Specific Immune Infiltration Research Articles

Activin levels correlate with lymphocytic infiltration in epithelial ovarian cancer.

The TGF-β superfamily member activin, a dimer of the gene products of INHBA and/or INHBB, has been implicated in immune cell maturation and recruitment, but its immune impact within epithelial ovarian cancer (EOC) is not well characterized. We sought to explore differences in activin (INHBA/ Inhibin-βA and INHBB/ Inhibin-βB) between malignant and ovarian tissues at the RNA and protein level and assess the relationship between activin and immune cells in EOC. Publicly available RNA sequencing data were accessed from GEO (#GSE143897) with normalization and quantification performed via DESeq2. Immune gene expression profile was further explored within the TCGA-OV cohort derived from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to evaluate activin A and T-cell markers CD8 and FoxP3 at the protein level. ELISA to activin-A was used to assess levels in the ascites of advanced EOC patients. Kaplan-Meier curves were generated to visualize survival outcomes. Gene expression levels of components of the activin signaling pathway were elevated within EOC when compared to a benign cohort, with differences in activin type I/II receptor gene profiles identified. Additionally, INHBA gene expression was linked to lymphocytic immune markers in EOC samples. Immunohistochemistry analysis revealed a positive correlation of CD8 and FOXP3 staining with activin A at the protein level in both primary and metastatic epithelial ovarian cancer samples. Furthermore, Activin-A (inhibin-βA) is significantly elevated in EOC patient ascites. INHBA expression is elevated within EOC, correlating with worse survival, with activin protein levels correlating with specific immune infiltration. Our findings suggest that activin-A may play a role in suppressing anti-tumor immunity in EOC, highlighting its potential as a therapeutic target.

Multiple omics integrative analysis identifies GARS1 as a novel prognostic and immunological biomarker: from pan-cancer to bladder cancer

Glycyl-tRNA synthetase (GARS1) is differentially expressed across cancers. In this study, the value of GARS1 in the diagnosis and prognosis of various cancers was comprehensively evaluated by multiple omics integrative pan-cancer analysis and experimental verification. Through Kaplan–Meier, ROC and multiple databases, we explored GARS1 expression and prognostic and diagnostic patterns across cancers. The GARS1 relative reaction network was identified in PPI, GO, KEGG, methylation models and the genetic mutation atlas. Further research on the GARS1 value in bladder urothelial carcinoma (BLCA) was conducted by regression and nomogram models. We further analyzed the correlation between GARS1 and immune markers and cells in BLCA. Finally, in vitro experiments were used to validate GARS1 the oncogenic function of GARS1 in BLCA. We found that GARS1 was highly expressed across cancers, especially in BLCA. GARS1 expression was correlated with poor survival and had high diagnostic value in most tumor types. GARS1 is significantly associated with tRNA-related pathways whose mutation sites are mainly located on tRNA synthetase. In addition, Upregulation of GARS1 was connected with immune cell infiltration and five key MMR genes. M2 macrophages, TAMs, Th1 and T-cell exhaustion, and marker sets associated with GARS1 expression indicated specific immune infiltration in BLCA. Finally, in vitro experiments validated that GARS1 expression promotes BLCA cell proliferation and metastasis and inhibits apoptosis. Overall, GARS1 can be a novel prognostic and immunological biomarker through multiple omics integrative pan-cancer analysis. The expression of GARS1 in BLCA was positively correlated with specific immune infiltration, indicating that GARS1 might be related to the tumor immune microenvironment.

Bioinformatics analysis reveals the landscape of immune cell infiltration and novel immune-related biomarkers in moyamoya disease.

