Tumoral CD105 promotes immunosuppression, metastasis, and angiogenesis in renal cell carcinoma

Abstract

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4⁺ and CD8⁺ T cells. Finally, while CD105 expression by endothelial cells is a well-established contributor to tumor angiogenesis, we also find that tumor cell-expressed CD105 significantly contributes to tumor angiogenesis in RCC.

Keywords: Angiogenesis; CD105; Endoglin; Renal cell carcinoma; Tumor microenvironment.

Fig. 1

CD105 is highly expressed in renal cell carcinoma. A Expression of CD105 between normal kidney tissues and primary tumor based on data obtained from TCGA. B–D Expression of CD105 between Renca and NIH-3T3. B mRNA expression of CD105. C Mean fluorescence intensity (MFI) of CD105. D Representative histograms of the expression of CD105

Fig. 2

Tumoral CD105 enhances tumor cell proliferation A protein and B mRNA expression of CD105 on Renca cells after treatment with a CD105-targeting CRISPR/Cas9 vector. C Comparison of growth rate between Control and crCD105 at 48 and 72 h. D Representative images of colony formation and statistical quantification of the number of colonies and proliferation ratio. The proliferation ratio is defined as the area covered by colonies/total area of well. E MFI of the proliferation marker ki67 and F MFI of CD44. G Representative image and statistical quantification of migrated cells in transwell migration assay. H Representative image and statistical quantification of scratch assay

Fig. 3

Tumoral CD105 promotes in vivo tumor growth and metastasis A Kaplan–Meier plot for tumorigenicity. B Longitudinal tumor growth rate of subcutaneously implanted Control and crCD105 tumors. C The individual tumor growth pattern of Control and D CD105-deficient tumors. E Representative image of diseased kidneys from both the Control and crCD105 intra-renal tumor challenge groups. F Final tumor mass of orthotopic tumors. G Incidence of metastasis between Control and crCD105. H Representative image of metastatic nodules. I quantification of metastasis in the lungs

Fig. 4

Tumoral CD105 regulates the functionality of TIL in the subcutaneous TME Gating strategy for tumor-infiltrating lymphocytes, and cytokine production is as presented in Supplementary Figure S3A A IFN-$\gamma$, B TNF-$\alpha ,$ C IL-2 producing CD8⁺T cells. D, E Multicytokine production by CD8⁺ T cells in the Control and crCD105 tumor tissues. F–H Population of F IFN-$\gamma$ and G TNF-$\alpha$ and H IL-2 producing CD4⁺ T cells. I, J Multicytokine production by CD4⁺ T cells in the Control and crCD105 tumor tissues

Fig. 5

Tumoral CD105 regulates the functionality of TIL in the orthotopic TME Gating strategy for tumor-infiltrating lymphocytes, and cytokine production is as presented in Supplementary Figure S3A. Production of A IFN-$\gamma$ and B TNF-$\alpha$ and C IL-2 by the tumor-infiltrating CD8⁺ T cells. D, E Multicytokine production by CD8⁺ T cells in the Control and crCD105 tumor tissues. F–H The population of CD4⁺ T cells producing F IFN-$\gamma$ G TNF-$\alpha$, and H IL-2. I-J Multicytokine production by CD4⁺ T cells in the Control and crCD105 TME. K tSNE plots of Gr-1. Population of L CD11b⁺ Gr1⁺MDSCs M CD11b + Gr1^lo cellsmMDSCs N myeloid population defined as CD11b⁺ Gr-1⁻ and O CD4⁺ foxp3⁺ Tregs in the TME of Control in comparison with the crCD105 Renca tumors

Fig. 6

Tumor cell-expressed CD105 mediates suppression of the TME Gating strategy for MDSCs and TAMS is as presented in Supplementary Fig. 3A and B A Representative dot plots for MDSCs. The population of B CD11b⁺Gr1⁺ MDSCs and C CD11b^hiGr1^lo cells mMDSCs. D Quantification and tSNE plots of myeloid cells in the TME of Control tumor tissues as compared to the CD105-deficient tumor tissues. E Population of dendritic cells and F M2-like cells. G Heat map showing the expression of markers of M2-TAMs and H Mmp9, an MDSC recruiting cytokine in the TME I frequency of CD4⁺Foxp3⁺ cells to the TME J heat map of markers of immunosuppression including Il-10,foxp3,pdcd1, and tgfb1 in the TME

Fig. 7

Tumor cell-expressed CD105 contributes to intra-tumoral vasculogenesis and angiogenesis A Representative images for tube formation. B Quantification of the tube formation assay C mRNA expression of Vegfa by tumor cells. D Experimental schedule for Matrigel angiogenesis assay. E Quantification of hemoglobin content. mRNA expression of F Cd31 and G Vegfa and H Vegfd between the tumor tissues from the CD105-deficient Renca cells and the Control Renca cells