Combining DR5 activation with FGFR1 and VEGFR2 inhibition via an engineered TRAIL-based multimodal fusion protein for dual antitumor and antivascular activity in gastric and colorectal cancer models.

Targeting both tumor cells and microenvironment is a promising anticancer strategy. FGFR1 and VEGFR2 represent important drivers of tumor progression and angiogenesis. We engineered multitarget proteins by fusing DR5‑specific TRAIL DR5-B with SRH and P9 peptides antagonizing VEGFR2 and FGFR1, respectively. SRH-DR5-B, DR5-B-P9, and SRH-DR5-B-P9 fusions demonstrated nanomolar affinity for receptors DR5, VEGFR2, and FGFR1 and reduced viability of FGFR1-positive gastric (MKN-45) and colon (HCT116, COLO 205) adenocarcinoma cells. DR5‑B-P9 and SRH‑DR5‑B‑P9 decreased phospho‑FGFR1 levels in FGF2‑stimulated NIH3T3 fibroblasts and MKN‑45 cells. In a co‑culture of MKN‑45, NIH‑3T3, and bEnd.3 cells, SRH‑DR5‑B‑P9 with gemcitabine showed greater cytotoxicity than DR5‑B. In MKN‑45 xenografts, SRH‑DR5‑B‑P9 inhibited tumor growth more efficiently and durably versus other proteins. Reduced vascular density in SRH‑DR5‑B and SRH‑DR5‑B‑P9‑treated tumors was demonstrated by CD31 immunostaining and by optoacoustic microangiography. PAS+/CD31- staining indicated suppressed vasculogenic mimicry, most pronounced with DR5‑B‑P9 and SRH‑DR5‑B‑P9. FLIM imaging revealed a glycolysis‑to‑oxidative phosphorylation shift in DR5‑B and SRH‑DR5‑B‑P9‑treated tumors, suggesting reprogrammed tumor bioenergetics. In HCT116 xenografts, SRH‑DR5‑B‑P9 suppressed tumor growth and CD31+ and PAS+/CD31- elements density more effectively than DR5‑B, confirming the results from MKN‑45 model. This supports a single‑fusion‑molecule strategy that activates DR5 while inhibiting FGFR1 and VEGFR2 for gastric and colorectal cancer therapy.