Polysaccharide extract of Spirulina sp. increases effector immune-cell killing activities against cholangiocarcinoma.

Cyanobacteria and algae serving as promising food supplements have recently garnered attention for their emerging potential in anti-cancer activity. Cholangiocarcinoma (CCA) or bile duct cancer is one of the top-leading cancers affecting people, particularly in Asian continent. With patients exhibiting no or minimal symptoms in the early stages, advanced CCA is often diagnosed, and primary treatments such as surgery may not be suitable. Discovery of natural bioactive compounds for cancer treatments have, thus, attracted attention as one of the effective means to combat CCA or to supplement primary treatments. In this work, ethanolic and polysaccharide extracts of cyanobacteria and algae were tested for their cytotoxicity against 2 CCA cell lines (KKU055 and KKU213A). The ethanolic extracts from Leptolyngbya sp. and Chlorella sp. demonstrated growth inhibition of both CCA cell lines, with IC50 values of 0.658 mg/mL and 0.687 mg/mL for KKU055, and 0.656 mg/mL and 0.450 mg/mL for KKU213A. In contrast, only the polysaccharide extracts from Sargassum spp. exhibited a remarkable cytotoxic effect, while the polysaccharide extract from Spirulina sp. showed slight effect only at a higher concentration (2 mg/mL). All tested extracts were further investigated for improving immune cell killing ability and showed that Spirulina sp. polysaccharide extract was able to improve the immune cell killing ability. This extract was then investigated for its effects on the immune cell population, which demonstrated to have positive impact on NK cell population. To further explore the potential use, synergistic effect of Spirulina sp. polysaccharide extract with an already-in-use chemotherapeutic drug, gemcitabine, on immune cell cytotoxicity was investigated. The results showed that the immune cell cytotoxicity was enhanced in the co-treatment compared to the use of each treatment separately. The most apparent difference was observed in KKU055 cells where % living cells were reduced from 78.96% (immune cell alone) to 20.93% when the combined gemcitabine and Spirulina sp. extracts were used.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods: Liver tissue samples were collected during 2008-2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results: CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells ("hot" cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions: These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with "hot" TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn "cold" into "hot" TIME in ICC.

The tumor immune microenvironment (TIME) plays a critical role in the immune response In many cancers, including intrahepatic cholangiocarcinoma (ICC). Molecular subtyping of the ICC microenvironment already revealed inter-tumoral heterogeneity with variant profiles of immune cell infiltrates. A recent study created an in-depth immune cell atlas of the TIME in biliary tract cancers and could demonstrate the relevance of specific immune cell subpopulations on patient outcome. We are able to provide a distinctive characterization of TIME, separating tumor epithelial- and stroma areas, in a large and representative ICC cohort using digitalized image analysis on tissue microarrays (TMA) as well as multiplex imaging mass cytometry (IMC). The study was designed for identification of immune cell prognosticators allocating institutional ICC patients into a discovery (2008­15) and a validation (2010­19) cohort. Immune cell subpopulations were correlated with clinicopathological characteristics and patient outcome. Our results highlight: i. The important role of CD4+ T cell infiltration in ICC patients; ii. ICC tumors with high density of immune cells associated with PD-L1 expression identifies a subset of patients with variant tumor biology; iii. Stimulation of STAT1 pathway may be a relevant target to turn "cold" into "hot" tumors.

Tumor-associated macrophages: orchestrators of cholangiocarcinoma progression.

Cholangiocarcinoma (CCA) is a rare but highly invasive cancer, with its incidence rising in recent years. Currently, surgery remains the most definitive therapeutic option for CCA. However, similar to other malignancies, most CCA patients are not eligible for surgical intervention at the time of diagnosis. The chemotherapeutic regimen of gemcitabine combined with cisplatin is the standard treatment for advanced CCA, but its effectiveness is often hampered by therapeutic resistance. Recent research highlights the remarkable plasticity of tumor-associated macrophages (TAMs) within the tumor microenvironment (TME). TAMs play a crucial dual role in either promoting or suppressing tumor development, depending on the factors that polarize them toward pro-tumorigenic or anti-tumorigenic phenotypes, as well as their interactions with cancer cells and other stromal components. In this review, we critically examine recent studies on TAMs in CCA, detailing the expression patterns and prognostic significance of different TAM subtypes in CCA, the mechanisms by which TAMs influence CCA progression and immune evasion, and the potential for reprogramming TAMs to enhance anticancer therapies. This review aims to provide a framework for deeper future research.

