Citations

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.

Abstract Background: Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy with limited treatment options. CCA exhibits a distinct desmoplastic and tolerogenic tumor immune microenvironment (TIME) characterized by elevated bile acid concentrations resulting from obstructive cholestasis and dysregulated bile acid metabolism. TGR5 is an essential component of bile acid signaling. We hypothesize that bile acids promote CCA progression by establishing an immunosuppressive TIME through TGR5 signaling, which can be therapeutically targeted using SBI-364, a novel potent small molecule TGR5 inhibitor. Methods: Single-cell RNA sequencing (scRNA-seq) of immune cells from human intrahepatic (iCCA) and perihilar (pCCA) CCA tumors were conducted. Therapeutic studies were performed using syngeneic murine models of iCCA (PMID: 38458319), a novel syngeneic model of pCCA, and patient-derived xenograft (PDX) models. In collaboration with Sanford Burnham Prebys, SBI-364, a first in-class competitive TGR5 antagonist, was assessed in vivo. Additionally, we performed co-culture experiments with bile acid +/- SBI-364-treated murine bone marrow-derived monocytic myeloid-derived suppressor cells (M-MDSCs) and CD8+ T cells to assess functional immunosuppressive effects. Results: Human scRNA-seq revealed significantly increased TGR5 (GPBAR1) expression predominantly in myeloid cell populations within the CCA TIME. SBI-364 treatment significantly reduced tumor burden in both syngeneic orthotopic iCCA models and a novel pCCA model that recapitulates the anatomic characteristics of perihilar CCA. In contrast, NOD/SCID mice bearing PDX535 (predicted sensitive) and PDX136 (predicted resistant), SBI-364 treatment did not achieve tumor reduction, suggesting that TGR5 inhibition predominantly attenuates tumor progression through modulation of bile acid-immune cell interactions. SBI-364 treated murine tumors had a significant reduction in immunosuppressive macrophages expressing interleukin-10 and M-MDSCs compared to vehicle treated tumors. Furthermore, bile acid-treated M-MDSCs exhibited enhanced suppression of CD8+ T cell proliferation and function, an effect that was abrogated by SBI-364 co-treatment. Conclusion: We demonstrate that TGR5 inhibition represents a promising therapeutic strategy across multiple immunocompetent preclinical CCA models. We demonstrate that the bile-acid TGR5 axis promotes CCA progression via immunosuppressive myeloid cells. Specifically, TGR5 signaling potentiates the immunosuppressive capacity of M-MDSCs, contributing to an immunosuppressive niche that facilitates tumor progression. These results establish TGR5 as a rational therapeutic target for overcoming immune evasion in cholangiocarcinoma. Citation Format: Enis Hikmet Ozmert, Binbin Li, Jingchun Yang, Hannah Elizabeth Stumpf, Jessica R. Willhite, Danielle Carlson, Jennifer L. Tomlinson, Jon Covel, Raghuveer Ramachandra, Steven Olson, Rory L. Smoot, Sumera Ilyas. Therapeutic targeting of TGR5 abrogates bile acid driven myeloid cell immunosuppression in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2896.

Abstract Cholangiocarcinoma (CCA), an aggressive cancer of the bile ducts, is classified into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) anatomic subtypes. Each subtype has a distinct epidemiology and pathogenesis. The tumor immune microenvironment (TIME) of each subtype is poorly understood. CCAs are poorly immunogenic tumors and systemic therapy has had subpar efficacy in CCA subtypes. Thus, we hypothesized that each anatomic subtype has a distinct TIME. To address this hypothesis, we completed single cell sequencing of tumor, liver, and bile duct from patients with either iCCA or pCCA/dCCA. 182,054 cells from paired liver (n=16), tumor (n=16) (9 iCCA, 7 pCCA/dCCA), and normal bile duct (n=9) samples were obtained from treatment-naïve patients undergoing resection from June 2020 to August 2023. The tissues were homogenized and flow sorted for immune (CD45+) populations immediately following resection. The cells then underwent single cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) with antibodies for 157 proteins. Multiple immunocompetent, syngeneic/orthotopic murine models of iCCA with distinct genetic drivers were used to assess immunotherapeutics (PMID: 38458319). Immune cells of interest were isolated by flow sorting and functionally validated via suppression assays and multiparameter flow cytometry. Additionally, a novel pCCA murine model was established by injection of an Pik3caH1047R/p27Kip1-derived extrahepatic biliary cancer cell line into the porta/hilar tissue plane. Compared to non-involved liver, the bile duct immune microenvironment composition closely resembled that of the tumor. Multimodal analysis demonstrated both human and murine iCCA tumors have a significant upregulation of regulatory T cells (Treg) expressing T cell immunoreceptor with Ig and ITIM domain (TIGIT) compared to controls. Suppression assay with intratumoral TIGIT+ Treg from mouse compared with TIGIT- Treg indicate TIGIT+ Treg are more potent in inhibiting proliferation of CD8+ T cells. Moreover, TIGIT blockade using a monoclonal antibody resulted in a significant reduction in tumor burden in multiple iCCA murine models, including SB1, a model resistant to anti-PD-(L)1 monotherapy. Meanwhile, human pCCA tumors had a significant increase in IgA+ plasma cells compared to iCCA tumors. An innovative genetically and anatomically relevant pCCA murine model was developed to interrogate this unique plasma cell population. In summary, these data suggest that iCCA and pCCA have distinct immune microenvironments which likely impacts the differences in response to ICB between the subtypes observed in phase III clinical trials. Additionally, the uninvolved, tumor-adjacent bile duct immune microenvironment resembles that of the tumor, suggesting a pre-malignant niche that facilitates tumor development. Citation Format: Hannah E. Stumpf, Jingchun Yang, Haidong Dong, Rory L. Smoot, Sumera I. Ilyas. Multimodal single cell analysis of cholangiocarcinoma subtypes reveals distinct immune microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6476.

