Single-cell RNA-sequencing atlas reveals an FABP1-dependent immunosuppressive environment in hepatocellular carcinoma.

BACKGROUND: Single-cell RNA sequencing, also known as scRNA-seq, is a method profiling cell populations on an individual cell basis. It is particularly useful for more deeply understanding cell behavior in a complicated tumor microenvironment. Although several previous studies have examined scRNA-seq for hepatocellular carcinoma (HCC) tissues, no one has tested and analyzed HCC with different stages. METHODS: In this investigation, immune cells isolated from surrounding normal tissues and cancer tissues from 3 II-stage and 4 III-stage HCC cases were subjected to deep scRNA-seq. The analysis included 15 samples. We distinguished developmentally relevant trajectories, unique immune cell subtypes, and enriched pathways regarding differential genes. Western blot and co-immunoprecipitation were performed to demonstrate the interaction between fatty acid binding protein 1 (FABP1) and peroxisome proliferator-activated receptor gamma(PPARG). In vivo experiments were performed in a C57BL/6 mouse model of HCC established via subcutaneous injection. RESULTS: FABP1 was discovered to be overexpressed in tumor-associated macrophages (TAMs) with III-stage HCC tissues compared with II-stage HCC tissues. This finding was fully supported by immunofluorescence detection in significant amounts of HCC human samples. FABP1 deficiency in TAMs inhibited HCC progression in vitro. Mechanistically, FABP1 interacted with PPARG/CD36 in TAMs to increase fatty acid oxidation in HCC. When compared with C57BL/6 mice of the wild type, tumors in FABP1-/- mice consistently showed attenuation. The FABP1-/- group's relative proportion of regulatory T cells and natural killer cells showed a downward trend, while dendritic cells, M1 macrophages, and B cells showed an upward trend, according to the results of mass cytometry. In further clinical translation, we found that orlistat significantly inhibited FABP1 activity, while the combination of anti-programmed cell death 1(PD-1) could synergistically treat HCC progression. Liposomes loaded with orlistat and connected with IR780 probe could further enhance the therapeutic effect of orlistat and visualize drug metabolism in vivo. CONCLUSIONS: ScRNA-seq atlas revealed an FABP1-dependent immunosuppressive environment in HCC. Orlistat significantly inhibited FABP1 activity, while the combination of anti-PD-1 could synergistically treat HCC progression. This study identified new treatment targets and strategies for HCC progression, contributing to patients with advanced HCC from new perspectives.

CHSY1 promotes CD8+ T cell exhaustion through activation of succinate metabolism pathway leading to colorectal cancer liver metastasis based on CRISPR/Cas9 screening.

BACKGROUND: The most common site of metastasis in colorectal cancer (CRC) is the liver and liver metastases occur in more than 50% of patients during diagnosis or treatment. The occurrence of metastasis depends on a series of events known as the invasive-metastasis cascade. Currently, the underlying genes and pathways regulating metastasis initiation in the liver microenvironment are unknown. METHODS: We performed systematic CRISPR/Cas9 screening using an in vivo mouse model of CRC liver metastasis to identify key regulators of CRC metastasis. We present the full results of this screen,which included a list of genes that promote or repress CRC liver colonization. By silencing these genes individually, we found that chondroitin sulfate synthase 1 (CHSY1) may be involved in CRC metastasis. We verified the function of CHSY1 and its involvement in liver metastasis of CRC through in vivo and in vitro experiments. RESULT: The results of TCGA and CRISPR/Cas9 showed that CHSY1 was overexpressed in CRC primary and liver metastasis tissues and indicated a worse clinical prognosis. In vitro and in vivo experiments confirmed that CHSY1 facilitated the liver metastasis of CRC and CHSY1 induced CD8+ T cell exhaustion and upregulated PD-L1 expression. The metabolomic analysis indicated that CHSY1 promoted CD8+ T cell exhaustion by activating the succinate metabolism pathway leading to liver metastasis of CRC. Artemisinin as a CHSY1 inhibitor reduced liver metastasis and enhanced the effect of anti-PD1 in CRC. PLGA-loaded Artemisinin and ICG probe reduced liver metastasis and increased the efficiency of anti-PD1 treatment in CRC. CONCLUSION: CHSY1 could promote CD8+ T cell exhaustion through activation of the succinate metabolic and PI3K/AKT/HIF1A pathway, leading to CRC liver metastasis. The combination of CHSY1 knockdown and anti-PD1 contributes to synergistic resistance to CRC liver metastasis. Artemisinin significantly inhibits CHSY1 activity and in combination with anti-PD1 could synergistically treat CRC liver metastases. This study provides new targets and specific strategies for the treatment of CRC liver metastases, bringing new hope and benefits to patients.