DAB2 + macrophages support FAP + fibroblasts in shaping tumor barrier and inducing poor clinical outcomes in liver cancer.

Background: Cancer-associated fibroblasts (CAFs) are the key components of the immune barrier in liver cancer. Therefore, gaining a deeper understanding of the heterogeneity and intercellular communication of CAFs holds utmost importance in boosting immunotherapy effectiveness and improving clinical outcomes. Methods: A comprehensive analysis by combing single-cell, bulk, and spatial transcriptome profiling with multiplexed immunofluorescence was conducted to unravel the complexities of CAFs in liver cancer. Results: Through an integrated approach involving 235 liver cancer scRNA-seq samples encompassing over 1.2 million cells, we found that CAFs were particularly increased in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). FAP + fibroblasts were identified as the dominant subtype of CAFs, and which were mainly involved in extracellular matrix organization and angiogenesis. These CAFs were enriched in the tumor boundary of HCC, but diffusely scattered within ICC. The DAB2 + and SPP1 + tumor-associated macrophages (TAMs) reinforce the function of FAP + CAFs through signals such as TGF-ß, PDGF, and ADM. Notably, the interaction between DAB2 + TAMs and FAP + CAFs promoted the formation of immune barrier and correlated with poorer patient survival, non-response to immunotherapy in HCC. High FAP and DAB2 immunohistochemical scores predicted shorter survival and higher serum AFP concentration in a local clinical cohort of 90 HCC patients. Furthermore, this communication pattern might be applicable to other solid malignancies as well. Conclusions: The interaction between DAB2 + TAMs and FAP + CAFs appears crucial in shaping the immune barrier. Strategies aimed at disrupting this communication or inhibiting the functions of FAP + CAFs could potentially enhance immunotherapy effectiveness and improve clinical outcomes.

Long-Term Survivor of Intrahepatic Cholangiocarcinoma for over 18 Years: Case Study with Longitudinal Histo-molecular and Tumor Immune Microenvironment Characterization and Systematic Review of the Literature.

BACKGROUND: Intrahepatic cholangiocarcinoma is a biliary neoplasm usually showing a dismal prognosis. In early stages, surgical resection is the best treatment option, significantly increasing the overall survival. This approach is also recommended in the case of relapsing disease. In this study, we report the case of a patient affected by intrahepatic cholangiocarcinoma with multiple relapses and still alive for over 18 years. We also provide a systematic review regarding long-survivor (> 60 months) of intrahepatic cholangiocarcinoma. CASE PRESENTATION: A 41-year-old woman with no pathological history was diagnosed with localized intrahepatic cholangiocarcinoma and surgically treated with left hepatectomy. After the first intervention, the patients underwent three further surgical resections because of locoregional recurrences. Histologically, there were some significant similarities among all neoplasms, including the tubule-glandular architecture, but also morphological heterogeneity. The tumor immune microenvironment remained stable across the different lesions. The molecular analysis with next-generation sequencing demonstrated that all neoplasms shared the same genomic profile, including NBN and NOTCH3 mutations and chromosomes 1 and 3 alterations. CONCLUSIONS: This case study highlights the essential role of a stringent follow-up after resection of intrahepatic cholangiocarcinoma for detecting early relapsing tumors. Moreover, it shows the importance of the molecular characterization of multiple tumors for understanding their real nature. The accurate study of long-surviving patients highlights the features that are critical for outcome improvement.

Expression and function of the major histocompatibility complex (MHC) class I chain-related A (MICA)*010 in NK cell killing activity.

