Immune Cells In Tumor Tissues Research Articles

Neoantigens and the tumor microenvironment play important roles in the prognosis of high-grade serous ovarian cancer

BackgroundDespite the consistency of patient age, disease stage and treatment options, the prognosis of different high-grade serous ovarian carcinoma (HGSOC) patients is different. Here, we sought to measure predictive biomarkers for distinct responses to platinum-based chemotherapy and immunotherapy benefits.MethodsSixteen HGSOC patients receiving debulking surgery and adjuvant first-line combination chemotherapy at Peking Union Medical College Hospital (PUMCH) were enrolled. Whole exome sequencing (WES) and RNA-seq were performed on tumor and normal tissues of these patients.ResultsThe tumor mutational burden (TMB) and intratumoral heterogeneity (ITH) of the platinum-resistant group were significantly higher than those of the platinum-sensitive group (P=0.0321 and P=0.0452, respectively). TMB, neoantigen and ITH had certain predictive value according to the area under the receiver operating characteristic (ROC) curve (AUC 0.7778 for TMB, 0.7619 for neoantigen, 0.7778 for ITH). The infiltration of other immune cells in tumor tissues was different between the two groups, but the difference was not significant. Univariate Cox proportional hazard analysis revealed poorer progression-free survival (PFS) for those patients who carried a higher number of neoantigens (P =0.0069), higher TMB (P =0.0083), and higher ITH (P =0.0249). Further Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis indicated the Differentially expressed genes (DEGs) in platinum-resistant and platinum-sensitive patients were mainly enriched in the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathway and focal adhesion pathway, which are associated with platinum resistance.ConclusionsHigher TMB, neoantigen and ITH may account for the worse prognosis of patients with platinum-based chemotherapy and higher TMB was observed in the platinum-resistant group, which could make the patients in the platinum-resistant group to be the better candidates for immunotherapy.

The correlation between infiltration of FoxP3+ Tregs, CD66b+ TANs and CD163+ TAMs in colorectal cancer.

IntroductionThe infiltration of immune cells in tumor tissue is affected by the tumor microenvironment. However, the relationship between the infiltration of regulatory T cells (Tregs), tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) in colorectal cancer (CRC) remains unclear.Material and methodsTissue microarray and immunohistochemistry were used to detect the infiltration of FoxP3+ Tregs, CD66b+ TANs and CD163+ TAMs in 249 CRC samples (training cohort) and 243 CRC samples (validation cohort). The relationship between two cells was evaluated by Spearman’s rank correlation coefficient and comparison between two groups was analyzed by Mann-Whitney U test.ResultsThe continuous variable positive cell numbers were non-normally distributed. Spearman correlation analysis showed that CD66b+ TAN level in cancer tissues was negatively related to FoxP3+ Treg level (correlation coefficient: –0.495, p < 0.05) and CD163+ TAM level (correlation coefficient: –0.266, p < 0.05), and FoxP3+ Treg level was positively related to CD163+ TAM level (correlation coefficient: 0.467, p < 0.05) in the training cohort. The numbers of FoxP3+ Tregs were significantly different between low and high CD66b+ TAN level groups (p < 0.001), as well as that of CD66b+ TANs in low and high CD163+ TAM level groups and CD163+ TAMs in different FoxP3+ Treg level groups. The results of the validation cohort were similar to those of the training cohort.ConclusionsThere is a negative correlation between infiltration of CD66b+ TANs and that of FoxP3+ Tregs or CD163+ TAMs, and a positive correlation between infiltration of FoxP3+ Tregs and CD163+ TAMs in CRC tissues.

Smart biomaterials to enhance the efficiency of immunotherapy in glioblastoma: State of the art and future perspectives.

