Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect analysis of vemurafenib and sorafenib on pericyte viability. Suppl. Figure 3 BRAFWT/WT-TPC1 cells viability assay. Suppl. Figure 4 ELISA analysis of cytokines secretion in pericytes, and PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT. Suppl. Figure 5 Effects of pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 6 Analysis of secreted TSP-1 levels in PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT treated with shTSP-1 or shGFP (control) pericyte secretome. Suppl. Figure 7 Effects of shTSP-1 pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 8 Viability assay in the presence of SRI31277 in PTC cells and pericytes. Suppl. Figure 9 Effects of the small molecule SRI31277 in PTC cells with BRAFWT/WT. Suppl. Figure 10 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compared to BRAFWT/V600E-PTC samples. Suppl. Figure 11 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compared to BRAFWT/WT-PTC samples. Suppl. Figure 12 THBS1 (TSP-1) gene regulatory networks pathways in PTC harboring BRAFWT/V600E and hTERT mutations compared to BRAFWT/WT-PTC clinical samples.
ARTICLE ABSTRACT
The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown.
We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment.
Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFβ1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFβ1 expression evoke gene-regulatory networks and pathways (TGFβ signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand–VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFβ1 axis reduces tumor cell growth and overcomes drug resistance.
Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFβ1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.