Objective: This study aimed to identify immune infiltration characteristics and new immunological diagnostic biomarkers in the cerebrovascular tissue of moyamoya disease (MMD) using bioinformatics analysis. Methods: GSE189993 and GSE141022 were downloaded from the GEO database. Differentially expressed gene and PPI analysis were performed. After performing WGCNA, the most significant module associated with MMD was obtained. Next, functional pathways according to GSEA, GO, and KEGG were enriched for the aforementioned core genes obtained from PPI and WGCNA. Additionally, immune infiltration, using the CIBERSORT deconvolution algorithm, immune-related biomarkers, and the relationship between these genes, was further explored. Finally, diagnostic accuracy was verified with ROC curves in the validation dataset GSE157628. Results: A total of 348 DEGs were screened, including 89 downregulated and 259 upregulated genes. The thistlel module was detected as the most significant module associated with MMD. Functional analysis of the core genes was chiefly involved in the immune response, immune system process, protein tyrosine kinase activity, secretory granule, and so on. Among 13 immune-related overlapping genes, 4 genes (BTK, FGR, PTPN11, and SYK) were identified as potential diagnostic biomarkers, where PTPN11 showed the highest specificity and sensitivity. Meanwhile, a higher proportion of eosinophils, not T cells or B cells, was demonstrated in the specific immune infiltration landscape of MMD. Conclusion: Immune activities and immune cells were actively involved in the progression of MMD. BTK, FGR, PTPN11, and SYK were identified as potential immune diagnostic biomarkers. These immune-related genes and cells may provide novel insights for immunotherapy in the future.

Immunogenic cell death related risk model to delineate ferroptosis pathway and predict immunotherapy response of patients with GBM.

Immunogenic cell death (ICD) is a type of cell death that leads to the regulation and activation of the immune response, which is marked by the exposure and delivery of damage‐associated molecular patterns (DAMPs) in the tumor microenvironment. Accumulating evidence has revealed the significance of ICD-related genes in tumor progression and therapeutic response. In this study, we obtained two ICD-related clusters for glioblastoma (GBM) by applying consensus clustering, and further constructed a risk signature on account of the prognostic ICD genes. Based on the risk signature, we found that higher risk scores were associated with worse patient prognosis. Besides, the results illustrated that ferroptosis regulators/markers were highly enriched the high-risk group, and ferroptosis were correlated with cytokine signaling pathway and other immune-related pathways. We also discovered that high-risk scores were correlated to specific immune infiltration patterns and good response to immune checkpoint blockade (ICB) treatment. In conclusion, our study highlights the significance of ICD-related genes as prognostic biomarkers and immune response indicators in GBM. And the risk signature integrating prognostic genes possessed significant potential value to predict the prognosis of patients and the efficacy of ICB treatment.

Effects of a natural nutritional supplement on immune cell infiltration and immune gene expression in exercise-induced injury.

Inflammatory immune response plays a key role in exercise-induced injury and healing; however, the relevant regulatory mechanisms of immune infiltration in exercise-induced injuries remain less studied. In the present study, a highly efficient system for screening immunity-related biomarkers and immunomodulatory ability of natural nutritional supplements was developed by integrating intelligent data acquisition, data mining, network pharmacology, and computer-assisted target fishing. The findings demonstrated that resting natural killer cells showed a higher rate of infiltration after exercise, whereas naive B cells and activated dendritic cells showed higher rate of infiltration before exercise. Four key genes, namely PRF1, GZMB, CCL4, and FASLG, were associated with exercise-induced injuries and inflammatory immune response. In total, 26 natural compounds including echinacoside, eugenol, tocopherol, and casuariin were predicted by using the HERB databases. Molecular docking analysis showed that GZMB, FASLG, and CCL4 bound to echinacoside. In vivo experiments in mice showed that after 30 min swimming, natural killer (NK) cells showed high infiltration rates, and the key genes (GZMB, PRF1, FASLG, and CCL4) were highly expressed; however, echinocandin significantly reduced the level of NK cells and decreased the expression of the four key genes post exercise. This natural nutritional supplement may act to protect against inflammatory injury after exercise by suppressing specific immune infiltration.

