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

Louise Gorline, Fillipe Luiz Rosa Do Carmo, Pierre Bourdely, Jérémie Bornères, Nathan Vaudiau, Aurélie Semervil, Mathias Vetillard, Aboubacar Sidiki K Coulibaly, Natacha Jugniot, Agathe Ok, et al.

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Louise Gorline, Fillipe Luiz Rosa Do Carmo, Pierre Bourdely, Jérémie Bornères, Nathan Vaudiau, et al.. Intratumoral delivery of FLT3L with CXCR3/CCR5 ligands promotes XCR1 + cDC1 infiltration and activates anti-tumor immunity. 2025. hal-05406964

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1 **Title:** Intratumoral delivery of FLT3L with CXCR3/CCR5 ligands promotes XCR1⁺
cDC1 infiltration and activates anti-tumor immunity.

3

4 **Authors:** Louise Gorline¹,² ³†, Fillipe Luiz Rosa do Carmo¹,² ³†, Pierre Bourdely⁴,⁵, Jérémie

5 Bornères¹,²,³, Nathan Vaudiau¹,²,³, Aurélie Semervil¹,²,³,⁵, Mathias Vetillard⁶, Aboubacar Sidiki

6 K Coulibaly¹,²,³, Natacha Jugniot¹,²,³, Agathe Ok⁶, Mathilde Bausart⁷, Oriane Fiquet⁸, Marine

7 Andrade⁸, Dicken Fardol⁹, Ikrame Haddar¹,²,³, Zeina Abou Nader⁵, Judith Weber⁵, Hannah

8 Theobald¹⁰, Matthieu Collin¹¹, Joseph Calmette¹¹, Giorgio Anselmi¹², Flavia Fico⁷, Florent

9 Ginhoux¹⁰, Laleh Majlessi¹³, Emmanuel L. Gautier⁹, Loredana Saveanu⁶, Julie Helft⁵, Marc

10 Dalod¹⁴, Mathilde Dusseaux⁸, James P. Di Santo³,¹⁵, Stéphanie Hugues⁷, Pierre

11 Guermonprez¹,²,³*

12

13 **Affiliations**

14 ¹Université de Paris Cité, Institut Pasteur, Immunology Department, Dendritic Cells and Adaptive Immunity Unit, 75015 Paris, France.

16 ²Université de Paris Cité, Institut Pasteur, CNRS UMR3738, Developmental Biology and Stem Cells, 75015 Paris, France.

18 ³Centre for Vaccine and Immunotherapy, Institut Pasteur, 75015 Paris, France.

19 ⁴Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology, VIB, Leuven.

21 ⁵Université Paris Cité, Institut Cochin, INSERM UMR 1016, CNRS UMR 8104, 75014 Paris, France.

23 ⁶Université Paris Cité, Bichat Medical School, INSERM UMR1149, CNRS EMR8252, 75018 Paris, France.

25 ⁷Department of Pathology and Immunology, Geneva Medical School, Geneva, Switzerland.

26 ⁸Human Disease Models Core Facility, Institut Pasteur, Université Paris Cité, 75015 Paris, France.

28 ⁹Sorbonne University, Inserm, Nutrition and Obesity: Systemic Approaches, 75013 Paris, France.

30 ¹⁰Institut Gustave Roussy, Inserm U1015, Université Paris-Saclay, Villejuif, France.

31 ¹¹Inserm Transfert, 75013 Paris, France.

32 ¹²Department of Cellular Immunology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

34 ¹³Université Paris Cité, Pasteur-TheraVectys Joint Laboratory, Institut Pasteur, Virology Department, 75015 Paris, France

36 ¹⁴Aix Marseille Univ, CNRS, INSERM, CIML, Centre d'Immunologie de Marseille-Luminy, France.

38 ¹⁵Institut Pasteur, Immunology Department, Immunité Innée, 75015 Paris, France.

39
40 †These authors contributed equally to this work.

41 *Corresponding author: pierre.guermonprez@pasteur.fr

42
43 Abstract

44 Tumor infiltration by XCR1+ conventional dendritic cells (cDC1) correlates strongly with
45 favorable prognosis and improved responses to immunotherapy. Yet, tumor-driven
46 immunosuppressive programs restrict efficient cDC1 recruitment, highlighting the need for
47 strategies increasing cDC1 access to the tumor microenvironment. Here, we establish a proof-
48 of-concept cell-based immunotherapy that enhances the infiltration of circulating cDC1
49 progenitors and supports their local expansion. Intratumoral engraftment of autologous
50 mesenchymal stromal cells engineered to express membrane bound FLT3L promotes cDC1
51 recruitment when combined with poly(I:C). We identify poly(I:C)-induced CXCL9 and CCL5
52 as essential chemokines controlling intratumoral cDC1 infiltration. Stromal cell-mediated local
53 delivery of FLT3L together with CXCL9 and CCL5 is sufficient to enhance cDC1 infiltration
54 in mice or humanized mice settings. Finally, this approach activates antitumor immunity and
55 partially overcomes resistance to immune checkpoint blockade. Collectively, our data support
56 the therapeutic potential of expanding intratumoral cDC1s through local and sustained delivery
57 of FLT3L, CXCL9, and CCL5.
58

58
## Introduction

60

61 An important factor limiting the development of anti-tumor immunity lies in the extent
62 of tumor infiltration by immune cells, including dendritic cells (DCs). DCs are sentinel cells of
63 the immune system initiating the tumor-immunity cycle (1, 2).

64 Tumor-associated DCs (TADCs) up-take antigens by phagocytosis and migrate to tumor
65 draining lymph nodes (tdLNs) after up-regulating CCR7. If engaged in immunogenic
66 maturation by the activation of proper innate signalling pathways, DCs can prime anti-tumor
67 CD4+ and CD8+ T cells in tdLNs (2, 3). Once activated, effector T lymphocytes can migrate
68 back to tumors *via* efferent lymph and then blood (2). All these activities define DCs as initiators
69 of the cancer-immunity cycle.

70 In addition to this virtuous cycle of T cell activation in tdLN, intratumoral DCs also
71 have local roles. Indeed, they participate to the recruitment of CXCR3+ effector T cells via the
72 release of CXCL9 chemokines (4–6). Also, intratumoral DCs shape niches supporting the
73 survival of tumor-infiltrating T cells (TILs) by delivering specific trophic cues. For instance,
74 CXCL16+ TADCs attract CXCR6+TCF1- effector and promote their survival by trans-
75 presenting IL15 (7). DCs establish local interactions with CD28+ T cells and preserve their
76 fitness (8). Finally, CCR7+ LAMP3+ DCs also populate Tertiary Lymphoid Structures (9), some
77 “stem immunity hubs” related-lymphoid aggregates where they colocalize with TCF1+CD8+
78 TILs (6, 10) and establish some tripartite interactions with CXCL13+CD4+ T cells and
79 TCF1+CD8+ TILs (11).

80 DCs are classified into two major subsets: XCR1+ type 1 conventional DCs (cDC1s),
81 which express IRF8 and an heterogenous population of CD11b+ DCs expressing IRF4 and
82 encompassing cDC2s and DC3s. cDC1s play a non-redundant role in the rejection of
83 immunogenic tumors (12) and are essential for the efficacy of immune checkpoint blockade
84 (ICB) therapies, as demonstrated in pre-clinical models (13, 14). Furthermore, cDC1 infiltration
85 in human tumors correlates with favorable clinical outcomes and response to ICB (15, 16).
86 Mechanistically, cDC1 would fine tune the CD8+ T cell response by maintaining the pool of
87 TCF1+ TILs along tumor development thereby fueling a reservoir of T cells potentially
88 promoting effector anti-tumor responses (17). However, cDC1 infiltration within tumors
89 remains generally limited (18–20). COX enzymes in tumor cells limit the activation of NK-
90 dependent DCs recruitment (16, 21). Also, oncogenic pathways such as β-catenin limit the
91 recruitment of cDC1s by limiting CCL4 production (19). Therefore, enhancing the recruitment
92 and activation of intratumoral cDC1s remains an unmet therapeutic need to overcome cancer-
93 induced immune suppression.

94 Mechanistically, recruitment of circulating DCs have been investigated in the context
95 of pre-clinical models of immunogenic tumors (15, 19, 21). Böttcher et al. have identified that
96 cDC1s recruitment depends on NK-cell derived CCR5 ligands (CCL4, CCL5), as well as XCR1
97 ligands (XCL1, XCL2) (21). It is unclear if infection activated CCR2-dependent pathways (22)
98 also control cDC1s recruitment.

99 The FLT3L-FLT3 signaling pathway is essential for the homeostasis of conventional
100 DCs, including cDC1s, in both mice (23, 24) and humans (25). Intratumoral FLT3L is an
101 important biomarker associated to the density of TADCs, particularly cDC1s, in human
102 melanoma (15). FLT3L-FLT3 pathway supports DC development and maintenance in both
103 lymphoid organs and peripheral tissues (24). Tissue FLT3L supports the proliferation of DC
104 precursors (pre-DCs) in bone marrow and peripheral sites (24, 26). Of note, radio-resistant cells
105 control homeostatic levels of FLT3L (27). Within secondary lymphoid organs, stromal cells
106 contribute to DC pool maintenance. For example, a CCL19+GREMLIN+ fibroblastic niche
107 support DCs survival at homeostasis (28) via FLT3L production (29). However, the cellular
108 sources of FLT3L within tumors are not very well known. Barry et al. have shown that NK
109 cells represent a substantial source of FLT3L (15) but other sources like T cells (30) or mast
110 cells (27) might participate to local FLT3L production.

111 Administration of recombinant soluble FLT3L is an effective approach to expand
112 circulating DCs (31) in humans but it faces questionable safety issues with general modification
113 of hematopoiesis and expansion of tolerogenic DCs (32). Furthermore, systemic recombinant
114 FLT3L might promote immune tolerance, as it leads to the expansion of regulatory T cells (33,
115 34). Lastly, combination therapies of FLT3L with innate agonists such as anti-CD40 (20) or
116 poly(I:C) (14) raise some toxicity concerns associated to systemic inflammation.

