Lipid droplet accumulation mediates macrophage survival and Treg recruitment via the CCL20/CCR6 axis in human hepatocellular carcinoma.

Metabolic changes play a crucial role in determining the status and function of macrophages, but how lipid reprogramming in macrophages contributes to tumor progression is not yet fully understood. Here, we investigated the phenotype, contribution, and regulatory mechanisms of lipid droplet (LD)-laden macrophages (LLMs) in hepatocellular carcinoma (HCC). Enriched LLMs were found in tumor tissues and were associated with disease progression in HCC patients. The LLMs displayed immunosuppressive phenotypes (with extensive expression of TREM2, PD-L1, CD206, and CD163) and attenuated the antitumor activities of CD8+ T cells. Mechanistically, tumor-induced reshuffling of cellular lipids and TNFα-mediated uptake of tumoral fatty acids contribute to the generation of triglycerides and LDs in macrophages. LDs prolong LLM survival and promote CCL20 secretion, which further recruits CCR6+ Tregs to HCC tissue. Inhibiting LLM formation by targeting DGAT1 and DGAT2, which catalyze the synthesis of triglycerides, significantly reduced Treg recruitment, and delayed tumor growth in a mouse hepatic tumor model. Our results reveal the suppressive phenotypes and mechanisms of LLM enrichment in HCC and suggest the therapeutic potential of targeting LLMs for HCC patients.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma.

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1ß-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Corrigendum: C-reactive protein is an indicator of the immunosuppressive microenvironment fostered by myeloid cells in hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2021.774823.].

Assessing the impact of the 2018 tetanus guidelines on knowledge and practices of emergency physicians in trauma patients: a national survey study.

Background: Tetanus remains a significant public health issue in China, with the approach of anti-tetanus prophylaxis in the emergency department resulting in both overuse, particularly of human tetanus immune globulin (TIG), and underuse with the tetanus vaccine. This is largely due to the absence of updated guidelines on tetanus prophylaxis before 2018. Our study aimed to evaluate the effects of the 2018 Chinese tetanus guidelines on the knowledge and practices of emergency physicians about tetanus prevention in trauma patients. Methods: From November 2019 to April 2020, we conducted a web-based survey involving 499 emergency physicians. The survey included a questionnaire covering knowledge, attitudes, and practices related to tetanus. We assessed the influence of the 2018 tetanus guidelines on the knowledge and practices of emergency physicians related to tetanus prevention for patients with trauma using multiple regression analysis. Results: The survey results showed that only 45.3% of the participants had received formal training on tetanus immunization, despite 53.3% reporting the availability of tetanus vaccines at their institutions. Physicians typically prescribed tetanus antitoxin or human TIG instead of tetanus toxoid (TT) to treat injuries, regardless of the patient's TT vaccination history. Among the respondents, those who were aware of the 2018 tetanus guidelines had higher mean scores on the general knowledge, risk knowledge, and treatment knowledge scales, with increases of 6%, 13%, and 9%, respectively, compared to those who were unaware of the guidelines. Awareness of the 2018 tetanus guidelines was associated with a high level of knowledge, as indicated by the general knowledge score, recommendation knowledge score, and total knowledge score, after adjusting for the effects of all variables on the knowledge, attitudes, and practices of the participants. A high level of education was also associated with a high level of knowledge indicated by the recommendation knowledge score and total knowledge score. Conclusions: Our study highlights a substantial gap in the attitudes, knowledge, and practices of emergency physicians in China regarding tetanus immunization. The results suggest an urgent need to promote the Chinese Expert Consensus Guidelines on tetanus to improve emergency physicians' knowledge and competence in tetanus prophylaxis. The findings underscore the importance of enhancing physicians' awareness of the latest guidelines to ensure appropriate and effective treatment for patients with tetanus-prone injuries.

Cholesterol Efflux Drives the Generation of Immunosuppressive Macrophages to Promote the Progression of Human Hepatocellular Carcinoma.

