Taurine enhances the antitumor efficacy of PD-1 antibody by boosting CD8+ T cell function.

The functional state of CD8+ T cells determines the therapeutic efficacy of PD-1 blockade antibodies in tumors. Amino acids are key nutrients for maintaining T cell antitumor immunity. In this study, we used samples from lung cancer patients treated with PD-1 blockade antibodies to assay the amino acids in their serum by mass spectrometry. We found that lung cancer patients with high serum taurine levels generally responded to PD-1 blockade antibody therapy, in parallel with the secretion of high levels of cytotoxic cytokines (IFN-γ and TNF-α). CD8+ T cells cultured with exogenous taurine exhibited decreased apoptosis, enhanced proliferation, and increased secretion of cytotoxic cytokines. High SLC6A6 expression in CD8+ T cells was positively associated with an effector T cell signature. SLC6A6 knockdown limited the function and proliferation of CD8+ T cells. RNA sequencing revealed that SLC6A6 knockdown altered the calcium signaling pathway, oxidative phosphorylation, and T cell receptor signaling in CD8+ T cells. Furthermore, taurine enhanced T cell proliferation and function in vitro by stimulation of PLCγ1-mediated calcium and MAPK signaling. Taurine plus immune checkpoint blockade antibody significantly attenuated tumor growth and markedly improved the function and proliferation of CD8+ T cells in a mouse tumor model. Thus, our findings indicate that taurine is an important driver for improving CD8+ T cell immune responses and could serve as a potential therapeutic agent for cancer patients.

Polarization of granulocytic myeloid-derived suppressor cells by hepatitis C core protein is mediated via IL-10/STAT3 signalling.

Myeloid-derived suppressor cells (MDSCs) have been described as suppressors of T-cell function in many malignancies. Impaired T-cell responses have been observed in patients with chronic hepatitis C virus infection (CHC), which is reportedly associated with the establishment of persistent HCV infection. Therefore, we hypothesized that MDSCs also play a role in chronic HCV infection. MDSCs in the peripheral blood of 206 patients with CHC and 20 healthy donors were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) of healthy donors cultured with hepatitis C virus core protein (HCVc) were stimulated with or without interleukin 10 (IL-10). Compared to healthy donors and certain CHC patients with sustained viral response (SVR), CHC patients without SVR presented with a dramatic elevation of G-MDSCs with the HLA-DR-/low CD33+ CD14- CD11b+ phenotype in peripheral blood. The frequency of G-MDSCs in CHC patients was positively correlated with serum HCVc, and G-MDSCs were induced from healthy PBMCs by adding exogenous HCVc. Furthermore, we revealed a potential mechanism by which HCVc mediates G-MDSC polarization; activation of ERK1/2 resulting in IL-10 production and IL-10-activated STAT3 signalling. Finally, we confirmed that HCVc-induced G-MDSCs suppress the proliferation and production of IFN-γ in autologous T-cells. We also found that the frequency of G-MDSCs in serum was associated with CHC prognosis. HCVc maintains immunosuppression by promoting IL-10/STAT3-dependent differentiation of G-MDSCs from PBMCs, resulting in the impaired functioning of T-cells. G-MDSCs may thus be a promising biomarker for predicting prognosis of CHC patients.

Dual TGF-ß and PD-1 blockade synergistically enhances MAGE-A3-specific CD8+ T cell response in esophageal squamous cell carcinoma.