Assessment of the immune landscapes of advanced ovarian cancer in an optimized in vivo model.

BackgroundOvarian cancer (OC) is typically diagnosed late, associated with high rates of metastasis and the onset of ascites during late stage disease. Understanding the tumor microenvironment and how it impacts the efficacy of current treatments, including immunotherapies, needs effective in vivo models that are fully characterized. In particular, understanding the role of immune cells within the tumor and ascitic fluid could provide important insights into why OC fails to respond to immunotherapies. In this work, we comprehensively described the immune cell infiltrates in tumor nodules and the ascitic fluid within an optimized preclinical model of advanced ovarian cancer.MethodsGreen Fluorescent Protein (GFP)‐ID8 OC cells were injected intraperitoneally into C57BL/6 mice and the development of advanced stage OC monitored. Nine weeks after tumor injection, mice were sacrificed and tumor nodules analyzed to identify specific immune infiltrates by immunohistochemistry. Ascites, developed in tumor bearing mice over a 10‐week period, was characterized by mass cytometry (CyTOF) to qualitatively and quantitatively assess the distribution of the immune cell subsets, and their relationship to ascites from ovarian cancer patients.ResultsTumor nodules in the peritoneal cavity proved to be enriched in T cells, antigen presenting cells and macrophages, demonstrating an active immune environment and cell‐mediated immunity. Assessment of the immune landscape in the ascites showed the predominance of CD8+, CD4+, B–, and memory T cells, among others, and the coexistance of different immune cell types within the same tumor microenvironment.ConclusionsWe performed, for the first time, a multiparametric analysis of the ascitic fluid and specifically identify immune cell populations in the peritoneal cavity of mice with advanced OC. Data obtained highlights the impact of CytOF as a diagnostic tool for this malignancy, with the opportunity to concomitantly identify novel targets, and define personalized therapeutic options.

Abstract PS17-45: Metastatic tissues display organ specific immune infiltration archetypes; lessons from a rapid autopsy tissue collection study

Abstract Immune composition in the tumor microenvironment (TME) of patient tumors has proven to play a central role in the development of metastases and response to therapy. Evidence has suggested that the metastatic TME is immune aberrant, however difficulty in obtaining biopsies of metastatic tumors has made assessment of the immune TME difficult. Here we utilize a rapid autopsy tissue collection protocol to assess the infiltration and composition of the immune TME in numerous metastatic tissue sites, paired disease-free tissue sites, and the associated tissue draining lymph nodes. Post-mortem tissues were collected from nine metastatic breast cancer patients shortly after death through City of Hope’s “Legacy Project for Rapid Tissue Donation” Program. The average post-mortem interval (PMI) for tissue collection was 6 hours. Collected specimens include metastatic lesions and paired non-cancer samples from every cancer-involved organ, disease-free specimens from non-involved major organs, distant and tumor-draining lymph nodes (both cancer-infiltrated and disease free), as well as blood and spleens. Immediately following collection, specimens were processed into single cell suspension for flow cytometry. Over 80 immune cell phenotypes were assessed, including CD8+ and CD4+ T cell subsets, B cell subsets, natural killer (NK) cells, tumor associated macrophages (TAMs), dendritic cell subsets, and other immune cells. Tumor infiltrated tissues were found to have comparable immune cell densities and composition compared to paired disease-free tissues of the same organ type. However, immune cell densities in metastatic tissues and disease-free tissues were significantly different between organ types, with lung immune infiltration consistently being greater than liver, brain, and skin tissues. Differences in immune composition between tissue sites were also observed. Notably, liver tissues favored the presence of IL-2 producing central memory CD8+ T cells, while lung tissues favored the presence of CD8+ tissue resident memory T cells and CD16+ NK cells. Relative to disease-free lung tissues, tumor infiltrated lungs contained diminished frequencies of CD8+ tissue resident memory T cells and altered B cell and monocyte phenotypes. Increased levels of targetable immune pathways were observed, including increased PD-L1 and CTLA-4 expressing cells in skin metastases, and increased GARP+ B cells in bone marrow metastases. These data suggest that immune monitoring and trafficking of metastatic tissues site is dictated by organ type, which can be altered in composition by tumor infiltration. Further studies such as these may reveal organ-specific mechanisms of response to therapeutic interventions. Identity of organ location for tumor metastases may guide choices for immunotherapeutic interventions. Citation Format: Colt Egelston, Weihua Guo, Eliza R. Bacon, Kena Ihle, Diana Simons, Christian Avalos, JIayi Tan, Jian Ye, Lei Wang, Minhui Lim, James R. Waisman, Peter P Lee. Metastatic tissues display organ specific immune infiltration archetypes; lessons from a rapid autopsy tissue collection study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-45.