GLIS1 intervention enhances anti-PD1 therapy for hepatocellular carcinoma by targeting SGK1-STAT3-PD1 pathway.

BACKGROUND: GLI-similar 1 (GLIS1) is one of of Krüppel-like zinc finger proteins, which are either stimulators or inhibitors of genetic transcription. Nevertheless, its effects on T cell were elusive. METHODS: In this study, we intend to explore the effects of GLIS1 on modulating the anticancer potency of CD8+ T cells in hepatocellular carcinoma (HCC). The expression of GLIS1 in CD8 peripheral blood mononuclear cell and CD8 tumor-infiltrating lymphocytes of HCC tissues was validated by quantificational real-time-PCR and flow cytometry. The anticancer potency of CD8+ T cells with GLIS1 knock down was confirmed in C57BL/6 mouse model and HCC patient-derived xenograft mice model. GLIS1-/- C57BL/6 mice was applied to explore the effects GLIS1 on tumor immune microenvironment. Chromatin immunoprecipitation and RNA transcriptome sequencing analysis were both performed in GLIS1-knock down of CD8+ T cells. RESULTS: GLIS1 was upregulated in exhausted CD8+ T cells in HCC. GLIS1 downregulation in CD8+ T cells repressed cancer development, elevated the infiltrate ability of CD8+ T cells, mitigated CD8+ T cell exhaustion and ameliorated the anti-PD1 reaction of CD8+ T cells in HCC. The causal link beneath this included transcriptional regulation of SGK1-STAT3-PD1 pathway by GLIS1, thereby maintaining the abundant PD1 expression on the surface of CD8+ T cells. CONCLUSION: Our study revealed that GLIS1 promoted CD8+ T cell exhaustion in HCC through transcriptional regulating SGK1-STAT3-PD1 pathway. Downregulating the expression of GLIS1 in CD8+ T cells exerted an effect with anti-PD1 treatment synergistically, revealing a prospective method for HCC immune therapy.

Idarubicin-loaded biodegradable microspheres enhance sensitivity to anti-PD1 immunotherapy in transcatheter arterial chemoembolization of hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is an image-guided locoregional therapy used for the treatment of patients with primary hepatocellular carcinoma (HCC). However, conventional TACE formulations such as epirubicin-lipiodol emulsion are rapidly dissociated due to the instability of the emulsion, resulting in insufficient local drug concentrations in the target tumor. To overcome these limitations, we used biodegradable Idarubicin loaded microspheres (BILMs), which were prepared from gelatin and carrageenan and could be loaded with Idarubicin (IDA-MS). The morphology and the ability to load and release IDA of BILMs were characterized in vitro. We evaluated tumor changes and side effects after TACE treatment with IDA-MS in VX2 rabbit and C57BL/6 mice HCC models. In addition, the effect of IDA-MS on the tumor immune microenvironment of HCC tumors was elucidated via mass spectrometry and immunohistochemistry. Result showed that IDA-MS was developed as a new TACE formulation to overcome the poor delivery of drugs due to rapid elimination of the anticancer drug into the systemic circulation. We demonstrated in rabbits and mice HCC models that TACE with IDA-MS resulted in significant tumor shrinkage and no more severe adverse events than those observed in the IDA group. TACE with IDA-MS could also significantly enhance the sensitivity of anti-PD1 immunotherapy, improve the expression of CD8+ T cells, and activate the tumor immune microenvironment in HCC. This study provides a new approach for TACE therapy and immunotherapy and illuminates the future of HCC treatment. STATEMENT OF SIGNIFICANCE: Conventional transarterial chemoembolization (TACE) formulations are rapidly dissociated due to the instability of the emulsion, resulting in insufficient local drug concentrations in hepatocellular carcinoma (HCC). To overcome these limitations, we used biodegradable microspheres called BILMs, which could be loaded with Idarubicin (IDA-MS). We demonstrated in rabbits and mice HCC models that TACE with IDA-MS resulted in significant tumor shrinkage and no more severe adverse events than those observed in the IDA group. TACE with IDA-MS could also significantly enhance the sensitivity of anti-PD1 immunotherapy, improve the expression of CD8+ T cells, and activate the tumor immune microenvironment in HCC. This study provides a new approach for TACE therapy and immunotherapy and illuminates the future of HCC treatment.

