Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

Potent CTLs can be induced against tumor cells in an environment of lower levels of systemic MFG-E8.

The direction and magnitude of immune responses are critically affected when dead cells are disposed of. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) promotes the engulfment of apoptotic normal and cancerous cells without inducing inflammation. We have previously reported that a certain proportion of the cancer cells express abundant MFG-E8, and that such expression is associated with the shorter survival of patients with esophageal cancer who had received chemotherapy before surgery. However, the influence of tumor-derived and systemically existing MFG-E8 on antitumor immune responses has not yet been fully investigated. Herein, we showed that CTL-dependent antitumor immune responses were observed in mice with no or decreased levels of systemic MFG-E8, and that such responses were enhanced further with the administration of anti-PD-1 antibody. In mice with decreased levels of systemic MFG-E8, the dominance of regulatory T cells in tumor-infiltrating lymphocytes was inverted to CD8+ T cell dominance. MFG-E8 expression by tumor cells appears to affect antitumor immune responses only when the level of systemic MFG-E8 is lower than the physiological status. We have also demonstrated in the clinical setting that lower levels of plasma MFG-E8, but not MFG-E8 expression in tumor cells, before the treatment was associated with objective responses to anti-PD-1 therapy in patients with non-small cell lung cancer. These results suggest that systemic MFG-E8 plays a critical role during the immunological initiation process of antigen-presenting cells to increase tumor-specific CTLs. Regulation of the systemic level of MFG-E8 might induce efficient antitumor immune responses and enhance the potency of anti-PD-1 therapy.

TMPRSS2 is a tumor suppressor and its downregulation promotes antitumor immunity and immunotherapy response in lung adenocarcinoma.

BACKGROUND: TMPRSS2, a key molecule for SARS-CoV-2 invading human host cells, has an association with cancer. However, its association with lung cancer remains insufficiently unexplored. METHODS: In five bulk transcriptomics datasets, one single-cell RNA sequencing (scRNA-seq) dataset and one proteomics dataset for lung adenocarcinoma (LUAD), we explored associations between TMPRSS2 expression and immune signatures, tumor progression phenotypes, genomic features, and clinical prognosis in LUAD by the bioinformatics approach. Furthermore, we performed experimental validation of the bioinformatics findings. RESULTS: TMPRSS2 expression levels correlated negatively with the enrichment levels of both immune-stimulatory and immune-inhibitory signatures, while they correlated positively with the ratios of immune-stimulatory/immune-inhibitory signatures. It indicated that TMPRSS2 levels had a stronger negative correlation with immune-inhibitory than with immune-stimulatory signatures. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor progression, and worse survival in LUAD. We further validated that TMPRSS2 was downregulated with tumor progression in the LUAD cohort we collected from Jiangsu Cancer Hospital, China. In vitro and in vivo experiments verified the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1. CONCLUSIONS: TMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a potential link between lung cancer and pneumonia caused by SARS-CoV-2 infection.

Immune response elicited in the tumor microenvironment upon rMV-SLAMblind cancer virotherapy.

Oncolytic virotherapy is a promising therapy for cancer. We previously established a recombinant measles virus (rMV-SLAMblind) that targets NECTIN4-expressing cancer cells and demonstrated its antitumor effects using a xenograft model in an immunodeficient mouse. In the current study, to investigate the immune response after rMV-SLAMblind therapy, we developed an immunocompetent cancer mouse model by introducing the NECTIN4 gene into mouse cancer cell lines. NECTIN4-expressing mouse cancer cells were successfully killed by rMV-SLAMblind in vitro. After transplantation of the NECTIN4-expressing tumor cells, rMV-SLAMblind significantly suppressed tumor growth in immunocompetent mice. Thus, this immunocompetent mouse cancer model could be a powerful tool in which to study the effect of rMV-SLAMblind therapy on the immune response. Using this model we found that rMV-SLAMblind elicited significant activation of natural killer cells, type 1 helper T cells and the tumor-specific CD8+ T-cell response in the tumor microenvironment. Immune cell depletion study revealed that CD8+ cells particularly played significant roles in the therapeutic efficacy of rMV-SLAMblind. Thus, rMV-SLAMblind exerts a therapeutic effect, not only directly by tumor cell killing, but also indirectly by efficient induction of antitumor immunity.

