Treatment of triple-negative breast cancer (TNBC) is challenging because of its “COLD” tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B ⁺ CD8 ⁺ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)–like endoplasmic reticulum kinase (PERK)–mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Rapid proliferation of cancer cells assisted by endothelial cell-mediated angiogenesis and acquired inflammation at the tumor microenvironment (TME) lowers the success rate of chemotherapeutic regimens. Therefore, targeting these processes using localized delivery of a minimally toxic drug combination may be a promising strategy. Here, we present engineering of a biocompatible self-assembled lithocholic acid-dipeptide derived hydrogel (TRI-Gel) that can maintain sustained delivery of antiproliferating doxorubicin, antiangiogenic combretastatin-A4 and anti-inflammatory dexamethasone. Application of TRI-Gel therapy to a murine tumor model promotes enhanced apoptosis with a concurrent reduction in angiogenesis and inflammation, leading to effective abrogation of tumor proliferation and increased median survival with reduced drug resistance. In-depth RNA-sequencing analysis showed that TRI-Gel therapy induced transcriptome-wide alternative splicing of many genes responsible for oncogenic transformation including sphingolipid genes. We demonstrate that TRI-Gel therapy targets the reversal of a unique intron retention event in β-glucocerebrosidase 1 (Gba1), thereby increasing the availability of functional Gba1 protein. An enhanced Gba1 activity elevates ceramide levels responsible for apoptosis and decreases glucosylceramides to overcome drug resistance. Therefore, TRI-Gel therapy provides a unique system that affects the TME via post-transcriptional modulations of sphingolipid metabolic genes, thereby opening a new and rational approach to cancer therapy.

Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show thatceramide synthase 2(CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event ofCERS2is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript ofCERS2in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript ofCERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.

We show that hydrogel-mediated combination therapy induces a synergistic effect on tumor regression.