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.

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.

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.

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.

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.

Expression and prognostic significance of MYL9 in esophageal squamous cell carcinoma.

OBJECTIVE: Myosin light chain 9 (MYL9) is necessary for cytoskeletal dynamics and experimental metastasis, but its expression in esophageal squamous cell carcinoma (ESCC) has not been addressed. We investigated the expression pattern and clinical significance of MYL9 in patients with ESCC. METHODS: We examined MYL9 expression using quantitative real-time PCR and western blotting in NE1 immortalized esophageal epithelial cells, ESCC cell lines, and paired ESCC tissues. MYL9 protein in 136 primary ESCC tissues and other types of solid tumor was detected using immunohistochemistry. The association between MYL9 expression and clinical parameters and survival was evaluated by statistical analysis. RESULTS: MYL9 was significantly upregulated in the ESCC cell lines as compared with NE1 cells. In the paired ESCC samples, MYL9 mRNA and protein expression was not significantly different between lesion tissues and the matched adjacent noncancerous tissues. In ESCC tissue, both intratumoral and peritumoral stroma were positive for MYL9. In the 136 ESCC samples, high MYL9 expression in the tumor cells significantly correlated with histological differentiation (p = 0.028), recurrence (p = 0.01), and vital status (p < 0.01). Patients with high MYL9 expression in the tumor cells had poorer overall survival (OS) and recurrence-free survival. Multivariate analysis revealed that high MYL9 expression in tumor cells was an independent and significant risk factor affecting OS after curative treatment (hazard ratio = 2.254, 95% confidence interval = 1.347-3.771, p = 0.002). CONCLUSIONS: MYL9 expression might be a promising prognostic marker and therapeutic target in ESCC.

Vascular CXCR4 Expression Promotes Vessel Sprouting and Sensitivity to Sorafenib Treatment in Hepatocellular Carcinoma.

Purpose: C-X-C chemokine receptor type 4 (CXCR4) is known to be involved in both developmental and adult angiogenesis; however, its role in tumor angiogenesis remains largely unknown. Here, the role of vascular CXCR4 in regulating vascular structure in hepatocellular carcinoma (HCC) was assessd, and the clinical value of CXCR4 was explored.Experimental Design: The expression of CXCR4 in HCC was determined by IHC and immunofluorescence. Characteristics of CXCR4+ cells were determined by in vitro and mice experiments. Kaplan-Meier survival analysis was used to determine the correlation of CXCR4 expression with prognosis.Results: We found that CXCR4 is selectively expressed on a fraction of tumor endothelial cells (TECs) in HCC tissues, but not on the hepatic endothelium in peritumoral area. High levels of CXCR4 on TECs tended to develop a sinusoidal vasculature in tumors and predicted poor prognosis for patients with HCC. CXCR4+ endothelial cells (EC) displayed the functional features of tip cells, with increased expression of tip cell-related markers. Functional studies revealed that CXCR4 could directly promote vessel sprouting in vitro and in vivo Interestingly, sorafenib treatment reduced the frequency of CXCR4+ ECs in culture and inhibited the formation of sinusoidal vasculature and growth of CXCR4High xenograft tumors. Moreover, high CXCR4 vascular density in resected tumor tissues before sorafenib treatment was associated with prolonged survival in patients with advanced HCC treated with sorafenib.Conclusions: These data revealed that CXCR4 is a novel HCC vascular marker for vessel sprouting and could serve as a potential therapeutic target and a predictive factor for sorafenib treatment in patients with HCC. Clin Cancer Res; 23(15); 4482-92. ©2017 AACR.

Distinct patterns and prognostic values of tumor-infiltrating macrophages in hepatocellular carcinoma and gastric cancer.