Prediction of radio-responsiveness with immune-profiling in patients with rectal cancer.

We evaluate whether the tumor immune infiltrate (TIL) could be used for prediction of responsiveness to preoperative chemoradiotherapy (PCRT) in rectal cancers. Using formalin-fixed paraffin-embedded slides of pretreatment biopsies, co-stain for CD4, CD8, CD274 (PD-L1), FOXP3, cytokeratin, and DAPI was performed with Opal multi staining kit (Perkin-Elmer, Waltham, MA). Multispectral imaging and digital analysis to visualize and quantify specific immune infiltrates were performed using the Vectra imaging system (Perkin-Elmer). The density (number of cells per mm2) and proportion of total TILs and specific cell types in the stroma were calculated by inForm™ 2.2.1 software (Perkin-Elmer). The density and proportion of total TILs and specific cell types in the stroma were calculated by inForm™ 2.2.1 software (Perkin-Elmer, Waltham, MA). Patients were classified as group with total regression (TR, n = 25) and group with residual disease (near total, moderate, and minimal regression, RD, n = 50). The mean density of T cell infiltration and CD274 (PD-L1)+ lymphocyte were significantly higher in TR (p = 0.005, p = 0.001). The proportion of CD4+ lymphocyte (p=0.042) and CD274 (PD-L1)+ lymphocyte (p = 0.002) were different between 2 groups. The TR group has lower CD4+ and higher CD274 (PD-L1)+ proportions than RD group. The ratio among CD4+, CD8+, CD274 (PD-L1)+, FOXP3+ T cell was different between groups. TR group showed lower CD4/ CD274 (PD-L1) (p = 0.007), CD8/ CD274 (PD-L1) (p = 0.02), and FOXP3/ CD274 (PD-L1) (p = 0.003) ratio than RD group. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of responsiveness to PCRT.

