persistence, with only a minor fraction\ndetectable beyond ten days. Future studies should aim at enhancing eMSC residency\nthrough tissue engineering strategies, including synthetic scaffolds or extracellular\nmatrix integration (Choi et al. 2017). Cell-intrinsic manipulation of stress pathway might\nalso offer some opportunities to improve the survival of eMSC in the TME. For instance,\nNQO1 expression might improve resistance to oxidative stress (Bae et al. 2022).\nWould these manipulations improve persistence, it would be important to determine\nwhether prolonged eMSC survival improves T and NK cell–dependent tumor control.\nWhile intratumoral delivery provides localized control, it may not be feasible for all\ncancer types. Developing systemic delivery approaches for eMSCs could extend\ntherapeutic reach to deep or inaccessible lesions while improving patient tolerability.\nIndeed, intravital imaging studies have shown that intravenously administered MSCs\nexhibit intrinsic tropism for sites of tissue remodeling, including wounds and solid\ntumors (Kidd et al. 2009; Kalimuthu et al. 2017). The molecular mechanisms underlying\nthis selective homing remain poorly defined but could be exploited to deliver eMSCs to\notherwise unreachable tumor sites.\nThird, eMSC-based therapies designed to stimulate DC-dependent antitumor\nimmunity must also incorporate cues that promote antigen release and DC maturation,\nthe two essential steps required to initiate the cancer-immunity cycle. In our study,\neMSCs expressing FLT3L and CXCL9/CCL5 effectively enhanced local DC infiltration\nbut did not efficiently drive DC maturation, potentially limiting migration to tumor-\ndraining lymph nodes and cross-priming of CD8⁺ T cells. Embedding maturation-\ninducing signals within eMSCs may overcome this limitation. In parallel, promoting\nlocal tumor cell death could enhance antigen availability for DC uptake. One promising\navenue involves combining eMSCs with oncolytic virotherapy, which induces\nimmunogenic cell death and antigen release. eMSCs have been widely explored as\ndelivery vehicles for oncolytic viruses, leveraging their tumor-homing capacity\n(Pereboeva and Curiel 2004; Hakkarainen et al. 2007). This approach has been\nimplemented for several viral platforms, including herpes simplex virus (Du et al. 2017),\nmyxoma virus (Josiah et al. 2010), Newcastle disease virus (Kazimirsky et al. 2016),\nmeasles virus (Sonabend et al. 2008), and adenovirus (Sonabend et al. 2008; Yoon et\nal. 2019). We propose that eMSC-based oncolytic virotherapy could be synergistically\ncombined with DC-targeting eMSCs, as suggested by Svensson-Arvelund et al., who\nobserved cooperation between recombinant FLT3L administration and NDV-mediated\noncolysis (Svensson-Arvelund et al. 2022). Moreover, type I IFN–releasing eMSCs\ncould complement DC-directed eMSCs to further enhance local immune activation\n(Ren et al. 2008; Choi et al. 2017; Zhang et al. 2022).\nIn summary, while technical and regulatory limitations remain, the translational\npotential of eMSC-based immunotherapies is considerable. Their inherent tumor\ntropism, compatibility with ex vivo genetic engineering, and capacity for localized,\nsustained delivery make eMSCs a promising cellular platform for next-generation\ncombination immunotherapies aimed at amplifying both innate and adaptive antitumor\nimmunity.”\nReviewer #2:\nThis manuscript by Dr. Guermonprez and colleagues presents an elegant and\ninnovative strategy for enhancing anti-tumor immunity through intra-tumoral delivery of\nengineered mesenchymal stromal cells expressing FLT3L and the chemokines CXCL9\nand CCL5. The experiments are well-designed, and the mechanistic insights into cDC1\nrecruitment and T cell priming are compelling. I particularly appreciated the use of both\nmouse and humanized models, which enhances the translational relevance of the\nfindings.\nWe sincerely thank the reviewer for their positive and encouraging comments. We\ngreatly appreciate their recognition of our experimental design, the mechanistic\ninsights into cDC1 recruitment and T cell priming, as well as the translational relevance\nprovided by the use of both mouse and humanized models.\nNevertheless, a few key points could be addressed to strengthen the manuscript:\n1- Cellular source of chemokines: The cellular source of CXCL9 and CCL5 following\npoly(I:C) treatment remains undefined. Further characterization of this axis would help\nclarify the underlying mechanism.\nWe thank the reviewer for bringing this interesting question. While identifying the\nprecise cellular source of CXCL9 and CCL5 following poly(I:C) treatment would indeed\nbe interesting, it is beyond the scope of the present study, which focuses on the\nfunctional impact of engineered eMSCs on cDC1 proliferation and maturation and T\ncell activation. We therefore did not investigate this aspect in the current work. Multiple\nstudies identify DCs and cDC1 specifically, as important producers of CXCL9\n(Spranger et al. 2017; Chow et al. 2019; Dangaj et al. 2019). However, multiple sources\nmight participate to CCL5 production including T cells, N cells and macrophages\n(Böttcher et al. 2018). These aspects have been added to the discussion. Please, find\nbelow in green the revised paragraphs, taking this comment into account.\n“To minimize the potential toxicities associated with poly(I:C), we replaced\npoly(I:C) by the co-expression of CXCL9 and CCL5 on eMSC-FLT3L. This substitution\nsimilarly enhanced the infiltration of cDC1s, T cells, and NK cells, while maintaining\nrobust antitumor activity. These results align with previous studies highlighting the role\nof CXCR3- and CCR5-mediated recruitment in sustaining immune effector infiltration\ninto the TME (Chow et al. 2019; Dangaj et al. 2019). Importantly, although CXCR3 and\nCCR5 are expressed on circulating pre-DCs (Cook et al. 2018) as well as on activated\nT and NK cells, our findings indicate that T and NK cells are dispensable for the\nCXCL9/CCL5-driven recruitment of DCs. It is known that intratumoral chemokines may\nderive from multiple sources, including NK cells and cDC1s themselves (Böttcher et\nal. 2018; Chow et al. 2019; Meiser et al. 2023) and are essential to support the\nsustained infiltration of cDC1 within the TME. Here, we show that this pathway of cDC1\nrecruitment associated to immunogenic tumors is activatable in the context of poorly\nimmunogenic tumors by eMSC-FLT3L-CCL5-CXCL9 engraftment. We demonstrate\nthat local chemokine release by eMSC increase the interaction of cDC1s with eMSC-\nFLT3L.”\n2- Safety considerations: Although systemic cytokine levels appear unaffected, the\nlong-term biodistribution, persistence, and potential immunogenicity of the eMSCs\nwarrant additional safety evaluation.\nWe agree that this is a key question, that was overlooked in the first version of the\nmanuscript.\ni) eMSC biodistribution: To further assess the long-term biodistribution and\npersistence of eMSCs, nano-luciferase–expressing eMSCs were generated\nand monitored for 8 days in the B16-OVA model using bioluminescence\nimaging. We observed that eMSCs persisted within the tumor for at least 8\ndays, although at very low levels, and that their activity remained restricted\nto the tumor site. These findings suggest that, once injected into the tumor,\neMSCs do not disseminate throughout the body but rather remain localized.\nThese data have been added to the figure 1F-H.\nPBS eMSC-NanoLuc\nD0\n4\nD4\n2\n1\ni.t.eMSC-NanoLuc\ni.v. Fluorofurimazine D8\nImaging Radiance\n(p/s/cm2/sr)\n108\n107\n106\n105\n0 4 8 Days post eMSC-NanoLuc\ninjection within B16-OVA tumors\necnaidar\negarevA\n)rs/2mc/s/p(\n601x\nRadiance\n(p/s/cm2/sr)\n701x\nHear L t iv K e i r dn S ey plee L n ung T s uu mo tdL M N uscle\necnaidar\negarevA\n)rs/2mc/s/p(\nG H\n3\nB16-OVA 1\n1.5 x 106\n1 x 106\n0.5 x 106\nexpressing membrane bound FLT3L does not alter intratumoral and systemic\ncDC1 populations.\nAltogether, we conclude that eMSC do not induce a detectable level of systemic\ninflammation.\n3000\ns.c.B16-OVA\nD7 D9 D11 2000\nC57BL/6 Serum 1000\ni.t.eMSC-FLT3L analysis\ni.t.poly(I:C)\ni.p. anti-CD40 0\nFigure 2: Intratumoral engraftment of autologous mesenchymal stromal cells\nexpressing membrane bound FLT3L stimulates anti-tumor immunity in the\npresence of poly(I:C).\n)L/U(\nTLA\nG\nE F ****\n****\n10\n****\n5 ****\n0\n-5\n-10\n-15\nDays post B16-OVA injection eMSC-FLT3L - + - + -\nPoly(I:C) - - + + -\nanti-CD40 - - - - +\n)%(\negnahc\nthgiew\nydoB\neMSC-FLT3L\npoly(I:C)\nUntreated eMSC-FLT3L + poly(I:C) anti-CD40 ** ** * *\n(F) Experimental design to evaluate the biodistribution and persistence of eMSCs\nwithin B16-OVA tumors. 1x106 eMSC-NanoLuc were injected i.t. on day 0. On days 0\n(4h after the injection), 4, and 8, mice received an intravenous injection of . μmoles\nof the fluorofurimazine substrate, followed by bioluminescence imaging using the IVIS\nsystem.\n(G) Bioluminescence imaging and quantification of the average radiance of eMSC-\nNanoLuc at days 0, 4 and 8 following their injection.\n(H) Bioluminescence imaging and quantification of the average radiance of eMSC-\nNanoLuc in individual organs of mice having received eMSC-NanoLuc. He:heart;\nLi:liver; Ki:kidney, Sp:spleen, Lu:lungs, Tu;tumor, tdLN:tumor-draining lymph node,\nMu:muscle.\nii) Systemic toxicity assessment: To further assess if eMSC immunotherapy\nwould induce a systemic, deleterious inflammatory response, we compared\nthe toxicity of our therapy with that induced by the administration of a high\n(toxic) dose of anti-CD40 antibody (Blake et al. 2021; Siwicki et al. 2021;\nSalomon et al. 2022). We assessed serum levels of ALT and AST, markers\nof hepatocellular injury, together with assessment of liver damage using H&E\nto assess liver necrotic regions. Changes in body weight were assessed over\ntime. We observed that treatment with anti-CD40 antibody induced\nsignificant toxicity (high levels of AST and ALT, liver damage), whereas\neMSC-based therapies did not appear to be toxic. These data have been\nadded to a new figure 2E-G and to the supplementary figure S2C-D.\n5 10 15\n(E) Experimental design to evaluate the toxicity of the therapy in B16-OVA bearing\nmice. On day 7, eMSC-FLT3L were i.t. injected. On day 9, mice were treated i.t. with\npoly(I:C) or i.p. with an anti-CD40 antibody. Serum was collected two days later.