531 Background: Chronic inflammation from autoimmune conditions is a well-recognized risk factor for cholangiocarcinoma in the Western world. Cholangiocarcinomas in the setting of autoimmune disease have earlier age-of-onset and worse prognosis. Currently, their biology is inadequately understood, and the risks and benefits of immunotherapy approaches in this population are unknown. We describe the clinical and genomic characteristics of a cohort of cholangiocarcinoma patients with autoimmune etiologies. Methods: In a prospective cohort of 43 patients with mismatch repair proficient cholangiocarcinoma, we performed whole-genome sequencing (WGS, n=43) and total RNA sequencing (RNA-seq, n=28). Among this cohort, 15 patients had cholangiocarcinoma with autoimmune etiologies, including primary sclerosing cholangitis (PSC, n=10), ulcerative colitis (UC, n=6), ankylosing spondylitis (AS, n=1), and Crohn’s disease (CD, n=3). Results: Patients with autoimmune conditions had a 6.46-fold higher Tumour Mutational Burden (TMB) ( P = 0.0010), characterized by higher load of indels ( P = 0.0006). TMB was greater than 10 mutations per megabase in 4 of 15 patients with autoimmune conditions and 0 of 28 patients in those without ( P = 0.0111). Autoimmune patients exhibited an enrichment in mutational signatures 5 ( P = 0.0154), 17 ( P = 0.0004), 28 ( P = 0.028) and 30 ( P = 0.0164). Despite a higher TMB, autoimmune tumours have similar patterns of driver mutations. Differential gene expression of bulk RNA-seq analysis revealed comparable transcriptional markers of immunogenicity, but autoimmune cancers are enriched in pathways involved with cell proliferation and cell-cycle checkpoints. One patient with PSC/UC was treated with gemcitabine, cisplatin, and durvalumab with a partial response, while another with Crohn’s disease had durvalumab added to gemcitabine and cisplatin at progression and experienced stable disease with a decreased CA19-9. Neither experienced immune-related adverse events. Conclusions: Cholangiocarcinoma with autoimmune etiologies have unique genomic features characterized by elevated TMB. Chemo-immunotherapy may be a novel therapeutic option in this subgroup of patients and warrants further study.

Abstract Cholangiocarcinoma (CCA) is an aggressive liver malignancy with increased incidence globally but poor prognosis. Cancer stemness plays critical roles on tumor initiation, recurrence, and resistance to therapy. However, the mechanism of stemness maintenance in CCA has been less explored. Previously, we have reported TM4SF1 is associated with poor survival in CCA and high expression of TM4SF1 tends to promote tumorigenesis. In this study, we found that the expression of TM4SF1 gene is upregulated in tumor tissues compared to adjacent normal tissues in CCA based on bulk RNA sequencing dataset from The Cancer Genome Atlas (TGCA) CCA cohort. There is a positive correlation between the expression of TM4SF1 and stemness genes, ITGB3, SOX9, ALDH1A3, and ITGB1. Interestingly, knockdown of TM4SF1 with siRNA in CCA cell line HuCCT-1 cells demonstrated a decrease in expression of cancer stem cell surface markers, CD24 and Prom1. Furthermore, with a CRISPR-based lentivirus construct to knockdown TM4SF1 gene in HuCCT-1 cells in vitro, we found that spheroid formation in TM4SF1 knockdown was attenuated in comparison to TM4SF1 wild type. In addition, the results from flow cytometry and RT-PCR showed a corresponding decrease in CD24 and CD133 expression with TM4SF1 knockdown when comparing to TM4SF1 wild type. In conclusion, TM4SF1 is associated with the stemness of CCA and could be a therapeutic target for CCA therapy to overcome therapy resistance in future. Citation Format: Xiao Bin Zhu, Jihye Golino, Xin Wang, Changqing Xie. TM4SF1 maintains cancer stemness in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5440.

CAR-T therapy is a promising new immunotherapy for cancers, but its efficacy for solid tumors requires improvement. A detailed understanding of the interplay between solid tumors and CAR-T cells is critical. Here we report temporal, multi-modal, single-cell profiling of patient-derived glioblastoma organoids with CAR-T treatment. We found that all tumor cell types responded to CAR-T cell activation and contributed to an initially anti-tumor, but subsequently pro-tumor and immune-inhibitory microenvironment, accompanied by CAR-T cell exhaustion. Unexpectedly, CAR-T treatment attenuated glioma stem-like states of both antigen-positive and antigen-negative neoplastic cells and reduced their proliferation via diffusible factors, including IFNγ. Analysis of samples from additional patients, including those in clinical trials, supported these findings. Our study reveals the dynamic interplay among different tumor cells and T cells in adaptive responses to immunotherapy and identifies previously unappreciated benefits of CAR-T therapy directly on antigen-negative neoplastic cells that may be leveraged to enhance therapeutic efficacy. ### Competing Interest Statement The authors have declared no competing interest.