PD-1 is highly expressed on tumor-infiltrated antigen-specific T cells and limit the antitumor function. Blocking of PD-1/PD-L1 signaling has shown unprecedented curative efficacies in patients with advanced cancer. However, only a limited population of patients benefited from such therapies. Our study aimed to explore biological properties, functional regulation and reversal of MAGE-A3-specific CD8+ T cells in patients with esophageal squamous cell carcinoma (ESCC). The underlying principle of deficiency and restoring MAGE-A3-specific CD8+ T cells function in tumor microenvironment (TME) was evaluated. MAGE-A3-specific CD8+ T cells could lyse HLA-A2+ /MAGE-A3+ tumor cells. Tetramer+ T cell frequency was higher in elder patients, but lower in patients with lymph node metastasis and late tumor stage (p < 0.05). CD107ahigh expression on functional T cells was an independent prognostic factor in Cox regression analysis. PD-1 was highly expressed on dysfunctional antigen-specific CD8+ T cells and tumor infiltrating T lymphocytes (p < 0.05). Myeloid-derived suppressor cells (MDSCs) derived-TGF-ß mediated PD-1high expression on CD8+ T cells, which led to be resistance to PD-1/PD-L1 blockade in TME. Dual PD-1/PD-L1 and TGF-ß signaling pathway blockades synergistically restored the function and antitumor ability of antigen-specific CD8+ T cells in vitro/vivo assay. The presence of functional MAGE-A3-specific CD8+ T cells had an independent prognostic impact on survival of patients with ESCC. Furthermore, MDSCs-derived TGF-ß increased PD-1 expression on T cells and decreased the sensitivity to PD-1/PD-L1 blockade. Combining T cell-based therapy with dual PD-1/PD-L1 and TGF-ß signaling pathway blockade could be considered a promising strategy for cancer treatment.

Selective effect of cytokine-induced killer cells on survival of patients with early-stage melanoma.

Adoptive immunotherapy using cytokine-induced killer (CIK) cells has shown potential antitumor ability against several kinds of cancers, including melanoma. However, little is known about the achievable outcome of CIK cells in melanoma patients at different pathological stages. Here we recruited 55 patients treated with conventional therapy plus CIK cells as the CIK group, and 49 patients treated with conventional therapy alone as the control group. The pathological characteristics were comparable between two groups, with a follow-up period up to 40 months. Survival data and immune responses were evaluated after CIK cell treatment. In this study, CIK cells were successfully generated from peripheral blood of melanoma patients after in vitro culture for 14 days. The cultured CIK cells not only produced high levels of pro-inflammatory cytokines upon in vitro stimulation but also efficiently killed human melanoma cell lines. No serious side events were observed in all patients treated with CIK cells. Furthermore, infusions of CIK cells improved the quality of life in some patients, including advanced cases. More importantly, the CIK group exhibited better survival rates compared to the control group among early-stage melanoma patients, in consistent with the increased frequency of peripheral CD4+ T cells. However, the patients with advanced-stage melanoma did not benefit from the CIK cell therapy in terms of survival rate. In conclusion, CIK cells combined with conventional treatments may prolong the survival of early-stage melanoma patients and improve the quality of life for some advanced cases in a safe way.

Impaired T cell function in malignant pleural effusion is caused by TGF-ß derived predominantly from macrophages.

Malignant pleural effusion (MPE) is an indication of advanced cancer. Immune dysfunction often occurs in MPE. We aimed to identify the reason for impaired T cell activity in MPE from lung cancer patients and to provide clues toward potential immune therapies for MPE. The surface inhibitory molecules and cytotoxic activity of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. Levels of inflammatory cytokines in MPE and PB were tested using ELISA. TGF-ß expression in tumor-associated macrophages (TAMs) was also analyzed. The effect of TAMs on T cells was verified in vitro. Lastly, changes in T cells were evaluated following treatment with anti-TGF-ß antibody. We found that expression levels of Tim-3, PD-1 and CTLA-4 in T cells from MPE were upregulated compared with those from PB, but levels of IFN-γ and Granzyme B were downregulated (p < 0.05). The amount of TGF-ß was significantly higher in MPE than in PB (p < 0.05). TGF-ß was mainly produced by TAMs in MPE. When T cells were co-cultured with TAMs, expression levels of Tim-3, PD-1 and CTLA-4 were significantly higher than controls, whereas levels of IFN-γ and Granzyme B were significantly decreased, in a dose-dependent manner (p < 0.05). In vitro treatment with anti-TGF-ß antibody restored the impaired T cell cytotoxic activity in MPE. Our results indicate that macrophage-derived TGF-ß plays an important role in impaired T cell cytotoxicity. It will therefore be valuable to develop therapeutic strategies against TGF-ß pathway for MPE therapy of lung cancer.