Glioblastoma multiform (GBM) is considered as the most lethal tumor among CNS malignancies. Although immunotherapy has achieved remarkable advances in cancer treatment, it has not shown satisfactory results in GBM patients. Biomaterial science, along with nanobiotechnology, is able to optimize the efficiency of immunotherapy in these patients. They can be employed to provide the specific activation of immune cells in tumor tissue and combinational therapy as well as preventing systemic adverse effects resulting from hyperactivation of immune responses and off-targeting effect. Advance biomaterials in this field are classified into targeting nanocarriers and localized delivery systems. This review will offer an overview of immunotherapy strategies for glioblastoma and advance delivery systems for immunotherapeutics that may have a high potential in glioblastoma treatment.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss‐of‐function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2‐deficient immune cells in tumor tissues. Myeloid‐specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single‐cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2‐deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2‐deficient mice relative to controls. Finally, treatment of Tet2‐deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2‐mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2‐mutated clonal hematopoiesis.

Prognostic and Immunological Role of Key Genes of Ferroptosis in Pan-Cancer.

Solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and apoptosis inducing factor mitochondria associated 2 (AIFM2) are the key regulators in ferroptosis. However, the expression patterns and prognostic roles of these genes in pan-cancer are still largely unclear. The expression patterns and prognostic roles of SLC7A11, GPX4, and AIFM2 and the relationships between the expression levels of these genes and immune infiltration levels in pan-cancer were analyzed by using TIMER, gene expression profiling interactive analysis (GEPIA), Oncomine, and Kaplan–Meier databases. Our results showed that both SLC7A11 and GPX4 were overexpressed in colorectal cancer, and SLC7A11 was overexpressed in lung cancer. High levels of SLC7A11 and AIFM2 were significantly linked with the shortened disease-free survival and overall survival (OS) in adrenocortical carcinoma (ACC), respectively. And high expression of SLC7A11, GPX4, and AIFM2 were significantly correlated with the shortened OS of acute myeloid leukemia patients. In esophageal carcinoma (ESCA), GPX4 expression was significantly associated with the infiltration of macrophage and myeloid dendritic cell, and AIFM2 expression was significantly associated with the infiltration of CD4+ T cell. Importantly, GPX4 expression was positively correlated with the expression levels of monocyte markers (CD14 and CD115) and M2 macrophage markers (VSIG4 and MS4A4A) both in ESCA and in head and neck squamous cell carcinoma (HNSC). In summary, SLC7A11, GPX4, and AIFM2 are dysregulated in many types of cancers, and are candidate prognostic biomarkers for many types of cancers, and can be used to evaluate the infiltration of immune cells in tumor tissues.

Changes of immune cells in patients with hepatocellular carcinoma treated by radiofrequency ablation and hepatectomy, a pilot study.

In this pilot study, we compared the dynamic changes of circulating immune cells between patients with hepatocellular carcinoma (HCC) who underwent radiofrequency ablation (RFA) and hepatectomy. Seventy-three patients were enrolled in this study. Flow cytometry assay was performed to determine the immune cells in the peripheral blood mononuclear cells (PBMCs) before treatment and on days 7, 14, and 28 after treatment. We found that in the RFA group, the circulating cluster of differentiation (CD)4+ cells, the CD4+/CD8+ ratio, and natural killer (NK) cells continued to increase, and the circulating CD8+ cells continued to decrease after the treatment. In contrast, in the surgery group, the circulating CD4+ cells and CD4+/CD8+ ratio decreased over the first seven postoperative days and then began to increase, and CD8+ cells decreased on the first 7 postoperative days and began to increase thereafter. The changes of immune cells in tumor tissues consisted of an increase in the number of CD4+ cells, CD8+ cells, CD3+ cells, and NK cells immediately after RFA. Our results show that postoperative immune function continued to improve after RFA, but after surgery, it decreased in the first week and started to improve thereafter. These findings are important for clinicians when selecting the appropriate therapy for HCC.

Targeting S1PR1 May Result in Enhanced Migration of Cancer Cells in Bladder Carcinoma.