BACKGROUND: Macrophages (Mφs) constitute a major component of the leukocyte infiltrate and perform distinct roles in different tumor microenvironments. This study aimed to characterize the distribution, composition and prognostic value of Mφs in hepatocellular carcinoma (HCC) and gastric cancer (GC). METHODS: Immunohistochemistry and immunofluorescence were used to identify Mφ subsets in HCC and GC tissues. Kaplan-Meier analysis and Cox regression models were applied to estimate the overall survival (OS) for HCC and GC patients. RESULTS: The results showed that the density of Mφs decreased in the intra-tumor region (IT) of HCC, but remarkably increased in the IT of GC, as compared with their non-tumor regions (NT). In HCC, most CD68+ Mφs were CD204+ and CD169+ cells in the NT region; however, there was a significant decrease in the percentage of CD169+ Mφ in the IT region. In contrast, CD68+ Mφs comprised a smaller percentage of CD204+ than the CD169+ subpopulation in the NT region, while more CD204+ but fewer CD169+ cells were present in the IT region of GC. The density of CD204+ Mφs correlated with poor prognosis in HCC, and CD169+ Mφs were associated with good survival in both HCC and GC. Moreover, the combination of low numbers of CD204+ and high numbers of CD169+ Mφs was associated with improved OS in both GC and HCC. CONCLUSIONS: Mφs display tissue-specific distributions and distinct composition patterns in HCC and GC tissues. Our results suggested that different types of tumors might use diverse strategies to reconstitute Mφ patterns to promote tumor progression.

GLUT1 and ASCT2 as Predictors for Prognosis of Hepatocellular Carcinoma.

An emerging hallmark of cancer is reprogrammed cellular metabolism, and several cancers involve increased glucose intake and glutamine addiction. Hepatocellular carcinoma (HCC) is one of the most fatal cancers, and its molecular basis needs to be delineated to identify biomarkers for its potential treatment without resection. Therefore, this study aimed to determine the metabolism status of HCC by evaluating the expression of the glucose transporter GLUT1 and glutamine transporter ASCT2. We enrolled 192 patients with surgically resected HCC in this study. Their tissue samples were subjected to immunohistochemistry to detect GLUT1 and ASCT2 expression. The prognostic value of GLUT1 and ASCT2 expression and their combined metabolic index was determined by Kaplan-Meier analysis and the Cox proportional hazards model. We found that GLUT1 and ASCT2 expression was significantly upregulated in tumor tissues as compared to adjacent non-tumor tissues and was positively associated with tumor size. Survival analysis revealed that patients with high GLUT1 or ASCT2 expression had poor overall survival (OS) and recurrence-free survival (RFS). In HCC patients, ASCT2 expression was an independent negative prognostic factor for OS (hazard ratio [HR], 1.760; 95% confidence interval [CI] = 1.124-2.755; p = 0.013) and the metabolic index was an independent negative prognostic factor for OS (HR = 1.672, 95% CI = 1.275-2.193, p < 0.001) and RFS (HR = 1.362, 95% CI = 1.066-1.740, p = 0.013). In conclusion, the tumor metabolism status determined by expression of GLUT1 and ASCT2 and their metabolic index is a promising prognostic predictor for HCC patients.

RASSF8 downregulation promotes lymphangiogenesis and metastasis in esophageal squamous cell carcinoma.

Lymphatic vessels are the major routes of human esophageal squamous cell carcinoma (ESCC) metastasis. Tumor cells secrete pro-lymphangiogenic factors to induce new lymphatic vessels, promoting lymph node metastasis. In this study, we show that RAS association domain family 8 (RASSF8) expression in ESCC clinical samples was inversely correlated with lymph node metastasis and patients survival. Tumor cells with low RASSF8 expression had higher apparent migratory ability, and promoted and lymphangiogenesis both in vitro and in vivo. RASSF8 downregulation enhanced VEGF-C expression and caused subcellular redistribution of p65 in ESCC. Our results show that RASSF8 acts as a tumor suppressor in ESCC and is a potential therapeutic target for preventing lymph node metastasis.

Circulating hematopoietic stem and progenitor cells are myeloid-biased in cancer patients.