Cholesterol is often enriched in tumor microenvironment (TME); however, its impact on disease progression varies in different tissues and cells. Monocytes/macrophages (Mφ) are major components and regulators of the TME and play pivotal roles in tumor progression and therapeutic responses. We aimed to investigate the profile, effects, and regulatory mechanisms of Mφ cholesterol metabolism in the context of human hepatocellular carcinoma (HCC). Here, we found that patients with high serum levels of cholesterol had shorter survival times and lower response rates to anti-PD-1 treatment. However, the cholesterol content in tumor-infiltrating monocytes/Mφ was significantly lower than that in their counterparts in paired nontumor tissues. The expression of the cholesterol efflux transporter, ABCA1, was upregulated in tumor monocytes/Mφ, and ABCA1 upregulation positively associated with decreased cellular cholesterol content and increased serum cholesterol levels. Mechanistically, autocrine cytokines from tumor-treated monocytes increased LXRα and ABCA1 expression, which led to the generation of immature and immunosuppressive Mφ. Although exogenous cholesterol alone had little direct effect on Mφ, it did act synergistically with tumor-derived factors to promote ABCA1 expression in Mφ with more immunosuppressive features. Moreover, high numbers of ABCA1+ Mφ in HCC tumors associated with reduced CD8+ T-cell infiltration and predicted poor clinical outcome for patients. Our results revealed that dysregulated cholesterol homeostasis, due to the collaborative effects of tumors and exogenous cholesterol, drives the generation of immunosuppressive Mφ. The selective modulation of cholesterol metabolism in Mφ may represent a novel strategy for cancer treatment.

Methyltransferase-like 3 promotes cervical cancer metastasis by enhancing cathepsin L mRNA stability in an N6-methyladenosine-dependent manner.

N6-methyladenosine (m6A) is a highly abundant RNA modification in eukaryotic cells. Methyltransferase-like 3 (METTL3), a major protein in the m6A methyltransferase complex, plays important roles in many malignancies, but its role in cervical cancer metastasis remains uncertain. Here, we found that METTL3 was significantly upregulated in cervical cancer tissue, and its upregulation was associated with a poor prognosis in cervical cancer patients. Knockdown of METTL3 significantly reduced cervical cancer cell migration and invasion. Conversely, METTL3 overexpression markedly promoted cervical cancer cell metastasis in vitro and in vivo. Furthermore, METTL3 mediated the m6A modification of cathepsin L (CTSL) mRNA at the 5'-UTR, and the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) bound to the m6A sites and enhanced CTSL mRNA stability. Our results indicated that METTL3 enhanced CTSL mRNA stability through an m6A-IGF2BP2-dependent mechanism, thereby promoting cervical cancer cell metastasis. These findings provide insights into a novel m6A modification pattern involved in cervical cancer development.

HHLA2 Expression is Associated with Poor Survival in Patients with Hepatocellular Carcinoma.

BACKGROUND: Human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) is a member of the B7 family; however, little is known regarding its expression and clinical relevance in hepatocellular carcinoma (HCC). METHODS: To better characterize HHLA2 expression in HCC, we analyzed its expression by in situ staining and further investigated its correlation with immune infiltration and patient prognosis. RESULTS: HHLA2 was primarily expressed in the peri-tumor region of HCC tissues and co-localized with CD68+ monocytes/macrophages. In vitro analysis and multi-immunofluorescence staining showed up-regulated HHLA2 expression in tumor-activated monocytes/macrophages, and HHLA2+ monocytes/macrophages expressed high levels of HLA-DR in HCC tissue. A correlation analysis showed that samples displaying high HHLA2 expression in the peri-tumor region had significant tumor infiltration of CD204+ and CD11b+ cells, and low expression of genes associated with an anti-tumor immune response. The high level of peri-tumoral HHLA2 expression was associated with a poor patient overall survival (OS; P = 0.008). A multivariate analysis revealed that HHLA2 expression in the peri-tumor region was an independent prognostic factor for OS (hazard ratio = 1.872, p = 0.003). Moreover, the expression of HHLA2 was negatively correlated with PD-L1, and patients exhibiting HHLA2 and programmed cell death-ligand 1(PD-L1) co-expression had the shortest survival time. CONCLUSION: HHLA2 expression represented an immunosuppressive microenvironment in HCC, and may serve as a potential target for immunotherapy.