Simple SummaryMetastasis is critical to the prognosis of patients with bladder cancer, and it is important to understand the mechanism of its occurrence. S1PR1 expression is thought to be associated with poor prognosis, but it is unknown whether it is associated with tumor metastasis. Analysis of clinical gene expression data suggests that endothelial or immune cells in tumor tissue may be the source of S1PR1 expression. Comparative analysis of clinical tumor tissues with bladder cancer cells suggests that S1PR1 expression is associated with cellular adhesion. In vitro experiments demonstrated that S1PR1 expression was negatively correlated with cancer cell motility, and that S1PR1 inhibition by FTY-720 may cause an increase in cancer cell motility, suggesting that the use of S1PR1 inhibition as a synergistic therapy requires additional observations and considerations.Clinical bladder tumor histological analysis shows that high expression of S1PR1 is associated with poor patient prognosis. However, there are no studies that describe the underlying mechanism. To investigate the relative distribution and actual function of S1PR1 in bladder tumors, we analyzed multiple clinical databases in combination with tumor purity and immune cell infiltration simulations, as well as databases of well-defined histological phenotypes of bladder cancer, and single-cell sequencing of adjacent normal tissues and bladder tumors, and further compared them with bladder cancer cell lines. The results showed that S1PR1 expression was generally higher in normal tissues than in bladder cancer tissues, and its distribution was mainly in endothelial cells or immune cells. The association between high S1PR1 expression and poor prognosis may be due to tumor invasion of adjacent normal tissues, where highly expressed S1PR1 may affect prognostic interpretation. The effect of S1PR1 itself on cancer cells was associated with cell adhesion, and in bladder cancer cells, S1PR1 expression was negatively correlated with cell motility. Moreover, the use of FTY-720 will cause an increased metastatic ability of bladder cancer cells. In conclusion, we suggest that the use of S1PR1-specific inhibition as a synergistic treatment requires more observation and consideration.

986P Antitumor effect of zolbetuximab combined with chemotherapeutic agents or an anti-mPD-1 antibody in syngeneic immune-competent mice

Zolbetuximab, a chimeric IgG1 monoclonal antibody, binds to Claudin 18.2 and mediates cancer death via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Phase III studies of zolbetuximab plus chemotherapy (fluoropyrimidine and oxaliplatin) are ongoing. Along with chemotherapy, immunotherapy is becoming standard of care for gastric cancer. The beneficial effect of zolbetuximab plus chemotherapy or anti-mouse PD-1 (anti-mPD-1) antibody is presented here. In this study, we examined the effect of zolbetuximab plus 5-fluorouracil + oxaliplatin (chemotherapy; Study 1) or an anti-mPD-1 antibody (Study 2) in a syngeneic mouse tumor model with mouse Claudin 18.2-expressing CLS-103 gastric carcinoma cells. Tumor cells were inoculated into immune-competent NMRI mice. Rituximab, PBS + 5% glucose, and anti-trinitrophenol antibody were used as isotype controls or vehicle (Table). Tumor volumes were assessed in both studies. In Study 1, flow cytometry was used to analyze the frequency of immune cells in tumor tissues. In Study 1, tumor growth was inhibited by 65%, 42%, or 93%, in the zolbetuximab, chemotherapy, or combination groups, respectively. Tumor volume was significantly lower with the combination versus zolbetuximab (P < 0.05) or chemotherapy alone (P < 0.01). The frequency of tumor-infiltrating CD8+ T-cells was significantly higher with the combination versus control (P < 0.05). In Study 2, tumor growth was inhibited by 50%, 60%, or 91%, in the zolbetuximab, the anti-mPD-1 antibody, or combination groups, respectively. Tumor volume was significantly lower with the combination versus zolbetuximab (P < 0.01) or anti-mPD-1 antibody (P < 0.05). Zolbetuximab combined with chemotherapy or an anti-mPD-1 antibody more potently inhibited tumor growth, compared with each treatment alone, in a syngeneic tumor model.Table: 986PStudy groupsStudy 1, n=15 each groupStudy 2, n=12 each groupGroup 1: control (rituximab + PBS + 5% glucose)Group 1: control (rituximab + anti-trinitrophenol antibody)Group 2: zolbetuximab (zolbetuximab + PBS + 5% glucose)Group 2: zolbetuximab (zolbetuximab + anti-trinitrophenol antibody)Group 3: chemotherapy (rituximab + 5-FU + oxaliplatin)Group 3: anti-mPD-1 antibody (rituximab + anti-mPD-1 antibody)Group 4: combination (zolbetuximab + 5-FU + oxaliplatin)Group 4: combination (zolbetuximab + anti-mPD-1 antibody)Abbreviations: 5-FU, 5-fluorouracil; anti-mPD-1, anti-mouse PD-1. Open table in a new tab

Correlation analysis of tumor mutation burden of hepatocellular carcinoma based on data mining.