MicroRNA-202 inhibits tumor progression by targeting LAMA1 in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies in the gastrointestinal tract. Emerging studies have indicated that microRNAs (miRNAs) are strongly implicated in the development and progression of ESCC. Here, we focused on the function and the underlying molecular mechanism of miR-202 in ESCC. The results showed that miR-202 was significantly down-regulated in ESCC tissues and cell lines. Overexpression of miR-202 in ECa-109 and KYSE-510 cells markedly suppressed cell proliferation and cell migration, and induced cell apoptosis. Furthermore, laminin α1 (LAMA1) expression was frequently positive in ESCC tissues and inversely correlated with miR-202 expression. Then we demonstrated that miR-202 targeted 3'-untranslated region (UTR) of LAMA1 and inhibited its protein expression. Additionally, LAMA1 overexpression rescued the proliferation inhibition and cell apoptosis elevation induced by miR-202. MiR-202 also inhibited the protein expression of p-FAK and p-Akt, which were all reversed by LAMA1 overexpression. Taken together, these findings suggest that miR-202 may function as a novel tumor suppressor in ESCC by repressing cell proliferation and migration, and its biological effects may attribute the inhibition of LAMA1-mediated FAK-PI3K-Akt signaling.

Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer.

Esophageal cancer is one of the most lethal solid malignancies. Mounting evidence demonstrates that cancer stem cells (CSCs) are able to cause tumor initiation, metastasis and responsible for chemotherapy and radiotherapy failures. As CSCs are thought to be the main reason of therapeutic failure, these cells must be effectively targeted to elicit long-lasting therapeutic responses. We aimed to enrich and identify the esophageal cancer cell subpopulation with stem-like properties and help to develop new target therapy strategies for CSCs. Here, we found esophageal cancer cells KYSE70 and TE1 could form spheres in ultra low attachment surface culture and be serially passaged. Sphere-forming cells could redifferentiate and acquire morphology comparable to parental cells, when return to adherent culture. The sphere-forming cells possessed the key criteria that define CSCs: persistent self-renewal, overexpression of stemness genes (SOX2, ALDH1A1 and KLF4), reduced expression of differentiation marker CK4, chemoresistance, strong invasion and enhanced tumorigenic potential. SB525334, transforming growth factor-beta 1(TGF-ß1) inhibitor, significantly inhibited migration and invasion of sphere-forming stem-like cells and had no effect on sphere-forming ability. In conclusion, esophageal cancer sphere-forming cells from KYSE70 and TE1 cultured in ultra low attachment surface possess cancer stem cell properties, providing a model for CSCs targeted therapy. TGF-ß1 promotes the migration and invasion of sphere-forming stem-like cells, which may guide future studies on therapeutic strategies targeting these cells.