Tumor Microenvironment and its Implications for Antitumor Immunity in Cholangiocarcinoma: Future Perspectives for Novel Therapies.

The incidence of cholangiocarcinoma (CCA) has been increasing over the past few years. Although there are surgery, chemotherapy and other conventional treatment methods, the effect is not as expected. At present, immunotherapy has become the research frontier of cancer treatment, and CCA tumor microenvironment (TME) is becoming a hot exploration direction of immunobiology. TME can affect tumor progression through changes in metabolism, secretion and immunity. Accordingly, understanding the role played by immune cells and stromal cells in TME is important for the study of CCA immunotherapy. This review will discuss the interactions between immune cells (including CD8+ T cells, CD4+ T cells, macrophages, natural killer cells, dendritic cells, myeloid suppressor cells, mast cells, and neutrophils) and stromal cells (including cancer-associated fibroblasts, endothelial cells) in the TME of CCA. In addition, we will also discuss current research results on TME of CCA and recent advances in immunotherapy.

Inhibition of APOC1 promotes the transformation of M2 into M1 macrophages via the ferroptosis pathway and enhances anti-PD1 immunotherapy in hepatocellular carcinoma based on single-cell RNA sequencing.

Single-cell RNA-sequencing (scRNA-seq) presents better insights into cell behavior in the context of a complex tumor microenvironment by profiling single-cell populations. However, the mechanisms underlying treatment failure in hepatocellular carcinoma (HCC) are poorly understood. In this study, we performed deep scRNA-seq on immune cells under the isolation in peripheral blood, cancer tissues, and nearby common tissues of four HCC cases and two non-cancer controls, and 212,494 cells were included in the analysis. We identified distinct immune cell subtypes, enriched pathways for differential genes, and delineated associated developmentally relevant trajectories. APOC1 was found over-expressed in tumor-associated macrophages (TAMs) of HCC tissues than in normal tissues. Inhibition of APOC1 reversed the M2 phenotype to the M1 phenotype via the ferroptosis pathway in TAMs from HCC. Tumors in APOC1 -/- C57BL/6 mice demonstrated consistent attenuation compared to wild-type (WT) mice. Mass spectrometry results revealed that the relative proportion of M2 macrophages, B cells, and CD4+ T cells in the APOC1 -/- group exhibited a downward expression compared with the WT group, whereas CD8+ T cells, M1 macrophages, and NK cells exhibited an upward trend. Finally, APOC1 was found to be negatively correlated with the expression of PD1/PD-L1 in human HCC samples. In conclusion, the present study demonstrated that inhibiting APOC1 can promote the transformation of M2 macrophages into M1 macrophages via the ferroptosis pathway, thereby reshaping the tumor immune microenvironment and improving the anti-PD1 immunotherapy for HCC, providing a new strategy for improving the therapeutic effect of anti-PD1, and bringing new hope to HCC patients.

Over-Expression of GUSB Leads to Primary Resistance of Anti-PD1 Therapy in Hepatocellular Carcinoma.