\n(F) Body weight loss of mice throughout the experiment. (n=3-4 mice per group, one\nexperiment, *p < 0.05, **p < 0.01, two-way ANOVA-test with Tukey’s multiple\ncomparisons test).\n(G) Level of ALT in serum collected 48h after poly(I:C) or anti-CD40 treatment.\nC * D\n**\nUntreated eMSC-FLT3L + poly(I:C) anti-CD40\n**\n**\nU/\nT\n(\nA S 1000\n0\neMSC-FLT3L - + - + -\nPoly(I:C) - - + + -\nanti-CD40 - - - - +\nSup Figure 2: eMSC-FLT3L + poly(I:C) immunotherapy is more effective than\nrecombinant FLT3L + poly(I:C) treatment, without inducing toxicity.\n(C) Level of AST in serum collected 48h after poly(I:C) or anti-CD40 treatment.\n(D) H&E staining of fixed liver tissue from mice treated with eMSC-FLT3L + poly(I:C)\ntherapy or anti-CD40. Necrotic lesions are shown with the dashed black lines.\n3- Model generalizability: The study would benefit from validation in additional tumor\nmodels to demonstrate generalizability beyond B16-OVA. This model is somewhat\npeculiar, as most OVA-expressing tumors are highly immunogenic and are often\nspontaneously rejected in immunocompetent mice. This limitation could be addressed\nin a follow-up study in which intra-tumoral delivery of FLT3L-expressing eMSCs is\ntested in other transplantable or spontaneous tumor models.\nWe thank the reviewer for this comment, and we agree that validating the therapeutic\nefficacy of the therapy in additional tumor models would strengthen the paper.\nTherefore, eMSC-FLT3L + poly(I:C) therapy was validated in three other tumor models:\nthe breast cancer E0771 (CRL-3461), the colon carcinoma MC38 (Corbett et al. 1975)\nand the lung cancer TC1 cell line (Lin et al. 1996). In each of this model, eMSC_FLT3L\nand poly(I:C) immunotherapy delayed tumor growth (see figure 2L, P, T) and improved\nsurvival (see figure 2N, R, V). These data have been added to a new figure, now\nfigure 2.\n25 20\n15\n10\n5\nexpressing membrane bound FLT3L stimulates anti-tumor immunity in the\npresence of poly(I:C).\n(K, O, S) Experimental design to evaluate anti-tumoral effects of eMSC-FLT3L and\npoly(I:C) in MC38 (K), TC-1 (O) and E0771 (S) tumor models.\n(L, P, T) Tumor growth curves (n=6-8 mice per group, one experiment)\n(M, Q, U) Individual tumor size variation compared to the day of the first eMSC-FLT3L\ninjection. (n=6-8 mice per group, one experiment, *p < 0.05, **p < 0.01 two-way\nANOVA-test with Šídák’s multiple comparisons test).\n(N, R, V) Survival curves. (n=6-8 mice per group, one experiment, *p < 0.05, log-rank\n(Mantel-Cox) test).\n4- Mechanistic synergy with checkpoint blockade: The authors should discuss more\nthoroughly the mechanism by which this therapy synergizes with checkpoint blockade.\nWe thank the reviewer for this valuable comment. In response, we have incorporated\na new paragraph into the discussion section of the revised manuscript. The revised\ntext, addressing this point, is in green below.\n“When combining eMSC-FLT3L immunotherapies to aPD-1/aCTLA-4 ICB\nimmunotherapy, we manage to partially overcome primary resistance to immune\ncheckpoint blockade and extend survival. We hypothesize that at least 3 mechanisms\nnoitairav\nezis\nromuT\n8D ot\nderapmoc\n71D\nta\n30 0.1141 100\n20\n50 10\n0 0\n0 20 40 60\n**\nDays post TC-1 injection\n0 1020304050\nDays post E0771 injection\n)³mm(\nemulov\nromuT\nUntreated\neMSC-FLT3L + poly(I:C)\n2000\n100 *\n1500\n1000\n50\n500\n0 0\n10 20 30 40 50\nDays post E0771 injection\nnoitairav\nezis\nromuT\nnoitairav\nezis\nromuT\n6D ot\nderapmoc\n71D\nta\n21D\not derapmoc\n32D\ta\nL M N\n100\n50\n0\n0 10 20 30 40\nDays post MC38 injection\nP Q R\nT U V\nlavivrus\ntnecreP\n2000 *\n1500\n1000\n500\n0\n0 10 20 30\nDays post MC38 injection\n)³mm(\nemulov\nromuT\n2500 2000\n1500\n1000\n500\n0\n0 20 40 60 Untreated\nDays post TC-1 injection eMSC-FLT3L + poly(I:C)\n)³mm(\nemulov\nromuT\n15\n10\n5\n*\neMSC-FLT3L + poly(I:C)\nlavivrus\ntnecreP\nlavivrus\ntnecreP\nK\ns.c.MC38\nD8 D13 D17 D24\nC57BL/6 D10 D20\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nO\ns.c.TC-1\nD6 D11 D14 D20\nC57BL/6 D8 D17\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nS\nC57BL/6 D12 D17 D21 D29\ns.c.E0771D14 D25 i.t.eMSC-FLT3L\ni.t.Poly(I:C)\n0\ncould account for this effect of eMSC-FLT3L immunotherapy:\ni) eMSCs might activate DCs engaging with poorly differentiated TCF1+ T\nSL\nCD8+ T cells enabling to generate ICB-responding pool within the tumors (Im\net al. 2016; Siddiqui et al. 2019). In support of this, multiple studies have\nidentified that mature DCs (and cDC1s in particular) participate to a niche\nsupporting TCF1+ poorly differentiated T cells (Schenkel et al. 2021; Meiser\net al. 2023; Magen et al. 2023; Chen et al. 2024). This might possibly happen\nvia the accumulation of mature DCs within TLS for instance (Mattiuz et al.\n2024).\nii) eMSC might promote the formation of a pool of activated DCs within the\ntumor engaging intratumoral effector CD8+ T or NK cells. Garris et al. have\nshown that ICB rapidly activates intratumoral DCs via the γ to become\nIL12/IL15+ DCs (Garris et al. 2018). By increasing DC density, eMSC could\npromote the generation of intratumoral IL12/IL15/CXCL16+ perivascular DCs\nwhich have been shown to play a key role in maintaining the fitness of\nintratumoral CXCR6+ CD8+ T cell effectors (Di Pilato et al. 2021).\niii) Lastly, eMSC might increase the number of migratory DCs cross-priming\nnovel CD8+ T cells in tumor draining lymph nodes. This might help\nbroadening and diversifying T cell response which is a feature of productive\nICB immunotherapy (Yost et al. 2019; Liu et al. 2022). In support of this\nnotion, we have evidenced that eMSC-FLT3L + poly(I:C) therapy increases\nthe flux of migratory DCs to tumor-draining lymph nodes” (see figure 4B-C).\n5- Clinical implications: The authors should discuss better the translational potential of\neMSC therapy, as the intratumor route of delivery would not be amenable to most\nhuman cancers.\nAddressing these points will enhance the impact of the study. However, given the\nnovelty and important implications of the work, I recommend the manuscript undergo\nminor revision prior to acceptance.\nWe agree with the comment. The discussion has been extensively modified,\nhighlighting practical and technological bottleneck in the road to translate the system\nto an immunotherapeutic intervention in clinical settings. Please, find below in green\nthe revised paragraphs, taking this comment into account.\n“The prospect of locally implanted, eMSCs acting as versatile, sustained\nfactories for therapeutic molecule delivery is highly attractive for cancer\nimmunotherapy. This is particularly relevant for chemokines, which generate spatially\norganized gradients governing leukocyte trafficking and tissue compartmentalization.\nNonetheless, several technological and translational challenges must be addressed to\nrealize the clinical potential of eMSCs.\nFirst, standardized and GMP-compatible protocols for autologous eMSCs\nisolation, engineering, and production must be established. Such methods should\nensure reproducible recovery of stromal populations of controlled complexity that are\namenable to ex vivo expansion, viral transduction, and post-transduction enrichment.\nAlthough the present study primarily employed murine embryonic fibroblasts as an\nMSC source, we demonstrate that adipose-derived stromal cells can be expanded,\ntransduced, and used to generate immunologically competent eMSCs. Other groups\nhave successfully implemented bone marrow–derived eMSCs for immunotherapy\n(Stagg et al. 2004; Ren et al. 2008; ułach et al. 2021; Zhang et al. 2022). Validation\nin human systems will be critical for clinical translation. Furthermore, the phenotypic\nheterogeneity resulting from ex vivo expansion and transduction remains to be fully\ncharacterized. Despite homogeneous transgene expression (e.g., via IRES-linked\nfluorescent reporters), we can not exclude that this cellular preparation is functionally\nheterogeneous and that only a subset of eMSCs may be functionally active in vivo.\nIntegration of single-cell RNA sequencing and spatial transcriptomics in eMSC-treated\ntumors could provide essential insight into stromal diversity, fate, and functional states.\nSecond, optimization of eMSC delivery methods could improve therapeutic\npersistence and efficacy. In our current model, intratumoral injection of cell\nsuspensions resulted in transient eMSC persistence, with only a minor fraction\ndetectable beyond ten days. Future studies should aim at enhancing eMSC residency\nthrough tissue engineering strategies, including synthetic scaffolds or extracellular\nmatrix integration (Choi et al. 2017). Cell-intrinsic manipulation of stress pathway might\nalso offer some opportunities to improve the survival of eMSC in the TME. For instance,\nNQO1 expression might improve resistance to oxidative stress (Bae et al. 2022).\nWould these manipulations improve persistence, it would be important to determine\nwhether prolonged eMSC survival improves T and NK cell–dependent tumor control.\nWhile intratumoral delivery provides localized control, it may not be feasible for all\ncancer types. Developing systemic delivery approaches for eMSCs could extend\ntherapeutic reach to deep or inaccessible lesions while improving patient tolerability.\nIndeed, intravital imaging studies have shown that intravenously administered MSCs\nexhibit intrinsic tropism for sites of tissue remodeling, including wounds and solid\ntumors (Kidd et al. 2009; Kalimuthu et al. 2017). The molecular mechanisms underlying\nthis selective homing remain poorly defined but could be exploited to deliver eMSCs to\notherwise unreachable tumor sites.