Phenotypic characterization and anti-tumor effects of cytokine-induced killer cells derived from cord blood.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cell therapy represents a feasible immunotherapeutic option for treating malignancies. However, the number of anti-tumor lymphocytes cannot be easily obtained from the cancer patients with poor immunity status, and older patients cannot tolerate repeated collection of blood. Cord blood-derived CIK (CB-CIK) cells have shown efficacy in treating the patients with cancer in several clinical trials. This study was conducted to evaluate the biological characteristics and anti-tumor function of CB-CIK cells. METHODS: The immunogenicity, chemokine receptors and proliferation of CB-CIK cells were analyzed by flow cytometry. The CIK cells on day 13 were treated with cisplatin and the anti-apoptosis capacity was analyzed. The function of CB-CIK cells against the human cancer was evaluated both in vitro and in vivo. RESULTS: Compared with peripheral blood-derived CIK (PB-CIK) cells, CB-CIK cells demonstrated lower immunogenicity and increased proliferation rates. CB-CIK cells also had a higher percentage of main functional fraction CD3(+)CD56(+). The anti-apoptosis ability of CB-CIK cells after treatment with cisplatin was higher than that of PB-CIK cells. Furthermore, CB-CIK cells were effective for secreting interleukin-2 and interferon-γ and a higher percentage of chemokine receptors CCR6 and CCR7. In addition, tumor growth was greatly inhibited by CB-CIK treatment in a nude mouse xenograft model. CONCLUSIONS: CB-CIK cells exhibit more efficient anti-tumor activity in in vitro analysis and in the preclinical model and may serve as a potential therapeutic approach for the treatment of cancer.

The host HLA-A*02 allele is associated with the response to pegylated interferon and ribavirin in patients with chronic hepatitis C virus infection.

Human leukocyte antigen (HLA) alleles are associated with both the progression of chronic hepatitis C (CHC) and the sustained virological response (SVR) to antiviral therapy. HLA-A*02 is the most common HLA allele in people of European/Caucasian descent and the Chinese and Japanese population. Therefore, we investigated whether HLA-A*02 expression is associated with disease outcome in Chinese CHC patients. Three hundred thirty-one treatment-naïve CHC patients were recruited in this study. The expression of HLA-A*02 was tested by FACS and LABType SSO assays. All patients were treated weekly with pegylated interferon plus ribavirin (PEG-IFN/RBV) according to a standard protocol. Virological response was assessed by TaqMan assay at the 4th, 12th, 24th, and 48th week of therapy, and again at the 24th week post-therapy. By the end of the study, 293 CHC patients, including 144 HLA-A*02-positive patients and 149 HLA-A*02-negative patients, were evaluable for analysis. There were no statistical differences in clinicopathological parameters between HLA-A*02-positive and negative patients before antiviral therapy (P > 0.05). The HLA-A*02-positive patients had a higher rapid virological response (RVR, 74.3 % versus 62.4 %, P = 0.03) and SVR (78.5 % versus 64.4 %, P = 0.01) and a lower relapse rate (4.2 % versus 11.9 %, P = 0.03) than HLA-A*02-negative patients. Multivariable logistic regression analysis showed that HLA-A*02 expression, liver fibrosis stages

Epigenetic inactivation of SPINT2 is associated with tumor suppressive function in esophageal squamous cell carcinoma.

Hepatocyte growth factor activator inhibitor type 2 (SPINT2), a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor suppressor gene silenced by promoter methylation. We aimed to investigate whether SPINT2 might act as an esophageal squamous cell carcinoma (ESCC) tumor suppressor gene. Four ESCC cell lines, Fifty-two ESCC tissues and twenty-nine neighboring non-cancerous tissues were included in this study. The expression of SPINT2 was monitored by real time PCR. Bisulfite genomic sequencing and methylation-specific PCR were used to analyze methylation status. The effect of SPINT2 on cell proliferation and apoptosis in EC109 and EC9706 cells was observed by CCK-8 assay and flow cytometric analysis. We found that silencing of SPINT2 was associated with promoter methylation in ESCC cell lines. The densely methylated SPINT2 promoter region was confirmed by bisulfite genomic sequencing. Ectopic expression of SPINT2 inhibited cell proliferation through inducing cell apoptosis in vitro. Furthermore, methylation-specific PCR analysis revealed that SPINT2 promoter methylation was prominent in carcinoma tissues (52.08%) compared with neighboring non-cancerous tissues (22.58%). Kaplan-Meier analysis showed that patients with SPINT2 hypermethylation had shorter survival time. The tumor suppressor gene of SPINT2 is commonly silenced by promoter hypermethylation in human ESCC and SPINT2 hypermethylation is correlated with poor overall survival, implicating SPINT2 is an underlying prognostic marker for human ESCC.