Immunotherapy treatments, particularly immune checkpoint blockade, can result in benefits in clinical settings. But many pre-clinical and clinical studies have shown that resistance to anti-PD1 therapy frequently occurs, leading to tumor recurrence and treatment failure, including in patients with hepatocellular carcinoma (HCC). In this study, 10 patients with HCC were remedied with anti-PD1, and pre-treatment biopsy samples were sequenced for 289 nanostring panel RNA to compare responsive and non-responsive tumors to identify possible pretreatment biomarkers or targets of anti-PD1 therapeutic responses. Fortunately, the expression of ß-Glucuronidase (GUSB) in the non-responding tumors was found to be remarkably higher than that in responding tumors. Results of the cell counting kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), transwell, wound healing test, and flow cytometry showed that GUSB facilitated proliferation, invasion, as well as migration of human HCC cells and downregulated PD-L1 expression by promoting miR-513a-5p. Additionally, as a GUSB inhibitor, amoxapine can reduce the progression of human HCC cells, and was an effective treatment for HCC and improved the sensitivity of anti-PD1 therapy. In summary, this study reveals that increased GUSB downregulates PD-L1 expression by promoting miR-513a-5p, leading to primary resistance to anti-PD1 treatment in HCC, and amoxapine enhances the sensitivity of anti-PD1 therapy by inhibiting GUSB, providing a new strategy and method for improving the efficacy of anti-PD1 therapy and bringing new prospects for therapy of HCC.

Comprehensive Analysis of VCAN Expression Profiles and Prognostic Values in HCC.

Background: Hepatocellular carcinoma (HCC) is the world's most common cause of cancer death. Therefore, more molecular mechanisms need to be clarified to meet the urgent need to develop new detection and treatment strategies. Methods: We used TCGAportal, Kaplan-Meier Plotter, the Cistrome DB Toolkit Database, MExpress, GEPIA2, and other databases to discuss the expression profiles, possible biological function, and potential prognostic value of versican (VCAN) in HCC. We conducted cell experiments such as Transwell migration and invasion assays, wound healing assay, and CCK8 experiment to explore the function of VCAN in HCC. Result: We selected three HCC transcriptome databases GSE124535, GSE136247, and GSE144269 and analyzed the overexpressed genes contained in them. The overlapping genes were found by the Venn map, and two interacting network modules were found by Mcode. Module 1 was mainly related to mitosis and cell cycle, and module 2 was mainly related to EMT, angiogenesis, glycolysis, and so on. We found that the seed gene in module 2 is VCAN. Data from TCGAportal showed that compared with normal tissues, the expression of VCAN was up-regulated in HCC tissues. The patients with high expression of VCAN had shorter distant recurrence-free survival and overall survival. Multiple possible VCAN interactions had also been identified. These results revealed that the level of VCAN was higher in the subtypes of HCC with higher malignant degree and was connected to the poor prognosis. In addition, the treatment of VCAN with DNA methyltransferase inhibitors and transcription factor inhibitors may improve the prognosis of patients with HCC. Conclusion: Our findings systematically elucidated the expression profile and different prognostic values of VCAN in HCC, which may provide new therapeutic targets and potential prognostic biomarkers for HCC patients.

A 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images: a prospective randomized controlled trial.