Reprogramming immunosuppressive myeloid cells by activated T cells promotes the response to anti-PD-1 therapy in colorectal cancer.

Overcoming local immunosuppression is critical for immunotherapy to produce robust anti-tumor responses. Myeloid-derived suppressor cells (MDSCs) are key regulators of immunosuppressive networks and promote tumor progression. However, it remains unclear whether and how tumor-infiltrating MDSCs are shaped in response to anti-PD-1 treatment and what their impact on therapeutic efficacy is in colorectal cancer (CRC). In this study, the levels of infiltrating MDSCs were significantly higher in the non-responding organoids and were selectively reduced in the responding group, with MDSCs showing increased apoptosis and attenuated functional activity after anti-PD-1 treatment. A negative correlation between T-cell activation and MDSC function was also observed in fresh human CRC tissues. Mechanistic studies revealed that autocrine IFN-α/ß upregulated TRAIL expression on activated T cells to elicit MDSC apoptosis via the TRAIL-DR5 interaction and acted synergistically with TNF-α to inhibit MDSC function of suppressing the T-cell response through the JNK-NMDAR-ARG-1 pathway. Moreover, blockade of IFN-α/ß and TNF-α abolished the therapeutic efficacy of anti-PD-1 treatment by preserving the frequency and suppressive activity of infiltrating MDSCs in a CRC mouse model. This result suggested that reprogramming MDSCs by IFN-α/ß and TNF-α from activated T cells was necessary for successful anti-PD-1 treatment and might serve as a novel strategy to improve the response and efficacy of anticancer therapy.

Retinoic Acid Synthesis Deficiency Fosters the Generation of Polymorphonuclear Myeloid-Derived Suppressor Cells in Colorectal Cancer.

Metabolism is reprogrammed in cancer to fulfill the demands of malignant cells for cancer initiation and progression. Apart from its effects within cancer cells, little is known about whether and how reprogramed metabolism regulates the surrounding tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSC) are key regulators of the TME and greatly affect tumor progression and therapeutic responses. In this study, our results revealed that retinol metabolism-related genes and enzymes were significantly downregulated in human colorectal cancer compared with adjacent colonic tissues, and tumors exhibited a defect in retinoic acid (RA) synthesis. Reduced ADH1-mediated retinol metabolism was associated with attenuated RA signaling and accumulated MDSCs in colorectal cancer tumors. Using an in vitro model, generating MDSCs from CD34+ myeloid precursors, we found that exogenous RA could abrogate the generation of polymorphonuclear MDSCs (PMN-MDSC) with negligible impact on myeloid differentiation. Mechanistically, RA could restrain the glycolytic capacity of myeloid cells, which in turn activated the AMP-activated protein kinase (AMPK) pathway, further impairing the suppressive capacity of myeloid cells. Supplementation with RA could significantly delay tumor growth, with reduced arginase-1-expressing myeloid cells and increased CD8+ and granzyme B+ T cells in both colitis-associated and implanted MC38 mouse colorectal cancer models. Our results indicated that the defect in ADH1-mediated RA synthesis could provide a possible mechanism that fosters the generation of PMN-MDSCs in colorectal cancer and that restoring RA signaling in the TME could serve as a promising therapeutic strategy to abrogate the generation of PMN-MDSCs.

C-Reactive Protein Is an Indicator of the Immunosuppressive Microenvironment Fostered by Myeloid Cells in Hepatocellular Carcinoma.