\nThird, eMSC-based therapies designed to stimulate DC-dependent antitumor\nimmunity must also incorporate cues that promote antigen release and DC maturation,\nthe two essential steps required to initiate the cancer-immunity cycle. In our study,\neMSCs expressing FLT3L and CXCL9/CCL5 effectively enhanced local DC infiltration\nbut did not efficiently drive DC maturation, potentially limiting migration to tumor-\ndraining lymph nodes and cross-priming of CD8⁺ T cells. Embedding maturation-\ninducing signals within eMSCs may overcome this limitation. In parallel, promoting\nlocal tumor cell death could enhance antigen availability for DC uptake. One promising\navenue involves combining eMSCs with oncolytic virotherapy, which induces\nimmunogenic cell death and antigen release. eMSCs have been widely explored as\ndelivery vehicles for oncolytic viruses, leveraging their tumor-homing capacity\n(Pereboeva and Curiel 2004; Hakkarainen et al. 2007). This approach has been\nimplemented for several viral platforms, including herpes simplex virus (Du et al. 2017),\nmyxoma virus (Josiah et al. 2010), Newcastle disease virus (Kazimirsky et al. 2016),\nmeasles virus (Sonabend et al. 2008), and adenovirus (Sonabend et al. 2008; Yoon et\nal. 2019). We propose that eMSC-based oncolytic virotherapy could be synergistically\ncombined with DC-targeting eMSCs, as suggested by Svensson-Arvelund et al., who\nobserved cooperation between recombinant FLT3L administration and NDV-mediated\noncolysis (Svensson-Arvelund et al. 2022). Moreover, type I IFN–releasing eMSCs\ncould complement DC-directed eMSCs to further enhance local immune activation\n(Ren et al. 2008; Choi et al. 2017; Zhang et al. 2022).\nIn summary, while technical and regulatory limitations remain, the translational\npotential of eMSC-based immunotherapies is considerable. Their inherent tumor\ntropism, compatibility with ex vivo genetic engineering, and capacity for localized,\nsustained delivery make eMSCs a promising cellular platform for next-generation\ncombination immunotherapies aimed at amplifying both innate and adaptive antitumor\nimmunity.”\nMinor specific points:\nIn the experiments shown in Figures 3B, S2, and S3, the observed effects on cDC1\nmaturation and T cell activation could be attributed to poly I:C treatment alone. The\nauthors should include a comparison between eMSC + poly I:C and poly I:C alone to\ndemonstrate that the presence of eMSCs is essential for enhancing cDC1 maturation\nand IFN-γ secretion by T cells.\nThe figures mentioned (now figure 4B, S3A and S4C-F) already include the\ncontrol condition “eMSC-Ctrl + poly(I:C)”, which can be considered equivalent to\n“poly(I:C) alone”, since eMSC-Ctrl do not exert any specific immunomodulatory\nactivity. These control cells express only a fluorescent reporter and do not express\nany additional ligands. Therefore, the observed effects cannot be attributed to\npoly(I:C) treatment alone but rather to the combination with the engineered eMSCs\nexpressing FLT3L.\nReviewer #3:\nThe manuscript by Gorline et al analyses the anti-cancer efficacy of engineered\nmesenchymal stem cells expressing factors that enhance DC and T/N cell\nactivation/infiltration in a mouse tumour model harbouring an ectopic immunodominant\nantigen. The use of humanized mice/human cells is highly appreciated and the authors\nperform a comprehensive analysis of the majority of relevant aspects of DC-mediated\ninduction of anti-cancer T cell immunity. The authors basically report the effective\nrepurposing of existing knowledge, the combination of a previously reported system\n(eMSCs) with known anti-cancer factors for the treatment of cancer in mice. Despite\nan eventual lack of novelty, the manuscript is clearly interesting from a translational\npoint of view.\nIn light of this, the choice of mouse cancer models that are most closely resembling\nhuman disease to demonstrate therapeutic efficacy may be of high relevance.\nUnfortunately, the manuscript practically exclusively relies on the B16-OVA cancer\nmodel. Moreover, several treatment controls are missing, which are relevant to\nunderstand if the efficacy of the new treatment strategy developed by the authors\noutcompetes previously used therapies. If those points are strengthened (see my\nsuggestions to do so below), the manuscript will be a valuable addition to current efforts\nto improve cancer immunotherapy.\nWe sincerely thank the reviewer for their thoughtful and detailed assessment of our\nwork. We appreciate their recognition of the comprehensive analysis we performed\nand the translational relevance of using humanized mice. We have carefully\nconsidered the reviewer’s suggestions and have conducted additional experiments\nand analyses to expand the relevance of our findings, as detailed below.\nMain comment 1 – only one pre-clinical cancer model expressing an ectopic immuno-\ndominant antigen is used in the manuscript.\nThe strongest suit of the present manuscript is its therapeutic relevance. The key\nfindings of every Figure should, therefore, be demonstrated in one or more cancer\nmodel that closely resembles human disease. Ideally not a cell line (for example\nspontaneous models, if possible, or at least engraftment of primary cancer cells) and\ncertainly not cancer cells expressing ectopic antigens.\nWe thank the reviewer for this comment, and we agree that validating the therapeutic\nefficacy of the therapy in additional tumor models would strengthen the paper.\nTherefore, eMSC-FLT3L + poly(I:C) therapy was validated in three other tumor models:\nthe breast cancer E0771 (CRL-3461), the colon carcinoma MC38 (Corbett et al. 1975)\nand the lung cancer TC1 cell line (Lin et al. 1996). In each of this model, eMSC_FLT3L\nand poly(I:C) immunotherapy delayed tumor growth (see figure 2L, P, T) and improved\nsurvival (see figure 2N, R, V). These data have been added to a new figure, now figure\n2.\n25 20\n15\n10\n5\nexpressing membrane bound FLT3L stimulates anti-tumor immunity in the\npresence of poly(I:C).\n(K, O, S) Experimental design to evaluate anti-tumoral effects of eMSC-FLT3L and\npoly(I:C) in MC38 (K), TC-1 (O) and E0771 (S) tumor models.\n(L, P, T) Tumor growth curves (n=6-8 mice per group, one experiment)\n(M, Q, U) Individual tumor size variation compared to the day of the first eMSC-FLT3L\ninjection. (n=6-8 mice per group, one experiment, *p < 0.05, **p < 0.01 two-way\nANOVA-test with Šídák’s multiple comparisons test).\n(N, R, V) Survival curves. (n=6-8 mice per group, one experiment, *p < 0.05, log-rank\n(Mantel-Cox) test).\nI believe the only Figure that didn’t use B16-OVA is Figure 1F-J with B16/F10 tumors,\nwhich is an experiment to “exclude” undesired effects. Probably in exactly such\n“excluding experiments”, the authors should be consistent in tumour model and use\nB16-OVA.\nnoitairav\nezis\nromuT\n8D ot\nderapmoc\n71D\nta\n30 0.1141 100\n20\n50 10\n0 0\n0 20 40 60\n**\nDays post TC-1 injection\n0 1020304050\nDays post E0771 injection\n)³mm(\nemulov\nromuT\nUntreated\neMSC-FLT3L + poly(I:C)\n2000\n100 *\n1500\n1000\n50\n500\n0 0\n10 20 30 40 50\nDays post E0771 injection\nnoitairav\nezis\nromuT\nnoitairav\nezis\nromuT\n6D ot\nderapmoc\n71D\nta\n21D\not derapmoc\n32D\ta\nL M N\n100\n50\n0\n0 10 20 30 40\nDays post MC38 injection\nP Q R\nT U V\nlavivrus\ntnecreP\n2000 *\n1500\n1000\n500\n0\n0 10 20 30\nDays post MC38 injection\n)³mm(\nemulov\nromuT\n2500 2000\n1500\n1000\n500\n0\n0 20 40 60 Untreated\nDays post TC-1 injection eMSC-FLT3L + poly(I:C)\n)³mm(\nemulov\nromuT\n15\n10\n5\n*\neMSC-FLT3L + poly(I:C)\nlavivrus\ntnecreP\nlavivrus\ntnecreP\nK\ns.c.MC38\nD8 D13 D17 D24\nC57BL/6 D10 D20\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nO\ns.c.TC-1\nD6 D11 D14 D20\nC57BL/6 D8 D17\ni.t.eMSC-FLT3L i.t.Poly(I:C)\nS\nC57BL/6 D12 D17 D21 D29\ns.c.E0771D14 D25 i.t.eMSC-FLT3L\ni.t.Poly(I:C)\nB16-OVA model and similar results were obtained, as shown in supplementary figure\nS1C. Overall, we conclude that eMSC_FLT3L does not impact on systemic FLT3L levels.\n20000\n15000\n10000\n5000\n(C) Circulating huFLT3L levels measured by ELISA in mice serum 4 days after eMSCs\ninjection. (n=3-4 mice per group in one experiment. *p < 0.05, Kruskal-Wallis test with\nDunn’s multiple comparisons).\nMajor comment 2 – missing controls (especially known factors as mono-treatment) and\nno repetition of experiments throughout the manuscript\nIntratumoral injection of PolyI:C and it’s clinically approved derivatives is a frequently\nexplored approach and known to be effective in pre-clinical mouse models – via a\nplethora of pathways (PMID: 31046839, 34824158, 34531246, 40079116).\nWe agree with the comment of the reviewer since multiple studies indeed highlighted\nsome effects of poly(I:C) as a monotherapy. Here is a detailed response to the\nquestions.\nThe authors only explore chemokine induction by polyI:C, which raises a few questions\non the effects of polyI:C (dependent or not on eMSC-FLT3L combination treatment):\nHow do eMSC-FLT3L compare with intratumoral injection of rec-huFLT3L in\ncombination with polyI:C?\nWe thank the reviewer for this insightful comment. Intratumoral injection of recombinant\nFLT3L also delayed tumor growth and improved survival, although these effects were\nmore pronounced with eMSC-FLT3L treatment, as shown with the tumor growth (see\nsupplementary figure S2A) and survival curves (see supplementary figure S2B).\n)Lm/gp(\nnoitartnecnoC\nC\n*\n*\nB16-OVA + + -\nB16-FLT3L - - +\neMSC-FLT3L - + -\nWe conclude that eMSC-FLT3L bring a significant although partial advantage upon\nrecombinant FLT3L for the local delivery of FLT3L within the TME. This advantage\nmight be more pronounced in slightly growing tumors and if eMSCs persistence within\nthe TME might be improved.\n100\n50\n0\n0 90\nSup Figure 2: eMSC-FLT3L + poly(I:C) immunotherapy is more effective than\nrecombinant FLT3L + poly(I:C) treatment, without inducing toxicity.\n(A) Tumor growth curves until day 15 (n=10 mice per group, two independent\nexperiments, ****p < 0.0001, two-way ANOVA-test with Tukey’s multiple comparisons\ntest).\n(B) Survival curves. (n=10 mice per group in two independent experiments, **p <\n0.01, ****p < 0.0001, log-rank (Mantel-Cox) test)\nFigure 4A-D and O-Q: the eMSC-FLT3L and, most importantly, the reference-control\npolyI:C mono-therapy groups are missing. That is important because many of those\neffects have previously been shown to be induced by polyI:C alone (see citations\nabove).\nWe thank the reviewer for this valuable comment. In response, we have repeated\nexperiments and have added the relevant controls. Quantifications for all experimental\ngroups (eMSC-Ctrl, eMSC-FLT3L, poly(I:C), and eMSC-FLT3L + poly(I:C)) have been\nadded to the figure 5D-E and previous figure quantifying the untreated and poly(I:C)\ngroups only (previously figure 4D-E) has been moved to the supplementary figure\nS5A-B. We conclude that the effect we observe is indeed uniquely induced by the\neMSC-FLT3L + poly(I:C) immunotherapy in our model.