117 In this work, we have reasoned that targeted, local interventions in the tumor
118 microenvironment (TME) could optimize local generation of immunogenic DCs (35), while
119 avoiding most systemic effects underpinning immune regulation or toxicity. Here, we explore
120 the therapeutic potential of delivering engineered autologous stromal cells within the TME.
121 Engrafted stromal cells are expressing chemotactic factors supporting DC infiltration,
122 differentiation, proliferation, and maintenance. We identify that stromal membrane bound form
123 of FLT3L combined with poly(I:C) or CXCL9 and CCL5 is sufficient to activate the local
124 recruitment and accrual of cDC1s, T and NK/ILC1 cells within tumors. Furthermore, we show
125 that these immunotherapies stimulate anti-tumor immunity and partially overcome resistance
126 to ICB. Altogether, these findings open an avenue to rewire the tumor microenvironment by
127 local cell-based therapeutic intervention.
128 **Results**

129

130 **Intratumoral delivery of engineered mesenchymal stromal cells expressing membrane**
131 **bound FLT3L does not alter intratumoral and systemic cDC1 populations.**

132 We have previously demonstrated that engineered stromal cells expressing human
133 FLT3L act as “synthetic niches” able to support the differentiation of human tissue DCs in
134 immunodeficient mice (36). In a first attempt to stimulate intratumoral cDC accumulation, we
135 generated stable, autologous, fibroblastic cell lines from embryonic fibroblasts of C57BL/6
136 mice. These cells are characterized as CD45⁻CD31⁺CD44⁺CD140a⁺gp38/PDPN⁺ (Fig.
137 Supplementary Fig. 1A) and termed as engineered mesenchymal stromal cells thereafter
138 (eMSC). eMSC-FLT3L express homogeneously the membrane form of human FLT3L, as
139 detected by flow cytometry (Fig. 1A). We found that eMSC-FLT3L but not eMSC-Control
140 (eMSC-Ctrl) stimulated the differentiation of cDC1s and cDC2s, when co-injected with CD45.1
141 bone-marrow hematopoietic stem cell progenitors (HSPCs) in basal membrane extract plugs in
142 the dermis of tumor free mice, showing that FLT3L expressed in eMSC-FLT3L was functional
143 (Fig. 1B-C, Supplementary Fig. 1B). On the contrary, recombinant FLT3L (rec-FLT3L) did
144 not stimulate cDC differentiation within the plugs. We next decided to inject eMSC-FLT3L
145 within subcutaneous B16 melanoma tumors expressing ovalbumin (B16-OVA), which have a
146 primary resistance to immune checkpoint blockade (ICB). First, we found that gp38⁺GFP⁺
147 eMSC-FLT3L were still detectable on site 14 days after intratumoral injection, validating their
148 persistence within the TME (Fig. 1D-E). To further evaluate the long-term biodistribution and
149 persistence of eMSCs, we engineered nano-luciferase-expressing eMSCs and monitored their
150 persistence using in vivo imaging for 8 days post engraftment in the B16-OVA model (Fig. 1F).
151 We observed that eMSCs remained within the tumor for at least 8 days (Fig. 1G), with their
152 activity confined to the tumor site (Fig. 1H). These observations indicate that, once injected,
153 eMSCs stay localized within the tumor and do not disseminate systemically. We next compared
154 intratumoral engraftment of eMSC-FLT3L to B16F10 cells overexpressing human FLT3L
155 (B16-huFLT3L) and assessed their impact on DC differentiation. In contrast with B16-
156 huFLT3L, B16F10 tumor bearing mice that had received intratumoral engraftment of eMSC-
157 FLT3L at day 7 did not display an elevation of systemic FLT3L serum levels at day 12 (Fig.
158 1I-J). Similar results were obtained using the B16-OVA model (Supplementary Fig. 1C).
159 However, we did not observe an increase of cDC1s in the tumor (Fig. 1K), the tumor draining
160 lymph node (tdLN) (Fig. 1L) nor in the spleen (Fig. 1M).

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

163
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

C

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

E

<figure><img src="image_6.png" /></figure>

F

<figure><img src="image_7.png" /></figure>

G

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Days post eMSC-NanoLuc
injection within B16-OVA tumors

H

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

I

<figure><img src="image_12.png" /></figure>

J

<figure><img src="image_13.png" /></figure>

K

<figure><img src="image_14.png" /></figure>

L

<figure><img src="image_15.png" /></figure>

M

<figure><img src="image_16.png" /></figure>
**Figure 1: Intratumoral delivery of engineered mesenchymal stromal cells expressing membrane bound FLT3L does not alter intratumoral and systemic cDC1 populations.**

(A) Expression of GFP (eMSC-Ctrl) and human membrane-bound FLT3 ligand in mouse embryonic fibroblasts (eMSC-FLT3L). φ: packaging signal essential for retroviral genome packaging; IRES: Internal Ribosome Entry Site.

(B) Experimental design to assess the ability of eMSC-FLT3L to drive DC differentiation *in vivo*.

(C) Representative flow cytometry plots and quantification at day 12 of CD45.1 and CD45.2 cDC1s in the synthetic niches containing HSPC only (n=3 plugs) or with eMSC-FLT3L, eMSC-Ctrl or rec-FLT3L (n=4 plugs), one experiment, one-way ANOVA-test with Tukey's multiple comparisons).

(D) Experimental design to evaluate the persistence of eMSC-FLT3L within B16-OVA tumors.

(E) Representative flow cytometry plots and quantification of the absolute number of eMSC-FLT3L-GFP/g tumor, 1 day (n=2 mice) and 14 days (n=3 mice) after eMSC-FLT3L-GFP injection, one experiment, one-tailed Mann-Whitney test.

(F) Experimental design to evaluate the biodistribution and persistence of eMSCs within B16-OVA tumors.

(G) Bioluminescence imaging and quantification of the average radiance of eMSC-NanoLuc at days 0, 4 and 8 following their injection, n=3 mice per group, one experiment.

(H) Bioluminescence imaging and quantification of the average radiance of eMSC-NanoLuc in individual organs of mice having received eMSC-NanoLuc, n=3 mice per group, one experiment. He:heart; Li:liver; Ki:kidney, Sp:spleen, Lu:lungs, Tu:tumor, tdLN:tumor-draining lymph node, Mu:muscle.

(I) Experimental design to evaluate cDC1s in tumors, tumor-draining lymph nodes and spleens.

(J) Circulating huFLT3L levels measured by ELISA at day 12 in mice bearing B16F10 and eMSC-Ctrl (n=3), B16F10 and eMSC-FLT3L (n=5) and B16-FLT3L (n=5), one experiment, one-way ANOVA-test with Tukey's multiple comparisons.

(K-M) Quantification of the absolute numbers of cDC1s in the tumors (K) (n=4 mice), in the skin tumor-draining lymph nodes (tdLN) (L) (n=3 mice for B16F10 and n=5 mice for the others) and in the spleens (M) (n = 5 mice), one experiment, one-way ANOVA-test with Tukey's multiple comparisons.

(C, J-M) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all data points displayed.

(E, G-H) Data are presented as mean values ± SD.

(C, E, G-H, J-M) Source data are provided as a Source Data file.

i.t.: intratumoral; i.v.: intravenous
202 Intratumoral delivery of eMSC-FLT3L synergizes with poly(I:C) to activate anti-tumor
203 immunity.

204 We next wondered if innate agonist administration would circumvent the inability of
205 eMSC-FLT3L to expand intratumoral DCs. We chose to use poly(I:C) because it had previously
206 been shown to stimulate anti-tumor immunity in combination with recombinant, soluble FLT3L
207 (14). We designed our intratumoral cell-based therapy as two injections of eMSC-FLT3L and
208 one injection of poly(I:C) within B16-OVA tumors (Fig. 2A). We found that only eMSC-
209 FLT3L in combination with poly(I:C) induced tumor regression as assessed by the tumor
210 growth (Fig. 2B) and the tumor weight (Fig. 2C). In addition, the combination therapy extended
211 the survival of mice. Importantly, 10% of the mice treated with the combination therapy
212 eliminated the tumor without relapses until day 90 (Fig. 2D). Interestingly, intratumoral
213 administration of recombinant FLT3L in combination with poly(I:C) also delayed tumor growth
214 (Supplementary Fig. 2A) and improved survival (Supplementary Fig. 2B) but less efficiently
215 than eMSC-FLT3L. Furthermore, mice treated with the therapy did not exhibit weight loss,
216 unlike those receiving anti-CD40 treatment as reported elsewhere (37–39) (Fig. 2E, F). In
217 addition, serum levels of ALT (Fig. 2G) and AST (Supplementary Fig. 2C), markers of
218 hepatocellular injury, remained unchanged in mice treated with eMSC-FLT3L + poly(I:C)
219 compared to the anti-CD40 group. Furthermore, mice having received the anti-CD40 antibody
220 exhibited necrotic lesions in the liver (Supplementary Fig. 2D). These results demonstrate the
221 lack of noticeable toxicity associated to immunotherapy by eMSC-FLT3L + poly(I:C)
222 engraftment. In a clinical translational point of view, we asked if adult, autologous fibroblasts
223 would represent a suitable option for eMSC-based immunotherapy. To test this, we generated
224 a CD45⁻CD31⁺ stromal fraction from adult murine visceral and inguinal adipose tissues. We
225 transduced them to express membrane bound human FLT3L (eAMSC-FLT3L) (Fig. 2H).
226 Following the same experimental plan as previously described (Fig. 2A), we found that
227 eAMSC-FLT3L displayed similar efficiency as eMSCs-FLT3L, when combined with
228 poly(I:C), as showed by the reduced tumor growth (Fig. 2I) and tumor weight (Fig. 2J). We
229 conclude that adult fibroblasts are amenable to engineering for local immunotherapy. Lastly, to
230 further validate the therapeutic efficacy of the eMSC-FLT3L + poly(I:C) strategy, we tested it
231 across multiple tumor models, including MC38 colon carcinoma (Fig. 2K-N), TC-1 lung
232 carcinoma transformed with HPV16 E6/E7 (Fig. 2O-R), and the orthotopic E0771 breast
233 carcinoma (Fig. 2S-V). In each model, treatment delayed tumor growth (Fig. 2L, P, T), reduced
234 individual tumor size variation relative to the day of the first intratumoral injection (Fig. 2M,
235 Q, U), and improved overall survival (Fig. 2N, R, V).
236 Altogether, these experiments show that local cell-based delivery of FLT3L within the
237 TME in combination with poly(I:C) is a potent therapy to control tumor growth in multiple
238 tumor contexts.
239
A

<figure><img src="image_17.png" /><figcaption>B16-OVA</figcaption></figure>

C57BL/6

Tumor analysis

∇i.t. eMSC-FLT3L

∇i.t. Poly (I:C)