BACKGROUND: C-reactive protein (CRP) is a widely used marker of systemic inflammation and predicts poor clinical outcomes in patients with hepatocellular carcinoma (HCC); however, its significance in the local immune response at the tumor site is not clear. METHODS: Serum CRP levels of 329 HCC patients were detected before resection. Paired paraffin-embedded tumor samples were used to quantify immune cell populations, such as CD11b+ myeloid cells, CD68+ macrophages (Mφs), CD15+ neutrophils, CD8+ T cells, and CD206+, CD204+, CD163+ and CD169+ Mφs, by immunohistochemistry. Enrichment scores for 34 types of immune cells based on transcriptome data from 24 HCC samples were calculated by xCell. Overall survival of patients was analyzed using the Kaplan-Meier method. RESULTS: Serum CRP levels were correlated with liver functions and tumor stages in patients with HCC. The densities of CD68+ tumor-associated macrophages (TAMs) and CD15+ tumor-associated neutrophils (TANs) were significantly higher in patients with elevated serum CRP levels than in those with low CRP levels (both p < 0.0001). Further analysis of TAM subtypes revealed that serum CRP levels were associated with CD204+ and CD163+ Mφ densities (p < 0.0001 and p = 0.0003, respectively). Moreover, transcriptome data showed that CRP expression was associated with the expression of myeloid cell infiltration-related genes in HCC tumors. The combination of serum CRP with TAMs or TANs in both the nontumor and intratumor regions could represent a powerful criterion for predicting patient prognoses. CONCLUSION: Serum CRP could serve as an indicator of an immunosuppressive TME in HCC, which could be of potential clinical application for treatment strategies targeting the TME.

Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma.

BACKGROUND & AIMS: Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). METHODS: Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. RESULTS: Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. CONCLUSIONS: Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. LAY SUMMARY: Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.

Glutamine Deprivation Promotes the Generation and Mobilization of MDSCs by Enhancing Expression of G-CSF and GM-CSF.

Solid tumors are often challenged by hypoxic and nutrient-deprived tumor microenvironments (TME) as tumors progress, due to limited perfusion and rapid nutrient consumption. While cancer cells can demonstrate the ability to survive in nutrient-deprived conditions through multiple intrinsic alterations, it is poorly understood how nutrient-deprived cancer cells co-opt the TME to promote cancer cell survival and tumor progression. In the present study, we found that glutamine deprivation markedly potentiated the expression of G-CSF and GM-CSF in mouse mammary cancer cells. The IRE1α-JNK pathway, which is activated by glutamine starvation, was found to be important for the upregulation of these cytokines. G-CSF and GM-CSF are well-known facilitators of myelopoiesis and mobilization of hematopoietic progenitor cells (HPC). Consistently, as tumors progressed, we found that several myeloid HPC compartments were gradually decreased in the bone marrow but were significantly increased in the spleen. Mechanistically, the HPC-maintaining capacity of the bone marrow was significantly impaired in tumor-bearing mice, with lower expression of HPC maintaining genes (i.e., CXCL12, SCF, ANGPT1, and VCAM1), and reduced levels of mesenchymal stem cells and CXCL12-producing cells. Furthermore, the mobilized HPCs that displayed the capacity for myelopoiesis were also found to accumulate in tumor tissue. Tumor-infiltrating HPCs were highly proliferative and served as important sources of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the TME. Our work has identified an important role for glutamine starvation in regulating the expression of G-CSF and GM-CSF, and in facilitating the generation of immunosuppressive MDSCs in breast cancer.

EZH2 negatively regulates PD-L1 expression in hepatocellular carcinoma.