[Expression and clinical significance of CCL5 in patients with esophageal carcinoma].

OBJECTIVE: To investigate the expression and significance of CCL5 in patients with esophageal carcinoma. METHODS: Using reverse transcriptase polymerase chain reaction (RT-PCR), the expressions of CCL5/CD8/granzyme B/perforin in tumor and corresponding adjacent tissues from esophageal carcinoma patients were examined. Flow cytometry (FACS) was used to detect the percentages of CD8(+) T cells and CCR5(+)CD8(+) T cells in TIL and PBMC from the patients. Transwell assay was performed to study the effect of CCL5 on the migration of T cells in vitro. T test and Spearman correlation analysis were performed. RESULTS: The mRNA expressions of CCL5 and perforin were 0.348 2 ± 0.300 1 and 0.181 9 ± 0.118 6, respectively, in the tumor samples, while their expressions in adjacent samples were 0.279 6 ± 0.138 0 and 0.118 0 ± 0.109 8, respectively, with no statistically significant differences between them (P > 0.05 for both). The mRNA expressions of CD8 and granzyme B were significantly higher in the tumor tissues than in adjacent tissues (0.464 9 ± 0.300 8 vs. 0.279 0 ± 0.173 4, 0.648 7 ± 0.516 0 vs. 0.469 7 ± 0.259 1; P < 0.05 for both). The relative expression of CCL5 was positively correlated with that of CD8, perforin and granzyme B (r(CD8) = 0.272, P = 0.034; r(perforin) = 0.305, P = 0.026; r(granzymeB) = 0.108, P = 0.012) in the tumor sites. FACS data revealed that the proportions of CD8(+) T cells in TIL and PBMC were (45.86 ± 16.09)% and (34.05 ± 15.07)%, respectively, showing a significant difference (P = 0.022). Similarly, CCR5(+)CD8(+) T cells fraction in TIL (48.12 ± 26.75)% was much higher than that in PBMC (19.53 ± 13.67) % (P < 0.001). Transwell assay showed that CCL5 protein enhanced the migration of T cells, supporting that CCL5 is crucial for CD8(+) T cells recruitment in vivo. Intriguingly, CCL5 expression was down-regulated in advanced patients (stage IIb-IV). The accumulation of CD8(+) T cells and CCR5(+)CD8(+) T cells was strongly reduced in advanced patients, suggesting that CCL5 expression may be involved in the local control of the disease and its reduction may be involved in disease progression. CONCLUSIONS: The current data indicate the involvement of CCL5 in the regulation of CD8(+) T cell entry into tumor lesions in esophageal carcinoma patients. This process may affect the disease status and potentially as a prognostic factor for cancer patients. Enhancing local CCL5 expression in tumor lesions may represent a novel strategy in esophageal cancer therapy.

Inactivation of MYO5B promotes invasion and motility in gastric cancer cells.

BACKGROUND: Loss of cell polarity and tissue disorganisation are hallmarks of cancer. MYO5B mutations disrupt epithelial cell polarity, suggesting that MYO5B may be involved in tumorigenesis. METHODS: We analyzed MYO5B expression in 70 gastric cancer tissues by immunohistochemistry using a tissue microarray method. Two related proteins, Rab11a and TfR, were also investigated. RESULTS: We found that the negative rate of MYO5B was 78.6 and 17.1% in gastric cancer and normal gastric tissues (P < 0.001), respectively. The MYO5B expression had a strong relationship with Rab11a expression (P = 0.002). We also found that inactivation by siRNA against MYO5B promoted the proliferation, invasion and migration of gastric cancer cells. CONCLUSION: The expression of MYO5B was downregulated in gastric cancer and inactivation of MYO5B may contribute to tumorigenesis. Therefore, MYO5B may become an important biomarker for gastric cancer in the future.