B

<figure><img src="image_18.png" /></figure>

Days post B16-OVA injection

C

<figure><img src="image_19.png" /></figure>

D

<figure><img src="image_20.png" /></figure>

Days post B16-OVA injection

E

<figure><img src="image_21.png" /><figcaption>B16-OVA</figcaption></figure>

7 i.t. eMSC-FLT3L

Serum analysis

7 i.t. poly (I:C)

7 i.p. anti-CD40

F

<figure><img src="image_22.png" /></figure>

Days post B16-OVA injection

eMSC-FLT3L

poly(I:C)

Untreated

eMSC-FLT3L + poly(I:C)

anti-CD40

<figure><img src="image_23.png" /></figure>

G

<figure><img src="image_24.png" /></figure>

Poly (I:C)

anti-CD40

5.66e-05

4.66e-05

4.95e-05

2.62e-05

H

Autologous
stromal cells:
adipose-derived

<figure><img src="image_25.png" /><figcaption>B16-OVA
bearing mice</figcaption></figure>

I

<figure><img src="image_26.png" /></figure>

Days post B16-OVA injection

J

<figure><img src="image_27.png" /></figure>

K

<figure><img src="image_28.png" /><figcaption>MC38</figcaption></figure>

∇i.t. eMSC-FLT3L

∇i.t. Poly (I:C)

L

<figure><img src="image_29.png" /></figure>

Days post MC38 injection

M

<figure><img src="image_30.png" /></figure>

N

<figure><img src="image_31.png" /></figure>

Days post MC38 injection

O

<figure><img src="image_32.png" /><figcaption>TC-1</figcaption></figure>

∇i.t. eMSC-FLT3L

∇i.t. Poly (I:C)

P

<figure><img src="image_33.png" /></figure>

Days post TC-1 injection

Q

<figure><img src="image_34.png" /></figure>

R

<figure><img src="image_35.png" /></figure>

Days post TC-1 injection

S

<figure><img src="image_36.png" /></figure>

C57BL/6

E0771

∇i.t. eMSC-FLT3L

∇i.t. Poly (I:C)

T

<figure><img src="image_37.png" /></figure>

Days post E0771 injection

U

<figure><img src="image_38.png" /></figure>

V

<figure><img src="image_39.png" /></figure>

Days post E0771 injection
241 **Figure 2: Intratumoral engraftment of autologous mesenchymal stromal cells expressing**
242 membrane bound FLT3L stimulates anti-tumor immunity in the presence of poly(I:C)

243 (A) Experimental design to evaluate anti-tumoral effects of eMSC-FLT3L+poly(I:C) in B16-244 OVA mice model.

245 (B-C) Tumor growth curves (n=10 mice) (B) and tumor weight (C) at day 15. n=9 (eMSC-Ctrl, 246 eMSC-FLT3L), n=10 (eMSC-Ctrl+poly(I:C), eMSC-FLT3L+poly(I:C)) mice per group, two 247 independent experiments, two-way ANOVA-test with Tukey's multiple comparisons, Kruskal-248 Wallis' test with Dunn's multiple comparisons.

249 (D) Survival curves. n=34 (eMSC-Ctrl), n=36 (eMSC-FLT3L+poly(I:C)) mice, six independent 250 experiments, log-rank (Mantel-Cox) test.

251 (E) Experimental design to evaluate the toxicity of the immunotherapy.

252 (F) Body weight loss of mice throughout the experiment. n=4 (eMSC-FLT3L+poly(I:C), anti-253 CD40), n=3 (other groups) mice per group, two-way ANOVA-test with Tukey's multiple 254 comparisons. Data are presented as mean values ± SD.

255 (G) Level of ALT in serum collected 48h after poly(I:C) or anti-CD40 treatment, one-way 256 ANOVA-test with Tukey's multiple comparisons. Data are presented as mean values ± SD. (F-257 G) n=4 mice (eMSC-FLT3L+poly(I:C)), n=3 mice (other ones) per group, one experiment.

258 (H) Experimental design. Adipocytes precursors (CD45-CD31-) were isolated from inguinal 259 and visceral fat of C57BL/6 mice and transduced with membrane-bound FLT3 ligand (eAMSC-260 FLT3L). These cells were injected following the experimental design in (A).

261 (I) Tumor growth curves, two-way ANOVA-test with Dunnett's multiple comparisons.

262 (J) Tumor weight at day 15, Kruskal-Wallis' test with Dunn's multiple comparisons. (I-J) n=5 263 (no therapy), n=8 (other groups) mice, one experiment.

264 (K, O, S) Experimental design to evaluate anti-tumoral effects of eMSC-FLT3L and poly(I:C) 265 in MC38 (K), TC-1 (O) and E0771 (S) tumor models.

266 (L, P, T) Tumor growth curves showing individual mice.

267 (M, Q, U) Individual tumor size variation compared to the day of the first eMSC-FLT3L 268 injection. Two-way ANOVA-test with Šídák's multiple comparisons.

269 (N, R, V) Survival curves. Log-rank (Mantel-Cox) test.

270 (L,N) n=7 mice per group, (M) n=6 (untreated), n=7 (eMSC-FLT3L+poly(I:C)) mice, one 271 experiment. (P-R) n=6 mice per group, one experiment. (T-V) n=8 mice per group, one 272 experiment.

273 (B, I) A line represents the mean and SEM is shown with the colored area.

274 (C, J) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all 275 data points displayed.

276 (B-D, F-G, I-J, L-N, P-R, T-V) Source data are provided as a Source Data file.

277 i.p.: intraperitoneal

278
279 **Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the infiltration of cDC1s,**
280 that are required for immunotherapy efficiency.

281 Having established the immunotherapeutic potential of eMSC-FLT3L + poly(I:C)
282 combination, we next sought to address if this intervention would modify cDC infiltration in
283 the TME. To do so, we analysed intratumoral cDCs 24h and 6 days after a poly(I:C) injection
284 (Fig. 3A). We observed that 24 hours post poly(I:C) injection (day 10), tumor cDC1s underwent
285 phenotypic maturation, as probed by CD40 and CD86 up-regulation within both eMSC-Ctrl
286 and eMSC-FLT3L deliveries (Fig. 3B-C). Poly(I:C) injection also induced a decrease in the
287 number of cDC1s in both eMSC-Ctrl and eMSC-FLT3L treated tumors (Fig. 3C). In contrast,
288 6 days post poly(I:C) injection (day 15) cDC1s underwent expansion in mice treated with both
289 poly(I:C) and eMSC-FLT3L but not in mice treated with poly(I:C) or eMSC-FLT3L alone (Fig.
290 3C). This shows that sustained cDC1 expansion at the tumor site, uniquely achieved by the
291 synergy of eMSC-FLT3L and poly(I:C), correlates with the efficiency of the therapy. Of note,
292 similar results were found for cDC2s (Supplementary Fig. 3A). In order to test the role of cDC1s
293 in the efficiency of the therapy, we injected B16-OVA in WT and in cDC1-deficient mice
294 (Xcr1<sup>cre</sup>xRosa<sup>lsl-DTA</sup> (Xcr1<sup>DTA</sup>) (40)) and treated the tumors with eMSC-FLT3L and poly(I:C)
295 immunotherapy. We found that the tumor control (Fig. 3D) and the survival extension (Fig. 3E)
296 provided by the therapy, were totally abrogated in the absence of cDC1s.

297 We conclude that the synergy between eMSC-FLT3L and poly(I:C) expands
298 intratumoral cDC1s, that are required for the therapeutic benefits of this intervention.
299
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<figure><img src="image_40.png" /></figure>

i.t. eMSC-FLT3L

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301 **Figure 3: Intratumoral delivery of eMSC-FLT3L and poly(I:C) stimulates the infiltration**
302 of cDC1s, that are required for immunotherapy efficiency.

303 (A) Experimental design to evaluate cDC1 infiltration in B16-OVA tumors.

304 (B-C) Representative flow cytometry plots at day 10 (B) and quantification (C) of the absolute
305 number of intratumoral cDC1/g tumor and the mean fluorescence intensity (MFI) of CD40 and
306 CD86. Results are shown as fold change to control (eMSC-Ctrl). Day10: n=6 (eMSC-Ctrl,
307 eMSC-Ctrl+poly(I:C)), n=7 (eMSC-FLT3L, eMSC-FLT3L+poly(I:C)) mice per group, two
308 independent experiments, one-way ANOVA-test with Tukey's multiple comparisons. Day 15:
309 n=8 (eMSC-Ctrl, eMSC-FLT3L), n=9 (eMSC-Ctrl+poly(I:C), eMSC-FLT3L+poly(I:C)) mice
310 per group, two independent experiments, one-way ANOVA-test with Tukey's multiple
311 comparisons. Box plots show median, 25th-75th percentiles, minimum-maximum whiskers,
312 with all data points displayed.

313 (D) Tumor growth curves of Xcr1DTA mice or C57BL/6 WT mice treated or not with eMSC-
314 FLT3L+poly(I:C). A line represents the mean and SEM is shown with the colored area, n=4
315 mice Xcr1DTA-No therapy and n=8 mice for the three other groups, representative of two
316 independent experiments, two-way ANOVA-test with Tukey's multiple comparisons.

317 (E) Survival curves. n=10 (C57BL/6-No therapy, Xcr1DTA-No therapy), n=15 (Xcr1DTA-eMSC-
318 FLT3L+poly(I:C)), n=16 (C57BL/6-eMSC-FLT3L+poly(I:C)) mice, two independent
319 experiments, log-rank (Mantel-Cox) test.

320 (C-E) Source data are provided as a Source Data file.

321
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 24 hours (day 10) and 6 days (day 15) after the poly(I:C) administration (Fig. 4A). At 24 hours post poly(I:C) injection, we observed an increase in the migration of mature (CD40^high, CD86^high) 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 T regulatory cells (Tregs) ratio increased (Fig. 4F-G, 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 OVA257-264 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.
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Gated on live CD45⁺CD3⁺

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**Figure 4:** Intratumoral delivery of eMSC-FLT3L and poly(I:C) increase mature migratory cDC1s in tumor-draining lymph nodes and enables cross-priming of tumor-specific CD8+ T cells.

(A) Experimental design to evaluate migratory cDC1s in the tumor-draining lymph nodes.

(B-C) Representative flow cytometry plots at day 10 (B) and quantification (C) of the absolute number of migratory cDC1s and the mean fluorescence intensity (MFI) of CD40 and CD86.