The major histocompatibility complex (MHC) class I chain-related A (MICA) plays an important role in stress cell recognition. High polymorphisms of MICA are relevant to NKG2D binding capacity, responses of NK cells and tumor progression. In this study, MICA genotyping of 97 cholangiocarcinoma patients was performed using PCR-SSP. MICA*010 was positively associated with a corrected p-value of < 0.001 (RR=2.16 (95 % CI, 1.48-3.14)). MICA*010 was previously reported as a non-expressed allele. Thus, the expression of MICA*010 on the cell surface was studied on both MICA*010 transfected cells (HEK 293 T and L929 cells) and stimulated primary monocytes obtained from homozygous MICA*010 individuals using different clones of antibodies (1H10, 1D10, 1C3.1, 1C3.2, 6D4 and 3H5) for detection. Surprisingly, the expression of MICA*010 could be observed on both transfected cells and stimulated monocytes and effectively bound to the NKG2D-Fc fusion protein. The functional study of various MICA alleles revealed the high relative killing activity of NK cells induced by the MICA*010 transfected C1R cells, not following the previously reported rule of the M129V substitution. The structural analysis highlighted the amino acid at position 36 as another important amino acid relevant to preserving the structural integrity of the MICA protein and NKG2D binding. Our data propose a new aspect of functional MICA contributing motifs and that MICA*010 has a potential effect on NK cell functions and might be applicable to other fields of immune responses.

Analysis of the Effector Functions of Vδ2 γδ T Cells and NK Cells against Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a rare disease characterized by malignant cells derived from the epithelial cells of the biliary duct system. Despite extensive treatments, the prognosis for CCA remains poor, emphasizing the critical need for the development of novel treatments. Considerable attention has been directed towards innate immune effector cells, which can recognize tumor cells independently of the major histocompatibility complex, laying the foundation for the development of off-the-shelf drugs. In this study, we cultured innate immune cells obtained from the peripheral blood of healthy adults and conducted a comparative analysis of the effector functions against CCA cell lines by Vδ2 γδ T cells and NK cells. This analysis was performed using standard short- and long-term cytotoxicity assays, as well as ELISA for IFN-γ. Vδ2 γδ T cells demonstrated cytotoxicity and IFN-γ production in response to CCA cells in a TCR-dependent manner, particularly in the presence of tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate, a bisphosphonate prodrug. In contrast, direct killing and antibody-dependent cellular cytotoxicity were relatively slow and weak. Conversely, NK cells displayed potent, direct cytotoxicity against CCA cells. In summary, both Vδ2 γδ T cells and NK cells show promise as innate immune effector cells for adoptive transfer therapy in the context of CCA.

Strategies for treating the cold tumors of cholangiocarcinoma: core concepts and future directions.

Cholangiocarcinoma (CCA) is a rare type of digestive tract cancer originating from the epithelial cells of the liver and biliary tract. Current treatment modalities for CCA, such as chemotherapy and radiation therapy, have demonstrated limited efficacy in enhancing survival rates. Despite the revolutionary potential of immunotherapy in cancer management, its application in CCA remains restricted due to the minimal infiltration of immune cells in these tumors, rendering them cold and unresponsive to immune checkpoint inhibitors (ICIs). Cancer cells within cold tumors deploy various mechanisms for evading immune attack, thus impeding clinical management. Recently, combination immunotherapy has become increasingly essential to comprehend the mechanisms underlying cold tumors to enhance a deficient antitumor immune response. Therefore, a thorough understanding of the knowledge on the combination immunotherapy of cold CCA is imperative to leverage the benefits of immunotherapy in treating patients. Moreover, gut microbiota plays an essential role in the immunotherapeutic responses in CCA. In this review, we summarize the current concepts of immunotherapy in CCA and clarify the intricate dynamics within the tumor immune microenvironment (TIME) of CCA. We also delve into the evasion mechanisms employed by CCA tumors against the anti-tumor immune responses. The context of combination immunotherapies in igniting cold tumors of CCA and the critical function of gut microbiota in prompting immune responses have also been annotated. Furthermore, we have proposed future directions in the realm of CCA immunotherapy, aiming to improve the clinical prognosis of CCA patients.

Investigation of transcriptional and immunological disparities among patient groups with varied prognostic risk factors in cholangiocarcinoma.