The aim of this study was to determine the relationship between tumor mutation burden (TMB) and prognosis of patients with hepatocellular carcinoma (HCC), and to explore the differential expression of genes in HCC by TMB and the relationship between immune cells, TMB, and HCC. Somatic variation data, gene transcriptional expression data and clinical information of patients with HCC were obtained from cancer genome map (TCGA) database. Analyze the characteristics of the gene mutation data of the sample, divide the high and low TMB groups and draw the survival curve at the same time, carry on the difference analysis to the gene of TMB, further carry on the univariate Cox regression analysis and Lasso regression analysis and construct the clinical model. Download the dataset GSE14520, from the Gene Expression Omnibus (GEO) database to verify the genes of the prognostic model. The differential genes were analyzed by gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes by (KEGG) enrichment analysis. Then the relative abundance of 22 immune cell types in HCC and normal control samples was calculated. Finally, the correlation between the scores of immune cells and Risk model was analyzed. Tumor protein p53 (TP53), catenin1 (CTNNB1), titin (TTN), mucin 16 (MUC16), and albumin (ALB) are the most common top 5 mutations in HCC. The prognosis of high level TMB group is worse than that of low TMB group. A total of 122 differentially expressed genes were screened by differential analysis of TMB genes. SQSTM1, ME1, BAMBI and PTTG1 are independent risk factors for poor prognosis of HCC. GO and KEGG analysis showed that the differential genes were mainly in extracellular matrix and immune response. There were significant differences in the distribution of Macrophages M0 and T cells CD4 native cells between HCC and normal tissues, which were correlated with the differential genes of TMB and correlated with prognosis. There is a negative correlation between TMB and the prognosis of patients with HCC. TMB has an effect on the differential expression of genes in HCC cells and the distribution of immune cells in tumor tissues.

Multiplex Immunofluorescence Tyramide Signal Amplification for Immune Cell Profiling of Paraffin-Embedded Tumor Tissues.

Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Tumor microenvironment-associated gene C3 can predict the prognosis of colorectal adenocarcinoma: a study based on TCGA.

Colorectal cancer is one of the most common malignancies. With continuous exploration of the interaction between tumor cells and the immune system, tumor immunotherapy has become a revolution. However, CRC remains one of the less effective tumors for immunotherapy. The tumor microenvironment plays an important role in tumorigenesis and progression. The aim of this study is to explore tumor microenvironment-related genes that can predict the prognosis of colorectal adenocarcinoma, and also to provide new ideas for the mechanism of tumor development as well as immunotherapy. After estimating Stromalscore and Immunescore of colorectal adenocarcinoma tumor samples according to RNA-Seq expression data downloaded from TCGA, we screened for TME-related differential genes. We filtered prognosis-related core genes by constructing protein-protein interaction networks and making one-factor cox analysis for prognosis. Finally, the relative content of 22 immune cells in tumor tissues was evaluated, and then immune cells associated with core genes were identified. We screened 773 differential genes related to the TME. Then we identified C3 as a core gene associated with prognosis. Single gene analysis showed that C3 expression was significantly higher in tumor tissues than in normal tissues (p < 0.001). High C3 expression was associated with lower overall survival (p = 0.046). Tumor immune cell analysis showed that mast cells resting, mast cells activated, T cells CD4 memory activated, eosinophils, and macrophages M0 were C3-associated immune cells. C3 has potential as a biomarker for colorectal adenocarcinoma and could provide new research ideas for the diagnosis and treatment of colorectal adenocarcinoma, especially for immunotherapy.

Preclinical analysis of novel prognostic transcription factors and immune-related gene signatures for bladder cancer via TCGA-based bioinformatic analysis.