Immunological Assessment of Recent Immunotherapy for Colorectal Cancer.

Colorectal cancer (CRC) is the third most prevalent malignancy with increased incidence and mortality rates worldwide. Traditional treatment approaches have attempted to efficiently target CRC; however, they have failed in most cases, owing to the cytotoxicity and non-specificity of these therapies. Therefore, it is essential to develop an effective alternative therapy to improve the clinical outcomes in heterogeneous CRC cases. Immunotherapy has transformed cancer treatment with remarkable efficacy and overcomes the limitations of traditional treatments. With an understanding of the cancer-immunity cycle and tumor microenvironment evolution, current immunotherapy approaches have elicited enhanced antitumor immune responses. In this comprehensive review, we outline the latest advances in immunotherapy targeting CRC and provide insights into antitumor immune responses reported in landmark clinical studies. We focused on highlighting the combination approaches that synergistically induce immune responses and eliminate immunosuppression. This review aimed to understand the limitations and potential of recent immunotherapy clinical studies conducted in the last five years (2019-2023) and to transform this knowledge into a rational design of clinical trials intended for effective antitumor immune responses in CRC.

Chemoimmunotherapy with doxorubicin and caffeine combination enhanced ICD induction and T-cell infiltration in B16F10 melanoma tumors.

Majority of chemotherapeutic agents can elicit antitumor immunity and modulate the composition, density, function, and distribution of tumor infiltrating lymphocytes (TILs), to influence differential therapeutic responses and prognosis in cancer patients. The clinical success of these agents, particularly anthracyclines like doxorubicin, not only depends on their cytotoxic activity but also by the enhancement of pre-existing immunity primarily through induction of immunogenic cell death (ICD). However, resistance for the induction of ICD either intrinsic or acquired is a major hurdle for most of these drugs. To enhance ICD by these agents, it has become clear that blockade of adenosine production or its signaling need to be specifically targeted as they represent highly resistant mechanisms. Given the prominent role of adenosine mediated immunosuppression and resistance to ICD induction in tumor microenvironment, combination strategies that involve ICD induction and adenosine signaling blockade are further warranted. In the present study, we investigated the antitumor effect of caffeine and doxorubicin combination therapy against 3-MCA-induced and cell-line induced tumors in mice. Our results demonstrated significant tumor growth inhibition by the combination therapy of doxorubicin and caffeine against both carcinogen-induced and cell-line induced tumor models. In addition, significant T-cell infiltration and enhanced ICD induction evidenced by increased intratumoral calreticulin and HMGB1 levels, was observed in B16F10 melanoma mice. The possible mechanism behind the observed antitumor activity might be due to the enhanced ICD induction and subsequent T-cell infiltration by the combination therapy. To prevent the development of resistance and to enhance the antitumor activity of ICD inducing drugs like doxorubicin, combination with adenosine-A2A receptor pathway inhibitors like caffeine might be a potential strategy.