Background: The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study, we aimed to investigate and characterize the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Methods: Cholangiocytes obtained from patients with PSC and without PSC by endoscopic retrograde cholangiography were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing. Results: Unsupervised clustering of all integrated single-cell RNA sequencing data identified 8 cholangiocyte clusters that did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, as noted by an increased number of differentially expressed genes by transcriptomics and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, genome sequencing identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. Conclusions: PSC and non-PSC patient-derived ECO respond differently to IL-17 stimulation, implicating this pathway in the pathogenesis of PSC.

Abstract The pathogenesis of primary sclerosing cholangitis (PSC) is unclear, although studies implicate IL-17A as an inflammatory mediator in this disease. However, a direct assessment of IL-17 signaling in PSC cholangiocytes is lacking. In this study we aimed to investigate the response of PSC extrahepatic cholangiocyte organoids (ECO) to IL-17A stimulation. Cholangiocytes obtained from PSC and non-PSC patients by endoscopic retrograde cholangiography (ERC) were cultured as ECO. The ECO were treated with vehicle or IL-17A and assessed by transcriptomics, secretome analysis, and genome sequencing (GS). Unsupervised clustering of all integrated scRNA-seq data identified 8 cholangiocyte clusters which did not differ between PSC and non-PSC ECO. However, PSC ECO cells demonstrated a robust response to IL-17 treatment, noted by an increased number of differentially expressed genes (DEG) by transcriptomics, and more abundant chemokine and cytokine expression and secretion. After rigorous filtering, GS identified candidate somatic variants shared among PSC ECO from unrelated individuals. However, no candidate rare variants in genes regulating the IL-17 pathway were identified, but rare variants regulating the MAPK signaling pathway were present in all PSC ECO. In conclusion, PSC and non-PSC patient derived ECO respond differently to IL-17 stimulation implicating this pathway in the pathogenesis of PSC.

Abstract Background: Cholangiocarcinoma (CCA) is a heterogeneous malignancy arising from the biliary epithelium. Its diverse molecular landscape and aggressive biology render many anti-cancer therapies ineffective. Nanovesicle technology provides an opportunity for therapeutic inhibition of oncogenic targets that have been previously classified as undruggable. EpCAM is an epithelial-specific, transmembrane glycoprotein with increased expression in human and murine CCA which can be used for nanovesicle targeting. As a proof of concept study, we designed and validated a novel strategy to direct therapeutic milk-derived nanovesicles (tMNVs) to CCA tumors. Methods: tMNVs were decorated with RNA nanoparticles containing a validated aptamer (EpDT3) against EpCAM conjugated to a cholesterol-triethylene-glycol (TEG) scaffold containing an Alexa647 fluorophore. Human and murine CCA cell lines were treated with aptamer directed tMNVs and assessed for nanovesicle uptake by fluorescent microscopy. CCA tumor tissue, derived from orthotopic implantation of a syngeneic CCA cell line, SB1, into a C57BL/6 mouse, was collected and treated with either aptamer-directed or bare tMNVs ex vivo, and compared with adjacent normal liver tissue. Flow cytometry was utilized to characterize tMNVs absorption profile. C57BL/6 mice who had previously undergone SB1 orthotopic and flank implantation were treated with aptamer-directed tMNVs by tail-vein injection and subsequently euthanized. Tissue was collected for biodistribution analyses by fluorescent microscopy. The experiment was repeated in NOD-scid mice following orthotopic implantation of patient derived xenograft (PDX) CCA tumor. Results: Both human and murine CCA cells treated with aptamer-directed tMNVs demonstrated high fluorescent signal consistent with tMNV absorption within 12 hours of application. Flow cytometry analysis showed aptamer-directed tMNVs were absorbed at a higher proportion by CCA tumors than bare tMNVs ex vivo. Aptamer-directed tMNVs also had better absorption by CCA tumors compared to adjacent normal liver tissue. Following treatment with aptamer-directed or bare tMNVs in vivo, fluorescent microscopy demonstrated that aptamer-directed tMNVs were significantly better absorbed in the orthotopic SB1 tumors, followed by the subcutaneous tumors. Minimal fluorescent signal was noted in the normal adjacent liver. Orthotopically implanted PDX tumors also demonstrated high fluorescent signals following intravenous treatment with aptamer-directed tMNVs. Conclusions: Utilizing a novel targeting strategy, we were able to design tMNVs capable of reliably and specifically targeting CCA in preclinical models. This work is foundational to the future application of nanovesicle technology in the CCA treatment paradigm. Citation Format: Mincheng Yu, Jennifer L. Tomlinson, Emilien J. Loeuillard, Ryan D. Watkins, Caitlin B. Conboy, Shohei Takaichi, Nathan W. Werneburg, Roberto Alva-Ruiz, Amro Abdelrahman, Danielle M. Carlson, Jingchun Yang, Sumera I. Ilyas, Gregory J. Gores, Tushar Patel, Rory L. Smoot. Novel strategy for aptamer-directed nanovesicle targeting in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 814.

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.