BACKGROUND: This study explores molecular features associated with better prognosis in cholangiocarcinoma (CCA). METHODS AND RESULTS: The transcriptomic and whole-exome sequencing data obtained from paired tissues of 70 were analyzed, grouping them based on progression-free survival (PFS), differentiation degree, and lymph node metastasis. Among the 70 patients, the TP53 gene mutation frequency was the highest (53%), while FLG gene mutation occurred exclusively in the long PFS group. In the comparison between long and short survival groups, the short PFS group exhibited higher monocyte infiltration levels (p = 0.0287) and upregulation of genes associated with cancer-related transcriptional misregulation, chemokine signaling, and cytokine-cytokine receptor interactions. Differences in immune cell infiltration and gene expression were significant across differentiation and lymph node metastasis groups. Particularly noteworthy was the marked increase in CD8 T cell and NK cell infiltration (p = 0.0291, 0.0459) in the lymph node metastasis group, significantly influences prognosis. Additionally, genes related to platinum resistance, Th17 cell differentiation, and Th1 and Th2 cell differentiation pathways were overexpressed in this group. In summary, higher monocyte infiltration levels in the short PFS group, along with elevated expression of genes associated with cancer-related pathways, suggest a poorer prognosis. The significant increase in CD8 T cell and NK cell infiltration reflects an enhanced anti-tumor immune response, underscoring the relevance of immune infiltration levels and gene expression in predicting outcomes for CCA patients. CONCLUSIONS: In this study, we elucidated the pertinent molecular mechanisms and pathways that influence the prognosis of CCAs through comprehensive multi-omics analysis.

Identification of autoantibodies as potential non-invasive biomarkers for intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) presents a challenging diagnosis due to its nonspecific early clinical manifestations, often resulting in late-stage detection and high mortality. Diagnosing iCCA is further complicated by its limited accuracy, often necessitating multiple invasive procedures for precise identification. Despite carbohydrate antigen 19-9 (CA19-9) having been investigated and employed for iCCA diagnosis, it demonstrates modest diagnostic performance. Consequently, the identification of novel biomarkers with improved sensitivity and specificity remains an imperative yet formidable task. Autoantibodies, as early indicators of the immune response against cancer, offer a promising avenue for enhancing diagnostic accuracy. Our study aimed to identify non-invasive blood-based autoantibody biomarkers capable of distinguishing iCCA patients from healthy individuals (CTRs). We profiled autoantibodies in 26 serum samples (16 iCCAs and 10 CTRs) using protein microarrays containing 1622 functional proteins. Leveraging machine learning techniques, we identified a signature composed of three autoantibody biomarkers (NDE1, PYCR1, and VIM) in conjunction with CA19-9 for iCCA detection. This combined signature demonstrated superior diagnostic performance with an AUC of 96.9%, outperforming CA19-9 alone (AUC: 83.8%). These results suggest the potential of autoantibody biomarkers to develop a complementary non-invasive diagnostic utility for routine iCCA screening.

HER2 amplification subtype intrahepatic cholangiocarcinoma exhibits high mutation burden and T cell exhaustion microenvironment.

OBJECTIVE: This study aimed to establish a uniform standard for the interpretation of HER2 gene and protein statuses in intrahepatic cholangiocarcinoma (ICC). We also intended to explore the clinical pathological characteristics, molecular features, RNA expression and immune microenvironment of HER2-positive ICC. METHODS: We analyzed a cohort of 304 ICCs using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to identify HER2 status. Comprehensive analyses of the clinicopathological, molecular genetic, and RNA expression characterizations of ICCs with varying HER2 statuses were performed using next-generation sequencing. We further investigated the tumor microenvironment of ICCs with different HER2 statuses using IHC and multiplex immunofluorescence staining. RESULTS: HER2/CEP17 ratio of ≥ 2.0 and HER2 copy number ≥ 4.0; or HER2 copy number ≥ 6.0 were setup as FISH positive criteria. Based on this criterion, 13 (4.27%, 13/304) samples were classified as having HER2 amplification. The agreement between FISH and IHC results in ICC was poor. HER2-amplified cases demonstrated a higher tumor mutational burden compared to non-amplified cases. No significant differences were observed in immune markers between the two groups. However, an increased density of CD8 + CTLA4 + and CD8 + FOXP3 + cells was identified in HER2 gene-amplified cases. CONCLUSION: FISH proves to be more appropriate as the gold standard for HER2 evaluation in ICC. HER2 gene-amplified ICCs exhibit poorer prognosis, higher mutational burden, and T cell exhaustion and immune suppressed microenvironment.