Bladder cancer (BLCA) is a common malignancy of human urinary tract, whose prognosis is influenced by complex gene interactions. Immune response activity can act as a potential prognostic factor in BLCA. The present study established a prognostic model, based on the identification of tumor transcription factors (TFs) and immune-related genes (IRGs), and further explored their therapeutic potential in BLCA. The enrichment scores of 29 IRG sets, identified in The Cancer Genome Atlas BLCA tumor samples, were quantified by single-sample Gene Set Enrichment Analysis. The abundance of infiltrated immune cells in tumor tissues was determined using the Estimating Relative algorithm. Tumor-related TFs and IRGs signatures were retrieved using Least Absolute Shrinkage and Selection Operator Cox regression analysis. A prognostic gene network was built using Pearson's correlation analysis as a means of predicting the regulatory relationship between prognostic TFs and IRGs. A nomogram was devised to also predict the overall survival (OS) rate of patients with BLCA. Based on the Genomics of Drug Sensitivity in Cancer data, potential therapeutic drugs were identified upon analyzing the relationship between the expression level of prognostic genes and respective IC50 values. In vitro experiments were implemented for further validation. Respective TF binding profiles were acquired from the JASPAR 2020 database. The elevated infiltration of CD8+ T Cells was correlated with an improved OS of patients with BLCA. An innovative prognostic model for BLCA was then constructed that composed of nine putative gene markers: CXCL13, prepronociceptin, microtubule-associated protein tau, major histocompatibility class I polypeptide-related sequence B, prostaglandin E2 receptor EP3 subtype, IL20RA, proepiregulin, early growth response protein 1 and FOS-related antigen 1 (FOSL1). Furthermore, a theoretical basis for the correlation between the prognostic TFs and IRGs was reported. For this, 10 potentially effective drugs targeting the TFs in the present model for patients with BLCA were identified. It was then verified that downregulation of FOSL1 can lead to an enhanced sensitivity of the TW37 in T24 bladder cancer cells. Overall, the present prognostic model demonstrated a robust capability of predicting OS of patients with BLCA. Hence, the gene markers identified could be applied for targeted therapies against BLCA.

Tumor infiltrated immune cell types support distinct immune checkpoint inhibitor outcomes in patients with advanced non-small cell lung cancer.

The evaluation of PD‐L1 expression alone has limitations in predicting clinical outcome in immune‐checkpoint inhibitors (ICI). This study aimed to evaluate the predictive and prognostic effects of the presence of various immune cells in pretreatment tissue samples and to identify determinants associated with response in patients with advanced non‐small cell lung cancer (NSCLC) treated with PD‐1 blockade. Immune cell distribution was heterogeneous and the most dominant immune cell type was T cells. Patients with durable clinical benefit (DCB) showed significantly higher PD‐L1 expression. The ratio of tumor/stroma region of T cell, B cell, and macrophage was significantly higher in patient with DCB. High intratumoral T‐ and B‐cell density (≥median) was associated with DCB in the low PD‐L1 expression (<50%) group. In univariate analyses, the overall survival (OS) benefit was shown according to intratumoral B‐cell density (p = 0.0337). The incidence of hyperprogressive disease (HPD) was 13.0%. The Chi‐square test revealed that HPD was significantly associated with intratumoral B‐cell density but not T‐cell or macrophage density. Our results demonstrate different predictive and prognostic values for infiltrating immune cells in tumor tissue, which may help in selecting patients for ICI.

Steatohepatitis Impairs T-cell–Directed Immunotherapies Against Liver Tumors in Mice

Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline-deficient diet or a choline-deficient l-amino acid-defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intraperitoneal injections of an antibody against OX40 (aOX40) on days 3, 7, and 10 after injection of the tumor cells. We measured tumor growth and analyzed immune cells in tumor tissues by flow cytometry. Mice were given N-acetylcysteine to prevent loss of CD4+ T cells from liver. Administration of M30 and aOX40 inhibited growth of tumors from intrahepatic injections of B16 or CT26 cells in mice on regular diet. However, M30 and/or aOX40 did not slow growth of liver tumors from B16 or CT26 cells in mice with diet-induced steatohepatitis (methionine and choline-deficient diet or choline-deficient l-amino acid-defined diet). Steatohepatitis did not affect the ability of M30 to slow growth of subcutaneous B16 tumors. In mice with steatohepatitis given N-acetylcysteine, which prevents loss of CD4+ T cells, M30 and aOX40 were able slow growth of hepatic tumors. Flow cytometry analysis of liver tumors revealed reduced CD4+ T cells and effector memory cells in mice with vs without steatohepatitis. Steatohepatitis reduces the abilities of immunotherapeutic agents, such as M30 and aOX40, to inhibit tumor liver growth by reducing tumor infiltration by CD4+ T cells and effector memory cells. N-acetylcysteine restores T-cell numbers in tumors and increases the ability of M30 and aOX40 to slow tumor growth in mice.

MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway.

Mucin1 (MUC1) upregulation in colon cancer has been linked to poor patient outcomes and advanced stage at diagnosis. This is partially due to MUC1-mediated inhibition of T-cell proliferation affecting efficient lysis by cytotoxic lymphocytes, which contributes to escape from immune surveillance. In the present study, human colorectal cancer tissues were collected, and MUC1-positive and MUC1-negative colon cancer mouse models were prepared; subsequently, the number and function of immune cells in tumor tissues were measured using flow cytometry. The present study revealed that MUC1, as a tumor-associated antigen, can recruit more tumor-infiltrating lymphocytes into the tumor microenvironment compared with MUC1-negative colon cancer, but that these cells could not serve antitumor roles. Conversely, the present study demonstrated that MUC1-positive colon cancer attracted more regulatory T cells (Treg cells), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) to the tumor site than MUC1-negative colon cancer. Furthermore, the data suggested that programmed death protein 1 (PD1)-programmed death ligand 1 (PDL1) expression is greater in MUC1-positive colon cancer. Blocking the PD1-PDL1 signaling pathway reduced the percentage of Treg cells, MDSCs and TAMs in the tumor microenvironment, enhanced T-cell cytotoxicity and inhibited tumor growth, prolonging the survival time of MUC1-positive tumor-bearing mice. Therefore, the present study elucidated the role of MUC1 in tumor immune escape and provides a foundation for the application of PDL1 inhibitors to MUC1-positive colon cancer.

HCA587 Protein Vaccine Induces Specific Antitumor Immunity Mediated by CD4+ T-cells Expressing Granzyme B in a Mouse Model of Melanoma.

The antigen HCA587 (also known as MAGE-C2), which is considered a cancer-testis antigen, exhibits upregulated expression in a wide range of malignant tumors with unique immunological properties, and may thus serve as a promising target for tumor immunotherapy. The study aimed to explore the antitumor effect of the HCA587 protein vaccine and the response of humoral and cell-mediated immunity. The HCA587 protein vaccine was formulated with adjuvants CpG and ISCOM. B16 melanoma cells were subcutaneously inoculated to C57BL/6 mice, followed by treatment with HCA587 protein vaccine subcutaneously. Mouse survival was monitored daily, and tumor volume was measured every 2 to 3 days. The tumor sizes, survival time and immune cells in tumor tissues were detected. And the vital immune cell subset and effector molecules were explored. After treatment with HCA587 protein vaccine, the vaccination elicited significant immune responses, which delayed tumor growth and improved animal survival. The vaccination increased the proportion of CD4+ T cells expressing IFN-γ and granzyme B in tumor tissues. The depletion of CD4+T cells resulted in an almost complete abrogation of the antitumor effect of the vaccination, suggesting that the antitumor efficacy was mediated by CD4+ T cells. In addition, knockout of IFN-γ resulted in a decrease in granzyme B levels, which were secreted by CD4+ T cells, and the antitumor effect was also significantly attenuated. The HCA587 protein vaccine may increase the levels of granzyme B expressed by CD4+ T cells, and this increase is dependent on IFN-γ, and the vaccine resulted in a specific tumor immune response and subsequent eradication of the tumor.