Selective reactivation of STING signaling to target Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a lethal skin cancer that metastasizes rapidly. Few effective treatments are available for patients with metastatic MCC. Poor intratumoral T cell infiltration and activation are major barriers that prevent MCC eradication by the immune system. However, the mechanisms that drive the immunologically restrictive tumor microenvironment remain poorly understood. In this study, we discovered that the innate immune regulator stimulator of IFN genes (STING) is completely silenced in MCCs. To reactivate STING in MCC, we developed an application of a human STING mutant, STINGS162A/G230I/Q266I, which we found to be readily stimulated by a mouse STING agonist, DMXAA. This STING molecule was efficiently delivered to MCC cells via an AAV vector. Introducing STINGS162A/G230I/Q266I expression and stimulating its activity by DMXAA in MCC cells reactivates their antitumor inflammatory cytokine/chemokine production. In response to MCC cells with restored STING, cocultured T cells expressing MCPyV-specific T cell receptors (TCRs) show increased cytokine production, migration toward tumor cells, and tumor cell killing. Our study therefore suggests that STING deficiency contributes to the immune suppressive nature of MCCs. More importantly, DMXAA stimulation of STINGS162A/G230I/Q266I causes robust cell death in MCCs as well as several other STING-silenced cancers. Because tumor antigens and DNA released by dying cancer cells have the potential to amplify innate immune response and activate antitumor adaptive responses, our finding indicates that targeted delivery and activation of STINGS162A/G230I/Q266I in tumor cells holds great therapeutic promise for the treatment of MCC and many other STING-deficient cancers.

Systemic Immunomodulatory Effects of Codonopsis pilosula Glucofructan on S180 Solid-Tumor-Bearing Mice.

The immune functions of the body are intricately intertwined with the onset and advancement of tumors, and immunotherapy mediated by bioactive compounds has exhibited initial effectiveness in overcoming chemotherapy resistance and inhibiting tumor growth. However, the comprehensive interpretation of the roles played by immunologic components in the process of combating tumors remains to be elucidated. In this study, the Codonopsis pilosula glucofructan (CPG) prepared in our previous research was employed as an immunopotentiator, and the impacts of CPG on both the humoral and cellular immunity of S180 tumor-bearing mice were investigated. Results showed that CPG administration of 100 mg/kg could effectively inhibit tumor growth in mice with an inhibitory ratio of 45.37% and significantly improve the expression of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), and Tumor Necrosis Factor-α (TNF-α). Additionally, CPG clearly enhanced B-cell-mediated humoral immunity and immune-cell-mediated cellular immunity, and, finally, induced S180 cell apoptosis by arresting cells in the G0/G1 phase, which might result from the IL-17 signaling pathway. These data may help to improve comprehension surrounding the roles of humoral and cellular immunity in anti-tumor immune responses.

Mexican Colorectal Cancer Research Consortium (MEX-CCRC): Etiology, Diagnosis/Prognosis, and Innovative Therapies.

In 2013, recognizing that Colorectal Cancer (CRC) is the second leading cause of death by cancer worldwide and that it was a neglected disease increasing rapidly in Mexico, the community of researchers at the Biomedicine Research Unit of the Facultad de Estudios Superiores Iztacala from the Universidad Nacional Autónoma de México (UNAM) established an intramural consortium that involves a multidisciplinary group of researchers, technicians, and postgraduate students to contribute to the understanding of this pathology in Mexico. This article is about the work developed by the Mexican Colorectal Cancer Research Consortium (MEX-CCRC): how the Consortium was created, its members, and its short- and long-term goals. Moreover, it is a narrative of the accomplishments of this project. Finally, we reflect on possible strategies against CRC in Mexico and contrast all the data presented with another international strategy to prevent and treat CRC. We believe that the Consortium's characteristics must be maintained to initiate a national strategy, and the reported data could be useful to establish future collaborations with other countries in Latin America and the world.

Correlation of INHBA Overexpression with Pathological Features, Antitumor Immune Response and Clinical Prognosis in Cervical Cancer.