BACKGROUND: Accumulating studies suggest that targeting epigenetic modifications could improve the efficacy of tumor immunotherapy; however, the mechanisms underlying this phenomenon remain largely unknown. Here, we investigated the ability of the epigenetic modifier, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), to regulate the expression of immune checkpoint inhibitor, programmed death-1 ligand 1 (PD-L1) in hepatocellular carcinoma (HCC). METHODS: Immunohistochemistry and multiplex immunofluorescence staining were performed to analyze the expression and correlation of EZH2 and PD-L1 in HCC tissues. Immunoblotting, quantitative real-time PCR, flow cytometry, chromatin immunoprecipitation, and dual-luciferase reporter gene assays were performed to evaluate the regulatory roles of EZH2 on PD-L1 expression. RESULTS: In vitro cell experiments revealed that EZH2 negatively regulated the PD-L1 expression of hepatoma cell lines in IFNγ-dependent manner. Mechanistic studies demonstrated that EZH2 could suppress PD-L1 expression by upregulating the H3K27me3 levels on the promoters of CD274 (encoding PD-L1) and interferon regulatory factor 1 (IRF1), an essential transcription factor for PD-L1 expression, without affecting the activation of the IFNγ-signal transducer and activator of transcription 1 (STAT1) pathway. Clinical samples from HCC patients with immune-activated microenvironments showed negative correlations between EZH2 and PD-L1 expression in hepatoma cells. Multivariate Cox analysis demonstrated that the combination of EZH2 and PD-L1 was an independent prognostic factor for both OS and RFS for patients with HCC. CONCLUSIONS: The epigenetic modificator EZH2 can suppress the expression of immune checkpoint inhibitor PD-L1 by directly upregulating the promoter H3K27me3 levels of CD274 and IRF1 in hepatoma cells, and might serve as a potential therapeutic target for combination of immunotherapy for immune-activated HCC.

Macrophages induce CD47 upregulation via IL-6 and correlate with poor survival in hepatocellular carcinoma patients.

CD47 is known to be involved in phagocyte-mediated tumor clearance; however, its expression, clinical significance, and regulatory mechanism in hepatocellular carcinoma (HCC) remain poorly understood. In the present study, we found that upregulation of CD47 expression on tumor cells was correlated with poor overall survival and recurrence-free survival in patients with HCC. Abundance of macrophages (Mϕs) infiltration was found in CD47+ tumor tissues. Mechanistic studies revealed that IL-6 derived from tumor-infiltrating Mϕs could upregulate CD47 expression on hepatoma cells through activation of the STAT3 pathway. Neutralization of CD47 or disruption of the IL-6-STAT3 axis reduced the ability of tumor cells to escape phagocytosis. Moreover, CD47 blockade could enhance Mϕ-mediated phagocytosis in the presence of chemotherapeutic drugs, and HCC patients with lower CD47 expression were more likely to benefit from adjuvant transcatheter arterial chemoembolization (TACE) treatment. These findings revealed that Mϕ-derived IL-6 was responsible for CD47 expression on hepatoma cells, which might be served as a potential prognostic marker and a predictor for patients who might benefit from adjuvant TACE treatment.

CD103+ tumor-infiltrating lymphocytes predict favorable prognosis in patients with esophageal squamous cell carcinoma.

As an indispensable factor in preventing the recirculation of tissue lymphocytes to the lymphatic and blood systems, the integrin CD103 has enabled the characterization of lymphocyte populations in non-lymphoid tissues and organs. However, the expression, distribution, and clinical significance of CD103+ tumor-infiltrating lymphocytes (TILs) in esophageal squamous cell carcinoma (ESCC) remain unclear. In the present study, we included tumor and adjacent non-tumor tissue specimens from 198 patients with ESCC who had undergone surgical resection. Immunohistochemistry and immunofluorescence were used to detect CD103+ TIL distribution, as well as the co-expression of CD103 and T cell markers and functional molecules. Kaplan-Meier analysis and the Cox proportional hazards model were used to estimate the prognostic value of CD103+ TILs. The results showed that CD103+ TILs were predominantly located in adjacent non-tumor tissues compared with tumor tissues (P < 0.0001). Immunofluorescence double staining revealed that CD8+ T cells, but not CD4+ T cells, comprised the majority of CD103-expressing cells. Most of these CD103-expressing cells co-expressed CTLA-4 and granzyme B rather than the exhaustion marker PD-1. High density of intratumoral CD103+ TIL is associated with longer overall survival (OS) and disease-free survival (DFS) in both the internal (OS, P = 0.0004 and DFS, P = 0.0002) and external (OS, P = 0.038 and DFS, P = 0.12) cohorts. Multivariate Cox analysis showed the density of CD103+ TILs was an independent positive prognostic factor for OS (hazards ratio [HR] = 0.406; P = 0.0003 in the internal cohort; HR = 0.328, P = 0.01, in the external cohort) and DFS (HR = 0.385; P = 0.0002 in the internal cohort; HR = 0.270, P = 0.003, in the external cohort). Our findings indicate that CD103+ TILs might play an important role in the tumor microenvironment, and intratumoral CD103+ TILs could serve as a promising prognostic marker in ESCC.