Clinical relevance and prognostic significance of PD-1/PD-Ls in non-metastatic bladder cancer: A role for PD-L2

BackgroundBladder cancer (BC) can be successfully treated by manipulating immune responses with intravesical Bacillus Calmette-Guerin instillation or targeting the PD-1/PD-L signaling pathway. In the present study we investigated the prognostic significance of the immune checkpoint inhibitor PD-1 and its ligands PD-L1 and PD-L2 on tumor cells and infiltrating lymphocytes, in the tumor microenvironment and draining lymph nodes in patients with non-metastatic BC. Patients and methodsCells were mechanically isolated from tissues and draining lymph nodes from 58 patients, and surface-stained for CD45, PD-1, PD-L1 and PD-L2. The cells were then analyzed with a flow cytometric method. ResultsApproximately 2% of CD45-negative tumor and stromal cells expressed PD-L1. Expression was not associated with the main clinicopathological characteristics of the disease or with survival. However, as tumors progressed the frequency of PD-L1+CD45hi cells and the mean expression of PD-1 on CD45hi cells increased remarkably on immune cells in tumor tissues and draining lymph nodes. In addition, frequency analysis showed that cell percentages as well as mean expression of PD-L2 on total CD45+ lymphocytes and their CD45hi subpopulation in tumor-draining lymph nodes was significantly associated with cancer-related death (P < 0.05). Multiple Cox regression also revealed that while CD45+ (hazard ratio: 0.596, 95 % CI 0.439–0.809, P = 0.001) was associated with improved survival, CD45neg (HR: 0.615, 95 % CI 0.454–0.831, P = 0.002), and PD-L2+CD45+ cells (hazard ratio: 1.472, 95 % CI 1.023–2.120, P = 0.038) in draining lymph nodes were associated with lower survival. ConclusionOur results suggest that in patients with BC, PD-1 and PD-L expression on immune cells, especially in draining lymph nodes, is valuable for predicting prognosis and survival, and possibly responsiveness to immunotherapy. However, expression of the inhibitor molecule or its ligands on tumor cells was not associated with prognosis. The results highlight the significance of PD-L2 as a second important suppressive molecule in tumors.

Correlations analysis between HCC mutation burden and patients’ prognosis based on data mining

Objective To study the correlations between tumor mutation burden (TMB) and the prognosis of hepatocellular carcinoma (HCC) patients, and to investigate the effect of TMB on differential expression genes of HCC and the proportion of invasive immune cells in tumor tissues. Methods The somatic variation data, gene transcriptional expression data and clinical information of HCC patients were obtained from the cancer genome atlas (TCGA) database. The R program language (version 3.6.1) maftools function package was used to analyze the gene mutation data characteristics of the samples. The TMB value of each sample was calculated using the full-exon sequencing data of patients with hepatocellular carcinoma on the VarScan2 platform, sorted by TMB value, and the median value was used to divide all samples into high TMB and low TMB groups. Kaplan-Meier method was used to draw the survival curves of two groups of patients and log-rank test was performed to determine the correlation between tumor mutation load and prognosis. The Limma function package of R language was used to screen the differentially expressed genes between the two groups (FDR=0.05 and logFC=1), and the clusterProfiler function package of R language was used to perform gene ontology (GO) enrichment analysis of the differential genes and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis (screening criteria were all P<0.05). Then the CIBERSORT tool was used to compare and analyze the difference in the proportion of invasive immune cells between the two groups. Results A total of 364 patients with HCC from TCGA database were included in the study. Mutations were found in 327(84%) samples, and there was a synergistic correlation between OBSCN and FLG mutations (P 0.05). A total of 198 with differentially expressed genes (28 up-regulated genes and 170 down-regulated genes) were screened between the high TMB group and the low TMB group. In GO analysis, it was found that the differentially expressed genes were mainly enriched in extracellular matrix tissues, extracellular structural tissues, extracellular matrix, extracellular matrix containing collagen, extracellular matrix structural components and other functions. In KEGG analysis, differential genes were highly enriched in extracellular matrix receptor interaction pathway and adhesive plaque pathway. In the correlation analysis of the proportion of infiltrating immune cells, CD4+ memory T cells were more infiltrating in the low TMB group (P<0.05). Monocytes showed a higher degree of infiltration in the high TMB group (P<0.05). Conclusion There was no correlation between TMB and the prognosis of HCC patients. TMB has significant influence on the differential expression genes of HCC and the proportion of invasive immune cells in tumor tissues. Key words: Carcinoma, hepatocellular; Tumor mutation burden; Prognosis