Background and Objectives: Cervical cancer (CC) is a malignant tumor occurring in the cervical epithelium, which is one of the most common cancer-caused deaths in females. Inhibin ß A (INHBA) is the most widely expressed biomarker of the transforming growth factor-ß (TGF-ß) family in tumor cells, and has predictive value for tumor development and prognosis. In this study, the expression of INHBA in CC tissue was examined to analyze the relationship between INHBA expression and pathological characteristics, anti-tumor immune response and clinical prognosis of CC. In addition, the factors affecting the prognosis of CC patients were explored. Materials and Methods: 84 patients with CC, who underwent surgical resection in our hospital from March 2016 to August 2017, were retrospectively picked. The tumor tissues and normal adjacent tissues of patients with CC were collected, and the expression of INHBA in CC tissues and adjacent tissues was detected using immunohistochemistry (IHC). The relationship between INHBA expression and clinicopathological characteristics of CC patients was analyzed. The relationship between INHBA expression and clinical prognosis was analyzed using the Kaplan-Meier (K-M) survival curve. The levels of anti-tumor immune-response-related factors (interferon-γ (IFN-γ), interleukin-10 (IL-10), tumor necrosis factor- α (TNF-α) and IL-2) were evaluated in patients with negative and positive expressions of INHBA. The patients were followed up for 60 months and were graded as a good prognosis group and poor prognosis group according to whether the patients died or had recurrence and metastasis. Relevant factors affecting the prognosis of the patients were analyzed. Results: INHBA was localized in the cytoplasm of cancer tissues. The positive expression rate in cancer tissues was 67.86%, which was much higher than the 28.57% in normal adjacent tissues (p < 0.05). Expression of INHBA was closely correlated with Federation of Gynecology and Obstetrics (FIGO) staging, differentiation and lymph node metastasis (p < 0.05). Compared with INHBA-negative expression group, the contents of IFN-γ, TNF-α and IL-2 were much lower, while the level of IL-10 was strongly elevated in the INHBA-positive expression group (p < 0.01). Eighty-four patients with CC were followed up for 36 months. The K-M survival curve showed that the patients with a positive expression of INHBA had a significantly shorter survival period than the patients with a negative expression of INHBA (p < 0.05). There were significant differences in FIGO staging, differentiation, lymph node metastasis and INHBA expression between patients with a good prognosis and poor prognosis (p < 0.05). Logistic regression analysis showed that FIGO stage, differentiation degree, lymph node metastasis and INHBA were the factors influencing the poor prognosis of patients with CC (p < 0.05). Conclusion: The abnormally high expression of INHBA in patients with CC was related to the pathological characteristics, anti-tumor immune response and survival time, and leaded to a poor prognosis. It was speculated that INHBA exerted an important reference role in tumor invasion and clinical prognosis evaluation, which could act as a new target for anti-tumor treatment of CC.

Targeting lysophosphatidic acid receptor with Ki16425 impedes T cell lymphoma progression through apoptosis induction, glycolysis inhibition, and activation of antitumor immune response.

Lysophosphatidic acid (LPA) is a small phospholipid that acts as an extracellular lipid mediator. It promotes cancer progression by altering a wide array of cellular processes, including apoptosis, survival, angiogenesis, invasion, and migration through binding with its cognate receptors. Intriguingly, our previous study showed that in vitro treatment of LPA induced survival of T lymphoma cells. Hence, the present investigation was designed to investigate the antitumor potential of Ki16425, an antagonist of LPA receptors, against T cell lymphoma. Our in vitro results showed inhibition of LPA-mediated survival and metabolic activity of T lymphoma cells by Ki16425. Further, in vivo experimental findings indicated the tumor retarding potential of Ki16425 against T cell lymphoma through apoptosis induction, glycolysis inhibition, and immunoactivation. The administration of Ki16425 triggered apoptosis by down-regulating the expression of Bcl2 and up-regulating p53, Bax, cleaved caspase-3, and Cyt c expression. Further, Ki16425 suppressed glycolytic activity with concomitantly decreased expression of GLUT3 and MCT1. Moreover, we also noticed an elevated level of NO and iNOS in tumor cells after Ki16425 administration which might also be responsible for apoptosis induction and suppressed glycolysis. Additionally, we observed an increased population of total leukocytes, lymphocytes, and monocytes along with increased thymocytes count and IL-2 and IFN-γ levels. Besides, we observed amelioration of tumor-induced kidney and liver damages by Ki16425. Taken together, this is the first study that demonstrates that LPA receptors could be potential future therapeutic targets for designing promising therapeutic strategies against T cell lymphoma.

