Role of TLR4 in the Activation of a Pro-Tumor Phenotype of Mesenchymal Stromal Cells in Mutiple Myeloma
Authors: Giallongo Cesarina , Tibullo Daniele , Parrinello Nunziatina Laura , Camiolo Giuseppina , La Cava Piera , Romano Alessandra , Puglisi Fabrizio , Sapienza Giuseppe , Conticello Concetta , Palumbo Giuseppe A. , Di Raimondo Francesco 
Published: October 2019

Abstract
Introduction We have already demonstrated that myeloma mesenchymal stromal cells (MM-MSC) promote cancer immune evasion through the activation of myeloid derived suppressor cells and we hypothesized that MM-MSC could be polarized stromal cells to better 'serve' cancer (Giallongo et al, Oncotarget 2016). Since it has been demonstrated that specific Toll-like receptors (TLR) can drive MSC activation status, including two distinct phenotypes defined MSC1 (TLR4-dependent) or MSC2 (TLR3-dependent), we evaluated the effect of TLR activation on MM-MSC
Methods Healthy Peripheral blood mononucleated cells (PBMC) were cultured with MSC for 6 days; then neutrophils were isolated using magnetic microbeads and their immunosuppressive activity was evaluated by their ability to suppress activation of CFSE+ T cells. Immunocompetent adult Zebrafish was used as in vivo model for myeloma cells engraftment. Tumor xenograft was measured by tomography 6 days post-injection.
Results Using specific agonists for TLR4 (LPS) or TLR3 (poly(I:C)) for 24 h, we observed that healthy MSC acquired the same immunological alteration of MM-MSC after a pre-treatment with LPS. Indeed, MSC1 polarization of HC-MSC induced neutrophils to become immunosuppressive. Moreover, wester blotting analysis confirmed the activation of TLR4/MyD88 pathway in MM-MSC but not in healthy control-MSC (HC-MSC).
To investigate if the polarization status of MM-MSC could promote tumor-growth in vivo, a mixtures of fluorescently labeled MM cells plus HC- or MM-MSC were implanted in zebrafish. After six days, the animals co-injected with plasma cells (PC) and MM-MSC showed enhanced tumor colonization and growth (calculated as tumor area) compared with zebrafish injected with PC and HC-MSC (control) (p<0.05). Flow cytometry detection of hCD138+ cells confirmed less MM cells in zebrafish injected with PC and HC-MSC (p=0.001). Therefore, we analyzed the expression of the master regulator transcription factors for Th1/Th2 (tbx21 and gata3) and Th1- and Th2-type cytokines to better assess in vivo the involvement of the immune escape mechanisms promoted by co-injection of PC with MM-MSC. As compared to control animals, gata3, IL-4 and IL-13 were significantly up-regulated in zebrafish injected with PC plus MM-MSC, revealing a Th2 responce. Next, we used TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4, to inhibit its signaling in MM-MSC before injection in zebrafish. Animals co-injected with PC and MM-MSC pre-treated with TAK-242 showed a reduction of 41% of tumor area compared to zebrafish injected with PC and MM-MSC (p<0.001). Moreover, the same animals showed the up-regulation of tbx21 and INFγ (Th1 response).
To examine if PC play a role in MSC polarization, before performing co-cultures with PBMC, we pre-treated HC-MSC with MM cell lines (U266, H929, MM1S). PC pre-treatment drove healthy MSC to activate neutrophils in immunosuppressive cells in vitro. Therefore, we investigated if PC activated TLR4 pathway in healthy MSC and found that PC induced nuclear translocation of NFkB and subsequently of IRF3 in HC-MSC, indicating the involvement of TLR4-MyD88-dependent and independent pathways in MSC commitment. Injecting zebrafish with MM cells and HC-MSC co-cultured or not for 24h with PC, we observed that animals injected with HC-MSC pre-treated with PC showed more tumor engraftment (p=0.001) and 15±2,8% more hCD138 (p=0.001). Inhibition of TLR4 signaling during co-culture in vitro of HC-MSC with MM cells led to a reduction of tumor growth (p=0.001) and hCD138 infiltrate (from 17,9±7% to 2,8±1,2%; p=0.0006).
Conclusion: TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment that circumvents the immune response and allows a better tumor engraftment.

Disclosures
Palumbo: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.