\nlavivrus\ntnecreP\nUntreated\nRecombinant FLT3L + poly(I:C)\neMSC-FLT3L + poly(I:C)\n30 60\nDays post B16-OVA injection\n**** **\n800\n200\neMSC-FLT3L + poly(I:C)\n0\n0 5 10 15\nDays post B16-OVA injection\n)³mm(\nezis\nromuT 0.0535\n****\n****\nA B\n*\n8 *\n**\n6\n4\n2\n0\neMSC-Ctrl + - + -\neMSC-FLT3L - + - +\nPoly(I:C) - - + +\nFigure 5: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the\ninfiltration of T and NK cells within tumor beds that are required for\nimmunotherapy efficiency and overcomes primary resistance to PD-1/CTLA-4\nblockade.\n(D-E) Representative flow cytometry plots (D) and quantification (E) of activated (T\nACT\n),\nexhausted (T ), and stem-like (T ) CD8+ T cells. (n=4 mice per group, one\nEX SL\nexperiment, *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons\ntest).\nRegarding the figure combining the eMSC-FLT3L + poly(I:C) therapy to the ICB\n(figure 5N-P, previously figure 4O-Q), we showed that in our model, eMSC-FLT3L\nand poly(I:C) monotherapies do not independently delay tumor growth. The purpose\nof this experiment was to test the combination of an effective therapy with\nimmunotherapies. Moreover, given that the efficacy of ICB therapy depends on\nenhanced intratumoral T-cell infiltration, and that only the eMSC-FLT3L + poly(I:C)\ncombination promoted an increase in CD8⁺ T cells, our subsequent analyses focused\non this combination in the context of ICB.\nFigure 5D – is the upregulation of CXCL9, CXCL11 and CCL5 unique to eMSC-FLT3L\n+ poly(I:C) or also found by polyI:C mono-treatment?\nsllec#\n)lrtC-CSMe/dlof(\nE T T T\nACT EX SL\n*\n**\n**\n✱\n+ - + - + - + -\n- + - + - + - +\n- - + + - - + +\nWe thank the reviewer for this comment. In our model, the upregulation of CXCL9,\nCXCL11 and CCL5 is more pronounced with eMSC-FLT3L + poly(I:C). However,\nadditional experiment including the poly(I:C) monotherapy identifies that poly(I:C) acts\nas an inducer of CXCL9 chemokine on its own (see supplementary figure S6A).\n1500\n1000\n500\n0\neMSC-Ctrl + - + -\neMSC-FLT3L - + - +\nPoly(I:C) - - + +\nSup Figure 6: poly(I:C) monotherapy is sufficient to upregulate CXCL9\nexpression in tumor homogenates.\n(A) Murine CXCL9 ELISA done on tumor homogenates 24h after poly(I:C) injection.\n(n=3-4, one experiment).\nNonetheless, FLT3L and chemokines both synergize for intratumoral cDC1 infiltration\nand expansion (see figure 3C, previously figure 2C).\nMany experiments using treatment with eMSC-FLT3L-CXCL9-CCL5 are missing the\neMSC-FLT3L control, for example:\nFigure 6D-I: are those untreated compared to treated tumors? The control of eMSC-\nFLT3L-treated tumours is missing, especially for Fig. 6H-I.\nUpon reviewer request, we have added the missing controls. Figure 7G-H (previously\nfigure 6F-G) and supplementary figure S7A (previously figure 6H), now compares\neMSC-FLT3L to eMSC-FLT3L-CXCL9-CCL5. As shown in the first figures, the\nuntreated, eMSC-Ctrl, and eMSC-FLT3L conditions did not affect tumor growth (see\nfigure 2B, previously figure 1L) or intratumoral dendritic cell infiltration (see figure 3C,\npreviously figure 2C). Here, we found that eMSC-FLT3L-CXCL9-CCL5 but not eMSC-\nFLT3L increase cDC1 density within the tumor. This highlights a cooperativity between\nchemokines enabling pre-cDC1 recruitment (see figure 7D-E, previously figure 6D-E)\nand growth factor signaling pathway FLT3L/FLT3 enabling survival and proliferation.\n9LCXC\neniruM\n)Lm/gp(\nA\nF\ns.c.B16-OVA\nD9 D11 D14D17\nC57BL/6 Tumor\ni.t.eMSC-FLT3L analysis\nCXCL9-CCL5 or\neMSC-FLT3L\nFigure 7: Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5\nstimulates cDC1 infiltration in mice and humanized mice.\n(F) Experimental design to evaluate cDC1 recruitment within tumors at a late time\npoint. On day 9, 11 and 14 after B16-OVA injection, eMSCs were i.t. injected. Tumors\nwere harvested on day 17 and analyzed by flow cytometry.\n(G-H) Quantification by flow cytometry of the absolute number of cDC1/g tumor (G)\nand their frequency in CD45+ live cells (H) at day 15, after 3 intratumoral injections of\neMSC-FLT3L-CXCL9-CCL5. Results are shown as fold change to control (Untreated).\n(n=16-17 mice for the untreated and eMSC-FLT3L-CXCL9-CCL5 groups, n=5 mice for\nthe eMSC-FLT3L group, three independent experiments, *p < 0.05, ***p < 0.001, ****p\n< 0.0001, one-way ANOVA with Tukey’s multiple comparisons test .\nSup Figure 7: eMSC-FLT3L-CXCL9-CCL5 are localized at the border of the tumor\nand attract hucDC2s in synthetic niches in reconstituted BRGSF mice.\n(A) Quantification by flow cytometry of the mean fluorescence intensity (MFI) of CD40\nand CD86 at day 15, after 3 intratumoral injections of eMSC-FLT3L-CXCL9-CCL5.\nResults are shown as fold change to control (Untreated). (n=16-17 mice for the\nromut\ng/1CDc#\n)detaertnU/dloF(\nG H\n4\n3\n2\n1\n0\nni\n1CDc%\nsllec\nevil\n+54DC\n)detaertnU/dloF(\n**** *\n8 0.0862 *\n6\n4\n2\n0\neMSC-FLT3L - + - - + -\neMSC-FLT3L-CXCL9-CCL5 - - + - - +\n2.5\n2.0\n1.5\n1.0\n0.5\n0\n04DC\nIFM\n)detaertnU/dloF(\nA\n***\n0.0975\n0.0976\n0\neMSC-FLT3L - + -\neMSC-FLT3L-CXCL9-CCL5 - - +\n68DC\nIFM\n)detaertnU/dloF(\n2.0\n1.5\n1.0\n0.5\nuntreated and eMSC-FLT3L-CXCL9-CCL5 groups, n=5 mice for the eMSC-FLT3L\ngroup, three independent experiments, ***p < 0.001, one-way ANOVA with\nTukey’s multiple comparisons test).\nTo further test this model, we have performed novel confocal imaging on explanted live\ntissue section in Xcr1cre x ROSAlsltdTOMATO mice, that received tumors and eMSC-\nFLT3L vs eMSC-FLT3L-CXCL9-CCL5 immunotherapies. As shown in figure 7I-J, we\nfound that chemokine expression by stromal cells significantly increase the interactions\nbetween eMSCs-FLT3L and cDC1s. We conclude that chemokine gradients organized\nby eMSCs lead to the efficient attraction of cDC1s at cellular sources of membrane\nbound FLT3L. This represents, we believe, an important addition to the study.\nI\n6\n4\n2\nstimulates cDC1 infiltration in mice and humanized mice.\n(I) Representative immunofluorescence images of YUMM-OVA tumors injected in\nXCR1tdTomato mice and treated with eMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5. The\nimages shown here represent masks for cDC1s (red) and eMSCs (yellow), generated\nfrom the maximum intensity projection using ImageJ.\n(J)Quantification of the number of cDC1s surrounding an eMSC, manually determined.\n(one experiment, two-tailed Mann-Whitney test, ****p < 0.0001).\nFigure 6L-O: highly appreciated, but probably eMSC-FLT3L would have been the\nappropriate control here? I understand this is very challenging to repeat, but could the\nauthors explain their choice of control here?\nWe thank the reviewer for this question, and we agree that including an eMSC-FLT3L\ncontrol group would have further strengthened the experiment. However, given the\ngnitcatnoc\ns1CDc#\nsCSMe\nhcae\nJ\n****\neMSC-FLT3L + -\neMSC-FLT3L\n- +\nCXCL9-CCL5\neMSC-FLT3L eMSC-FLT3L-CXCL9-CCL5\nXCR1tdTomato mice XCR1tdTomato mice\n100µm 100µm\ntechnical difficulty and resource intensity of this model, additional replicates were not\nfeasible. Importantly, as shown in figure 3C (previously figure 2C), both eMSC-Ctrl\nand eMSC-FLT3L exhibited comparable capacities to enhance intratumoral dendritic\ncell frequencies. Therefore, we used eMSC-Ctrl as the control condition in this\nexperiment.\nFinally, the majority of experiments have only been performed once. Every experiment\n(especially in vivo tumour-treatments) have to be performed 2 independent times to\ngenerate robust and reproducible data.\nWe agree with this comment. Nonetheless, the key findings of the manuscript are\nsupported by experiments that were independently repeated several times, confirming\nthe robustness and reproducibility of the results. Experiments corresponding to the\nmain figures that were initially performed once have since been repeated, and the\npooled data are now presented. For example, the quantification of intratumoral and\ntumor-draining lymph node–associated cDC1 and cDC2 numbers and phenotypes at\nday 10 (see figure 3C,4C and supplementary figure S3A, previously figure 2C, 3C\nand S2A), were confirmed in independent experiments.\n+ - + -\n- + - +\n- - + +\nFigure 3: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the\ninfiltration of cDC1s, that are required for immunotherapy efficiency.\n(C) Quantification of the absolute number of intra-tumoral cDC1/g tumor and the mean\nfluorescence intensity (MFI) of CD40 and CD86. Results are shown as fold change to\ncontrol (eMSC-Ctrl). (n=6-7 mice per group, two independent experiments for day 10,\nn=8-9 mice per group, two independent experiments for day 15, *p < 0.05, **p < 0.01,\n***p < 0.001, ****p < 0.0001, one-way ANOVA-test with Tukey’s multiple\ncomparisons).\nromut\ng/1CD#\n04DC\nIFM\n)lrtC-CSMe/dlof(\n)lrtC-CSMe/dlof(\nDay10 Day 15\n**\n***\n**\n16\n**\n12\n8\n4\n0\n15\n10\n5\n0\neMSC-Ctrl\neMSC-FLT3L\nPoly(I:C)\n68DC\nIFM\n)lrtC-CSMe/dlof(\nC\n***\n****\n****\n**\n**\n*\n100 **\n80\n60\n40\n20\n0\n+ - + -\n- + - +\n- - + +\n+ - + -\n- + - +\n- - + +\nSup Figure 3: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the\ninfiltration of cDC2s in the tumor.\n(A) Quantification of the absolute number of intratumoral cDC2/g tumor and the mean\nfluorescence intensity (MFI) of CD40 and CD86. Results are shown as fold change to\ncontrol (eMSC-Ctrl). (n=6-7 mice per group, two independent experiments for day 10,\nn=8-9 mice per group, two independent experiments for day 15, *p < 0.05, **p < 0.01,\none-way ANOVA-test with Tukey’s multiple comparisons).\nromut\ng/2CDc#\n04DC\nIFM\n)lrtC-CSMe/dlof(\n)lrtC-CSMe/dlof(\nDay10 Day 15\n6\n4\n2\n0\n2\n1\n0.5\n0\n**\n**\neMSC-Ctrl\neMSC-FLT3L\nPoly(I:C)\n68DC\nIFM\n)lrtC-CSMe/dlof(\nA\n*\n**\n**\n10\n8\n0.0762\n2.5 *\n*\n1.5\n*\n10\n*\n8\n6\n4\n2\n0\n+ - + -\n- + - +\n- - + +\nFigure 4: Intratumoral delivery of eMSC-FLT3L and poly(I:C) increase mature\nmigratory cDC1s in tumor-draining lymph nodes and enables cross-priming of\ntumor-specific CD8+ T cells.\n(C) Quantification of the absolute number of migratory cDC1s and the mean\nfluorescence intensity (MFI) of CD40 and CD86. Results are shown as fold change to\ncontrol (eMSC-Ctrl). (n=7-8 mice per group, two independent experiments, *p < 0.05,\n***p< < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons\ntest).