Background: Anesthesia, nerve block, therapeutic injections, and biopsies all require an acupuncture intervention. However, traditional two-dimensional (2D) ultrasound-guided needle puncture is often challenging and therefore requires the use of three-dimensional (3D) ultrasound images to accurately identify and evaluate the patient's anatomical structure. Methods: In this study, a 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images was described. A total of 190 cases requiring puncture were randomly divided into control (conventional 2D ultrasound instrument) and experimental (novel 3D ultrasound imedis9000) groups. The advantages and disadvantages of the two puncture methods were prospectively analyzed in the 190 cases, and the feasibility of electromagnetic navigation real-time positioning was compared to ultrasound imaging. Results: This study included 190 cases from two centers that required puncture treatment and were randomly assigned to the control (conventional 2D ultrasound instrument; n=95) or the experimental (novel 3D ultrasound imedis9000; n=95) groups. Percutaneous vascular puncture, percutaneous biopsy, percutaneous bile duct puncture, thoracic paravertebral nerve block, and sciatic nerve block operations were performed separately. The results indicated that the puncture time and number of trials in the experimental group were significantly lower than those in the control group. No significant difference was identified in the basic vital signs between the two groups before and after surgery. The success rate of the novel 3D ultrasound imedis9000 was 100%, and the success rate of the conventional 2D ultrasound instrument was 95.7%. Furthermore, the results also showed that the novel 3D ultrasound imedis9000 and the matching coaxial positioning channel puncture needle had low pain, good toughness and strength, and great convenience. Conclusions: The new 3D multi-modal intelligent intervention system using electromagnetic navigation real-time positioning and ultrasound images has significant advantages compared with conventional 2D ultrasound in terms of puncture time, number of trials, operation difficulty, and convenience, and is worthy of further promotion and use in clinics. Trial Registration: Beijing Municipal Drug Administration, 20190015.

The Effect of Anlotinib Combined with anti-PD-1 in the Treatment of Gastric Cancer.

Background: Protein tyrosine kinase (PTK) signaling pathway has been confirmed to be involved in the proliferation, differentiation and migration of tumor cells. Anlotinib, as a multi-target tyrosine kinase inhibitor, which can inhibit the expression of vascular endothelial growth factor receptor (VEGFR), has been confirmed to have significant therapeutic effects on non-small cell lung cancer, medullary thyroid carcinoma, and soft tissue sarcoma, but the therapeutic effect on gastric cancer (GC) is still unclear. Methods: Anlotinib was screened out of 880 drugs through Cell Counting Kit 8 (CCK-8) technology. TCGA was used to detect the expression of VEGFR in GC, and Kaplan-Meier Plotter was used to analyze the correlation between the expression of VEGFR and the survival rate of GC patients. The impacts exerted by anlotinib to GC cell proliferating, migrating and invading processes were assessed through wound healing assay, transwell assay, and proliferation assay in vitro. In vivo experiments of GC were performed in C57/B6 mouse model to evaluate the function of anlotinib and PD-1 antibody. Results: It was found from more than compunds that anlotinib has a significant inhibitory effect on GC cells. In vitro experiments show that anlotinib can significantly inhibit the proliferation, invasion and proliferation of GC cells. The expression level of VEGFR is related to the prognosis and survival of GC. GC patients with low expression of VEGFR have better survival. Anlotinib can inhibit the expression of PD-L1, and achieve better therapeutic effects after combined with PD-1 antibody. Conclusion: The present study reveals that anlotinib down regulates PD-L1. The combination of anlotinib and PD-1 monoclonal antibody is beneficial to GC therapy.

Treatment of patients with cancer using PD­1/PD­L1 antibodies: Adverse effects and management strategies (Review).

In 2020, there were an estimated 19.3 million new cancer cases and close to 10 million cancer deaths worldwide. Cancer remains one of the leading causes of death. In recent years, with the continuous improvement of our understanding of tumor immunotherapy, immunotherapeutics, such as immune checkpoint inhibitors, have gradually become a hot spot for tumor treatment. Amongst these, programmed cell death protein 1/programmed cell death protein ligand 1 (PD­1/PD­L1) related inhibitors, such as nivolumab and pembrolizumab, atezolizumab, avelumab and durvalumab have been shown to exhibit a high level of efficacy in several types of tumors. It has been confirmed that these inhibitors play an important role in the anti­tumor process, significantly improving the survival rate of patients and delaying the progress of the underlying cancer. However, its method of therapeutic interference and potential for damaging the immune system has caused concern regarding its suitability. As these adverse effects are caused by an immune response to endogenous tissues, they are designated as immune­related adverse events (irAEs). In this review, the typical irAEs reported in recent years and the management strategies adopted are highlighted, to serve as a reference in assessing the clinical response to these adverse reactions.

Inhibition of PARP Potentiates Immune Checkpoint Therapy through miR-513/PD-L1 Pathway in Hepatocellular Carcinoma.