Immunosuppressive Immature Myeloid Cell Generation Is Controlled by Glutamine Metabolism in Human Cancer.

Tumor-associated myeloid cells are one of the prominent components of solid tumors, serving as major immune regulators for the tumor microenvironment (TME) and an obstacle for immune-checkpoint blocking (ICB) therapy. However, it remains unclear how metabolic processes regulate the generation of suppressive myeloid cells in the TME. Here, we found that hematopoietic precursor cells are enriched in the tissues of several types of human cancer and can differentiate into immature myeloid cells (IMC). Tumor-infiltrating IMCs are highly immunosuppressive, glycolytic, and proliferative, as indicated by high levels of M-CSFR, Glut1, and Ki67. To elucidate the role of metabolism in regulating the generation of IMCs, we induced suppressive IMCs from hematopoietic precursor cells with GM-CSF and G-CSF in vitro We found that the generation of suppressive IMCs was accompanied by increased glycolysis, but not affected by glucose deprivation due to alternative catabolism. Generation of IMCs relied on glutaminolysis, regardless of glucose availability. Glutamine metabolism not only supported the expansion of IMCs with glutamine-derived α-ketoglutarate but also regulated the suppressive capacity through the glutamate-NMDA receptor axis. Moreover, inhibition of glutaminase GLS1 enhanced the therapeutic efficacy of anti-PD-L1 treatment, with reduced arginase 1+ myeloid cells, increased CD8+, IFNγ+ and granzyme B+ T cells, and delayed tumor growth in an ICB-resistant mouse model. Our work identified a novel regulatory mechanism of glutamine metabolism in controlling the generation of suppressive IMCs in the TME.

Combined prognostic value of the cancer stem cell markers CD47 and CD133 in esophageal squamous cell carcinoma.

BACKGROUND: Treatments based on the inhibition of pivotal signals of cancer stem cells (CSCs) are on a promising track. Recent studies have shown that targeting CSCs with broader immune-based therapeutic methods, for example, the anti-CD47 treatment, may serve as a more potent strategy for eliminating these intractable cells. We aimed to explore the prognostic effects of CD47/CD133 and the potential therapeutic significance of CD47 in esophageal squamous cell carcinoma (ESCC). METHODS: Immunohistochemistry was employed to identify the characteristics of CD47 and CD133 in 26 pairs of tumor tissues and adjacent non-tumor tissues and 136 ESCC tissues. Kaplan-Meier analysis and Cox proportional hazards models were built for estimating the prognostic values of CD47 and CD133 expression and their combined stemness index. Sphere formation assays were undertaken to explore the effects of CD47 inhibition on primary human ESCC CSCs. RESULTS: Results conclude that CD47 and CD133 expression is increased in tumor tissues as compared to adjacent non-tumor tissues. A positive correlation between CD47/CD133 expression and differentiation was found in 136 ESCC patients. Survival analysis indicated that patients with high CD47 or CD133 expression exhibited poor overall survival and progression-free survival (PFS). The combination of high CD47 and CD133 expression was a reliable independent prognostic factor for both OS (HR = 1.940, 95% CI = 1.399-2.690, P < 0.0001) and progression-free survival (HR = 1.883, 95% CI = 1.384-2.562, P < 0.0001). Notably, CD47+ CD133+ ESCC cells were observed to possess the characteristics of CSCs, and anti-CD47 treatment veritably eliminated the CSCs pool. CONCLUSIONS: The stemness index determined by the expression of CD47 and CD133 is a promising prognostic predictor, and CD47 is a potential therapeutic target for CSCs in ESCC patients.

Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.