Chemotherapeutic and targeted agents can modulate the tumor microenvironment and increase the efficacy of immune checkpoint blockades.

The development of immune checkpoint blockade (ICB)-based immunotherapy has dramatically changed methods of cancer treatment. This approach triggers a durable treatment response and prolongs patients' survival; however, not all patients can benefit. Accumulating evidence demonstrated that the efficacy of ICB is dependent on a robust antitumor immune response that is usually damaged in most tumors. Conventional chemotherapy and targeted therapy promote the antitumor immune response by increasing the immunogenicity of tumor cells, improving CD8+ T cell infiltration, or inhibiting immunosuppressive cells in the tumor microenvironment. Such immunomodulation provides a convincing rationale for the combination therapy of chemotherapeutics and ICBs, and both preclinical and clinical investigations have shown encouraging results. However, the optimal drug combinations, doses, timing, and sequence of administration, all of which affect the immunomodulatory effect of chemotherapeutics, as well as the benefit of combination therapy, are not yet determined. Future studies should focus on these issues and help to develop the optimal combination regimen for each cancer.

High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice.

BACKGROUND AND AIMS: The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. METHODS: We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlr-/-mice), and mice with conditional deletion of nature killer (NK) cells (NKdele mice). Some C57BL/6J and NKdele mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE-/- mice), which spontaneously develop high serum cholesterol. C57BL/6J and NKdele mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. RESULTS: C57BL/6J mice on HCD and ApoE-/- mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE-/- mice had increased numbers of NK cells compared to tumors from ND-fed mice. NKdele mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr-/- mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr-/- mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. CONCLUSIONS: Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC.

Identification of Subtypes of Barrett's Esophagus and Esophageal Adenocarcinoma Based on DNA Methylation Profiles and Integration of Transcriptome and Genome Data.

BACKGROUND & AIMS: Esophageal adenocarcinomas (EACs) are heterogeneous and often preceded by Barrett's esophagus (BE). Many genomic changes have been associated with development of BE and EAC, but little is known about epigenetic alterations. We performed epigenetic analyses of BE and EAC tissues and combined these data with transcriptome and genomic data to identify mechanisms that control gene expression and genome integrity. METHODS: In a retrospective cohort study, we collected tissue samples and clinical data from 150 BE and 285 EAC cases from the Oesophageal Cancer Classification and Molecular Stratification consortium in the United Kingdom. We analyzed methylation profiles of all BE and EAC tissues and assigned them to subgroups using non-negative matrix factorization with k-means clustering. Data from whole-genome sequencing and transcriptome studies were then incorporated; we performed integrative methylation and RNA-sequencing analyses to identify genes that were suppressed with increased methylation in promoter regions. Levels of different immune cell types were computed using single-sample gene set enrichment methods. We derived 8 organoids from 8 EAC tissues and tested their sensitivity to different drugs. RESULTS: BE and EAC samples shared genome-wide methylation features, compared with normal tissues (esophageal, gastric, and duodenum; controls) from the same patients and grouped into 4 subtypes. Subtype 1 was characterized by DNA hypermethylation with a high mutation burden and multiple mutations in genes in cell cycle and receptor tyrosine signaling pathways. Subtype 2 was characterized by a gene expression pattern associated with metabolic processes (ATP synthesis and fatty acid oxidation) and lack methylation at specific binding sites for transcription factors; 83% of samples of this subtype were BE and 17% were EAC. The third subtype did not have changes in methylation pattern, compared with control tissue, but had a gene expression pattern that indicated immune cell infiltration; this tumor type was associated with the shortest time of patient survival. The fourth subtype was characterized by DNA hypomethylation associated with structure rearrangements, copy number alterations, with preferential amplification of CCNE1 (cells with this gene amplification have been reported to be sensitive to CDK2 inhibitors). Organoids with reduced levels of MGMT and CHFR expression were sensitive to temozolomide and taxane drugs. CONCLUSIONS: In a comprehensive integrated analysis of methylation, transcriptome, and genome profiles of more than 400 BE and EAC tissues, along with clinical data, we identified 4 subtypes that were associated with patient outcomes and potential responses to therapy.