\nThe involvement of cDC1s in driving the eMSC-FLT3L + poly(I:C) immunotherapy was\nalso confirmed (see figure 3D-E, previously figure 2D-E).\n1CDc\nyrotargiM#\n04DC\nIFM\n68DC\nIFM\n)lrtC-CSMe/dlof(\n)lrtC-CSMe/dlof(\n)lrtC-CSMe/dlof(\nC\nDay 10 Day 15\n*\n5\n0.0679\n4\n3\n2\n1\n0\n*\n*\n0.0649\n5 0.0594\n4\n3\n2\n1\n0\n****\n****\n***\n8 ***\n6\n4\n2\n0\neMSC-Ctrl + - + - + - + -\neMSC-FLT3L - + - + - + - +\nPoly(I:C) - - + + - - + +\nD\nXCR1DTA - eMSC-FLT3L + poly(I:C)\nXCR1DTA - No therapy C57BL6 - No therapy\nC57BL6 - eMSC-FLT3L + poly(I:C)\nDays post B16-OVA injection\nFigure 3: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the\ninfiltration of cDC1s, that are required for immunotherapy efficiency.\n(D) Tumor growth curves of XCR1DTA mice or C57BL/6 WT mice treated or not with\neMSC-FLT3L + poly(I:C). (A line represents the mean and SD is shown with the\ncolored doted lines, n=4 mice XCR1DTA - No therapy and n=8 mice for the three other\ngroups, representative of two independent experiments, ****p < 0.0001 two-way\nANOVA-test with Tukey’s multiple comparisons).\n(E) Survival curves. (n=10-16 mice per group, two independent experiments, *p < 0.05,\nlog-rank (Mantel-Cox) test).\nFurthermore, instead of showing only representative plots from independent\nexperiments, we have pooled the data from individual experiments when applicable.\nFor example, in Figure 4I (previously figure 3H), we now present the combined\nabsolute numbers of tetramer-positive CD8⁺ T cells in the tumor-draining lymph nodes\nat day 15.\nFigure 4: Intratumoral delivery of eMSC-FLT3L and poly(I:C) increase mature\nmigratory cDC1s in tumor-draining lymph nodes and enables cross-priming of\ntumor-specific CD8+ T cells.\n)³mm(\nezis\nromuT\n4000\n100\n3000\n2000 50 * 1000 * * * * * * \n* *\n0\n0 10 20\n****\nlavivrustnecreP\n****\n*\nE\n** * * * * *\n0\n0 20 40 60\nDays post B16-OVA injection\nXCR1DTA-Notherapy\nXCR1DTA-eMSC-FT3L+poly(I:C)\nC57BL6-Notherapy\nC57BL6-eMSC-FT3L+poly(I:C)\n+remarteT#\n)lrtC-CSMe/dloF(\n*** **\nI\n** 25\n20\n15\n10\n5\n0\neMSC-Ctrl + - + -\neMSC-FLT3L - + - +\nPoly(I:C) - - + +\n(I) Quantification (I) of OVA-specific CD8+ Tetramer+ T cells at day 15. Results are\nshown as fold change to control (eMSC-Ctrl). (n=8-9 mice per group, two independent\nexperiments, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons).\nThe figure legends have been updated to indicate the number of experimental repeats\nand the number of independent mice analyzed.\nMajor comment 3 – some conclusions should be revised and may require further testing\nThe unique induction of activated and non-exhausted CD8+ T cells in the eMSC-\nFLT3L+ PolyI:C-treated group is not clear, because a lot of data from the other groups\nare omitted/not shown. Also, it is not clear if the efficacy of eMSC-FLT3L+ PolyI:C\ntreatment is CD8+ T cell dependent or Treg cell dependent. Please consider the\nfollowing for a better understanding of the effects of the therapy:\nWe agree with the comment of the reviewer. Here is a detailed response to the\nquestions.\nFigure 3F – please add a quantification of all experimental groups – not only the\nuntreated and combo.\nWe thank the reviewer for this valuable comment. In response, we have repeated\nexperiments and have added the relevant controls. Quantifications for all experimental\ngroups (eMSC-Ctrl, eMSC-FLT3L, poly(I:C), and eMSC-FLT3L + poly(I:C)) have been\nadded to the figure 4D-E) and previous figure quantifying the untreated and poly(I:C)\ngroups only (previously figure 3D-F) have been moved to the supplementary figure\nS4A-B. We conclude that the effect we observe is indeed uniquely induced by the\neMSC-FLT3L + poly(I:C) immunotherapy in our model.\n15\n10\n5\nmigratory cDC1s in tumor-draining lymph nodes and enables cross-priming of\ntumor-specific CD8+ T cells.\n(D) Representative flow cytometry plots of CD8 T cells in the tumor-draining lymph\nnode at day 15.\n(E) Quantification of the absolute number of activated, exhausted and stem-like CD8\nT cells. Results are shown as fold change to control (eMSC-Ctrl). (n=12-14 mice per\ngroup, three independent experiments, **p < 0.01, ***p < 0.001, Kruskal-Wallis test\nwith Dunn’s multiple comparisons. n=4 mice per group, one experiment, **p < 0.01,\nKruskal-Wallis test with Dunn’s multiple comparisons).\nsllec\nT#\nTCA\n)lrtC-CSMe/dlof(\nE\n***\n**\n**\n60\n40\n20\n0\neMSC-Ctrl + - + -\neMSC-FLT3L - + - +\nPoly(I:C) - - + +\nsllec\nT#\nXE\n)lrtC-CSMe/dlof(\n25\n20\n15\n10\n5\n0\nsllec\nT#\nLS\n)lrtC-CSMe/dlof(\n**\nFigure 3G – please add a quantification of Tregs and IFNgamma-producing CD8+ T\ncells for all experimental groups – not only the ratio with CD8 T cells\nThis quantification was already shown in the previous version of the manuscript and\ncan now be found in the supplementary figure S4C-D (previously figure 3A-B).\n15\n*\n10\n5\neMSC-Ctrl + - + -\neMSC-FLT3L - + - +\nPoly(I:C) - - + +\nSup Figure 4: Intratumoral delivery of eMSC-FLT3L and poly(I:C) increase\nproliferative stem-like CD8+ T cells, IFNγ+CD8+ and IFNγ+CD4+ but not T\nregulatory cells.\n(C-D) Quantification of the absolute number of IFNγ+CD8+ T cells (C), CD4+Foxp3+ T\nregulatory cells (Tregs) (D) at day 15. (n=5 mice per group in one experiment, *p <\n0.05, one-way ANOVA-test with Dunnett’s multiple comparison test).\nIn Figure 4K-N, CD8 blockade should be tested alone to confirm an involvement of CD8\nT cells. Also, to distinguish total CD4 from Treg cells, please block Tregs specifically\nusing Foxp3-ko mice or a blocking antibody (for example anti-CD25).\nWe thank the reviewer for this insightful comment and, as suggested by the reviewer,\nwe performed additional experiments to address this. By selectively depleting CD8⁺ T\ncells, we confirmed their involvement in the therapeutic efficacy. In contrast, depletion\nof all CD4⁺ T cells or regulatory T cells (Tregs) alone did not significantly affect tumor\ngrowth (see supplementary figure 5E) or survival (see supplementary figure 5F),\nindicating that the efficacy of this therapy is primarily CD8⁺ T cell dependent.\nsllec\n+8DC+γNFI#\n)lrtC-CSMe/dloF(\n0\nsgerT#\n)lrtC-CSMe/dloF(\nC\nD\n4\n*\n3\n2\n1\n1500\naCD8\naCD25\n500\naCD4\n0\n0 10 20\nSup Figure 5: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates\ninfiltration and proliferation of CD4+ T cells but not of T regulatory cells.\n(E) Tumor growth curves. (n=7 mice per group, one experiment, ****p < 0.0001,\ntwo-way ANOVA-test with Tukey’s multiple comparisons).\n(F) Survival curves. (n=7 mice per group, one experiment, *p < 0.05, log-rank\n(Mantel-Cox) test).\nAdditional comments\nIn their explanations on conventional DCs (cDCs) the authors should probably adhere\nto the currently established nomenclature as much as possible and use the\nabbreviations cDC1 and cDC2 (instead of DC1 and DC2) to prevent confusion for the\nreader.\nWe agree with this comment. The terms DC1 and DC2 have been replaced with cDC1\nand cDC2 throughout the manuscript.\nDefine what “rec-huFLT3L” is. I understand recombinant human Flt3L.\nYes, rec-huFLT3L means recombinant human FLT3L. This modification has been\nadded to the figure legend and the text in the revised manuscript.\nThe authors assess “Intra-tumoral delivery of eMSC-FLT3L” in Figure 1A-J. However,\nthe experimental setup is a co-injection of tumour cells and MSCs from the\nbeginning. The caveat of this strategy should be stated.\nThere seems to be a misunderstanding regarding the different experimental setups\nshown in the figures.\n)³mm(\nemulov\nromuT\nDays post\nB16-OVA injection\n**** ****\n****\n100\n50\n0\n0 10 20 30 40\nDays post B16-OVA injection\nlavivruS\nfo\nytilibaborP\nF\naCD8\nIsotype * aCD4\naCD25\n5 15\nIn figure 1B–C (previously figure 1B-C), eMSCs were co-injected with murine\nhematopoietic cells embedded in an extracellular matrix, but no tumor was present\nat that stage. This specific experiment was designed to test if eMSC-FLT3L would\ninduce DC differentiation from co-injected DC progenitors.\nIn contrast, in the experiment shown in figures 1D (previously 1D), 1F, 1I (previously\n1F) or 2A (previously 1K), and later on, tumor cells were injected first, and\neMSCs were administered later, once the tumors became palpable (between 50 and\n100 mm3). Thus, this setup does not correspond to a co-injection of tumor cells and\neMSCs. To ensure clarity and prevent any confusion, we have added the indication\n“C57BL/6 bearing B16-OVA” in figures 1D and 1F. These panels refer to the\npersistence experiments, where the timeline is based on the day of eMSC injection\ninstead of the day following B16-OVA tumor inoculation, which can be confusing.\nIn summary, our experimental setting for immunotherapy is based on the\nintratumoral injection of eMSC-FLT3L in already growing tumors.\nAs the treatment with eMSC-FLT3L is of a transient nature, how to the authors\nenvisage clinical translation? Please discuss and explain.\nThis is indeed a key issue. To answer this, we have first added additional experiments\naddressing the persistence and biodistribution of eMSCs using in vivo imaging. Nano-\nluciferase–expressing eMSCs were generated and monitored for 8 days in the B16-\nOVA model using bioluminescence imaging. We observed that eMSCs persisted within\nthe tumor for at least 8 days, although at very low levels, and that their activity remained\nrestricted to the tumor site. These findings suggest that, once injected into the tumor,\neMSCs do not disseminate throughout the body but rather remain localized. These data\nhave been added to the figure 1F-H.\nPBS eMSC-NanoLuc\nD0\n4\nD4\n2\n1\ni.t.eMSC-NanoLuc\ni.v. Fluorofurimazine D8\nImaging Radiance\n(p/s/cm2/sr)\n108\n107\n106\n105\n0 4 8 Days post eMSC-NanoLuc\ninjection within B16-OVA tumors\nFigure 1: Intratumoral delivery of engineered mesenchymal stromal cells\nexpressing membrane bound FLT3L does not alter intratumoral and systemic\ncDC1 populations.\n(F) Experimental design to evaluate the biodistribution and persistence of eMSCs\nwithin B16-OVA tumors. 1x106 eMSC-NanoLuc were injected i.t. on day 0. On days 0\n(4h after the injection) , 4, and 8, mice received an intravenous injection of\n0.44μmoles of the fluorofurimazine substrate, followed by bioluminescence imaging\nusing the IVIS system.