BACKGROUND: Recent clinical studies have suggested that programmed death ligand 1 (PD-L1) expression in a tumour could be a potential biomarker for PD-L1/PD-1 blockade therapies. METHODS: To better characterise PD-L1 expression in hepatocellular carcinoma (HCC), we analysed its expression patterns in 453 HCC patients by double staining for CD68 and PD-L1 using the Tyramide Signal Amplification Systems combined with immunohistochemistry. We also investigated its correlation with clinical features, prognosis and immune status. RESULTS: The results showed that PD-L1 expression on tumour cells (TCs) was negatively associated with patients' overall survival (OS; P = 0.001) and relapse-free survival (RFS; P = 0.006); however, PD-L1 expression on macrophages (Mφs) was positively correlated with OS (P = 0.017). Multivariate analysis revealed that PD-L1 expression on TCs and Mφs were both independent prognostic factors for OS (hazard ratio (HR) = 1.168, P = 0.004 for TC-PD-L1; HR = 0.708, P = 0.003 for Mφ-PD-L1). Further studies showed that Mφ-PD-L1+ tumours exhibited an activated immune microenvironment, with high levels of CD8+ T-cell infiltration and immune-related gene expression. CONCLUSION: Our study provided a novel methodology to evaluate PD-L1 expression in the tumour microenvironment, which might help to select patients who would benefit from anti-PD-1/PD-L1 immunotherapies.

Monocytes/Macrophages promote vascular CXCR4 expression via the ERK pathway in hepatocellular carcinoma.

We recently identified CXCR4 as a novel vascular marker for vessel sprouting in hepatocellular carcinoma (HCC) tissues. Thus, CXCR4+ endothelial cells (ECs) could serve as a potential predictor for patients who may benefit from sorafenib treatment; however, the mechanism that regulates vascular CXCR4 expression in HCC remains largely unknown. Here, we revealed a large number of monocytes/macrophages (Mo/Mϕ) to be selectively enriched in the perivascular areas of CXCR4+ vessels in HCC samples. The depletion of Mo/Mϕ with gadolinium chloride (GdCl3) or zoledronic acid (ZA) treatment significantly reduced vascular CXCR4 expression in HCC tumors. This phenomenon was also confirmed in CCR2-KO mice, which exhibited reduced infiltration of inflammatory Mo/Mϕ in tumor tissues. Mechanistic studies revealed that inflammatory cytokines derived from tumor conditioned Mo/Mϕ, especially TNF-α, could up-regulate CXCR4 expression on ECs. TNF-α-induced activation of the Raf-ERK pathway, but not Notch signaling, was responsible for the expression of CXCR4. Moreover, the combination treatment of sorafenib with ZA was associated with improved anti-tumor efficacy by significantly reducing vascular CXCR4 expression. These findings revealed that Mo/Mϕ could regulate CXCR4 expression in the tumor vasculature. Thus, the inhibition of Mo/Mϕ inflammation might enhance the treatment efficacy of sorafenib in HCC.

The Bidirectional Regulation between MYL5 and HIF-1α Promotes Cervical Carcinoma Metastasis.

Myosin light chains (MLC) serve important regulatory functions in a wide range of cellular and physiological processes. Recent research found that MLC are also chromatin-associated nuclear proteins which regulate gene transcription. In this study, the MLC member myosin regulatory light chain 5 (MYL5) expression was upregulated in late stage cervical cancer patients, positively correlated with pelvic lymph node metastasis, and identified as a poor survival indicator. MYL5 overexpression promoted metastasis in cervical cancer in vitro and in vivo models, whereas MYL5 silencing had the converse effect. We demonstrated a bidirectional regulation between MYL5 and hypoxia inducible factor-1α (HIF-1α). HIF-1α activates MYL5 via binding to the hypoxia response element (HRE) in the promoter of MYL5, and MYL5 could sustain HIF-1α expression by tethering to recognition sequence AGCTCC in the HIF-1α promoter region. Clinical data confirmed a positive correlation between MYL5 and HIF-1α. In summary, our data show that MYL5 may act as a prognosis predictive factor in cervical carcinoma, and strategies that inhibit the interaction of MYL5 and HIF-1α may benefit the cervical carcinoma patients with metastasis.