Septin 9 expression regulates 'don't eat me' signals and identifies an immune-epithelial class of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

Development of a subunit vaccine against the cholangiocarcinoma causing Opisthorchis viverrini: a computational approach.

Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.

Small duct and large duct type intrahepatic cholangiocarcinoma reveal distinct patterns of immune signatures.

PURPOSE: Dedicated gene signatures in small (SD-iCCA) and large (LD-iCCA) duct type intrahepatic cholangiocarcinoma remain unknown. We performed immune profiling in SD- and LD-iCCA to identify novel biomarker candidates for personalized medicine. METHODS: Retrospectively, 19 iCCA patients with either SD-iCCA (n = 10, median age, 63.1 years (45-86); men, 4) or LD-iCCA (n = 9, median age, 69.7 years (62-85); men, 5)) were included. All patients were diagnosed and histologically confirmed between 04/2009 and 01/2021. Tumor tissue samples were processed for differential expression profiling using NanoString nCounter® PanCancer Immune Profiling Panel. RESULTS: With the exception of complement signatures, immune-related pathways were broadly downregulated in SD-iCCA vs. LD-iCCA. A total of 20 immune-related genes were strongly downregulated in SD-iCCA with DMBT1 (log2fc = -5.39, p = 0.01) and CEACAM6 (log2fc = -6.38, p = 0.01) showing the strongest downregulation. Among 7 strongly (log2fc > 2, p ≤ 0.02) upregulated genes, CRP (log2fc = 5.06, p = 0.02) ranked first, and four others were associated with complement (C5, C4BPA, C8A, C8B). Total tumor-infiltrating lymphocytes (TIL) signature was decreased in SD-iCCA with elevated ratios of exhausted-CD8/TILs, NK/TILs, and cytotoxic cells/TILs while having decreased ratios of B-cells/TILs, mast cells/TILs and dendritic cells/TILs. The immune profiling signatures in SD-iCCA revealed downregulation in chemokine signaling pathways inclulding JAK2/3 and ERK1/2 as well as nearly all cytokine-cytokine receptor interaction pathways with the exception of the CXCL1/CXCR1-axis. CONCLUSION: Immune patterns differed in SD-iCCA versus LD-iCCA. We identified potential biomarker candidate genes, including CRP, CEACAM6, DMBT1, and various complement factors that could be explored for augmented diagnostics and treatment decision-making.

Tumor-infiltrating T lymphocytes: A promising immunotherapeutic target for preventing immune escape in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is becoming more common and deadly worldwide. Tumor-infiltrating T cell subtypes make distinct contributions to the immune system; collectively, they constitute a significant portion of the tumor microenvironment (TME) in CCA. By secreting cytokines and other chemicals, regulatory T cells (Tregs) decrease activated T cell responses, acting as immunosuppressors. Reduced CD8+ T cell activation results in stimulating programmed death-1 (PD-1), which undermines the immunological homeostasis of T lymphocytes. On the other hand, cancer cells are eliminated by activated cytotoxic T lymphocyte (CTL) through the perforin-granzyme or Fas-FasL pathways. Th1 and CTL immune cell infiltration into the malignant tumor is also facilitated by γδ T cells. A higher prognosis is typically implied by CD8+ T cell infiltration, and survival is inversely associated with Treg cell density. Immune checkpoint inhibitors, either singly or in combination, provide novel therapeutic strategies for CCA immunotherapy. Furthermore, it is anticipated that immunotherapeutic strategies-such as the identification of new immune targets, combination treatments involving several immune checkpoint inhibitors, and chimeric antigen receptor-T therapies (CAR-T)-will optimize the effectiveness of anti-CCA treatments while reducing adverse effects.

Trastuzumab, a monoclonal anti-HER2 antibody modulates cytotoxicity against cholangiocarcinoma via multiple mechanisms.