High Mobility Group Protein B1 Decreases Surface Localization of PD-1 to Augment T-cell Activation.

High-mobility group protein B1 (HMGB1) is a danger signaling molecule that has been found to trigger an effective antitumor immune response. However, the mechanisms underlying its antitumor effects are not fully understood. Here, we found that HMGB1 release induced by chemotherapy in patients with non-small cell lung cancer was negatively correlated with PD-1 expression on CD8+ T cells. In vitro analysis indicated that treatment with HMGB1 led to a significant decrease in the level of expression of PD-1 on CD8+ T cells. Further analysis demonstrated that HMGB1 reduced PD-1 expression by inducing dynamin-mediated internalization of the protein, leading to early endocytosis in the cytoplasm, and subsequently degradation in the lysosomes. In a xenograft model, HER2-targeted chimeric antigen receptor (CAR) T cells had enhanced function in the presence of HMGB1. These data identify a role for HMGB1 as a negative regulator of PD-1 signaling in lung cancer and the observed antitumor effect of HMGB1 on CAR T cells may provide a theoretical foundation for a new immunotherapy combination.

Corrigendum: Chloroquine Inhibits Stemness of Esophageal Squamous Cell Carcinoma Cells Through Targeting CXCR4-STAT3 Pathway.

[This corrects the article DOI: 10.3389/fonc.2020.00311.].

NEDD9 promotes cancer stemness by recruiting myeloid-derived suppressor cells via CXCL8 in esophageal squamous cell carcinoma.

OBJECTIVE: Esophageal squamous cell carcinoma (ESCC) has high morbidity and mortality rates worldwide. Cancer stem cells (CSCs) may cause tumor initiation, metastasis, and recurrence and are also responsible for chemotherapy and radiotherapy failures. Myeloid-derived suppressor cells (MDSCs), in contrast, are known to be involved in mediating immunosuppression. Here, we aimed to investigate the mechanisms of interaction of CSCs and MDSCs in the tumor microenvironment. METHODS: ESCC tissues and cell lines were evaluated. Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) was knocked down and overexpressed by lentiviral transfection. Quantitative PCR, Western blot, immunohistochemistry, cell invasion, flow cytometry, cell sorting, multiplex chemokine profiling, and tumor growth analyses were performed. RESULTS: Microarray analysis revealed 10 upregulated genes in esophageal CSCs. Only NEDD9 was upregulated in CSCs using the sphere-forming method. NEDD9 expression was correlated with tumor invasion (P = 0.0218), differentiation (P = 0.0153), and poor prognosis (P = 0.0373). Additionally, NEDD9 was required to maintain the stem-like phenotype. Screening of chemokine expression in ESCC cells with NEDD9 overexpression and knockdown showed that NEDD9 regulated C-X-C motif chemokine ligand 8 (CXCL8) expression via the ERK pathway. CXCL8 mediated the recruitment of MDSCs induced by NEDD9 in vitro and in vivo. MDSCs promoted the stemness of ESCC cells through NEDD9 via the Notch pathway. CONCLUSIONS: As a marker of ESCC, NEDD9 maintained the stemness of ESCC cells and regulated CXCL8 through the ERK pathway to recruit MDSCs into the tumor, suggesting NEDD9 as a therapeutic target and novel prognostic marker for ESCC.

ClC-3/SGK1 regulatory axis enhances the olaparib-induced antitumor effect in human stomach adenocarcinoma.

