Targeting tumor heterogeneity using an injectable gelatin hydrogel for the dual elimination of cancer cells and cancer stem cells.

Breast cancer relapse and therapeutic resistance recurrence are mainly due to the presence of breast cancer-associated cancer stem cells (bCSCs), which are inadequately eradicated by conventional chemotherapeutic agents. To mitigate these challenges, we engineered an injectable in situ-forming gelatin hydrogel capable of the sustained, localized co-delivery of resveratrol (RES) and salinomycin (SAL), thereby simultaneously targeting both cancer cells and bCSCs. Further, the gelatin hydrogel gels under physiological conditions without UV or toxic crosslinking agents, and it provides tunable mechanical strength and degradation kinetics. The 10% optimized gelatin hydrogel showed the controlled, sustained release of RES and SAL, with minimal initial burst and prolonged release extending to 30 days. In vitro cytotoxicity studies showed that treatment with RES or SAL alone resulted in approximately 40%-50% reduction in cancer cell viability; in contrast, the combination hydrogel (GelVS-RES + SAL) resulted in nearly 90% cell death. Mammosphere assays revealed a pronounced decrease in both the number and size of the mammospheres, indicating the strong suppression of bCSCs' intrinsic self-renewal capacity. Correspondingly, the gene expression levels of key bCSC-linked stemness biomarkers were decreased by approximately 3-fold compared with those observed in the individual drug, free-drug, and control conditions. Flow cytometry analysis further demonstrated a significant increase in apoptosis within the bCSC-enriched cell fraction upon exposure to GelVS-RES + SAL. In vivo evaluation using a 4T1 murine breast cancer model confirmed that the localized, sustained delivery of RES and SAL from the injectable gelatin hydrogel produced a synergistic antitumor effect, markedly inhibiting tumor progression while substantially mitigating systemic toxicity relative to free-drug administration. Collectively, the sustained co-delivery from the in situ injectable gelatin hydrogel efficiently targets both the bulk tumor population and bCSCs, highlighting its potential as a therapeutic strategy to diminish bCSC-driven recurrence and improve overall breast cancer treatment efficacy.