Results are shown as fold change to control (eMSC-Ctrl). Day 10: n=8 (eMSC-Ctrl, eMSC-FLT3L), n=7 (eMSC-Ctrl+poly(I:C), eMSC-FLT3L+poly(I:C)) mice per group, two independent experiments, one-way ANOVA-test with Tukey's multiple comparisons. Day 15:

n=8 (eMSC-Ctrl, eMSC-FLT3L+poly(I:C)), n=9 (eMSC-FLT3L, eMSC-Ctrl+poly(I:C)), two independent experiments, for the MFI CD40, n=4 mice per group, representative of two independent experiments, one-way ANOVA-test with Tukey's multiple comparisons.

(D) Representative flow cytometry plots of CD8 T cells in the tumor-draining lymph nodes at day 15.

(E) Quantification of the absolute number of activated (T<sub>ACT</sub>), exhausted (T<sub>EX</sub>) and stem-like (T<sub>SL</sub>) CD8 T cells. Results are shown as fold change to control (eMSC-Ctrl). T<sub>ACT</sub>: n=12

(eMSC-Ctrl, eMSC-FLT3L+poly(I:C)), n=13 (eMSC-FLT3L), n=14 (eMSC-Ctrl+poly(I:C)) mice per group, three independent experiments, Kruskal-Wallis test with Dunn's multiple comparisons. T<sub>EX</sub> and T<sub>SL</sub>: n=4 mice per group, Kruskal-Wallis test with Dunn's multiple comparisons.

(F-G) Representative flow cytometry plots (F) and quantification (G) of the ratio of IFNγ<sup>+</sup>CD8<sup>+</sup> T cells over T regulatory cells at day 15. n=4 (eMSC-Ctrl), n=5 (other groups) mice per group, one experiment, Kruskal-Wallis test with Dunn's multiple comparisons.

(H-I) Representative flow cytometry plots (H) and absolute number quantification (I) of OVA-specific CD8<sup>+</sup> Tetramer<sup>+</sup> T cells at day 15. Results are shown as fold change to control (eMSC-Ctrl), n=8 (eMSC-Ctrl, eMSC-FLT3L+poly(I:C)), n=9 (eMSC-FLT3L, eMSC-Ctrl+poly(I:C)) mice per group, two independent experiments, Kruskal-Wallis test with Dunn's multiple comparisons.

(C, E, G, I) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all data points displayed. Source data are provided as a Source Data file.
388

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.

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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 (T<sub>ACT</sub>) (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 (T<sub>EX</sub>) and CD8⁺CD44⁺TIM3⁺TCF1⁺ stem-like T cells (T<sub>SL</sub>) (Fig. 5D-E). Of note, the intratumoral abundance of T<sub>SL</sub> is associated to responses to immunotherapy in multiple cancer types (41–43). Interestingly, expansion of T<sub>SL</sub> cells was associated with a significant increase in cell cycling activity, as probed by Ki67 staining (Supplementary Fig. 5A-B). By contrast, T<sub>EX</sub> 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<sub>257-264</sub> 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<sub>257-264</sub>-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 by-pass resistance to immune checkpoint blockade (ICB). To test this, we submitted B16-OVA to aPD-1/αCTLA-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.

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bioRxiv preprint doi: https://doi.org/10.1101/2025.05.19.654717; this version posted December 2, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_63.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>G</figcaption></figure>

5h ex-vivo SIINFEKL restimulation
Live CD45$^+$CD11b$^low$ MHCII$^+$CD8$^+$ T cells

<figure><img src="image_69.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_73.png" /><figcaption>L</figcaption></figure>

<figure><img src="image_74.png" /><figcaption>M</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>N</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>O</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>P</figcaption></figure>

<figure><img src="image_78.png" /></figure>
**Figure 5:** 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 and overcomes primary resistance to PD-1/CTLA-4 blockade.

(A) Experimental design to evaluate lymphocyte infiltration within tumor.

(B-C) Representative flow cytometry plots (B) and quantification (C) of NK1.1 and ILC1 subsets. Dots represent the absolute number of cells/g tumor, colours represent the expression level of Ki67. n=4 mice per group, one experiment.

(D-E) Representative flow cytometry plots (D) and quantification (E) of activated (T<sub>ACT</sub>), exhausted (T<sub>EX</sub>), and stem-like (T<sub>SL</sub>) CD8<sup>+</sup> T cells. n=4 mice per group, one experiment, one-way ANOVA with Tukey's multiple comparisons test.

(F) Representative flow cytometry plots and quantification of OVA-specific CD8<sup>+</sup>Tetramer<sup>+</sup> T cells/g tumor. n=12 (eMSC-Ctrl+poly(I:C)), n=13 (eMSC-Ctrl, eMSC-FLT3L+poly(I:C)), n=14 (eMSC-FLT3L) mice per group, three independent experiments, Kruskal-Wallis' test with Dunn's multiple comparisons.

(G) Representative flow cytometry plots and quantification of IFNγ<sup>+</sup>CD44<sup>+</sup>CD8<sup>+</sup> T cells after 5h *ex-vivo* restimulation with SIINFEKL. n=4 (eMSC-Ctrl+poly(I:C), eMSC-FLT3L+poly(I:C)), n=5 (eMSC-Ctrl, eMSC-FLT3L) mice per group, one experiment, Kruskal-Wallis' test with Dunn's multiple comparisons.

(H) Experimental design to evaluate T cell memory generation.

(I) Representative flow cytometry plots and quantification of CD44<sup>+</sup>CD8<sup>+</sup>Tetramer<sup>+</sup> cells in the skin-draining lymph nodes after *in vivo* SIINFEKL restimulation. n=4 naïve, n=6 survivor mice, two-tailed Mann-Whitney test.

(J) Representative flow cytometry plots of Tet<sup>+</sup>CD8<sup>+</sup> T cells (red dots) overlaid on total CD8<sup>+</sup> T cells of survivor mice.

(K) Experimental design to assess the role of CD4, CD8 and NK cells in the anti-tumor effect induced by eMSC-FLT3L+poly(I:C).

(L) Tumor growth curves. Two-way ANOVA-test with Tukey's multiple comparisons.

(M) Survival curves. Log-rank (Mantel-Cox) test.

(L-M) n=7 mice per group, one experiment.

(N) Experimental design to evaluate the synergy of eMSC-FLT3L+poly(I:C) with immune checkpoint blockade.

(O) Tumor growth curves. Two-way ANOVA-test with Tukey's multiple comparisons.

(P) Survival curves. Log-rank (Mantel-Cox) test.

(O-P) n=7 (aPD1-aCTL4, eMSC-FLT3L+poly(I:C)), n=8 (No therapy, eMSC-FLT3L+poly(I:C)+aPD1-aCTLA4) mice per group, one experiment.

(E-G) Results are shown as fold change to control (eMSC-Ctrl).

(E-G, I) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all data points displayed.

(L-O) A line represents the mean and SEM is shown with the colored area.
491 (C, E-G, I, L-M, O-P) Source data are provided as a Source Data file.

492
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, 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). 3 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 CCR5 (44)
(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
precursors (4, 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.
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▽ i.t. eMSC-FLT3L

▽ i.t. muHSPC CD45.1

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▽ i.t. eMSC-FLT3L

▽ i.t. Poly(I:C)

△ i.p. Antagonist of CXCR3 and/or CCR5

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**Figure 6: CXCR3 and CCR5 ligands are induced by poly(I:C) and control its impact on cDC1 and T cell infiltration.**

(A) Experimental design to assess the ability of eMSC-FLT3L to drive DC differentiation in B16-OVA tumors.

(B-C) Quantification of the absolute number of CD45.1 cDC1/g tumor (B) and CD45.2 cDC1/g tumor (C). n=5 (HSPC+eMSC-FLT3L), n=4 (other groups) mice per group, one experiment, Kruskal-Wallis' test with Dunn's multiple comparisons.

(D) Volcano plot for the chemokine multiplex array, where each dot represents one gene, plotted based on log2 fold change and -log10 of adj p-value. Genes in blue are up-regulated in tumors treated with eMSC-FLT3L, while genes in red are up-regulated in tumors treated with eMSC-FLT3L+poly(I:C).

(E) Individual selected chemokines of interest, based on up-regulated chemokines in eMSC-FLT3L+poly(I:C) found in volcano plot. Values were obtained with a Legendplex. n=5 mice per group, one experiment, two-tailed Mann-Whitney test. Data are presented as mean values ± SD.

(F) Experimental design to block chemokine receptors CXCR3 and/or CCR5.

(G) Tumor growth curves. A line represents the mean and SEM is shown with the colored area. n=9 (antagonist groups), n=10 (untreated), n=11 (eMSC-FLT3L+poly(I:C)) mice per group, two independent experiments, two-way ANOVA-test with Tukey's multiple comparisons.

(H) Tumor weight at day 15. n=9 mice per group, two independent experiments, Kruskal-Wallis test with Dunn's multiple comparisons.

(I) Quantification of the absolute number of cDC1/g tumor. Results are shown as fold change to control (untreated). n=10 (untreated), n=9 (other groups) mice per group, two independent experiments, one-way ANOVA test with Dunnett's multiple comparisons.

(J) Quantification of the frequency of activated CD8+ T cells in live CD45+ cells in the tumor-draining lymph node. n=4 mice per group, one experiment, one-way ANOVA test with Dunnett's multiple comparisons.

(B-C, H-J) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all data points displayed.

(B-C, E, G, H-J) Source data are provided as a Source Data file.
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 produced 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. 24h 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 Xcr1<sup>cre</sup>xRosa<sup>LsL-tdTomato</sup> mice bearing melanoma tumors. In these mice, cDC1s are genetically labelled 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<sup>+</sup> cDC1s and membrane bound FLT3L-expressing eMSCs.

Since CCR5 and CXCR3 are expressed by activated T and NK cells (4), 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*<sup>-/-</sup>), murine NK cells (*IL2rγc*<sup>-/-</sup>) -and macrophage phagocytosis of human cells (*Sirpa*<sup>NOD</sup>) (47). BRGS mice were injected subcutaneously with basal membrane extract plugs containing either eMSC-Ctrl (left flank), or eMSC-huFLT3L-CCL5-CXCL9 (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 tissues (24). To test this, we used BRGSF mice deficient in FLT3 (BRGSF Flt3-
(1-)) (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. 17-weeks 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.
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<figure><img src="image_102.png" /><figcaption>BALBc Rag2<sup>+</sup>IL2rγ<sup>+</sup> Sirpa<sup>NOD</sup>Flt3<sup>+</sup> mice</figcaption></figure>

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618 **Figure 7: Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5**
619 stimulates cDC1 infiltration in mice and humanized mice.