Currently, only a few available targeted drugs are considered to be effective in stomach adenocarcinoma (STAD) treatment. The PARP inhibitor olaparib is a molecularly targeted drug that continues to be investigated in BRCA-mutated tumors. However, in tumors without BRCA gene mutations, particularly in STAD, the effect and molecular mechanism of olaparib are unclear, which largely restricts the use of olaparib in STAD treatment. In this study, the in vitro results showed that olaparib specifically inhibited cell growth and migration, exerting antitumor effect in STAD cell lines. In addition, a ClC-3/SGK1 regulatory axis was identified and validated in STAD cells. We then found that the down-regulation of ClC-3/SGK1 axis attenuated olaparib-induced cell growth and migration inhibition. On the contrary, the up-regulation of ClC-3/SGK1 axis enhanced olaparib-induced cell growth and migration inhibition, and the enhancement effect could be attenuated by SGK1 knockdown. Consistently, the whole-cell recorded chloride current activated by olaparib presented the same variation trend. Next, the clinical data showed that ClC-3 and SGK1 were highly expressed in human STAD tissues and positively correlated (r = 0.276, P = 0.009). Furthermore, high protein expression of both ClC-3 (P = 0.030) and SGK1 (P = 0.006) was associated with poor survival rate in STAD patients, and positive correlations between ClC-3/SGK1 and their downstream molecules in STAD tissues were demonstrated via the GEPIA datasets. Finally, our results suggested that olaparib inhibited the PI3K/AKT pathway in STAD cells, and up-regulation of ClC-3/SGK1 axis enhanced olaparib-induced PI3K/AKT pathway inhibition. The animal experiments indicated that olaparib also exerted antitumor effect in vivo. Altogether, our findings illustrate that olaparib exerts antitumor effect in human STAD, and ClC-3/SGK1 regulatory axis enhances the olaparib-induced antitumor effect. Up-regulation of the ClC-3/SGK1 axis may provide promising therapeutic potential for the clinical application of olaparib in STAD treatment.

Chloroquine Inhibits Stemness of Esophageal Squamous Cell Carcinoma Cells Through Targeting CXCR4-STAT3 Pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. Recent studies have shown that cancer stem cells (CSCs) are present in ESCC, are thought to lead to aggressive tumor behavior and the prognosis. The CXC chemokine receptor 4 (CXCR4), is regarded as a putative CSCs marker in various malignancies. Here, we demonstrate that CXCR4 played a key role in ESCC progression and CXCR4 positive ESCC cells possessed stem-like properties. Furthermore, the anti-malarial agent chloroquine (CQ) targeted CXCR4-positive ESCC cells via STAT3 pathway. Therefore, CQ with anti-CSCs effects may be an effective adjunct to current ESCC chemotherapy regimens.

Decitabine enhances tumor recognition by T cells through upregulating the MAGE-A3 expression in esophageal carcinoma.

Cancer testis (CT) antigens are expressed in various types of tumors and represent the potential targets for T cell-based immunotherapy. Analysis of CT gene expression and DNA methylation have indicated that certain CT genes are epigenetically regulated and studies have confirmed that certain CT antigens are regulated by DNA methylation. In this study, we explored the epigenetic regulation of MAGE-A3 and improved the clinical outcome of MAGE-A3-specific T cell therapy in esophageal squamous cell carcinoma (ESCC). We used molecular profiling datasets in The Cancer Genome Atlas to analyze CT gene expression in ESCC and its regulation by DNA methylation. We performed quantitative reverse transcription PCR (qRT-PCR), immunohistochemistry and bisulfite sequencing in ESCC cell lines and ESCC tissues. Functional assays, such as flow cytometry, cytotoxicity assays and ELISA, were performed to determine the demethylation agent, decitabine (5-aza-2'-deoxycytidine, DAC)-treated cancer cell improved antigen specific T cells response. ESCC tumor cell-xenograft mouse model and enzyme-linked immunospot (ELISPOT) assays were used to determine the function of DAC treatment in enhancing anti-MAGE-3 T cell responses in ESCC. Furthermore, we performed qRT-PCR and flow cytometry in the peripheral blood mononuclear cells (PBMC) of myelodysplastic syndromes (MDS) patients. MAGE-A3, one of the CT antigens, expressed at various levels in ESCC and was interfered by DNA methylation. We observed an efficient increase in MAGE-A3 expression in tumor cells and tissues after the treatment of decitabine and the expression of MAGE-A3 was affected by DNA methylation. Functional assays showed enhanced secretion of IFN-γ and cytolysis of MAGE-A3 antigen-specific T cells by DAC-treated target cells. In the tumor cell-xenograft mouse model and ELISPOT assays, DAC increased the expression of MAGE-A3 and T cell mediated tumor clearance in ESCC as well. Notably, the proportions of MAGE-A3-responsive T cells were elevated in DAC-treated patients with MDS, indicating DAC dismissed the epigenetic inhibition of MAGE-A3. DAC would probably improve the clinical outcome of MAGE-A3-specific T cell therapy by augmenting the expression of target gene.

