Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. METHODS: A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). RESULTS: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had a preponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. CONCLUSIONS: Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. IMPACT AND IMPLICATIONS: Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma.

BACKGROUND & AIMS: Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). METHODS: Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. RESULTS: In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. CONCLUSIONS: Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Progression in Cholangiocarcinoma.

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in humans, challenging the concept of TRAIL as a potent anticancer agent. Herein, we demonstrate that TRAIL + cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). In multiple immunocompetent syngeneic, orthotopic murine models of CCA, implantation of TRAIL + murine cancer cells into Trail-r -/- mice resulted in a significant reduction in tumor volumes compared to wild type mice. Tumor bearing Trail-r -/- mice had a significant decrease in the abundance of MDSCs due to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent NF-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of CD45 + cells in murine tumors from three distinct immunocompetent CCA models demonstrated a significant enrichment of an NF-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis due to enhanced expression of cellular FLICE inhibitory protein (cFLIP), an inhibitor of proapoptotic TRAIL signaling. Accordingly, cFLIP knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. In summary, our findings define a noncanonical TRAIL signal in MDSCs and highlight the therapeutic potential of targeting TRAIL + cancer cells for the treatment of a poorly immunogenic cancer.