620 (A) Constructions to produce eMSC-CCL5, eMSC-CXCL9 and eMSC-FLT3L-CXCL9-CCL5.

621 (B-C) ELISA quantification of CCL5 (B) and CXCL9 (C) released by transduced eMSCs.
622 CCL5: n=6 (untransduced), n=3 (FLT3L, CXCL9, CCL5), n=2 (FLT3L-CXCL9-CCL5),
623 CXCL9: n=6 (untransduced), n=3 (FLT3L, CCL5, FLT3L-CXCL9-CCL5), n=8 (CXCL9)
624 technical replicates, one experiment. Data are presented as mean values ± SD.

625 (D) Experimental design to evaluate eMSC-FLT3L-CXCL9-CCL5 ability to recruit pre-cDC1s
626 within tumors. IRF8-GFP mice bearing two tumors were injected with eMSC-FLT3L in the left
627 and eMSC-FLT3L-CXCL9-CCL5 in the right tumor.

628 (E) Representative flow cytometry plots and frequencies of intratumoral pre-cDC1s upon
629 CD45+ live cells. n=14 mice, three independent experiments, two-tailed paired t-test.

630 (F) Experimental design to evaluate cDC1 recruitment within tumors.

631 (G-H) Flow cytometry quantification of the absolute number of cDC1/g tumor (G) and their
632 frequency in CD45+ live cells (H). Results are shown as fold change to control (Untreated). G:
633 n=15 (Untreated), H: n=16 (Untreated), G-H: n=17 (eMSC-FLT3L-CXCL9-CCL5), n=5
634 (eMSC-FLT3L) mice per group, three independent experiments, one-way ANOVA with
635 Tukey's multiple comparisons test. Box plots show median, 25th-75th percentiles, minimum-
636 maximum whiskers, with all data points displayed.

637 (I) Representative images of YUMM-OVA tumors injected in Xcr1<sup>tdTomato</sup> mice and treated
638 with eMSC-FLT3L or eMSC-FLT3L-CXCL9-CCL5. Images represent masks for cDC1s (red)
639 and eMSCs (yellow), generated from the maximum intensity projection.

640 (J) Violin plots showing the quantification of the number of cDC1s contacting an eMSC. n=159
641 (eMSC-FLT3L), n=109 (eMSC-FLT3L-CXCL9-CCL5) individual eMSC, one experiment,
642 two-tailed Mann-Whitney test.

643 (K) Experimental design to evaluate cDC1 recruitment in tissues in BALB/c Rag2<sup>-/-</sup> IL2rγc<sup>-/-</sup> Sirpa<sup>NOD</sup> Flt3<sup>+/-</sup> or <sup>-/-</sup> (BRGS and BRGSF) mice.

645 (L) Flow cytometry quantification of the absolute number of cDC1/plug in BRGS Flt3<sup>-/-</sup> or
646 Flt3<sup>+/-</sup> mice. n=8 plugs, one experiment, two-tailed paired t-test.

647 (M) Experimental design to evaluate human cDC1 recruitment in tissues of BRGSF humanized
648 mice.

649 (N) Gating strategy used to identify human cDC1s in plugs. Lineage: NKp46, CD3, CD19,
650 CD56, CD66b, CD203c, CD20.

651 (O-P) Quantification of the absolute number (N) and frequencies (O) of human cDC1/plug.
652 n=16 plugs, three independent experiments, two-tailed Wilcoxon matched-pairs signed rank
653 test.

654 (B-C, E, G-J, L, O-P) Source data are provided as a Source Data file.

655
656
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 late 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.
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684 **Figure 8: Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5**
685 stimulates the infiltration of activated CD4+ and CD8+ T cells.

686 (A) Experimental scheme of a migration assay. eMSC-CXCL9, eMSC-CCL5 and eMSC-Ctrl
687 were plated in the lower compartment of Boyden chambers, 24 hours before the migration
688 assay. *Ex-vivo* polarized CD45.1 T cells were then transferred to the upper compartment of the
689 culture system and incubated at 37°C for 3 h.

690 (B-C) Quantification of the migration index of CD4+ (B) and CD8+ (C) T cells. The migration
691 index was obtained by counting the number of T cells that migrated to the bottom chambers,
692 regarding the number of T cells initially put on top of the chambers. n=3 technical replicates,
693 one experiment, one-way ANOVA test with Tukey's multiple comparisons. Data are
694 represented as floating bars with individual data points and a line at median.

695 (D) Experimental design for adoptive Th1-CD45.1 T cells transfer in B16-OVA-bearing mice.

696 (E-F) Quantification of the absolute number of CD45.1 CD4+ T cells/g tumor (E) and CD45.1
697 CD8+ T cells/g tumor (F). n=5 mice per group, one experiment, one-way ANOVA test with
698 Dunnett's multiple comparisons.

699 (G-H) Quantification of the absolute number of endogenous CD44+CD4+ T cells/g tumor (G)
700 and endogenous CD44+CD8+ T cells/g tumor (H). n=5 mice per group, one experiment, one-
701 way ANOVA test with Dunnett's multiple comparisons.

702 (E-H) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all
703 data points displayed.

704 (I) Experimental design to assess cDC1 intratumoral infiltration after administrating eMSC-
705 FLT3L-CCL5-CXCL9.

706 (J) Quantification of the absolute number and the frequencies in CD45+ live cells of NK, ILC1,
707 CD4+, CD8+CD44+ activated (TACT), CD8+CD44+PD1+Tim3+ exhausted (TEx),
708 CD8+CD44+PD1+Tim3- stem-like (TSL) T cells. Dots represent the absolute number of cells/g
709 tumor while the colors represent the frequencies of cells in CD45+ live cells. n=11 (No therapy),
710 n=12 (eMSC-FLT3L-CXCL9-CCL5) mice per group, two independent experiments, statistics
711 are shown for the quantification of the absolute numbers, two-tailed unpaired t-test.

712 (B-C, E-H, J) Source data are provided as a Source Data file.

713
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 late time point.

Finally, we wanted to assess if 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 overcome resistance to ICB.

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Figure 9: Intratumoral delivery of eMSC co-expressing FLT3L, CXCL9 and CCL5 is sufficient to activate anti-tumor immunity

(A) Experimental design to assess the anti-tumor effect of eMSC-FLT3L-CCL5-CXCL9.

(B) Tumor growth curves. A line represents the mean and SEM is shown with the colored area. n=5, representative of four independent experiments, two-way ANOVA-test with Šídák's multiple comparisons.

(C) Tumor size at day 18. n=23 (untreated), n=25 (eMSC-FLT3L-CXCL9-CCL5) mice per group, four independent experiments, two-tailed unpaired t-test.

(D) Tumor weight at day 18. n=11 (untreated), n=12 (eMSC-FLT3L-CXCL9-CCL5) mice per group, two independent experiments, two-tailed unpaired t-test.

(C-D) Box plots show median, 25th-75th percentiles, minimum-maximum whiskers, with all data points displayed.

(E) Experimental design to evaluate the synergy of eMSC-FLT3L-CXCL9-CCL5 with immune checkpoint blockade.

(F) Tumor growth curves. A line represents the mean and SEM is shown with the colored area, n=6 (untreated), n=9 (aPD-1-aCTLA-4, eMSC-FLT3L-CXCL9-CCL5+aPD-1-aCTLA-4), n=8 (eMSC-FLT3L-CXCL9-CCL5), two-way ANOVA-test with Tukey's multiple comparisons.

(G) Survival curves. n=6 (untreated), n=9 (aPD-1-aCTLA-4, eMSC-FLT3L-CXCL9-CCL5+aPD-1-aCTLA-4), n=8 (eMSC-FLT3L-CXCL9-CCL5), one experiment, log-rank (Mantel-Cox) test.

(B-D, F-G) Source data are provided as a Source Data file.

751
752
753
754 **Discussion**

755 In this study, we developed an immunotherapeutic platform based on the intratumoral
756 engraftment of ex vivo-engineered, autologous mesenchymal stromal cells (eMSCs). In the
757 first part, we demonstrate a potent therapeutic synergy between eMSCs expressing a
758 membrane-bound form of FLT3 ligand (FLT3L) and the adjuvant poly(I:C). These findings are
759 consistent with previous reports showing that combined administration of recombinant FLT3L
760 and poly(I:C) enhances cDC1 expansion and maturation, promotes tumor regression, and
761 improves responses to immune checkpoint blockade (14, 49, 50). However, systemic delivery
762 or secretion of FLT3L has been shown to perturb hematopoiesis and induce peripheral and
763 intratumoral Treg expansion (33, 34). Moreover, the requirement for repeated injections of
764 poly(I:C) and/or FLT3L in these studies raises safety and feasibility concerns for clinical
765 translation. This calls for the development of delivery methods limiting FLT3L protein
766 production to the TME. Here, we show that intratumoral delivery of eMSCs expressing
767 membrane bound FLT3L elicits potent anti-tumor immunity while restricting FLT3L activity
768 to the tumor site, thereby avoiding systemic toxicities. Treated mice exhibited no elevation in
769 serum FLT3L levels or Treg frequencies, and no evidence of weight loss or liver injury,
770 suggesting that this localized, cell-based strategy mitigates systemic adverse effects commonly
771 associated with DC-targeted immunotherapies (38). Autologous eMSC-FLT3L proposes an
772 alternative option to the intratumoral injection of recombinant FLT3L (14), viral vectors
773 encoding FLT3L (13), CAR-T cell transduced FLT3L (51) and Herpes Virus oncolytic virus
774 encoding FLT3L (52). Indeed, local retention of eMSC has the advantage to restrict expression
775 of FLT3L to the TME, a feature not afforded by CAR T cell mediated systemic delivery (51).
776 In addition, our system, in contrast to viral vectors, is not susceptible to immunity.

This study expands the therapeutic scope of engineered mesenchymal stromal cells (eMSCs) to include modulation of the dendritic cell (DC) lineage. Previous studies have shown that IL-2-based, T cell-targeting eMSCs confer substantial benefits by promoting the expansion of non-exhausted PD-1+TIM-3- CD8+ T cells (53, 54). Likewise, eMSCs engineered to express type I interferons or IL-12 have been reported to enhance the infiltration and cytotoxic effector functions of intratumoral CD8+ T cells (55-58) and NK cells (59). It remains to be determined whether eMSCs that act directly on lymphocytes could synergize with DC-targeting eMSCs such as those developed in this study. Exploring such combinatorial strategies may further amplify both antigen presentation and effector responses, providing a versatile platform for next-generation cell-based immunotherapies.