Background: The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is involved in DNA damage repair and cell death. However, the association between PARP's biological activities and the immune microenvironment in hepatocellular carcinoma (HCC) is unclear. The present study will explore whether combining a PARP inhibitor with anti-PD1 might improve the anti-HCC impact and explain how it works. Method: The PARP inhibitor olaparib was screened out of 867 drugs through Cell Counting Kit 8 (CCK-8) assay. The expression of PARP was verified through the TCGA and TISIDB databases. The impacts exerted by PARP inhibitor olaparib to HCC cells were assessed via wound healing, Transwell, and proliferation assay. In vivo, experiments were performed in a C57BL/6 mouse model to evaluate the function of PARP inhibitor olaparib combination with anti-PD1 in HCC and mice tumors were further detected by immunohistochemically staining. Result: Olaparib was selected as the research object on the basis of drug screening. The results of the TCGA and Human Protein Atlas databases revealed that PARP was significantly upregulated in carcinoma cell cluster of HCC tissues compared to normal tissues. Higher expression of PARP showed a poorer prognosis based on Kaplan-Meier Plotter. qRT-PCR experiments confirmed that olaparib could increase PD-L1 expression through inhibiting miR-513 in HCC cells. In vivo, experiment confirmed that the combination of olaparib and anti-PD1 could enhance the immunotherapy effect of HCC. Conclusion: The present study reveals that inhibition of PARP potentiates immune checkpoint therapy through the miR-513/PD-L1 pathway in HCC and the combination of PARP inhibitor olaparib and anti-PD1 is beneficial to HCC therapy.

MGP promotes CD8+ T cell exhaustion by activating the NF-κB pathway leading to liver metastasis of colorectal cancer.

Matrix Gla protein (MGP) was originally reported as a physiological suppressor of ectopia calcification and has also been reported to be associated with cancer. However, the relation between the biological functions of MGP and the immune response in colorectal cancer (CRC) remains unclear. Here, we investigated the regulatory role of MGP in the immune microenvironment of CRC. MGP expression in CRC samples was assessed by single-cell RNA sequencing and the Gene Expression Omnibus (GEO) database, and confirmed by quantitative real-time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry analysis of human CRC samples. The effect of MGP on proliferation and invasion of CRC cells was evaluated by in vitro assays involving MGP knockdown and overexpression. Luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay were performed to identify transcriptional regulatory sites of the nuclear factor kappa-B (NF-κB) and programmed cell death ligand 1 (PD-L1). In vivo experiments were performed in mouse model of CRC liver metastasis established via spleen injection. The results revealed that MGP was significantly upregulated in cancer cell clusters from the primary CRC or liver metastases, compared with that in the corresponding paracancerous tissues via single-cell RNA sequencing. MGP enriched intracellular free Ca2+ levels and promoted NF-κB phosphorylation, thereby activated PD-L1 expression to promote CD8+ T cell exhaustion in CRC. The luciferase reporter assay and ChIP-qPCR assay indicated that the transcriptional regulation of NF-κB upregulated PD-L1 expression. In vivo, MGP inhibition significantly decreased the rate of CRC liver metastasis, which was further reduced after combined therapy with αPD1 (anti-PD1). In conclusions, this study revealed that MGP can facilitate CD8+ T cell exhaustion by activating the NF-κB pathway, leading to liver metastasis of CRC. The combination of MGP knockdown and αPD1 can synergistically resist liver metastasis of CRC.

Potential molecular mechanisms and clinical progress in liver metastasis of breast cancer.

Breast cancer is the most common malignant tumor in women and the leading cause of cancer death in women. About 30% of breast cancer patients have metastasis every year, which greatly increases the mortality rate of breast cancer. The main target organs for metastasis are bone, brain, liver and lung. The breast cancer liver metastasis (BCLM) mechanism is not fully clarified. This is a complex process involving multiple factors, which is not only related to the microenvironment of the primary tumor and liver, but also regulated by a variety of signaling pathways. Clarifying these mechanisms is of great help to guide clinical treatment. With the in-depth study of BCLM, a variety of new treatment schemes such as targeted therapy and endocrine therapy provide new ideas for the cure of BCLM. In this review, we will summarize the molecular mechanism and treatment of BCLM.