\n(G) Bioluminescence imaging and quantification of the average radiance of eMSC-\nNanoLuc at days 0, 4 and 8 following their injection.\n(H) Bioluminescence imaging and quantification of the average radiance of eMSC-\nNanoLuc in individual organs of mice having received eMSC-NanoLuc. He:heart;\nLi:liver; Ki:kidney, Sp:spleen, Lu:lungs, Tu;tumor, tdLN:tumor-draining lymph node,\nMu:muscle.\nFrom this experiment, we conclude that engraftment of eMSCs leads to persistence\nover 8 days and we hope that optimization of engraftment methods might extend this\npersistence.\nIn addition, as this point is highly important in the study, the discussion has been\nextensively modified, highlighting practical and technological bottleneck in the road to\ntranslate the system to an immunotherapeutic intervention in clinical settings. Please,\nfind below in green the revised paragraphs, taking this comment into account.\necnaidar\negarevA\n)rs/2mc/s/p(\n601x\nRadiance\n(p/s/cm2/sr)\n701x\nHear L t iv K e i r dn S ey plee L n ung T s uu mo tdL M N uscle\necnaidar\negarevA\n)rs/2mc/s/p(\nG H\n3\nB16-OVA 1\n1.5 x 106\n1 x 106\n0.5 x 106\n0\n“The prospect of locally implanted, eMSCs acting as versatile, sustained\nfactories for therapeutic molecule delivery is highly attractive for cancer\nimmunotherapy. This is particularly relevant for chemokines, which generate spatially\norganized gradients governing leukocyte trafficking and tissue compartmentalization.\nNonetheless, several technological and translational challenges must be addressed to\nrealize the clinical potential of eMSCs.\nFirst, standardized and GMP-compatible protocols for autologous eMSCs\nisolation, engineering, and production must be established. Such methods should\nensure reproducible recovery of stromal populations of controlled complexity that are\namenable to ex vivo expansion, viral transduction, and post-transduction enrichment.\nAlthough the present study primarily employed murine embryonic fibroblasts as an\nMSC source, we demonstrate that adipose-derived stromal cells can be expanded,\ntransduced, and used to generate immunologically competent eMSCs. Other groups\nhave successfully implemented bone marrow–derived eMSCs for immunotherapy\n(Stagg et al. 2004; Ren et al. 2008; ułach et al. 2021; Zhang et al. 2022). Validation\nin human systems will be critical for clinical translation. Furthermore, the phenotypic\nheterogeneity resulting from ex vivo expansion and transduction remains to be fully\ncharacterized. Despite homogeneous transgene expression (e.g., via IRES-linked\nfluorescent reporters), we can not exclude that this cellular preparation is functionally\nheterogeneous and that only a subset of eMSCs may be functionally active in vivo.\nIntegration of single-cell RNA sequencing and spatial transcriptomics in eMSC-treated\ntumors could provide essential insight into stromal diversity, fate, and functional states.\nSecond, optimization of eMSC delivery methods could improve therapeutic\npersistence and efficacy. In our current model, intratumoral injection of cell\nsuspensions resulted in transient eMSC persistence, with only a minor fraction\ndetectable beyond ten days. Future studies should aim at enhancing eMSC residency\nthrough tissue engineering strategies, including synthetic scaffolds or extracellular\nmatrix integration (Choi et al. 2017). Cell-intrinsic manipulation of stress pathway might\nalso offer some opportunities to improve the survival of eMSC in the TME. For instance,\nNQO1 expression might improve resistance to oxidative stress (Bae et al. 2022).\nWould these manipulations improve persistence, it would be important to determine\nwhether prolonged eMSC survival improves T and NK cell–dependent tumor control.\nWhile intratumoral delivery provides localized control, it may not be feasible for all\ncancer types. Developing systemic delivery approaches for eMSCs could extend\ntherapeutic reach to deep or inaccessible lesions while improving patient tolerability.\nIndeed, intravital imaging studies have shown that intravenously administered MSCs\nexhibit intrinsic tropism for sites of tissue remodeling, including wounds and solid\ntumors (Kidd et al. 2009; Kalimuthu et al. 2017). The molecular mechanisms underlying\nthis selective homing remain poorly defined but could be exploited to deliver eMSCs to\notherwise unreachable tumor sites.\nThird, eMSC-based therapies designed to stimulate DC-dependent antitumor\nimmunity must also incorporate cues that promote antigen release and DC maturation,\nthe two essential steps required to initiate the cancer-immunity cycle. In our study,\neMSCs expressing FLT3L and CXCL9/CCL5 effectively enhanced local DC infiltration\nbut did not efficiently drive DC maturation, potentially limiting migration to tumor-\ndraining lymph nodes and cross-priming of CD8⁺ T cells. Embedding maturation-\ninducing signals within eMSCs may overcome this limitation. In parallel, promoting\nlocal tumor cell death could enhance antigen availability for DC uptake. One promising\navenue involves combining eMSCs with oncolytic virotherapy, which induces\nimmunogenic cell death and antigen release. eMSCs have been widely explored as\ndelivery vehicles for oncolytic viruses, leveraging their tumor-homing capacity\n(Pereboeva and Curiel 2004; Hakkarainen et al. 2007). This approach has been\nimplemented for several viral platforms, including herpes simplex virus (Du et al. 2017),\nmyxoma virus (Josiah et al. 2010), Newcastle disease virus (Kazimirsky et al. 2016),\nmeasles virus (Sonabend et al. 2008), and adenovirus (Sonabend et al. 2008; Yoon et\nal. 2019). We propose that eMSC-based oncolytic virotherapy could be synergistically\ncombined with DC-targeting eMSCs, as suggested by Svensson-Arvelund et al., who\nobserved cooperation between recombinant FLT3L administration and NDV-mediated\noncolysis (Svensson-Arvelund et al. 2022). Moreover, type I IFN–releasing eMSCs\ncould complement DC-directed eMSCs to further enhance local immune activation\n(Ren et al. 2008; Choi et al. 2017; Zhang et al. 2022).\nIn summary, while technical and regulatory limitations remain, the translational\npotential of eMSC-based immunotherapies is considerable. Their inherent tumor\ntropism, compatibility with ex vivo genetic engineering, and capacity for localized,\nsustained delivery make eMSCs a promising cellular platform for next-generation\ncombination immunotherapies aimed at amplifying both innate and adaptive antitumor\nimmunity.”\nFigure 4 is missing panel M\nThis error has been corrected.\nFigure 4D – add percentages to representative FACS plot please\nPercentages have been added to the representative FACS plots (see supplementary\nfigure S5A).\nResults from Figure 4H-J should be reinterpreted: it is clear that tumour-experienced\nmice will develop a T cell memory and it is not clear how much the therapy contributed\nto that. For confirmation, tumour-resected mice would be necessary. And many more\nmarkers than displayed in Figure 4J would be necessary to confirm a resident-memory\nphenotype. I do not think this is a necessary message of the manuscript, but please\nomit any statements like “We conclude that eMSC-FLT3L + poly(I:C) immunotherapy\ninduce local, systemic and long- term immunity against tumor antigens in the CD8+ T\ncell compartment” throughout the manuscript.\nWe agree with the comment and have rephrased accordingly to distinguish between\n+\nthe induction of CD8 T cells and actual anti-tumor immunity, as follows: “We conclude\n+\nthat eMSC-FLT3L + poly(I:C) immunotherapy induces a sustained memory CD8 T\ncell response.”\nFigure 7D-F: could the authors also show non-transferred, endogenous T cell\ninfiltration into tumours of this experiment? That would be more relevant than\nquantifying adoptively transferred T cells.\nThe goal of this experiment was to selectively address if eMSC-derived chemokines\nwould be able to increase the recruitment of activated T cells from the blood stream.\nTo address this, it was important to selectively track blood derived activated T cells\npost intra-veinous transfer. Nonetheless and as requested, we have added the\nrequested quantifications of endogenous T cell accumulation in the figure 8G-H.\neMSC-ctrl + - - - + - - -\neMSC-CCL5 - + - + - + - +\neMSC-CXCL9 - - + + - - + +\nFigure 8: Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5\nstimulates the infiltration of activated CD4+ and CD8+ T cells.\n(G-H) Quantification of the absolute number of endogenous CD44+CD4+ T cells/g\ntumor (G) and endogenous CD44+CD8+ T cells/g tumor (H). (n=5 mice per group, one\nexperiment, one-way ANOVA test with Dunnett’s multiple comparisons).\nFigure 7G-H: at day 17, tumours already have a significantly different size (according\nto Figure 8). Hence, the differences in T cell infiltration could be a consequence rather\nthan a cause. T cell infiltration into tumours should be analyzed at an earlier time-point,\nwhen tumours have equal sizes between groups.\nThis comment would be valid in absolute numbers. However, we have expressed\nresults in absolute numbers normalized by tumor weight. This method enables to\nquantify T cell accrual independently of tumor size. In addition, kinetic constraints\nguided the choice of this time point. At this early time point, an immune response within\nthe tumor is unlikely to be detectable. The sequence of events, from dendritic cell\nactivation, migration into the tumor, and T-cell priming, to the subsequent infiltration of\nactivated T cells, requires sufficient time, by which point tumors may already differ in\nsize between groups.\nThe authors state: From these results, we conclude that the recruitment of DC1\ninduced by eMSC-FLT3L-CXCL9-CCL5 is independent on B, T and N /ILCs cells but\ndependent on FLT3 signalling. Yet, FLT3L overexpression alone is not sufficient to\ninduce cDC1 recruitment by itself. This is puzzling and the authors should comment on\nthis. To decipher this and understand if Flt3L is actually required (but not sufficient),\nthe authors could consider to show if eMSC-CXCL9-CCL5 (without FLT3L expression)\nare able to recruit cDC1s to tumours (but that is not vital for the manuscript).