787 An important finding of this study is that elevation of FLT3L levels alone in the TME
788 is not sufficient to induce DC recruitment and bring immunotherapeutic benefits. We found that
789 poly(I:C) addition synergizes with eMSC-FLT3L. Therefore, we next investigated the
790 mechanisms underlying the enhanced efficacy of the eMSC-FLT3L + poly(I:C) therapy and
791 identified a marked upregulation of multiple chemokines within the tumor microenvironment
792 (TME), notably CXCL9 and CCL5, which serve as ligands for CXCR3 and CCR5, respectively.
793 Pharmacological blockade experiments demonstrated that both CXCR3 and CCR5 are essential
794 for the intratumoral accumulation of cDC1s and for the establishment of effective antitumor
795 immunity. To minimize the potential toxicities associated with poly(I:C), we replaced poly(I:C)
796 by the co-expression of CXCL9 and CCL5 on eMSC-FLT3L. This substitution similarly
797 enhanced the infiltration of cDC1s, T cells, and NK cells, while maintaining robust antitumor
798 activity. These results align with previous studies highlighting the role of CXCR3- and CCR5-
799 mediated recruitment in sustaining immune effector infiltration into the TME (4, 46).
800 Importantly, although CXCR3 and CCR5 are expressed on circulating pre-DCs (45) as well as
801 on activated T and NK cells, our findings indicate that T and NK cells are dispensable for the
802 CXCL9/CCL5-driven recruitment of DCs. It is known that intratumoral chemokines may derive
803 from multiple sources, including NK cells and cDC1s themselves (4, 6, 21) and are essential to
804 support the sustained infiltration of cDC1 within the TME. Here, we show that this pathway of
805 cDC1 recruitment associated to immunogenic tumors is activatable in the context of poorly
806 immunogenic tumors by eMSC-FLT3L-CCL5-CXCL9 engraftment. We demonstrate that local
807 chemokine release by eMSC increase the interaction of cDC1s with eMSC-FLT3L.
808 Translatability of the approach to human cDC1s is demonstrated because eMSC-FLT3L-CCL5-
809 CXCL9 treatment supports the local recruitment of human cDC1s in the dermis of BRGSF
810 immunodeficient mice reconstituted with human CD34+ hematopoietic stem cells. However,
811 the identity of the circulating progenitor population responding to this therapy remains to be
812 elucidated, as several candidate precursors have been proposed as human equivalents of murine
813 pre-DCs (31, 60, 61).

814 When combining eMSC-FLT3L immunotherapies to aPD-1/aCTLA-4 ICB
815 immunotherapy, we manage to partially overcome primary resistance to immune checkpoint
816 blockade and extend survival. We hypothesize that at least 3 mechanisms could account for this
817 effect of eMSC-FLT3L immunotherapy:

818 i) eMSCs might activate DCs engaging with poorly differentiated TCF1⁺T<sub>SL</sub>CD8⁺ T cells enabling to generate ICB-responding pool within the tumors (62, 43). In support of this, multiple studies have identified that mature DCs (and cDC1s in particular) participate to a niche supporting TCF1⁺ poorly differentiated T cells (17, 6, 11, 10). This might possibly happen via the accumulation of mature DCs within TLS for instance (63).

824 ii) eMSC might promote the formation of a pool of activated DCs within the tumor
825 engaging intratumoral effector CD8⁺ T or NK cells. Garris et al. have shown that
826 ICB rapidly activates intratumoral DCs via the IFNγR to become IL12/IL15⁺
827 DCs (64). By increasing DC density, eMSC could promote the generation of
828 intratumoral IL12/IL15/CXCL16⁺ perivascular DCs which have been shown to
829 play a key role in maintaining the fitness of intratumoral CXCR6⁺ CD8⁺ T cell effectors (65).

831 iii) Lastly, eMSC might increase the number of migratory DCs cross-priming novel CD8⁺ T cells in tumor draining lymph nodes. This might help broadening and diversifying T cell response which is a feature of productive ICB immunotherapy (66, 67). In support of this notion, we have evidenced that eMSC-FLT3L + poly(I:C) therapy increases the flux of migratory DCs to tumor-draining lymph nodes.

837 Altogether, this study provides a proof-of-concept for the implementation of eMSC in tissue engineering approaches purported to induce anti-tumor immunity via increased DC infiltration within tumors, based on synergistic activation of chemokine receptors (CXCR3, CCR5) and the FLT3 receptor tyrosine kinase.

841
842 The prospect of locally implanted, eMSCs acting as versatile, sustained factories for therapeutic molecule delivery is highly attractive for cancer immunotherapy. This is particularly relevant for chemokines, which generate spatially organized gradients governing leukocyte trafficking and tissue compartmentalization. Nonetheless, several technological and translational challenges must be addressed to realize the clinical potential of eMSCs.

847 First, standardized and GMP-compatible protocols for autologous eMSCs isolation, engineering, and production must be established. Such methods should ensure reproducible recovery of stromal populations of controlled complexity that are amenable to ex vivo expansion, viral transduction, and post-transduction enrichment. Although the present study 849 primarily employed murine embryonic fibroblasts as an MSC source, we demonstrate that 851 adipose-derived stromal cells can be expanded, transduced, and used to generate 853 immunologically competent eMSCs. Other groups have successfully implemented bone 854 marrow-derived eMSCs for immunotherapy (53, 59, 57, 58). Validation in human systems will 855 be critical for clinical translation. Furthermore, the phenotypic heterogeneity resulting from ex 856 vivo expansion and transduction remains to be fully characterized. Despite homogeneous 857 transgene expression (e.g., via IRES-linked fluorescent reporters), we cannot exclude that this 858 cellular preparation is functionally heterogeneous and that only a subset of eMSCs may be 859 functionally active in vivo. Integration of single-cell RNA sequencing and spatial 860 transcriptomics in eMSC-treated tumors could provide essential insight into stromal diversity, 861 fate, and functional states.

862 Second, optimization of eMSC delivery methods could improve therapeutic persistence 863 and efficacy. In our current model, intratumoral injection of cell suspensions resulted in 864 transient eMSC persistence, with only a minor fraction detectable beyond ten days. Future 865 studies should aim at enhancing eMSC residency through tissue engineering strategies, 866 including synthetic scaffolds or extracellular matrix integration (56). Cell-intrinsic 867 manipulation of stress pathway might also offer some opportunities to improve the survival of
eMSC in the TME. For instance, NQO1 expression might improve resistance to oxidative stress (54). Would these manipulations improve persistence, it would be important to determine whether prolonged eMSC survival improves T and NK cell-dependent tumor control. While intratumoral delivery provides localized control, it may not be feasible for all cancer types. Developing systemic delivery approaches for eMSCs could extend therapeutic reach to deep or inaccessible lesions while improving patient tolerability. Indeed, intravital imaging studies have shown that intravenously administered MSCs exhibit intrinsic tropism for sites of tissue remodeling, including wounds and solid tumors (68, 69). The molecular mechanisms underlying this selective homing remain poorly defined but could be exploited to deliver eMSCs to otherwise unreachable tumor sites.

Third, eMSC-based therapies designed to stimulate DC-dependent antitumor immunity must also incorporate cues that promote antigen release and DC maturation, the two essential steps required to initiate the cancer-immunity cycle. In our study, eMSCs expressing FLT3L and CXCL9/CCL5 effectively enhanced local DC infiltration but did not efficiently drive DC maturation, potentially limiting migration to tumor-draining lymph nodes and cross-priming of CD8+ T cells. Embedding maturation-inducing signals within eMSCs may overcome this limitation. In parallel, promoting local tumor cell death could enhance antigen availability for DC uptake. One promising avenue involves combining eMSCs with oncolytic virotherapy, which induces immunogenic cell death and antigen release. eMSCs have been widely explored as delivery vehicles for oncolytic viruses, leveraging their tumor-homing capacity (70, 71). This approach has been implemented for several viral platforms, including herpes simplex virus (72), myxoma virus (73), Newcastle disease virus (74), measles virus (75), and adenovirus (75, 76). We propose that eMSC-based oncolytic virotherapy could be synergistically combined with DC-targeting eMSCs, as suggested by Svensson-Arvelund et al., who observed cooperation between recombinant FLT3L administration and NDV-mediated oncolysis (77). Moreover, type I IFN-releasing eMSCs could complement DC-directed eMSCs to further enhance local immune activation (59, 56, 58).

In summary, while technical and regulatory limitations remain, the translational potential of eMSC-based immunotherapies is considerable. Their inherent tumor tropism, compatibility with ex vivo genetic engineering, and capacity for localized, sustained delivery make eMSCs a promising cellular platform for next-generation combination immunotherapies aimed at amplifying both innate and adaptive antitumor immunity.