Immunological effect of irreversible electroporation on hepatocellular carcinoma.

BACKGROUND: This study intends to investigate the immunological effects of tumor ablation with irreversible electroporation (IRE). METHODS: We evaluated the systemic immune response in patients with hepatocellular carcinoma (HCC) after IRE treatment. Furthermore, we analyzed the tumor infiltrating T lymphocytes and the level of serum cytokines in IRE and control groups of tumor-bearing mice. RESULTS: We observed that IRE induced an increase in WBC, neutrophil and monocyte counts and a decrease in lymphocyte count 1 day post-IRE and returned to baseline values within 7 days in the patients. Meanwhile, circulating CD4+ T cell subsets, but not CD8+, decreased 1 day post-IRE. The activated T cells and natural killer (NK) cells increased, and regulatory T (Treg) cells decreased. Furthermore, a significant increase in cytotoxic CD8+ T cells infiltration was observed on ablative tumors in mice. The level of serum IFN-γ also significantly increased in the IRE group. CONCLUSIONS: Our study demonstrated that IRE upregulated activated T cells and downregulated Tregs in the peripheral blood of patients. Meanwhile, the results from the animal model indicated that IRE could induce antitumor adaptive immunity dominated by the infiltration of cytotoxic CD8+ T cells into the tumors, accompanied by reduced Tregs.

CXCL12 Signaling in the Tumor Microenvironment.

Tumor microenvironment (TME) is the local environment of tumor, composed of tumor cells and blood vessels, extracellular matrix (ECM), immune cells, and metabolic and signaling molecules. Chemokines and their receptors play a fundamental role in the crosstalk between tumor cells and TME, regulating tumor-related angiogenesis, specific leukocyte infiltration, and activation of the immune response and directly influencing tumor cell growth, invasion, and cancer progression. The chemokine CXCL12 is a homeostatic chemokine that regulates physiological and pathological process such as inflammation, cell proliferation, and specific migration. CXCL12 activates CXCR4 and CXCR7 chemokine receptors, and the entire axis has been shown to be dysregulated in more than 20 different tumors. CXCL12 binding to CXCR4 triggers multiple signal transduction pathways that regulate intracellular calcium flux, chemotaxis, transcription, and cell survival. CXCR7 binds with high-affinity CXCL12 and with lower-affinity CXCL11, which binds also CXCR3. Although CXCR7 acts as a CXCL12 scavenger through ligand internalization and degradation, it transduces the signal mainly through ß-arrestin with a pivotal role in endothelial and neural cells. Recent studies demonstrate that TME rich in CXCL12 leads to resistance to immune checkpoint inhibitors (ICI) therapy and that CXCL12 axis inhibitors sensitize resistant tumors to ICI effect. Thus targeting the CXCL12-mediated axis may control tumor and tumor microenvironment exerting an antitumor dual action. Herein CXCL12 physiology, role in cancer biology and in composite TME, prognostic role, and the relative inhibitors are addressed.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth and Invasion of Colon Tumors in Mice.