Cholangiocarcinoma (CCA) is an aggressive and fatal cancer. The prognosis is very poor and no optimal chemotherapy has been established. Human epidermal growth factor receptor 2 (HER2, neu, and erbB2) is highly-expressed in breast cancer and is expressed in many other tumors but poorly expressed in CCA. The anti-HER2 antibody, trastuzumab, has been used for the treatment of HER2-positive breast and gastric cancer. In this study, we examined the surface expression of HER2 on seven Thai liver-fluke-associated CCA cell lines by flow cytometry, and found all of these CCA cells were weakly positive for HER2. MTT assay revealed that trastuzumab directly suppressed the growth of CCA. By using FcR-bearing recombinant Jurkat T-cell-expressing firefly luciferase gene under the control of NFAT response elements, we defined the activities of antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). ADCC was confirmed by using expanded NK cells. ADCP was confirmed by using mouse peritoneal macrophages and human monocyte-derived macrophages as effector cells. Rabbit serum was administered to test the complement-dependent cytotoxicity (CDC) activity of trastuzumab. Finally, we evaluated the efficacy of trastuzumab in in vivo patient-derived cell xenograft and patient-derived xenograft (PDX) models. Our results showed that a distinct population of CCA (liver-fluke-associated CCA) expressed HER2. Trastuzumab demonstrated a potent inhibitory effect on even HER2 weakly positive CCA both in vitro and in vivo via multiple mechanisms. Thus, HER2 is a promising target in anti-CCA therapy, and trastuzumab can be considered a promising antibody immunotherapy agent for the treatment of CCA.

Targeting CD73 limits tumor progression and enhances anti-tumor activity of anti-PD-1 therapy in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Patients with intrahepatic cholangiocarcinoma (iCCA) respond poorly to immune checkpoint blockades (ICBs). In this study, we aimed to dissect the potential mechanisms underlying poor response to ICBs and explore a rational ICB-based combination therapy in iCCA. METHODS: scRNA-seq dataset GSE151530 was analyzed to investigate the differentially expressed genes in malignant cells following ICBs therapy. RNA-seq analysis and western blot assays were performed to examine the upstream and downstream signaling pathways of CD73. Subcutaneous tumor xenograft models were utilized to investigate the impact of CD73 on iCCA growth. Plasmid AKT/NICD-induced spontaneous murine iCCAs were used to explore the therapeutic efficacy of CD73 enzymatic inhibitor AB680 combined with PD-1 blockade. Time-of-flight mass cytometry (CyTOF) was conducted to identify the tumor-infiltrating immune cell populations and their functional changes in murine iCCAs treated with AB680 in combination with PD-1 antibody. RESULTS: scRNA-seq analysis identified elevated CD73 expression in malignant cells in response to ICBs therapy. Mechanistically, ICBs therapy upregulated CD73 expression in malignant cells via TNF-α/NF-κB signaling pathway. In vivo studies revealed that CD73 inhibition suppressed the growth of subcutaneous tumors, and achieved synergistic depression effects with gemcitabine and cisplatin (GC). Adenosine produced by CD73 activates AKT/GSK3ß/ß-catenin signaling axis in iCCA cells. CD73 inhibitor AB680 potentiates anti-tumor efficacy of PD-1 antibody in murine iCCAs. CyTOF analysis showed that AB680 combined with anti-PD-1 therapy promoted the infiltration of CD8+ T, CD4+ T cells, and NK cells in murine iCCAs, while simultaneously decreased the proportions of macrophages and neutrophils. Moreover, AB680 combined with anti-PD-1 significantly upregulated the expression of Granzyme B, Tbet and co-stimulatory molecule ICOS in infiltrating CD8+ T cells. CONCLUSIONS: CD73 inhibitor AB680 limits tumor progression and potentiates therapeutic efficacy of GC chemotherapy or anti-PD-1 treatment in iCCA. AB680 combined with anti-PD-1 therapy effectively elicits anti-tumor immune response.

A predictive radiotranscriptomics model based on DCE-MRI for tumor immune landscape and immunotherapy in cholangiocarcinoma.