900
901 Methods

902

903 Mice

904 Mice were housed at the INSERM U1149 CRI, Medicine school site Bichat or at the Paris
905 Institut Pasteur animal house facilities. Wild type C57BL/6J and CD45.1 (CByJ.SJL(B6)
906 Pptprc<sup>a</sup>/J) mice were purchased from Janvier and kept in our facility. Irf8-GFP (B6.Cg
907 Irf8<sup>tm2.1Hm</sup>/J), Xcr1<sup>cre</sup> (B6 Xcr1<sup>tm1Ciphe</sup>), ROSA26-LSL-RFP (B6.Cg Gt(ROSA)26Sor<sup>tm1Hif/J</sup>) and
908 ROSA26-LSL-DTA (B6.129P2 Gt(ROSA)26Sor<sup>tm1(DTA)Lky</sup>/J) (78) mice were kindly provided
909 by Dr. Marc Dalod ROSA36-LSL-tdTomato (B6.Cg Gt(ROSA)26Sor<sup>tm14(CAG-tdTomato)Hze/J</sup>) were
910 kindly provided by Dr. Tessa Bergsbaken. Xcr1<sup>DTA</sup>, Xcr1<sup>RFP</sup>, Xcr1<sup>tdTomato</sup> mice were generated
911 as described (40). OT-I Rag2<sup>-/-</sup> CD45.1 mice were kindly provided by Dr. Sebastian Amigorena.
912 BALB/c Rag2<sup>-/-</sup> IL2rγ<sup>-/-</sup> Sirpa<sup>NOD</sup> (BRGS), BALB/c Rag2<sup>-/-</sup> IL2rγ<sup>-/-</sup> Sirpa<sup>NOD</sup> Flt3<sup>-/-</sup> (BRGSF)
913 mice and BRGSF-HIS mice were provided by the Human Disease model Core facility (Institut
914 Pasteur, Paris). The study was approved by the local ethics committee (Comité d’éthique Paris
915 Nord n°121 or CEEA89) and the French Ministry of Education and Research under the
916 authorization number APAFIS#15373 and APAFIS #48865. Animal care and treatment were
917 conducted with national and international laws and policies (European Economic Community
918 Council Directive 86/609; OJL 358; December 12, 1987). All experiments were performed in
919 specific pathogen-free animal facilities, under controlled environmental conditions with
920 ambient temperature maintained at 22 ± 2°C, a relative humidity between 40-60% and on a
921 12hr light/dark cycle, in accordance with the Federation of European Laboratory Animal
922 Science Association (FELASA) guidelines, institutional guidelines and the French law.
923

924

924 BRGSF humanized immune system mice (BRGSF-HIS)

925 Briefly, cord blood CD34⁺ cells (CRB de la Banque de Sang de Cordon, AP-HP, Paris) were
926 isolated with affinity matrices according to the manufacturer's instructions (Miltenyi Biotec)
927 and subsequently phenotyped for CD34 and CD38 expression. Newborn (5-9 days old) BRGSF
928 mouse pups received sublethal irradiation (3 Gy) and were injected intra-hepatically with 3×10⁵
929 CD34⁺ human cord blood cells. 12 and 16 weeks after graft, reconstitution of the human
930 immune system was confirmed by analysis of human CD45, CD19, CD3, CD4, CD8 expression
931 on live leukocytes in the blood using flow cytometry. All animal studies were approved by the
932 Pasteur Institute Safety Committee in accordance with French and European Guidelines
933 (Committee for Ethics in Animal Experimentation 240006) and validated by the French
934 Ministry of Education and Research (APAFIS #49211-2024042612197924 v3).
935

936

936 Tumor cell lines

937 B16-OVA and YUMM-OVA cell lines were generated by lentiviral transduction using a
938 pCDH_CMV7-OVA-EFI-G418 plasmid. TC-1-Luc cells were kindly provided by Dr.
939 Alexandre Boissonnas, E0771 cells were kindly provided by Dr. Stéphanie Hugues and MC38
940 cells were kindly provided by Dr. Philippe Bousso. B16F10, B16-FLT3L (79), B16-OVA and
941 TC-1-Luc cell lines were cultured in RPMI Medium 1640 (ThermoFisher Scientific)
942 supplemented with GlutaMAX, 10% heat-inactivated fetal bovine serum (FBS) (ThermoFisher
943 Scientific), 50µM β-mercaptoethanol (ThermoFisher Scientific) and 100U/mL penicillin-
944 streptomycin (ThermoFisher Scientific). YUMM-OVA, MC38, and E0771 cell lines were
945 cultured in DMEM (ThermoFisher Scientific) supplemented with GlutaMAX, 10% heat-
946 inactivated FBS (ThermoFisher Scientific), 50µM β-mercaptoethanol (ThermoFisher
947 Scientific) and 100U/mL penicillin-streptomycin (ThermoFisher Scientific). Cells were
948 maintained at 37°C and 5% CO2.
949

950

Tumor models

950

951 For the grafted tumor model, tumor cells were harvested during the exponential growth phase
952 using 0.05% Trypsin-EDTA (Gibco), washed twice in PBS, and counted using trypan blue
953 exclusion. 8- to 12-week-old male (B16-OVA, MC38, YUMM-OVA) and female (E0771, TC-
954 1) mice were subcutaneously inoculated with 5×10⁵ to 1×10⁶ tumor cells suspended in 100µL
955 of not supplemented RPMI or DMEM medium. Tumor dimensions were recorded three times
956 per week using a digital caliper, and tumor volume (V) was calculated using the formula
957 V=(L×W×W)/2, with volume expressed in mm³. Mice were monitored daily, and endpoints
958 were established based on signs of distress, including pain, lethargy, dehydration, ulceration,
959 or tumor necrosis. To generate survival curves, mice were sacrificed at ethical endpoints, the
960 main one being the tumor volume (2000mm³).
961

961

962

Generation of engineered mesenchymal stromal cells

963

964

Generation of mesenchymal stromal cells

965 Mesenchymal stromal cells (MSC) were generated from mouse embryonic fibroblasts (MEFs)
966 from individual C57/BL6 embryos at embryonic day 13 and were kindly provided by Dr.
967 Loredana Saveanu. Practically, the embryo body was digested with 0.05% trypsin and 0.02%
968 EDTA in Hanks balanced salt solution at 37°C for 30 minutes. The single cell suspension
969 obtained after digestion was resuspended in high glucose DMEM medium containing 10% FCS,
970 2mM L-glutamine, 100U/ml penicillin and 100µg/ml streptomycin and seeded on 0.1% gelatin
971 (Sigma G1890) coated tissue culture dishes at 5x10⁴ cells/cm². Cells growing on the gelatin-
972 coated dishes were harvested and cell passages were repeated in DMEM medium containing
973 10% FCS, 2mM L-glutamine, 100U/ml penicillin and 100µg/ml streptomycin.
974

975
## 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 to 8mL 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 40min digestion at 37°C, the digestion was mechanically ended with a syringe and a needle of 18G. Cell suspension was then filtered on a 100µm 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 1,2µg plasmid, 21mM 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 Dr. Laleh Majlessi.

## In Vitro experiments

### T cells isolation, activation and polarization

CD4⁺ and CD8⁺ T cells were isolated from the draining lymph nodes and spleens of 10- to 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
1013 Miltenyi magnet and LS columns (Miltenyi Biotec). For *in vitro* stimulation, purified T cells 1014 were cultured on plates coated with anti-CD3 (2µg/mL; clone 17A2) and anti-CD28 (2µg/mL; 1015 clone 3751; BioLegend) in the presence of recombinant IL-2 (5µg/mL; BioLegend). To 1016 promote Th1 differentiation, cultures were supplemented with recombinant IL-12 (10ng/mL; 1017 BioLegend) and blocking antibody anti-IL-4 (5µg/mL; clone MP4-25D2; BioLegend). 1018

1019

**Migration assay**

1020 Cell migration assays were conducted using 24-well transwell inserts with 5-µm pore 1021 polycarbonate membranes (Corning). Engineered mesenchymal stromal cells (eMSCs) 1022 expressing CCL5 or CXCL9 were seeded at a density of 250,000 cells per well in the lower 1023 chamber. Once the eMSCs reached confluency, the culture medium was replaced with serum- 1024 free DMEM. A total of 100,000 CD45.1 Th1-polarized T cells were resuspended in serum-free 1025 DMEM, and 100µL of the cell suspension was carefully added to the upper chamber of the 1026 Transwell insert. The upper chamber was then gently placed into the lower chamber using 1027 sterile forceps, ensuring proper immersion in the culture medium. The plate was incubated at 1028 37°C for 3 hours. Following incubation, the supernatant was collected to quantify T-cell 1029 migration by flow cytometry. 1030

1031

In Vivo experiments
---

1032

1033 **HSPCs differentiation in membrane extract plugs**

1034 Immunomagnetic negative selection was used to isolate mouse stem and progenitor cells 1035 (HSPC) from CD45.1 mice using the Mouse Hematopoietic Progenitor Cell Isolation Kit 1036 (StemCell), following the manufacturer's instructions. A total of 10⁴ hematopoietic stem and 1037 progenitor cells (HSPCs) per plug were subcutaneously injected alongside engineered 1038 mesenchymal stromal cells (eMSCs) at a 1:50 ratio (HSPC/eMSC) in 200 µL of ice-cold 1039 Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher). 1040 Mice were euthanized on day 12 of differentiation via cervical dislocation, and GelTrex plugs 1041 were excised for further analysis. The plugs were sectioned and enzymatically digested in 1042 HBSS (Life Technologies) containing 1% FBS, 0.37U/mL Collagenase D (Roche), 10µg/mL 1043 DNase I (Roche), and 1mg/mL Dispase (Sigma-Aldrich) at 37°C for 30 minutes. Following 1044 digestion, the tissue was smashed on a 100-µm strainer (Corning), and the resulting cell 1045 suspension was collected and resuspended in FACS buffer for flow cytometry analysis. For 1046 experiments involving recombinant FLT3L, human FLT3L was used at a concentration of 1047 1048 1049 1050

1048
1049 **In Vivo Treatment with Engineered Stromal Cells**

1050 Intratumoral injections of eMSCs started when the tumors were between 50 and 100mm³. Mice
1051 were treated with 5×10⁵ to 1×10⁶ eMSC-FLT3L, in combination with 25µg/mouse of poly I:C
1052 LMW (InVivoGen). We designed our intratumoral cell-based therapy as two injections of
1053 eMSC-FLT3L (day 7 and day 12) and one injection of poly(I:C) (day 9). Mice that were later
1054 treated with 5×10⁵ to 1×10⁶ eMSC-FLT3L-CXCL9-CCL5 received three injections of eMSCs.
1055 In the latest experiments, mice developed tumors with the desired injectable size few days later
1056 than the initial schedule. Thus, the start of injections was postponed but the number and
1057 frequencies of injections was kept.

1058

1059 **In Vivo bioluminescence**

1060 A total of 1×10⁶ eMSC-NanoLuc cells were administered intratumorally into palpable B16-
1061 OVA tumors. Mice were imaged using the IVIS SpectrumCT 2 In Vivo Imaging System 4 h
1062 after injection and again at days 4 and 8. Prior to imaging, mice were anesthetized and received
1063 an intravenous injection of 0.44 µmoles fluorofurimazine substrate (Promega). On day 8, mice
1064 were sacrificed, and various organs were collected and imaged ex vivo to assess
1065 bioluminescence. Photon flux was quantified using the Living Image 4.8.2 software. Regions
1066 of interest (ROIs) were quantified as average radiance (photons/s/cm²/sr) represented as color-
1067 scaled images superimposed on grayscale photos of mice using Living Image software.

1068

1069 **In Vivo Treatment with recombinant FLT3L**

1070 Intratumoral injections of recombinant FLT3L started when the tumors were between 50 and
1071 100mm³. Mice were treated 4 times (every two days) with 10µg/mouse of recombinant FLT3L
1072 (Celldex).

