ESTRO 2024 - Abstract Book

S5204

Radiobiology - Microenvironment

ESTRO 2024

To investigate the function of FGFR2+ fibroblasts, we conducted a preliminary analysis using the TCGA database and found that patients with increased expression of FGFR2 were often presented with progressive disease (PD) after radiotherapy (5/14, 35.71 %) compared with those with reduced expression of FGFR2 (0/5, 0 %) (P < 0.0001) (Figure 1A). Our clinical ESCC tumor tissues showed that all patients with high infiltration of FGFR2+ fibroblasts experienced disease recurrence after radiotherapy, whereas only 50 % of patients with low infiltration FGFR2+ fibroblasts experienced recurrence (Figure 1B). In addition, survival analysis showed that high infiltration of FGFR2+ fibroblasts was associated with poorer prognosis (P < 0.001) (Figure 1C). Based on these findings, we speculated that FGFR2+ fibroblasts might contribute to radioresistance. In clinical samples, we observed that FGFR2+ fibroblasts were always located near or surrounding cancer stem cell nests (Figure 1D) and the proportion of CD133+ tumor cells positively correlated with the infiltration of FGFR2+ fibroblasts (r = 0.4380, P < 0.001) (Figure 1E). In addition, we discovered that in the TCGA database the levels of the CSC markers, CD133, ALHD1A1, SOX2, and KIT, were much higher in patients with a high level of FGFR2 than in those with low expression (Figure 1F). As expected, patients with a high proportion of CD133+ tumor cells showed poor survival outcomes (Figure 1G). Then we isolated fibroblasts from fresh ESCC tissues and collected supernatants from cultured FGFR2+ and FGFR2- fibroblasts. Flow cytometric analysis showed that the FGFR2+ fibroblast-derived culture medium led to an increase in the percentage of CD133+ and CD90+ cancer cells, which was greater than that obtained with the FGFR2- fibroblast-derived culture medium (Figure 2A). The sphere formation assay showed that the self-renewal ability of cancer cells was upregulated by FGFR2+ fibroblasts (Figure 2B). Finally, RT-qPCR analysis revealed the overexpression of CD133, CD90, NANOG, NOTCH1, SNAI2, and SOX2 in tumor cells in the presence of FGFR2+ fibroblast-derived culture medium (Figure 2C). The above results suggested FGFR2+ fibroblasts might increase CSC proportions and contribute to CSC properties sustaining. As cancer stem cells are the key factors in radioresistance, we conducted clonogenic assays after radiation using FGFR2+ fibroblasts and observed that the presence of FGFR2+ fibroblasts augmented the survival of tumor cells after radiation (SF2: 79.7 % vs. 90.1 %) (Figure 2D). Furthermore, we detected γH2AX expression in tumor cells after ionizing radiation (IR) and found that tumor cells cultured with FGFR2+ fibroblasts had fewer γH2AX foci per nucleus than those in the control group at 24 h after IR (Figure 2E). Consistent with the in vitro assay results, in vivo experiments suggested that FGFR2+ fibroblasts enhanced the radioresistance of tumor cells (Figure 2F). Immunohistochemistry was performed to observe the proportion of CD133+ tumor cells in the excised tumor tissues and the results showed that more CD133+ tumor cells were represented in the FGFR2+ group (Figure 2G). Based on this finding, we hypothesized that FGFR2+ fibroblasts promote radioresistance by increasing the proportion of CSCs. 3. FGFR2+ fibroblasts promoted radioresistance 2. FGFR2+ fibroblasts increased the proportion of cancer stem cells

4. BMP5 secreted by FGFR2+ fibroblasts increased cancer stemness and promoted tumor cells radioresistance

As a surface receptor, FGFR2 is rarely secreted to affect neighboring tumor cells; therefore, FGFR2+ fibroblasts potentially have at least one secreted factor that influences tumor progression. We found that BMP5 increased the percentage of CD133+ tumor cells from 4.66 % to 13.7 % . We then found that BMP5 was overexpressed in isolated FGFR2+ fibroblasts. Besides that, the clonogenic survival assay after IR showed that BMP5 promoted cancer cell radioresistance.