This study aims to determine the predictive role of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) derived radiomic model in tumor immune profiling and immunotherapy for cholangiocarcinoma. To perform radiomic analysis, immune related subgroup clustering was first performed by single sample gene set enrichment analysis (ssGSEA). Second, a total of 806 radiomic features for each phase of DCE-MRI were extracted by utilizing the Python package Pyradiomics. Then, a predictive radiomic signature model was constructed after a three-step features reduction and selection, and receiver operating characteristic (ROC) curve was employed to evaluate the performance of this model. In the end, an independent testing cohort involving cholangiocarcinoma patients with anti-PD-1 Sintilimab treatment after surgery was used to verify the potential application of the established radiomic model in immunotherapy for cholangiocarcinoma. Two distinct immune related subgroups were classified using ssGSEA based on transcriptome sequencing. For radiomic analysis, a total of 10 predictive radiomic features were finally identified to establish a radiomic signature model for immune landscape classification. Regarding to the predictive performance, the mean AUC of ROC curves was 0.80 in the training/validation cohort. For the independent testing cohort, the individual predictive probability by radiomic model and the corresponding immune score derived from ssGSEA was significantly correlated. In conclusion, radiomic signature model based on DCE-MRI was capable of predicting the immune landscape of chalangiocarcinoma. Consequently, a potentially clinical application of this developed radiomic model to guide immunotherapy for cholangiocarcinoma was suggested.

Targeting PD-L1 in cholangiocarcinoma using nanovesicle-based immunotherapy.

This study demonstrates the potential of using biological nanoparticles to deliver RNA therapeutics targeting programmed death-ligand 1 (PD-L1) as a treatment strategy for cholangiocarcinoma (CCA). RNA therapeutics offer prospects for intracellular immune modulation, but effective clinical translation requires appropriate delivery strategies. Milk-derived nanovesicles were decorated with epithelial cellular adhesion molecule (EpCAM) aptamers and used to deliver PD-L1 small interfering RNA (siRNA) or Cas9 ribonucleoproteins directly to CCA cells. In vitro, nanovesicle treatments reduced PD-L1 expression in CCA cells while increasing degranulation, cytokine release, and tumor cell cytotoxicity when tumor cells were co-cultured with T cells or natural killer cells. Similarly, immunomodulation was observed in multicellular spheroids that mimicked the tumor microenvironment. Combining targeted therapeutic vesicles loaded with siRNA to PD-L1 with gemcitabine effectively reduced tumor burden in an immunocompetent mouse CCA model compared with controls. This proof-of-concept study demonstrates the potential of engineered targeted nanovesicle platforms for delivering therapeutic RNA cargoes to tumors, as well as their use in generating effective targeted immunomodulatory therapies for difficult-to-treat cancers such as CCA.

Single-cell analysis reveals hypoxia-induced immunosuppressive microenvironment in intrahepatic cholangiocarcinoma.

The role of hypoxia in the tumor microenvironment of intrahepatic cholangiocarcinoma (iCCA) remains unclear. Here, we generated a comprehensive atlas of the entire tumor microenvironment and delineated the multifaceted cell-cell interactions to decipher hypoxia-induced pro-tumor immune suppression. We discovered hypoxia is significantly associated with iCCA progression via the activation of HIF1A expression. Moreover, hypoxia-dependent PPARγ-mediated fatty acid oxidation in APOE+ TAMs promoted M2 macrophage polarization by activating the HIF1A-PPARG-CD36 axis. These polarized APOE+ TAMs recruited Treg cell infiltration via the CCL3-CCR5 pair to form an immunosuppressive microenvironment. APOE+ TAMs tended to co-localize spatially with Treg cells in the malignant tissue based on spatial transcriptome data and immunofluorescence analysis results. We identified tumor-reactive CXCL13+ CD8-PreTex with specific high expression of ENTPD1 and ITGAE, which acted as precursors of CD8-Tex and had higher cytotoxicity, lower exhaustion, and more vigorous proliferation. Consequently, CXCL13+ CD8-PreTex functioned as a positive regulator of antitumor immunity by expressing the pro-inflammatory cytokines IFNG and TNF, associated with a better survival outcome. Our study reveals the mechanisms involved in hypoxia-induced immunosuppression and suggests that targeting precursor-exhausted CXCL13+CD8+ T cells might provide a pratical immunotherapeutic approach.