\ndetavitca\nsuonegodnE#\nsllec\nT\n+4DC\n)romut\ng/601x(\n0.0806\n0.0997\n8\n6\n4\n2\n0\ndetavitca\nsuonegodnE#\nsllec\nT\n+8DC\n)romut\ng/601x(\nG H\n4\n3\n2\n1\ndeficiency is known to impact on the amount of bone marrow and circulating pre-\ncDC1s. Therefore, the interesting experiment suggested by the reviewer should be\ndone in WT, FLT3 proficient mice. We predict that chemokines should lead to pre-\ncDC1s recruitment in tissues regardless of their local expansion. (Although it might be\nvery difficult to quantify given the rarity of these cells, see figure 7E, previously figure\n6E).\nFigure 8E-F: a tumor model that is less susceptible to aPD1/aCTLA4 blockade should\nbe used to evaluate synergy with eMSC-FLT3L-CXCL9-CCL5 treatment. I don’t think\nthis is necessary for the manuscript and the data could be removed.\nOther reviewers didn’t ask for it to be removed, therefore we have left it and wait for\neditor instructions regarding this specific topic.\nREFERENCES\nae, Joon eom, ongchao iu, asey oore, et al. . “ -2 Delivery by Engineered\nMesenchymal Stem Cells Re-Invigorates CD8+ T Cells to Overcome\nmmunotherapy esistance in ancer.” Nature Cell Biology 24 (12): 1754–65.\nhttps://doi.org/10.1038/s41556-022-01024-5.\nlake, tephen J., Jane James, eargal J. yan, et al. . “ he mmunotoxicity, ut\nNot Anti-Tumor Efficacy, of Anti-CD40 and Anti-CD137 Immunotherapies Is\ndependent on the ut icro iota.” Cell Reports Medicine 2 (12): 100464.\nhttps://doi.org/10.1016/j.xcrm.2021.100464.\nöttcher, Jan ., duardo onavita, ro ir hakravarty, et al. . “ ells\nStimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting\nancer mmune ontrol.” Cell 172 (5): 1022-1037.e14.\nhttps://doi.org/10.1016/j.cell.2018.01.004.\nhen, Jonathan ., inda . ieman, axwell purrell, et al. . “ uman ung\nCancer Harbors Spatially Organized Stem-Immunity Hubs Associated with\nesponse to mmunotherapy.” Nature Immunology 25 (4): 644–58.\nhttps://doi.org/10.1038/s41590-024-01792-2.\nhoi, ung ugh, aniel W. tuckey, ara ignatta, et al. . “ umor esection\nRecruits Effector T Cells and Boosts Therapeutic Efficacy of Encapsulated Stem\nells xpressing β in lio lastomas.” Clinical Cancer Research : An Official\nJournal of the American Association for Cancer Research 23 (22): 7047–58.\nhttps://doi.org/10.1158/1078-0432.CCR-17-0077.\nChow, Melvyn T., Aleksandra J. Ozga, Rachel L. Servis, et al. 2019. “ ntratumoral\nActivity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-\nPD- herapy.” Immunity 50 (6): 1498-1512.e5.\nhttps://doi.org/10.1016/j.immuni.2019.04.010.\nook, tuart J., uintin ee, lex h Wong, et al. . “ ifferential hemokine\nReceptor Expression and Usage by Pre-cDC1 and Pre-c .” Immunology and\nCell Biology 96 (10): 1131–39. https://doi.org/10.1111/imcb.12186.\nCorbett, T. H., D. P. Griswold, B. J. Roberts, J. C. Peckham, and F. M. Schabel. 1975.\n“ umor nduction elationships in evelopment of ransplanta le ancers of\nthe Colon in Mice for Chemotherapy Assays, with a Note on Carcinogen\ntructure.” Cancer Research 35 (9): 2434–39.\nanga , enarda, arine ruand, li ée J. rimm, et al. . “ ooperation etween\nConstitutive and Inducible Chemokines Enables T Cell Engraftment and\nmmune ttack in olid umors.” Cancer Cell 35 (6): 885-900.e10.\nhttps://doi.org/10.1016/j.ccell.2019.05.004.\nDarrasse-Jèze, Guillaume, Stephanie Deroubaix, Hugo Mouquet, et al. 2009.\n“ eed ack ontrol of egulatory ell omeostasis y endritic ells in ivo.”\nThe Journal of Experimental Medicine 206 (9): 1853–62.\nhttps://doi.org/10.1084/jem.20090746.\nDi Pilato, M., R. Kfuri- u ens, J. . ruessmann, et al. . “ ositions\nCytotoxic T Cells to Receive Critical Survival Signals in the Tumor\nicroenvironment.” Cell 184 (17): 4512-4530 e22. 34343496.\nhttps://doi.org/10.1016/j.cell.2021.07.015.\nDu, Wanlu, Ivan Seah, Oumaima Bougazzoul, et al. 2017. “ tem ell-Released\nOncolytic Herpes Simplex Virus Has Therapeutic Efficacy in Brain Metastatic\nelanomas.” Proceedings of the National Academy of Sciences of the United\nStates of America 114 (30): E6157–65.\nhttps://doi.org/10.1073/pnas.1700363114.\nDuong, Ellen, Tim B. Fessenden, Emi Lutz, et al. 2022. “ ype nterferon ctivates\nMHC Class I-Dressed CD11b+ Conventional Dendritic Cells to Promote\nProtective Anti-Tumor CD8+ T ell mmunity.” Immunity 55 (2): 308-323.e9.\nhttps://doi.org/10.1016/j.immuni.2021.10.020.\narris, hristopher ., ean . rlauckas, ainer . ohler, et al. . “ uccessful\nAnti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk\nInvolving the Cytokines IFN-γ and - .” Immunity 49 (6): 1148-1161.e7.\nhttps://doi.org/10.1016/j.immuni.2018.09.024.\nHakkarainen, Tanja, Merja Särkioja, Petri Lehenkari, et al. 2007. “ uman\nMesenchymal Stem Cells Lack Tumor Tropism but Enhance the Antitumor\nctivity of ncolytic denoviruses in rthotopic ung and reast umors.”\nHuman Gene Therapy 18 (7): 627–41. https://doi.org/10.1089/hum.2007.034.\nammerich, inda, homas . arron, an an padhyay, et al. . “ ystemic\nClinical Tumor Regressions and Potentiation of PD1 Blockade with in Situ\naccination.” Nature Medicine 25 (5): 814–24. https://doi.org/10.1038/s41591-\n019-0410-x.\nm, e Jin, asao ashimoto, ichael Y. erner, et al. . “ efining ells\nThat Provide the Proliferative Burst after PD- herapy.” Nature 537 (7620):\n417–21. https://doi.org/10.1038/nature19330.\nJosiah, Darnell T., Dongqin Zhu, Fernanda Dreher, John Olson, Grant McFadden, and\nannah aldas. . “ dipose-Derived Stem Cells as Therapeutic Delivery\nehicles of an ncolytic irus for lio lastoma.” Molecular Therapy: The\nJournal of the American Society of Gene Therapy 18 (2): 377–85.\nhttps://doi.org/10.1038/mt.2009.265.\nKalimuthu, Senthilkumar, Ji Min Oh, Prakash Gangadaran, et al. 2017. “ n ivo\nTracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell\nigration y ptical olecular maging.” Stem Cells International 2017 (1):\n8085637. https://doi.org/10.1155/2017/8085637.\nKazimirsky, Gila, Wei Jiang, Shimon Slavin, Amotz Ziv-Av, and Chaya Brodie. 2016.\n“ esenchymal tem ells nhance the ncolytic ffect of ewcastle isease\nirus in lioma ells and lioma tem ells via the ecretion of .” Stem\nCell Research & Therapy 7 (1): 149. https://doi.org/10.1186/s13287-016-0414-\n0.\nidd, hannon, rika paeth, Jennifer . em inski, et al. . “ irect vidence of\nMesenchymal Stem Cell Tropism for Tumor and Wounding Microenvironments\nsing in ivo ioluminescent maging.” Stem Cells (Dayton, Ohio) 27 (10):\n2614–23. https://doi.org/10.1002/stem.187.\nułach, atalia, welina ilny, omas ichoń, et al. . “ esenchymal tromal\nCells as Carriers of IL-12 Reduce Primary and Metastatic Tumors of Murine\nelanoma.” Scientific Reports 11 (1): 18335. https://doi.org/10.1038/s41598-\n021-97435-9.\nai, Junyun, herly ardiana, mran . ouse, et al. . “ doptive ellular herapy\nwith T Cells Expressing the Dendritic Cell Growth Factor Flt3L Drives Epitope\npreading and ntitumor mmunity.” Nature Immunology 21 (8): 914–26.\nhttps://doi.org/10.1038/s41590-020-0676-7.\nee, Jaeyop, aëlle reton, hiago Yukio ikuchi liveira, et al. . “ estricted\nDendritic Cell and Monocyte Progenitors in Human Cord Blood and Bone\narrow.” The Journal of Experimental Medicine 212 (3): 385–99.\nhttps://doi.org/10.1084/jem.20141442.\nLin, K. Y., F. G. Guarnieri, K. F. Staveley- ’ arroll, et al. . “ reatment of\nEstablished Tumors with a Novel Vaccine That Enhances Major\nistocompati ility lass resentation of umor ntigen.” Cancer Research 56\n(1): 21–26.\niu, aolin, ueda u, aichao eng, et al. . “ emporal ingle-Cell Tracing\nReveals Clonal Revival and Expansion of Precursor Exhausted T Cells during\nAnti-PD- herapy in ung ancer.” Nature Cancer 3 (1): 108–21.\nhttps://doi.org/10.1038/s43018-021-00292-8.\nonghi, . aula, hristine rumpfheller, Juliana doyaga, et al. . “ endritic ells\nRequire a Systemic Type I Interferon Response to Mature and Induce CD4+\nh mmunity with oly as d uvant.” The Journal of Experimental Medicine\n206 (7): 1589–602. https://doi.org/10.1084/jem.20090247.\nagen, ssaf, auline amon, athalie iaschi, et al. . “ ntratumoral endritic\nCell-CD4+ T Helper Cell Niches Enable CD8+ T Cell Differentiation Following\nPD- lockade in epatocellular arcinoma.” Nature Medicine 29 (6): 1389–\n99. https://doi.org/10.1038/s41591-023-02345-0.\nMattiuz, Raphaël, Jesse Boumelha, Pauline Hamon, et al. 2024. “ endritic ells ype\ntructures in ancer.” reprint, ecem er .\nhttps://doi.org/10.1101/2024.12.27.628014.\neiser, hilippa, orit . nolle, nna irsch erger, et al. . “ istinct\nStimulatory cDC1 Subpopulation Amplifies CD8+ T Cell Responses in Tumors\nfor Protective Anti- ancer mmunity.” Cancer Cell 41 (8): 1498-1515.e10.\nhttps://doi.org/10.1016/j.ccell.2023.06.008.\na, akaaki, ark . ong, i or eler, et al. . “ vercoming rimary and\nAcquired Resistance to Anti-PD-L1 Therapy by Induction and Activation of\nTumor- esiding c s.” Nature Communications 11 (1): 5415.\nhttps://doi.org/10.1038/s41467-020-19192-z.\nPereboeva, Larisa, and David T. Curiel. . “ ellular ehicles for ancer ene\nherapy.” BioDrugs 18 (6): 361–85. https://doi.org/10.2165/00063030-\n200418060-00003.\nRen, C., S. Kumar, D. Chanda, et al. 2008. “ ancer ene herapy sing\nMesenchymal Stem Cells Expressing Interferon-Beta in a Mouse Prostate\nancer ung etastasis odel.” Gene Therapy 15 (21): 1446–53.\nhttps://doi.org/10.1038/gt.2008.101.\nRyu, Chung Heon, Sang- oon ark, oon . ark, et al. . “ ene herapy of\nIntracranial Glioma Using Interleukin 12-Secreting Human Umbilical Cord\nBlood- erived esenchymal tem ells.” Human Gene Therapy 22 (6): 733–\n43. https://doi.org/10.1089/hum.2010.187.\nalmon, élène, Juliana doyaga, dee ahman, et al. . “ xpansion and\nActivation of CD103(+) Dendritic Cell Progenitors at the Tumor Site Enhances\nTumor Responses to Therapeutic PD- and nhi ition.” Immunity 44 (4):\n924–38. https://doi.org/10.1016/j.immuni.2016.03.012.\nalomon, an, agar otem, Yonatan at enelen ogen, et al. . “ ispecific\nAntibodies Increase the Therapeutic Window of CD40 Agonists through\nelective endritic ell argeting.” Nature Cancer 3 (3): 287–302.\nhttps://doi.org/10.1038/s41508-022-00329-6.\nSánchez-Paulete, Alfonso R., Álvaro Teijeira, José I. Quetglas, et al. 2018.\n“ ntratumoral mmunotherapy with and s lt ncoded in ecom inant\nSemliki Forest Virus-Derived Vectors Fosters Dendritic Cell-Mediated T-Cell\nCross- riming.” Cancer Research 78 (23): 6643–54.\nhttps://doi.org/10.1158/0008-5472.CAN-18-0933.\nchenkel, Jason ., e ecca . er st, avid anner, et al. . “ onventional\nType I Dendritic Cells Maintain a Reservoir of Proliferative Tumor-Antigen\nSpecific TCF-1+ CD8+ T Cells in Tumor- raining ymph odes.” Immunity 54\n(10): 2338-2353.e6. https://doi.org/10.1016/j.immuni.2021.08.026.