Tegaserod Maleate Inhibits Breast Cancer Progression and Enhances the Sensitivity of Immunotherapy.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide. The challenge in managing this heterogeneous malignancy is that BC is highly aggressive and is always associated with chemical resistance, radiation resistance, hormone therapy resistance, and targeted therapy resistance. Therefore, there is an urgent need to find effective drugs to treat BC. METHODS: Based on the Selleck drug library approved by FDA, we screened 800 drugs for anti-BC cells and found that tegaserod maleate (TM), a 5-hydroxytryptamine 4-receptor (HTR4) partial agonist had the best anti-BC effect, which was further verified. The effects of different concentrations of TM on cell proliferation, invasion, and migration were evaluated in vitro using CCK8, plate cloning, transwell, and scratch assays. The UALCAN database, Kaplan-Meier Plotter database, Human Protein Atlas, and GEPIA2 were used to explore the correlation between HTR4 expression and BC patients' clinicopathological data as well as immune response. In vivo experiments demonstrated the effect of the TM and immunotherapy drug (anti-PD1/anti-TIGIT) combination on BC tumor growth in mice. RESULTS: TM significantly inhibited the proliferation, invasion, and migration of BC cells, and the higher the concentration, the better the inhibition effect. HTR4 was significantly downregulated in BC tissues compared to paracancerous tissues. The downregulation of HTR4 was correlated with clinicopathological data and positively correlated with BC prognosis. Interestingly, the GEPIA2 database suggested that there was a strong positive correlation between the expression of HTR4 and effector T cells, effector memory T cells, and exhausted T cells. In vitro experiments showed that TM, anti-PD1, and anti-TIGIT could all inhibit the growth and weight of BC tumors as compared with the control group. However, when anti-PD1 or anti-TIGIT was used simultaneously with TM, the inhibition of tumors significantly exceeded that in the control group. Moreover, the combination of anti-TIGIT and TM has the best inhibitory effect. CONCLUSION: TM inhibited the progression of breast cancer, and its combination with anti-TIGIT could effectively inhibit tumor growth and improve the sensitivity of immunotherapy in breast cancer.

Macrophage GSK3ß-deficiency inhibits the progression of hepatocellular carcinoma and enhances the sensitivity of anti-PD1 immunotherapy.

BACKGROUND: Glycogen synthase kinase 3ß (GSK3ß) was originally discovered to regulate glycogen synthesis and show a relationship to tumors. However, the biological functions of GSK3ß in tumor-associated macrophages (TAMs) in cancers including hepatocellular carcinoma (HCC) remain unclear. METHODS: The enrichment of GSK3ß in tumor tissues was assessed by Gene Expression Omnibus (GEO) database. The in vitro and in vivo assays assisted in evaluating how GSK3ß in TAMs affected HCC in terms of proliferation, invasion and migration. Immunofluorescence was used to assess GSK3ß expression in TAMs in the anti-PD1 therapy non-responsive HCC group and the responsive group. Western blot and coimmunoprecipitation were performed to demonstrate the interaction between GSK3ß and PD-L1. We carried out in vivo experiments in a C57BL/6 mouse model of HCC established through subcutaneous injection. RESULTS: GEO single-cell RNA sequencing data suggested that GSK3ß was highly enriched in TAMs of HCC. According to in vitro and in vivo experiments, reducing GSK3ß in TAMs inhibits the cancer cell proliferation, invasion, and migration. The immunofluorescence and immunohistochemistry results confirmed that the GSK3ß is significantly upregulated in TAMs of the anti-PD1 therapy non-responsive group in comparison with the responsive group. In vitro and in vivo experiments confirmed that reduced GSK3ß in TAMs are capable of enhancing the sensitivity of anti-PD1 immunotherapy for HCC by decreasing PD-L1 ubiquitination. Mass spectrometry results suggested that high expression of CD14+GSK3ß+ in the peripheral blood mononuclear cell (PBMC) can predict non-responsive to anti-PD1 treatment. Moreover, escitalopram is confirmed to act as GSK3ß inhibitor that can increase the sensitivity of anti-PD1 immunotherapy for HCC. CONCLUSIONS: This study revealed that macrophage GSK3ß deficiency can inhibit the development of HCC by inhibiting the M2 phenotype and enhance the sensitivity of anti-PD1 immunotherapy for HCC by decreasing PD-L1 ubiquitination. The expression of CD14+GSK3ß+ in PBMC can noninvasively predict anti-PD1 sensitivity in HCC patients, which provides novel strategies to predict anti-PD1 sensitivity, increase anti-PD1 therapeutic effect, and bring new hope for HCC patients.