Objective Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. Design Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. Results We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29 + ( Itgb1 , integrin β1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29 + Tregs as the prominent niche-filling subpopulation in the liver, and CD29 + Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29 + and CD29 − hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29 + Tregs was in part IL2-independent. Conclusion We propose that CD29 + Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Functional tumor-specific CD8+ T cells are essential for an effective anti-tumor immune response and the efficacy of immune checkpoint inhibitor therapy. In comparison to other organ sites, we found higher numbers of tumor-specific CD8+ T cells in primary, metastatic liver tumors in murine tumor models. Despite their abundance, CD8+ T cells in the liver displayed an exhausted phenotype. Depletion of CD8+ T cells showed that liver tumor-reactive CD8+ T failed to control liver tumors but was effective against subcutaneous tumors. Similarly, analysis of single-cell RNA sequencing data from patients showed a higher frequency of exhausted tumor-reactive CD8+ T cells in liver metastasis compared to paired primary colon cancer. High-dimensional, multi-omic analysis combining proteomic CODEX and scRNA-seq data revealed enriched interaction of SPP1+ macrophages and CD8+ tumor-reactive T cells in profibrotic, alpha-SMA rich regions in the liver. Liver tumors grew less in Spp1-/- mice and the tumor-specific CD8+ T cells were less exhausted. Differential pseudotime trajectory inference analysis revealed extrahepatic signaling promoting an intermediate cell (IC) population in the liver, characterized by co-expression of VISG4, CSF1R, CD163, TGF-βR, IL-6R, SPP1. scRNA-seq of a third data set of premetastatic adenocarcinoma showed that enrichment of this population may predict liver metastasis. Our data suggests a mechanism by which extrahepatic tumors facilitate the formation of liver metastasis by promoting an IC population inhibiting tumor-reactive CD8+ T cell function.

Cholangiocarcinoma (CCA), a heterogeneous malignancy of bile duct epithelial cells, is characterized by aggressiveness, difficult diagnosis, and poor prognosis due to limited understanding and lack of effective therapeutic strategies. Genetic and epigenetic alterations accumulated in CCA cells can cause the aberrant regulation of oncogenes and tumor suppressors. Epigenetic alterations with histone modification, DNA methylation, and noncoding RNA modulation are associated with the carcinogenesis of CCA. Mutation or silencing of genes by various mechanisms can be a frequent event during CCA development. Alterations in histone acetylation/deacetylation at the posttranslational level, DNA methylation at promoters, and noncoding RNA regulation contribute to the heterogeneity of CCA and drive tumor development. In this review article, we mainly focus on the roles of epigenetic regulation in cholangiocarcinogenesis. Alterations in epigenetic modification can be potential targets for the therapeutic management of CCA, and epigenetic targets may become diagnostic biomarkers of CCA.

Cholangiocarcinoma (CCA) is a heterogenous malignancy that arises from the biliary epithelium and has a poor clinical prognosis. The Hippo/yes-associated protein (YAP) pathway has been reported to affect various aspects of tumorigenesis, with high expression of YAP1 being negatively associated with survival in CCA patients. Thus, we investigated the antitumoral effect of verteporfin, a YAP1 pathway inhibitor, in YAP1/AKT hydrodynamic tail vein injected murine models. We also used flow cytometry and single-cell RNA sequencing (scRNA-seq) to analyze the change in the immune cell profile and malignant cell stemness following verteporfin treatment. Our results demonstrated reduced liver weight and tumor formation in verteporfin-treated groups compared to that of a vehicle-treated group. Immune cell profiling through flow cytometry showed that relative to the vehicle, verteporfin induced a higher ratio of tumor-associated macrophage (TAM) M1/M2 and increased the percentage of activated CD8 T cell population (CD8+CD25+ and CD8+CD69+). scRNA-seq analysis showed significantly increased TAM M1 populations following verteporfin treatment and decreased proportions of stem-like cells within the malignant cell population. In summary, this study indicates that in CCA YAP/AKT murine models, verteporfin reduces tumorigenesis by polarizing anti-tumoral TAM and activating CD8 T cells and decreasing stem-like malignant cell proportions in the tumor microenvironment.