Cancer is associated with a profound perturbation in myelopoiesis that results in the accumulation of myeloid-derived suppressor cells (MDSCs) to promote disease progression. Recent studies in mice suggest that tumor-derived factors could regulate the differentiation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and subsequently contribute to dysregulation of hematopoiesis. However, the nature and role of HPSCs in patients with cancer remain unknown. Here we show, in detailed studies of the peripheral blood from 133 untreated patients with seven different types of tumors, that the composition of circulating HSPCs was significantly altered in patients with solid tumors. The frequencies of circulating granulocyte-monocyte progenitors (GMPs) were increased four to seven fold in all types of tumors examined, and the circulating hematopoietic precursors exhibited myeloid bias with a skew toward granulocytic differentiation in patients with solid tumors. These myeloid precursors are selectively enriched in tumor tissues, and the high levels of circulating GMPs were positively correlated with disease progression. By using cord blood-derived CD34(+) cells, we developed an in vitro short-term culture model to effectively induce the rapid generation of MDSCs. We found that, among the factors produced by various tumors, GM-CSF, granulocyte colony-stimulating factor, and IL-6 could not only promote the myeloid-biased differentiation, but also induce the differentiation of myeloid precursors into functional MDSCs. These findings suggest that the altered circulating HSPCs may serve as an important link between dysregulated bone marrow hematopoiesis and accumulated MDSCs in patients with cancer.

Increased circulating Th17 cells after transarterial chemoembolization correlate with improved survival in stage III hepatocellular carcinoma: a prospective study.

Transarterial chemoembolization (TACE) has therapeutic effects in patients with unresectable hepatocellular carcinoma (HCC), but its impact on the cellular immune response during disease progression is largely unknown. Here we conducted a prospective study to evaluate the effect of TACE on immune status and to identify prognostic immune markers governing treatment success. In this study, 51 stage III HCC patients, 28 stage I HCC patients (TNM classification) and 20 healthy donors were enrolled. Flow cytometry and cytometric bead array were used to evaluate the circulating immune cell subsets, including CD4(+) T cells (Th1, Th17 and Treg cells), CD8(+) T cells, NK cells, and NKT cells, and plasma cytokines before TACE and 30 days after TACE. Interestingly, among those immune parameters, the frequency of circulating Th17 cells was higher in stage III HCC patients than in stage I HCC patients (P = 0.015) and healthy donors (P<0.001). Moreover, an increased frequency of circulating Th17 cells was observed 30 days after TACE (Th17 D30 ) compared with the baseline level (P = 0.036). Kaplan-Meier analysis demonstrated that Th17 D30 was positively associated with overall survival (OS; P = 0.007) and time to progression (TTP; P = 0.009). Multivariate Cox analysis revealed that Th17 D30 was an independent prognostic factor for OS (HR = 0.317, P = 0.032) and TTP (HR = 0.304, P = 0.010). These results provide a potential prognostic marker for stage III HCC patients undergoing TACE and may be useful for identifying patients who can benefit from adjuvant immunotherapies.

An in situ molecular signature to predict early recurrence in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: To develop an in situ molecular signature to predict postsurgical recurrence in hepatocellular carcinoma (HCC) patients. METHODS: Immunohistochemistry was performed using tissue microarrays containing both tumoral and peri-tumoral regions of the advancing tumor edge from 336 HCC patients (289 were positive for hepatitis B virus) who underwent curative resection. Forty-nine variables were analyzed in the training set (n=151) using support vector machine and stepwise algorithms to develop a classifier to predict recurrence within 1 year, which was mainly caused by invasion or metastasis from the primary tumors. The classifier was further validated in an independent cohort of 185 patients (71 internal and 114 external). RESULTS: The final signature was composed of eight IHC features: CD80(T), B7-DC(T), HLA-DR(P), FasL(P), Bcl-2(T), Ki-67(T), cyclin D1(T), and CK19(T). In the independent test set, this classifier reliably predicted recurrence within 1 year (sensitivity, 69.1%; specificity, 65.0%) with an odds ratio of 4.149 (95% CI, 2.189-7.864). Based on a multivariate logistic model, the in situ molecular signature provided significant predictive power independent of tumor number, tumor size, vascular invasion and BCLC classification (p=0.001). The highest potential clinical impact of the classifier was observed in early-stage (BCLC classification 0-A) patients (p<0.0001), and the classifier was also predictive of the time-to-recurrence and overall survival (both p<0.0001). CONCLUSIONS: This in situ molecular classifier could provide a novel approach to identify patients who are at greatest risk for postsurgical recurrence of HCC and may benefit from intensive clinical follow-up or chemopreventive strategies.