1073

1074 **Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) assay**
1075 **and hematoxylin-eosin coloration**

1076 Serum was collected 48h after poly(I:C) intratumoral or anti-CD40 (100µg/mice, clone FGK
1077 4.5) intraperitoneal injection. Blood was allowed to clot at room temperature for 30min and
1078 centrifuged two times at 1000g for 10min. ALT and AST levels were measured using the
1079 ALT/GPT kit (ThermoFisher Diagnostics) following the manufacturer's instructions. Samples
1080 were run on a Konelab analyzer (ThermoFisher Scientific). Liver of the same mice were
1081 harvested and put in 4% paraformaldehyde (ThermoFisher Scientific) overnight. The next day,
1082 livers were transferred in 70% ethanol, paraffin embedded, cut, and stained with hematoxylin
1083 and eosin (H&E) at the histology platform of the Pasteur Institute. Slides were scanned using
1084 the Axioscan 7 (Zeiss).

1085
1086 Immune Checkpoint Blockade (ICB) Treatment

1087 Mice received intraperitoneal (i.p.) injections of 200µg anti-PD-1 (clone RMP1-14, 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 intra-peritoneally at treatment initiation and subsequently every two days until reaching 5 injections.

1092

1093 CD4, CD8, CD25 and NK cells depletion

1094 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 three days at a dose of 200µg, until reaching 4 to 6 injections.

1101

1102 CXCR3- and CCR5-antagonists' treatments

1103 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).
1104
1105 Administration started at the initiation of treatment and continued every two days until day 15,
1106
1107 where samples were collected for flow cytometry analysis.

1108

1109 In Vivo re-challenge OVA peptide in mice

1110 Survivor mice that fully rejected B16-OVA tumors treated with eMSC-FLT3L + poly(I:C)
1111 underwent *in vivo* restimulation via subcutaneous injection of 1µg OVA 257-264 peptide
1112 (SIINFEKL, IBA Lifesciences) dissolved in 100µl phosphate-buffered saline (PBS). This
1113 experiment was conducted 90 days after primary tumor injection. The injection site was located
1114 in the dorsal flank region. Three days post-restimulation, mice were euthanized, and inguinal
1115 draining lymph nodes were excised for subsequent analysis.

1116

1117 Adoptive T cell transfer

1118 1x10⁵ eMSCs were injected in B16-OVA tumors at day 7, 9 and 12. At day 9, 2x10⁶ Th1-
1119 polarized CD45.1 T cells (previously described) were intravenously administered.

1120

1121 Synthetic niches (plugs)

1122 1x10⁶ eMSC-Ctrl or eMSC-FLT3L-CXCL9-CCL5 were injected subcutaneously (s.c.) in
1123 200µL solution, containing 150µL of Geltrex LDEV-Free Reduced Growth Factor Basement
1124 Membrane Matrix and 50µL of PBS. The synthetic niches (plugs) were harvested and analyzed
1125 by flow cytometry 10 days after the injection.

1126

1127 Mouse tissues processing

1128 Murine tumors, tumor-draining lymph nodes (LNs), spleens, or synthetic niches were harvested
1129 and subjected to enzymatic digestion. Tissues were incubated in 3mL of digestion buffer,
1130 comprising Hank's Balanced Salt Solution (HBSS, ThermoFisher Scientific) supplemented
1131 with calcium and magnesium (ThermoFisher Scientific), 75µg/mL Collagenase D (Roche), and
1132 0.02mg/mL DNase I (ThermoFisher Scientific), at 37°C for 30 minutes. Single-cell suspensions
1133 were then generated by smashing the digested tissues through 70µm cell strainers (Corning).
1134 Spleen red blood cells were lysed using ACK lysis buffer (ThermoFisher Scientific). The
1135 absolute number of live immune cells in each tissue cell suspension was assessed using
1136 AccuCheck Counting Beads (ThermoFisher Scientific) along with anti-CD45 and DAPI
1137 stainings on BD LSR-Fortessa X20, BD Symphony A5 (BD Biosciences) or spectral SONY
1138 ID7000 (SONY).

1139

1140 Flow cytometry analysis and fluorescent-activated cell sorting

1141 After tissue processing and cell counting, Fc-receptor were blocked using FcBlock for 20min
1142 at 4°C. Viability was assessed using 7-AAD (Biolegend), Zombie dyes (BioLegend), or
1143 Live/dead fixable stains (ThermoFisher Scientific). Extracellular staining was performed in
1144 FACS buffer (PBS (Life Technologies), 3% FBS (ThermoFisher Scientific), and 2 mM EDTA
1145 (Life Technologies)) during 30min at 4°C. Labelling with H2K<sup>b</sup>-SIINFEKL tetramers (MBL)
1146 was performed at room temperature for 30min. For intra-cellular stainings, single cell
1147 suspensions were then fixed during 30min and stained during 30min using a
1148 fixation/permeabilization kit (eBiosciences, transcription factor staining buffer). For intra-
1149 cellular IFNγ staining, single-cell suspensions were first restimulated *ex vivo* with 2µM
1150 OVA257-264 peptide (SIINFEKL, IBA Lifesciences) for 5 hours at 37°C in 5% CO2, followed
1151 by a 4-hour incubation with 5µg/mL Brefeldin A (Sigma Aldrich). Cells were subsequently
1152 labelled with surface antibodies, fixed either with the Cytofix/Cytoperm kit (BD, cytokines
1153 stainings) or the Transcription Factor Staining Buffer Set (Thermo Fisher scientific, nuclear
1154 intracellular stainings), and stained with an intracellular cytokine staining (ICS) antibody panel.
1155 A comprehensive list of antibodies used is provided in Supplementary Table 1. Flow cytometry
1156 data were acquired using BD LSR-Fortessa X20, BD Symphony A5 (BD Biosciences) or
1157 ID7000 Spectral (Sony). Cells sorting was performed using a BD FACSMelody™ Cell Sorter
1158 equipped with BD FACSDiva software. Data were analyzed with FlowJo software.

1159

1160

1161
1162 **Multiplex chemokines analysis**

1163 Dissected tumor samples were weighted and homogenized in 1mL of 1x phosphate-buffered saline (PBS) supplemented with protease inhibitor, using a Fisherbrand™ 150 handheld homogenizer. Homogenates were then centrifuged at 14000g for 15 minutes, 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.

1172

1173 **Immunofluorescence**

1174 1x10⁶ YUMM-OVA tumor cells were injected in the flank of Xcr1<sup>RFP</sup> or Xcr1<sup>tdTomato</sup> 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. (80). 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 1mm. 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 20X 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.

1190

1191 **Statistical Analysis**

1192 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
1200 means ± SD or SEM, box and whiskers, or violin plots as described in the figure legends. SD:
1201 standard deviation; SEM: standard error of the mean.

1202

**Data availability**

1204

1205 Source data are provided as a Source Data file.

1206

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1569 Acknowledgements

1570
1571 PG is a CNRS investigator (Centre National de la Recherche Scientifique). This work was
1572 supported by Fonds National Suisse (SINGERGIA SNF CRSII5_202246/1, PG, SH), INSERM
1573 transfert CO-POC grant (PG), Agence Nationale pour la Recherche (ANR-17-CE11-0001-01
1574 and ANR-18-IDEX-0001, PG), La Ligue contre le cancer (PG), Institut National du Cancer
1575 INCA (PL-BIO22-147, PG), Fondation pour la Recherche Médicale (EQU202203014687, PG),
1576 Fondation pour la Recherche sur le Cancer ARC (PJA2021060003913, PG) and the Pasteur
1577 Institute. We would like to thank the staff of the flow cytometry and the animal facility
1578 platforms from the CRI (Centre de Recherche sur l'Inflammation, Faculté de Médecine, Bichat)
1579 and from the Pasteur Institute for all the work, help and support in most of the experiments
1580 presented in this paper. We also like to thank the staff from the histopathology core facility
1581 from the Pasteur Institute for the liver H&E images. We thank Rémy Yim (Human Disease
1582 Models Core Facility, Institut Pasteur, Université Paris Cité, 75015 Paris, France), the human
1583 disease models core facility (Institut Pasteur, Paris) and the CRB de la Banque de Sang de
1584 Cordon, AP-HP, Paris for the experiments on the BRGSF reconstituted mice. We thank the
1585 units U1149 and U1016 of Institut National de la Santé et de la Recherche Médicale. We thank
1586 unit UMR3738 of Centre National de la Recherche at Pasteur Institute. We also would like to
1587 thank Dr. Laleh Majlessi for providing the NanoLuciferase vector, Dr. Alexandre Boissonnas
1588 (Sorbonne Université, INSERM U1135, CNRS, Centre d'Immunologie et des Maladies
1589 Infectieuses, Cimi-Paris, Paris 75013, France), Dr. Philippe Bousso (Dynamics of Immune
1590 Responses Unit, Institut Pasteur, Université de Paris Cité, INSERM U1223, F-75015 Paris,
1591 France), Dr. Loredana Saveanu and Dr. Laurie Menger (Inserm U1015, Gustave Roussy Cancer
1592 Campus, Villejuif 94800, France) for providing us various cell lines, Dr. Marc Dalod, Dr. Tessa
1593 Bergsbaken (Department of Pathology, Immunology and Laboratory Medicine, Center for
1594 Immunity and Inflammation, Rutgers New Jersey Medical School, Rutgers-The State
1595 University of New Jersey, Newark, NJ) and Dr. Sebastian Amigorena (Institut Curie, PSL
1596 University, INSERM U932, Immunity and Cancer, Paris, France) for providing us mice strains
1597 and Dr. Geneviève Marcelin (Sorbonne Université, INSERM U1269, Nutrition and obesities:
1598 systemic approach research group, Nutriomics, Paris F-75013, France) for providing us
1599 protocols. Graphics were created in BioRender. Guermonprez, P. (2025)
1600 https://BioRender.com/o76t012.

1601

1602 Author contributions

1603
1604 Conceptualization: SH, PG
1605 Investigation: LG, FLRDC, PB, JB, NV, AS, MV, ASKC, NJ, AO, MB, OF, MA, DF, IH,
1606 ZAN, JW, HT, GA, FF
1607 Resources: FG, LM, ELG, LS, JH, MDalod, MDusseaux, JPDS, SH, PG
1608 Writing: LG, SH, PG

1609 Supervision: SH, PG

1610 Funding acquisition: MC, JC, JH, SH, PG

1611

1612 Competing interests

1613

1614 The authors declare no competing interests.