Cholangiocarcinoma is a malignancy of the bile ducts that is driven by activities of cancer stem-like cells and characterized by a heterogeneous tumor microenvironment. To better understand the transcriptional profiles of cancer stem-like cells and dynamics in the tumor microenvironment during the progression of cholangiocarcinoma, we performed single-cell RNA analysis on cells collected from three different timepoints of tumorigenesis in a YAP/AKT mouse model. Bulk RNA sequencing data from TCGA (The Cancer Genome Atlas program) and ICGC cohorts were used to verify and support the finding. In vitro and in vivo experiments were performed to assess the stemness of cancer stem-like cells. We identified Tm4sf1high malignant cells as cancer stem-like cells. Across timepoints of cholangiocarcinoma formation in YAP/AKT mice, we found dynamic change in cancer stem-like cell/stromal/immune cell composition. Nevertheless, the dynamic interaction among cancer stem-like cells, immune cells, and stromal cells at different timepoints was elaborated. Collectively, these data serve as a useful resource for better understanding cancer stem-like cell and malignant cell heterogeneity, stromal cell remodeling, and immune cell reprogramming. It also sheds new light on transcriptomic dynamics during cholangiocarcinoma progression at single-cell resolution.

The prognosis of cholangiocarcinoma remains poor. The heterogeneity of the tumor ecosystem of cholangiocarcinoma plays a critical role in tumorigenesis and therapeutic resistance, thereby affecting the clinical outcome of patients with cholangiocarcinoma. Recent progress in single-cell RNA sequencing (scRNA-seq) has enabled detailed characterization of intratumoral stromal and malignant cells, which has vastly improved our understanding of the heterogeneity of various cell components in the tumor ecosystem of cholangiocarcinoma. It also provides an unprecedented view of the phenotypical and functional diversity in tumor and stromal cells including infiltrating immune cells. This review focuses on examining tumor heterogeneity and the interaction between various cellular components in the tumor ecosystem of cholangiocarcinoma derived from an scRNA-seq dataset, discussing limitations in current studies, and proposing future directions along with potential clinical applications.

Mucosal-associated invariant T (MATT) cells represent an abundant innate-like T cell subtype in the human liver. MATT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MATT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MATT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MATT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MATT cells was identified as a key regulatory element of MATT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MATT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MATT cells in HCC patients.

PDF file - 52K, Clinicopathology data for whole human diagnostic cancer specimens.

Cholangiocarcinoma is an aggressive type of liver cancer with few effective treatment options. Therefore, there is great need to better understand the biology of this malignancy to further development of novel treatment options. Cancer stem cells (CSCs) are thought to the underlying reason for cancer initiation, metastasis, and relapse. However, due to their elusive character and differences in identification among different types of cancer, it remains a challenge to study such cells. Additionally, characterization of the tumor microenvironment such as interactions with immune cells remain largely unknown. Here, we employ a fluorescent reporter system to track and isolate stem-like cancer cells of cholangiocarcinoma cell lines. Following verification of a stem-like signature (upregulated expression of stemness markers, resistance to chemotherapy, increased spheroid formation, and tumorigenesis capabilities despite inoculation of a small number of cells), we analyzed the interaction of these cells with macrophages via direct and indirect coculture assays. We noted direct coculturing increased stemness among CSC populations and induced both M1 (CD80 and HLA-DR) and M2 (CD163) tumor associated macrophage polarization. These studies suggest that there is a bi-directional crosstalk between macrophages and CSCs that promotes stemness renewal and tumor associated macrophage polarization.

Cancer stem cells (CSCs) are responsible for long-term maintenance of tumors and thought to play a role in treatment resistance. The interaction between stemness and immunogenicity of CSCs in the intrahepatic cholangiocarcinoma (iCCA) is largely unknown. Here, we used single-cell transcriptomic data to study immunogenicity of malignant cells in human iCCA. Using an established computerized method CytoTRACE, we found significant heterogeneity in stemness/differentiation states among malignant cells. We demonstrated that the high stemness malignant cells express much lower levels of major histocompatibility complex II molecules when compared to low stemness malignant cells, suggesting a role of immune evasion in high stemness malignant cells. In addition, high stemness malignant iCCA cells exhibited significant expression of certain cytokine members, including CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, TNFRSF12A, and IL6ST, indicating communication with surrounding immune cells. These results indicate that high stemness malignant cells retain their intrinsic immunological feature that facilitate the escape of immune surveillance.