Macrophage-derived CCL22 promotes an immunosuppressive tumor microenvironment via IL-8 in malignant pleural effusion.

Immune dysfunction often occurs in malignant pleural effusion (MPE). In our previous study, TGF-ß derived predominantly from macrophages plays an important role in impairing T cell cytotoxicity in MPE. Therefore, we aimed to investigate whether other immunoregulatory cells and factors mediated TGF-ß secretion from macrophages, involved in the immunosuppressive microenvironment of MPE, and to provide clues for potential immune therapy for MPE as well. We found that CCL22 level in MPE was significantly higher than that in non-malignant pleural effusion. The high level of CCL22 was closely associated with poor survival in MPE patients with lung cancer. CCL22 was dominantly produced by tumor-associated macrophages (TAMs) in MPE. Meanwhile, TAM-derived TGF-ß mediated CCL22 expression in TAMs via c-Fos. CCL22 promoted the recruitment of regulatory T cells (Tregs) in MPE. Lastly, Treg-secreted high level of IL-8 further induced TGF-ß production from TAMs, and promoted the immunosuppressive tumor microenvironment in MPE. Our results indicate that macrophage-derived CCL22 plays an important role in the immunosuppressive tumor microenvironment via IL-8 in MPE.

Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC.

BACKGROUND: Chemotherapy combined with immunotherapy becomes the main trend in lung cancer intervention; however, how chemotherapy promotes the immune function remains elusive. Therefore, we sought to determine how chemotherapy promotes the immune function. METHODS: We determined in 100 NSCLC patients the expression of CD8, functional markers (IFN-γ, Granzyme B, and Perforin) and specific chemokines by quantitative real-time reverse transcriptase-PCR. Functional experiments were carried out to check whether docetaxel (DOC), a chemotherapeutic agent, modifies the expression of HMGB1 and CXCL11, and influences the infiltration properties of CD8+ T cells to the tumor microenvironment. The mechanism of the release of HMGB1 and CXCL11 was determined by flow cytometry, immunofluorescence and western blotting. In in vivo experiment, we confirmed how DOC enhanced the recruitment of HER2-CAR T cells to tumor sites. RESULTS: We found that DOC upregulated the expression of chemokine receptor ligand CXCL11 in tumor microenvironment and subsequently enhanced CD8+ T cell recruitment. DOC treatment significantly increased HMGB1 release in an ROS-dependent manner. Recombinant protein HMGB1 stimulated the secretion of CXCL11 via NF-κB activation in vitro. Tumors from DOC-treated mice exhibited higher expression of HMGB1 and CXCL11, more HER2-CAR T cell infiltration, and reduced progression, relative to control. Increased HMGB1 and CXCL11 expressions were positively correlated with prolonged overall survival of lung cancer patients. CONCLUSIONS: Our results demonstrate that DOC induces CD8+ T cell recruitment to the tumor microenvironment by enhancing the secretion of HMGB1 and CXCL11, thus improving the anti-tumor efficacy, indicating that modulating the HMGB1-CXCL11 axis might be helpful for NSCLC treatment.