\nSee, Peter, Charles-Antoine Dutertre, Jinmiao Chen, et al. 2017. “ apping the uman\nDC Lineage through the Integration of High- imensional echniques.” Science\n(New York, N.Y.) 356 (6342): eaag3009.\nhttps://doi.org/10.1126/science.aag3009.\nSiddiqui, Imran, Karin Schaeuble, Vijaykumar Chennupati, et al. 2019. “ ntratumoral\nTcf1+PD-1+CD8+ T Cells with Stem-like Properties Promote Tumor Control in\nesponse to accination and heckpoint lockade mmunotherapy.” Immunity\n50 (1): 195-211.e10. https://doi.org/10.1016/j.immuni.2018.12.021.\nSiwicki, Marie, Nicolas A. Gort-Freitas, Marius Messemaker, et al. 2021. “ esident\nupffer ells and eutrophils rive iver oxicity in ancer mmunotherapy.”\nScience Immunology 6 (61): eabi7083.\nhttps://doi.org/10.1126/sciimmunol.abi7083.\nSonabend, Adam M., Ilya V. Ulasov, Matthew A. Tyler, Angel A. Rivera, James M.\nathis, and acie . esniak. . “ esenchymal tem ells ffectively\neliver an ncolytic denovirus to ntracranial lioma.” Stem Cells (Dayton,\nOhio) 26 (3): 831–41. https://doi.org/10.1634/stemcells.2007-0758.\nSpranger, Stefani, Daisy Dai, Brendan Horton, and Thomas F. Gajewski. 2017.\n“ umor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell\nrafficking and doptive ell herapy.” Cancer Cell 31 (5): 711-723.e4.\nhttps://doi.org/10.1016/j.ccell.2017.04.003.\nStagg, John, Laurence Lejeune, André Paquin, and Jacques Galipeau. . “ arrow\nStromal Cells for Interleukin- elivery in ancer mmunotherapy.” Human\nGene Therapy 15 (6): 597–608. https://doi.org/10.1089/104303404323142042.\nSvensson-Arvelund, Judit, Sara Cuadrado-Castano, Gvantsa Pantsulaia, et al. 2022.\n“ xpanding ross-Presenting Dendritic Cells Enhances Oncolytic Virotherapy\nand Is Critical for Long-Term Anti- umor mmunity.” Nature Communications 13\n(1): 7149. https://doi.org/10.1038/s41467-022-34791-8.\nSwee, Lee Kim, Nabil Bosco, Bernard Malissen, Rhodri Ceredig, and Antonius Rolink.\n. “ xpansion of eripheral aturally ccurring egulatory ells y ms-\nlike yrosine inase igand reatment.” Blood 113 (25): 6277–87.\nhttps://doi.org/10.1182/blood-2008-06-161026.\nTheisen, Derek J., Jesse T. Davidson, Carlos G. Briseño, et al. 2018. “W Y s\nRequired for Cross- resentation in esponse to iral and umor ntigens.”\nScience (New York, N.Y.) 362 (6415): 694–99.\nhttps://doi.org/10.1126/science.aat5030.\nVillani, Alexandra-Chloé, Rahul Satija, Gary Reynolds, et al. 2017. “ ingle-Cell RNA-\nSeq Reveals New Types of Human Blood Dendritic Cells, Monocytes, and\nrogenitors.” Science (New York, N.Y.) 356 (6335): eaah4573.\nhttps://doi.org/10.1126/science.aah4573.\nWan, Duo, Qi Zhang, Zhenrong Yang, et al. 2025. “ ngineered ncolytic irus -\nnhances ntitumor mmunity via endritic ell ctivation.” Molecular\nTherapy Oncology 33 (2): 200975.\nhttps://doi.org/10.1016/j.omton.2025.200975.\nYoon, A.- um, JinWoo ong, Yan i, et al. . “ esenchymal tem ell-Mediated\nDelivery of an Oncolytic Adenovirus Enhances Antitumor Efficacy in\nepatocellular arcinoma.” Cancer Research 79 (17): 4503–14.\nhttps://doi.org/10.1158/0008-5472.CAN-18-3900.\nYost, athryn ., nsuman . atpathy, aniel . Wells, et al. . “ lonal\nReplacement of Tumor-Specific T Cells Following PD- lockade.” Nature\nMedicine 25 (8): 1251–59. https://doi.org/10.1038/s41591-019-0522-3.\nZhang, ao, Yu Wang, ing i, et al. . “ esenchymal tromal ells quipped y\nα mpower ells with otent nti- umor mmunity.” Oncogene 41 (13):\n1866–81. https://doi.org/10.1038/s41388-022-02201-4.\nREVIEWERS' COMMENTS\nReviewer #1:\nThe authors have greatly addressed each of my comments. I have no more requests\nnor additional clarifications.\nWe sincerely thank the reviewer for the positive feedback and are pleased that our\nrevisions have satisfactorily addressed the comments.\nReviewer #2:\nThe authors have extensively addressed my comments and those of the other\nreviewers. I have no additional suggestions.\nWe sincerely thank the reviewer for his careful evaluation and for acknowledging the\nrevisions. We greatly appreciate your time and constructive feedback throughout the\nreview process.\nReviewer #3:\nI would like to congratulate the authors for strongly improving the manuscript and\nerasing most of my comments. Additional analyses and controls strengthen the\nscientific soundness of the data and conclusions. Also, it is appreciated that the authors\nvalidated the efficacy of eMSC-FLT3L + poly(I:C) in 3 more grafted cancer models.\nYet, the key findings in the remaining paper are unfortunately still limited to B16-OVA.\nHowever, more importantly, in light of the new data in response to my major comment\n2, the comparison of Recombinant FLT3L + poly(I:C) (which are known and previously\ndescribed treatments) and eMSC-FLT3L + poly(I:C) and mono-treatments, the\ntherapeutic improvement of using eMSC-FLT3L (over recombinant Flt3l or polyI:C\nalone) is unfortunately overall less convincing in the revised version of the manuscript.\nIndeed, this direct comparison was not tested in more physiological cancer models\n(without ectopic antigen), where the therapeutic efficacy may actually be equal. Hence,\nbased on the provided data in Supplementary Figure 2 (that shows no significant\ndifference between Recombinant FLT3L + poly(I:C) and eMSC-FLT3L + poly(I:C)), I\nam unfortunately not convinced that there is a strong therapeutic advantage of using\neMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5 of the other treatment (combinations) of\npolyI:C and rec-Flt3L. In my point of view, this should be better shown experimentally\nin at least a second physiological cancer model (not only B16-OVA). Other advantages\nof eMSC-FLT3L compared with rec-Flt3L could also be more closely explored or at\nleast explained in detail to justify the use of an adoptive transfer of cells versus a\nrecombinant protein/adjuvant in clinical practice (e.g. eventual toxicity, Treg induction\nin a comparative experiment, etc) – but those experiments are just a suggestion.\nErasing this last doubt would really strengthen the relevance of the presented\nmanuscript, that otherwise is very interesting.\nWe sincerely thank the reviewer for the positive and encouraging feedback. We are\nglad that the additional analyses and controls have reinforced the robustness of our\nfindings. Your insights were very helpful in improving the manuscript.\nRegarding eMSC-FLT3L + poly(I:C) vs recombinant protein FLT3L + poly(I:C):\nThe data presented in Supplementary Fig. 2 show that both strategies achieve\ncomparable tumor control. However, in terms of survival, eMSC_FLT3L + poly(I:C)\ndemonstrates improved efficacy, with some surviving mice (vs. untreated, ****),\nwhereas recombinant FLT3L + polyI:C does not result in surviving animals (vs.\nuntreated, **). We therefore conclude that eMSC-FLT3L provides a modest but\nmeasurable advantage over recombinant FLT3L, which is further supported by the\nabsence of systemic effects on FLT3L levels (Fig. 1J). Overall, these data demonstrate\nbenefit–risk ratio that favors eMSC-FLT3L over recombinant FLT3L.\nWe agree with Reviewer 3 on the need to extend these analyses to additional tumor\nmodels, including autochthonous models. We plan to undertake these experiments in\nsubsequent studies, which will include careful titration of recombinant FLT3L and\nevaluation of alternative injection schedules. For instance, intratumoral route was used\nfor recombinant FLT3L. We would need to also compare systemic delivery route (more\namenable in clinical practice).\nAs noted in the revised Discussion section, we believe that cell delivery methods must\nbe carefully optimized to ensure reliable implantation of eMSC-FLT3L within tumors.\nAs this work is beyond the scope of the current manuscript, we consider it premature\nto draw conclusions regarding the relative clinical utility of eMSC-FLT3L versus\nrecombinant FLT3L. Nevertheless, we believe that the current study provides a\nvaluable proof-of-concept demonstrating the feasibility of using eMSCs to stimulate\ncDC infiltration in solid tumors."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have greatly addressed each of my comments. I have no more requests nor additional clarifications.", "reviewer_2": "The authors have extensively addressed my comments and those of the other reviewers. I have no additional suggestions.", "reviewer_3": "I would like to congratulate the authors for strongly improving the manuscript and erasing most of my comments. Additional analyses and controls strengthen the scientific soundness of the data and conclusions. Also, it is appreciated that the authors validated the efficacy of eMSC-FLT3L + poly(I:C) in 3 more grafted cancer models. Yet, the key findings in the remaining paper are unfortunately still limited to B16-OVA.\nHowever, more importantly, in light of the new data in response to my major comment 2, the comparison of Recombinant FLT3L + poly(I:C) (which are known and previously described treatments) and eMSC-FLT3L + poly(I:C) and mono-treatments, the therapeutic improvement of using eMSC-FLT3L (over recombinant Flt3l or polyI:C alone) is unfortunately overall less convincing in the revised version of the manuscript. Indeed, this direct comparison was not tested in more physiological cancer models (without ectopic antigen), where the therapeutic efficacy may actually be equal. Hence, based on the provided data in Supplementary Figure 2 (that shows no significant difference between Recombinant FLT3L + poly(I:C) and eMSC-FLT3L + poly(I:C)), I am unfortunately not convinced that there is a strong therapeutic advantage of using eMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5 of the other treatment (combinations) of polyI:C and rec-Flt3L. In my point of view, this should be better shown experimentally in at least a second physiological cancer model (not only B16-OVA). Other advantages of eMSC-FLT3L compared with rec-Flt3L could also be more closely explored or at least explained in detail to justify the use of an adoptive transfer of cells versus a recombinant protein/adjuvant in clinical practice (e.g. eventual toxicity, Treg induction in a comparative experiment, etc) – but those experiments are just a suggestion.\nErasing this last doubt would really strengthen the relevance of the presented manuscript, that otherwise is very interesting."}, "author_response": ""}]

Decision letter