Cholangiocarcinoma (CCA) is a heterogenous cancer originated from the biliary epithelium in the biliary tree system. The incidence of CCA has gradually increased across nation/worldwide and overall prognosis is poor with only 10% of 5-year survival rate. Recently developed immune checkpoint inhibitor-based immunotherapy has less benefit on CCA patients. We investigated antitumoral effect of verteporfin in combination of anti-PD-1 in YAP/Akt and Notch/Akt hydrodynamic injected mouse model. Our result showed reduced liver weight in verteporfin treated mice group compared to vehicle treated mice group, both in anti-PD-1 and isotype control groups. Single cell RNA sequence showed there is reduced expression of stemness related cells from data observed in verteporfin treated group. B cell, CD8+ T cell, TAM M1 populations were increased in verteporfin treatment group and similar pattern was observed in anti-PD1 treated verteporfin group. In conclusion, verteporfin reduced tumorigenesis in CCA via control of cancer stemness in CCA animal models with anti-PD-1 immunotherapy. Citation Format: Jihye Lee, Jing Bian, Xin Wang, Meggie Cam, Changqing Xie. Verteporfin synergizes antitumoral effect of anti-PD-1 in mice cholangiocarcinoma models [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B16.

Cancer stem cells (CSCs) are a subset of the tumor population that play critical roles in tumorigenicity, metastasis, and relapse. A key feature of CSCs is their resistance to numerous therapeutic strategies which include chemotherapy, radiation, and immune checkpoint inhibitors. In recent years, there is a growing body of literature that suggests a link between CSC maintenance and autophagy, a mechanism to recycle intracellular components during moments of environmental stress, especially since CSCs thrive in a tumor microenvironment that is plagued with hypoxia, acidosis, and lack of nutrients. Autophagy activation has been shown to aid in the upkeep of a stemness state along with bolstering resistance to cancer treatment. However, recent studies have also suggested that autophagy is a double-edged sword with anti-tumorigenic properties under certain circumstances. This review summarizes and integrates what has been published in the literature in terms of what role autophagy plays in stemness maintenance of CSCs and suggests that there is a more complex interplay between autophagy and apoptosis which involves multiple pathways of regulation. Future cancer therapy strategies are needed to eradicate this resistant subset of the cell population through autophagy regulation.

Abstract Tumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. Hepatocellular carcinoma (HCC) is clinically and biologically heterogeneous, which is partly attributed to the presence of hepatic cancer stem cells (CSCs). CSC surface makers can have overlapping expression or exclusive expression of different subpopulations of hepatic CSCs, and these cells may contain different oncogenic changes. The existence of various CSC surface-marked subpopulations might pose a problem for CSC targeted therapeutics. Little is known about whether there are shared or distinct pathways in different subpopulations. Moreover, it remains unknown whether heterogeneity exists within certain subpopulations. Thus, we propose to profile the global transcriptome of surface-marked CSCs (EpCAM+, CD133+ and CD24+) at the single-cell level using index flow cytometric sorting and single-cell RNA sequencing technology. We statistically compared mRNA transcriptomes of individual single cells of triple positive (EpCAM+/ CD133+ /CD24+) CSCs and triple negative (EpCAM-/ CD133- /CD24-) cells. Further, we compared the triple positive and the triple negative cells at the single cell level in terms of their self-renew capability in both normoxic and hypoxic conditions. Our results show that at the single cell level, the transcriptomic profiles show a high-degree of heterogeneity in HCC cell lines, meanwhile, there is a dramatic difference between triple positive and triple negative cells in their transcriptomic profiles. There is a high-degree of intra-tumor and inter-tumor heterogeneity observed in single cell transcriptomes of HCC patients’ samples. Triple positive CSC single cells show continuous rather than discrete stemness-related gene expression patterns. In parallel, the self-renewal capability of triple positive cells also exhibits heterogeneity, while triple negative cells show little self-renewal capability. Thus, our single cell analysis study reveals molecular and biological heterogeneity of cancer stem cells in HCC, which may provide insight into the underlying mechanisms of how CSCs contribute to tumor heterogeneity and underscore key pathways and novel targets for hepatic CSC therapy. Citation Format: Hongping Zheng, Yotsawat Pomyen, Maria Hernandez, Caiyi Li, Ferenc Livak, Tim Greten, Anuradha Budhu, Xin Wang. Single cell analysis reveals cancer stem cell heterogeneities in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 936. doi:10.1158/1538-7445.AM2017-936