Correlation analysis of the prognosis of HPV positive oropharyngeal cancer patients with T cell infiltration and neoantigen load

Oropharyngeal cancer is one type of head and neck squamous cell carcinoma that is commonly associated with human papillomavirus (HPV) infection. Its incidence is increasing year by year. However, in the clinical treatment, it is found that the overall prognosis of HPV positive oropharyngeal cancer patients is better than that of HPV negative patients. But till now, the underlying mechanism that leads to this phenomenon has not been fully elucidated. This research analyzed the immune cell infiltration and function, as well as neoantigen loads between HPV positive and negative patients by bioinformatic analysis using the TCGA database, and found that the overall survival rate of HPV positive patients was significantly higher than those in the HPV negative group. Analysis of the relative abundance of immune cells in tumor tissues showed that CD8 + T cells in HPV positive patients were significantly increased compared to those in HPV negative patients, and the expression levels of effector molecules, like IFN-γ and Granzyme B, were significantly upregulated. Meanwhile, the analysis of tumor neoantigen load (TNB) showed that the TNB of HPV positive patients was lower than that of the HPV negative group. This study provides some basic theoretical support for the treating HPV-related oropharyngeal cancer.

Abstract 2104: Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells

Abstract The purpose of our study was to develop near-infrared fluorophore-labeled antibodies to overcome autofluorescence interference in the visible spectrum, thereby enabling enhanced multiplexed identification of immune cells in human tumor tissue sections. Towards multiplexing, we conjugated each monoclonal primary antibody to a unique hapten tag. Detection of hapten-tagged primary antibodies was accomplished using high-affinity anti-hapten rabbit monoclonal secondary antibodies, which were each conjugated to a near-infrared fluorophore with Emax = 594, 650, 731, or 800 nm. For identification of immunosuppressive cells in tumor tissues, we used formalin-fixed, paraffin-embedded human tissue microarrays comprising malignant breast or prostate tumor core sections. Microarrays contained 12-60 patient biopsy cores with duplicate sections per case mounted on standard glass slides. Two antibody panels to detect immunosuppressive cell phenotypes were used in our study. The T regulatory panel targeted the antigens CD4, CD8, FOXP3, and LAG-3, allowing for phenotypic identification of CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+ FOXP3+ Tregs, and highly immunosuppressive CD4+ FOXP3+ LAG-3+ Tregs. The macrophage panel targeted the antigens CD3, CD20, CD68, and CD163 for identification of CD3+ T cells, CD20+ B cells, CD68+ M1 macrophages, and highly immunosuppressive CD163+ M2 macrophages. Widefield fluorescence microscopy was used to image the stained tumor microarrays, utilizing a high-radiance mercury arc lamp, high-sensitivity digital CMOS camera, and single-band optical filter sets to detect near-infrared fluorescence. Triplicate images were taken per tissue core. Prior to image analysis, a spectral unmixing algorithm was created using a reference set of single-stained tissue slides in order to deconvolute minor spectral overlap of near-infrared fluorophores. The algorithm was then applied to the multiplexed digital raw images, and immunophenotyping analysis was conducted to enumerate cells of interest. Image analysis results indicated unique patterns of distribution of immune cells in tumor tissues correlated with tumor grade and stage. In breast cancer tissues, higher levels of LAG-3+ immunosuppressive cells were found in tumors of the aggressive, treatment-resistant "triple-negative" tumor subtype. In prostate cancer tissues, an increase in CD68+ macrophages and loss of immunostimulatory CD3+ T cells and CD20+ B cells were found to be associated with advanced tumor grade and Gleason score. The use of near-infrared fluorophores and hapten-tagged detection antibodies facilitated enhanced, rapid multiplexed phenotyping of immunosuppressive cells in tumor tissue. These findings demonstrate our approach to be a potentially paradigm-shifting technology for detection of immune infiltrates in patient samples. Citation Format: Amy Flor, Annalise Vaccarello, Matt Levin, Helen Snyder, David Schwartz, Stephen Kron. Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2104.