BACKGROUND & AIMS: Neutrophils are among the most prevalent immune cells in the microenvironment of colon tumors; they are believed to promote growth of colon tumors, and their numbers correlate with outcomes of patients with colon cancer. Trials of inhibitors of neutrophil trafficking are underway in patients with cancer, but it is not clear how neutrophils contribute to colon tumorigenesis. METHODS: Colitis-associated colon cancer was induced in mice with conditional deletion of neutrophils (LysMCre;Mcl1fl/fl) and wild-type littermates (LysMCre;Mcl1wt/wt, control mice) by administration of azoxythmethane and/or dextran sulfate sodium. Sporadic colon tumorigenesis was assessed in neutrophil-deficient and neutrophil-replete mice with conditional deletion of colon epithelial Apc (Cdx2-CreERT2;Apcfl/fl). Primary colon tumor tissues from these mice were assessed by histology, RNA sequencing, quantitative polymerase chain reaction, and fluorescence in situ hybridization analyses. Fecal and tumor-associated microbiota were assessed by 16s ribosomal RNA sequencing. RESULTS: In mice with inflammation-induced and sporadic colon tumors, depletion of neutrophils increased the growth, proliferation, and invasiveness of the tumors. RNA sequencing analysis identified genes that regulate antimicrobial and inflammatory processes that were dysregulated in neutrophil-deficient colon tumors compared with colon tumors from control mice. Neutrophil depletion correlated with increased numbers of bacteria in tumors and proliferation of tumor cells, tumor-cell DNA damage, and an inflammatory response mediated by interleukin 17 (IL17). The 16s ribosomal RNA sequencing identified significant differences in the composition of the microbiota between colon tumors from neutrophil-deficient vs control mice. Administration of antibiotics or a neutralizing antibody against IL17 to neutrophil-deficient mice resulted in development of less-invasive tumors compared with mice given vehicle. We found bacteria in tumors to induce production of IL17, which promotes influx of intratumor B cells that promote tumor growth and progression. CONCLUSIONS: In comparisons of mice with vs without neutrophils, we found neutrophils to slow colon tumor growth and progression by restricting numbers of bacteria and tumor-associated inflammatory responses.

Prognostic Significance of PD-1, PD-L1 and CD8 Gene Expression Levels in Gastric Cancer.

INTRODUCTION: Anti-programmed cell death 1 (PD-1) therapies have shown promising clinical activity against gastric cancer (GC). We evaluated the clinical significance of immune-related gene expression in GC tissues to better understand the tumor immune microenvironment. METHODS: PD-1, PD-1 ligand 1 (PD-L1) and CD8 mRNA levels and clinicopathological factors, including survival, were examined by quantitative RT-PCR in 155 GC patients who underwent surgery. PD-1 and PD-L1 expression in tumor tissue from 24 GC patients was investigated by immunohistochemical analysis. RESULTS: PD-1, PD-L1 and CD8 mRNA levels were significantly lower in tumor tissue than in normal tissue (p < 0.0001, p < 0.05, and p < 0.0001). GC patients with low PD-1, PD-L1 and CD8 mRNA levels had significantly poorer overall survival (OS) than those with high PD-1, PD-L1 and CD8 mRNA levels, respectively (p < 0.001, p < 0.01 and p < 0.05). Low PD-1, PD-L1 and CD8 mRNA levels were more significantly associated with poor prognosis in undifferentiated-type GC patients than in differentiated-type GC patients (PD-1: differentiated p = 0.0071 vs. undifferentiated p = 0.0024; PD-L1: p = 0.6527 vs. p < 0.0001; CD8: p = 0.4465 vs. p < 0.05). Multivariate analysis showed that lymph node metastasis, peritoneal dissemination, distant metastasis, low PD-1 mRNA levels and low CD8 mRNA levels were independent prognostic factors for worse OS (low PD-1 mRNA level: OR 2.16, 95% CI 1.10-4.58, p < 0.05; low CD8 mRNA level: OR 2.55, 95% CI 1.12-6.90, p < 0.05). PD-1 and PD-L1 mRNA levels in immune cells were significantly associated with PD-1 and PD-L1 protein levels (both p < 0.05), respectively. CONCLUSIONS: PD-1, PD-L1 and CD8 mRNA levels may reflect antitumor immunity in GC, and low PD-1 and CD8 mRNA levels are potential predictive biomarkers for poor prognosis in GC patients who underwent surgery.