Various Uses of PD1/PD-L1 Inhibitor in Oncology: Opportunities and Challenges.

The occurrence and development of cancer are closely related to the immune escape of tumor cells and immune tolerance. Unlike previous surgical, chemotherapy, radiotherapy and targeted therapy, tumor immunotherapy is a therapeutic strategy that uses various means to stimulate and enhance the immune function of the body, and ultimately achieves the goal of controlling tumor cells.With the in-depth understanding of tumor immune escape mechanism and tumor microenvironment, and the in-depth study of tumor immunotherapy, immune checkpoint inhibitors represented by Programmed Death 1/Programmed cell Death-Ligand 1(PD-1/PD-L1) inhibitors are becoming increasingly significant in cancer medication treatment. employ a variety of ways to avoid detection by the immune system, a single strategy is not more effective in overcoming tumor immune evasion and metastasis. Combining different immune agents or other drugs can effectively address situations where immunotherapy is not efficacious, thereby increasing the chances of success and alternative access to alternative immunotherapy. Immune combination therapies for cancer have become a hot topic in cancer treatment today. In this paper, several combination therapeutic modalities of PD1/PD-L1 inhibitors are systematically reviewed. Finally, an analysis and outlook are provided in the context of the recent advances in combination therapy with PD1/PD-L1 inhibitors and the pressing issues in this field.

Targeting Immune Cells in the Tumor Microenvironment of HCC: New Opportunities and Challenges.

Immune associated cells in the microenvironment have a significant impact on the development and progression of hepatocellular carcinoma (HCC) and have received more and more attention. Different types of immune-associated cells play different roles, including promoting/inhibiting HCC and several different types that are controversial. It is well known that immune escape of HCC has become a difficult problem in tumor therapy. Therefore, in recent years, a large number of studies have focused on the immune microenvironment of HCC, explored many mechanisms worth identifying tumor immunosuppression, and developed a variety of immunotherapy methods as targets, laying the foundation for the final victory in the fight against HCC. This paper reviews recent studies on the immune microenvironment of HCC that are more reliable and important, and provides a more comprehensive view of the investigation of the immune microenvironment of HCC and the development of more immunotherapeutic approaches based on the relevant summaries of different immune cells.

STK39 enhances the progression of Cholangiocarcinoma via PI3K/AKT pathway.

Serine/threonine kinase 39 (STK39) is overexpressed in various tumor tissues and plays an essential role in tumor progression. In this study, we investigated the clinical value, as well as the potential functions and mechanisms of STK39 in cholangiocarcinoma (CCA). The results showed that STK39 was overexpressed in CCA and negatively associated with the prognosis of patients with CCA. Functionally, STK39 knockdown suppressed cell proliferation, migration, and invasion, while STK39 overexpression facilitated tumor aggressiveness. The tumor-promoting effects of STK39 in CCA were also validated by in vivo experiments. Mechanistically, RNA-seq analysis identified that STK39 enhanced the progression of CCA by activating PI3K/AKT signaling pathway. Furthermore, overexpression of STK39 could induce gemcitabine resistance in CCA cells. Moreover, the increased expression of STK39 may be mediated by the dysregulation of miR-26a-5p. In summary, STK39 could be served as a valuable prognostic candidate and a potential therapeutic target of CCA.