Tumoral Endoglin (CD105) Regulates the Tumor Microenvironment, Tumorigenicity and Therapeutic Response in Renal Cell Carcinoma

Abstract

<h3>Purpose/Objective(s)</h3> Endoglin (CD105) is a transmembrane glycoprotein that serves as a co-receptor for the TGF-β signaling cascade. Due to its elevated expression on tumor associated vasculature of highly vascular cancers, it is categorized as a tumor associated antigen (TAA). Although CD105 has been largely studied as an endothelial cell marker and mediator of angiogenesis in various cancers including Renal cell carcinoma (RCC), the effect of its expression by tumor cells has not been fully elucidated in immune-competent models. Here we address the implication of tumoral CD105 on the tumor microenvironment (TME) and response to a <i>Listeria-monocytogenes</i> based immunotherapeutic vaccine directed against CD105 based on the hypothesis that "<i>Tumoral CD105 contributes to the aggressive phenotype of RCC and further drives response to immunotherapy"</i>. <h3>Materials/Methods</h3> A mutant version of Renca that is deficient in CD105(CD105KO-Renca) was generated from murine Renca cell line using CRISPR/cas9. In an immunocompetent model syngeneic to male BALB/C mice, subcutaneously implanted control Renca (Scr-Renca) or CD105 KO-Renca tumors were monitored for tumor growth. Tumor tissues from either group were subjected to flow cytometry and gene expression using RT-qPCR. The contribution of tumoral CD105 to neovascularization was determined by matrigel plug angiogenesis assay followed by hemoglobin quantification. Lastly, we elucidated how tumoral expression of CD105 affects the response to a <i>Listeria-</i>based vaccine directed against CD105 (Lm-LLO-CD105) in a subcutaneous immunocompetent mouse model. <h3>Results</h3> CD105KO-Renca displays increased potential for early tumor formation, but the tumor progression is reduced compared to the Scr-Renca. Deletion of tumoral CD105 impacted the TME by increasing the population of multicytokine (IFNγ⁺ IL-2⁺TNFα⁺) producing CD8⁺ and CD4⁺ T cells and reducing infiltration of myeloid derived suppressor cells (MDSCs) and M2-macrophages. Further, tumoral CD105 contributes to angiogenesis as observed by a reduction in the hemoglobin content and CD31mRNA and VEGFa mRNA in Renca-CD105KO tumor tissues. Finally, Lm-LLO-CD105 was able to control tumor growth in the Scr-Renca tumor bearing mice but not in the CD105KO-Renca tumor bearing mice. Lm-LLO-CD105 increases the CD8⁺ T cell infiltration, production of cytokines and reduces the population of CD4⁺Foxp3⁺Tregs in the TME of Scr-Renca but not in the CD105-KO-Renca tumors. <h3>Conclusion</h3> Tumoral CD105 serves as an independent prognostic that is important for both tumor progression and modulation of the RCC's immunological phenotype. Furthermore, CD105 deficient RCC did not respond to an immunotherapeutic strategy that is dependent on CD8⁺ T cell recruitment. Altogether, our data suggests that tumoral CD105 is an important mediator of tumor progression and response to immunotherapy.