Cai, Meina

ABSTRACT Cholangiocarcinoma (CCA) is a lethal biliary cancer in which chemoresistance is nearly universal, and its tumor immune microenvironment is dominated by immunosuppressive tumor-associated macrophages (TAMs) that exclude cytotoxic CD8 + T cells. How tumor cells sustain this immunosuppressive state during chemotherapy is undefined. Here we demonstrate that therapy-induced senescent-like (Sen-L) cancer cells accumulate after gemcitabine/cisplatin in human and murine CCA, are predominantly cancer cells, and predict shorter survival. Genetic elimination of Sen-L cancer cells reduces tumor burden, lowers TAM abundance, and restores intratumoral CD8 + T cells, establishing them as causal drivers. Growth differentiation factor 15 (GDF-15) is the dominant Sen-L-secreted factor and reprograms macrophages to suppress CD8 + T cells through the non-canonical receptor TGFBR2 and STAT6, and p16-restricted Gdf15 silencing phenocopies Sen-L elimination. Combined with chemotherapy, Sen-L elimination improves survival beyond chemotherapy alone. These findings establish Sen-L cancer cells and their GDF-15 output as causal, targetable drivers of macrophage-mediated immune evasion in CCA. SIGNIFICANCE STATEMENT Therapy-induced senescent-like cancer cells, not stromal cells, are the dominant senescent-like and immunosuppressive population in cholangiocarcinoma, and their elimination restores antitumor immunity. GDF-15 is their dominant secreted effector and engages a non-canonical macrophage receptor, TGFBR2, identifying a cancer-cell-to-macrophage axis and a Sen-L-elimination strategy to restore chemosensitivity. Abstract Figure Graphical abstract. Senescent-like CCA cells promote tumor immunosuppression through TAMs polarization by GDF-15

Abstract Background & Objectives Patients with cholangiocarcinoma (CCA) commonly progress within months on first-line gemcitabine/cisplatin–based therapy with immune checkpoint inhibition (ICI), consistent with the emergence of treatment-induced adaptive resistance. Cellular senescence and its secretory program (senescence-associated secretory phenotype, SASP) can remodel the tumor immune microenvironment. We hypothesized that gemcitabine/cisplatin induces tumor-cell senescence that promotes immunosuppression and progression and prioritized SASP mediators that are mechanistically linked to this phenotype. Method We quantified senescence and lineage markers in chemotherapy-naïve (n = 18) and gemcitabine/cisplatin treated (n = 23) human cholangiocarcinoma specimens and analyzed single-nucleus RNA sequencing in 9 human CCA tumors. In murine orthotopic CCA models, senescent tumor cells were ablated using p16 or p21 promoter restricted inducible apoptosis (ATTAC), followed by immune profiling by flow cytometry in endpoint cohorts, or tested alone and in combination with gemcitabine/cisplatin-based therapy and immune checkpoint inhibition (ICI) for survival cohorts. Conditioned media from proliferating versus senescent tumor cells were applied to bone marrow-derived macrophages (BMDMs) to assess polarization and suppression on CD8+ T-cell proliferation. SASP candidates were screened by cytokine arrays and Growth/Differentiation Factor 15 (GDF-15) was validated by expression and genetic perturbation. Results Senescent cells were enriched in the tumor compartment in human cholangiocarcinoma and increased after chemotherapy, as assessed by p16, p21and telomere-associated DNA foci (TAF). In mice, ablation of p16+ or p21+ senescent tumor cells reduced tumor burden, prolonged survival, and improved response to gemcitabine/cisplatin-based regimens. Chemotherapy-induced senescence increased immunosuppressive tumor-associated macrophages (TAMs), an effect reversed by senescent tumor cell ablation. Mechanically, GDF-15 was the top enriched secreted factor from senescent human CCA cells, tracked with senescence associated transcriptional programs, and decreased after senescent tumor cell ablation. GDF-15 promoted immunosuppressive macrophage phenotype switching and macrophage-mediated suppression on CD8+ T cell proliferation. In comparison, senescence-restricted GDF-15 knockdown reduced tumor growth and macrophage-mediated immunosuppression. Conclusion Chemotherapy-induced tumor cell senescence promotes an immunosuppressive microenvironment through GDF-15 linked macrophage remodeling. Targeting senescent tumor cells or intercepting senescence associated GDF-15 signaling may improve response to first-line therapy in CCA.

Background and Aims: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the biliary epithelium with limited therapeutic options and poor long-term survival rates. To address the limitations of CCA treatment, we investigated cell-targeted nanovesicles as a delivery platform for transcriptome-targeting therapeutics. Approach and Results: Milk-derived nanovesicles were loaded with short interfering RNAs targeting Yes-associated protein (YAP), the downstream effector of the Hippo pathway; LCK, an upstream regulator of YAP; and tafazzin, a protein critical for the integrity of the inner mitochondrial membrane. These transcriptome-targeting nanovesicles were decorated with a lipid-coupled RNA aptamer to epithelial cell adhesion molecule, including a tracking fluorophore. In vitro studies were conducted using multiple CCA cell lines. In vivo studies were performed using C57BL/6 and non-obese diabetic/severe combined immunodeficient mice to evaluate delivery and efficacy in both an immunocompetent syngeneic murine and a patient-derived xenograft model. We demonstrated that transcriptome-targeting nanovesicles were selectively taken up by liver tumor cells, which was augmented by the incorporation of a targeting aptamer, and that milk-derived nanovesicles loaded with short interfering RNA effectively downregulated target gene expression, both in vitro and in vivo. Downstream effects of target gene inhibition were observed, including downregulation of YAP-TEAD target genes and an increase in reactive oxygen species production at the mitochondrial level. Administration of transcriptome-targeting nanovesicles targeting YAP, LCK, and tafazzin inhibited CCA growth and further synergized with chemotherapy in 2 preclinical CCA models. Conclusions: Herein, we show that aptamer-directed, nanovesicle-mediated targeting of YAP, LCK, and tafazzin potentiates chemosensitivity in 2 CCA models when delivered using aptamer-guided milk-derived nanovesicles.