Abstract 1845: Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. The vast majority of the human HNSCCs contain p53 mutations, some of which acquire oncogenic gain-of-function (GOF) activities. Previous mouse models in which either the p53R172H GOF mutation or deletion of the p53 gene were co-activated with oncogenic K-ras showed that p53R172H can predispose to oral tumor initiation, accelerate tumor growth and promote progression to SCC. However, the impact of p53 gain- and loss-of-function (LOF) mutations in metastasis could not be assessed because of the low rates of progression to SCC in that model. To overcome this limitation, we used the tobacco-surrogate 4NQO to induce oral lesions that may progress to advance carcinomas following stepwise changes that resemble the gradual accumulation of histological and molecular abnormalities observed during human oral cancer progression. To examine the role of p53 mutations during SCC malignant progression we applied 4NQO to mice in which the p53R172H mutation or homozygous deletion of p53 were activated in oral epithelial cells. We observed that oral tumors appeared faster in the presence of p53 mutations than in mice with wtp53. No significant difference was observed between the p53R172H mutation and p53 deletion. However, survival was shorter in mice expressing p53R172H than in mice with loss of p53. Notably, regional and distant metastasis were only observed in mice with oral tumors expressing p53R172H. Therefore, these mice are excellent tools for pre-clinical studies designed to target early stages of tumor development or latest stages of progression and metastasis. As immunotherapy is becoming adopted as part of the standard treatment for head and neck cancer patients, we conducted a detailed characterization of the immune infiltrates of the lesions that developed in these mice in order to assess their immunogenicity. We observed a progressive increase in T-cell infiltration from normal oral mucosa to oral premalignant lesions (OPL) to SCC, in the presence or absence of p53 mutations. Similarly, the expression of immune checkpoint proteins, including PD1, PDL1 and CTLA4 increased during oral tumor progression, regardless of the p53 status. Interestingly, we observed a sharp increase in the presence of immune infiltrates expressing the co-stimulatory OX40 in SCCs relative to premalignant lesions, with different extent depending on the nature of the p53 mutation. The significance of these observations is currently being analyzed. In summary, p53R172H promotes metastasis in oral tumors induced by 4NQO. In addition, T-cell infiltration and activation of immune checkpoints during oral tumor progression suggest that this is an excellent model to test immunotherapy-based strategies. Furthermore, our findings suggest that OX40-based therapies may be tailored to patients according to their p53 status. Citation Format: Jin Wang, Yuanyuan Zhang, Bingbing Wang, Adel K. El-Naggar, Jeffrey N. Myers, Carlos Caulin. Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2017-1845

Imaging Human Immune Cell Infiltration in a Xenograft Graft-Versus-Host Disease Model

Graft versus host disease (GvHD) is one of the major complications of hematopoetic stem cell transplantation (HSCT), and is characterized by massive alloreactive T cell infiltration into target organs and tissues. Currently, non-invasive tools to diagnose and follow up disease are lacking. Here we show non-invasive imaging of infiltrating immunocytes in a human xenograft model for chronic GvHD, using a single domain camelid-derived antibody fragment (VHH) specific for human Class II MHC products present on myeloid cells, B cells and activated T cells. NSG (NOD-scid gamma) mice reconstituted with human fetal thymus, bone marrow and liver (BLT mice) spontaneously develop a GvHD-like condition, characterized by alopecia, blepharitis and target organ infiltration by activated human T cells. In diseased animals, we show an increased PET signal in the liver, attributable to infiltration of activated Class II MHC positive T cells. Non-invasive imaging of specific immune infiltration and activation could thus be of importance for diagnosis and treatment evaluation of GvHD and holds promise for other diseases characterized by inflammation. DisclosuresNo relevant conflicts of interest to declare.

Abstract P5-04-07: Multispectral imaging allows visualization and quantification of multiple immunologic cell types in breast tumor tissues

Abstract Background: Multiple studies have identified tumor infiltrating lymphocytes (TIL) in breast cancer and shown them to have prognostic and predictive significance. TIL are comprised of CD8+ and CD4+ T cells, regulatory T cells (Treg) and B cells. To date, the majority of studies have identified TIL using H&E staining. Studies utilizing IHC to stain for TIL generally have been limited to evaluating a single immune cell phenotype such as CD8+ T cells, while studies evaluating multiple immune cell types have generated single cell suspensions from tumors for analysis by flow cytometry to determine relative percentages of specific immune cell populations. Although these studies have confirmed that immune infiltrates in tumors are heterogeneous, they have not addressed the spatial relationship between tumor and immune cells, which could be critical to anti-tumor immune effects. This study was undertaken to demonstrate the feasibility of multispectral imaging and multiplexed immunofluorescence to visualize and quantify specific immune infiltrates in the tumor and surrounding stroma on single FFPE tissue sections. Methods: Single FFPE slides from 9 HER2+ breast cancer patients receiving neoadjuvant chemotherapy were stained with primary antibodies targeting cytokeratin, CD8, CD4, FoxP3 (Treg), CD20 (B-cells) and PD-L1 (T cell inhibitory molecule). Tyramide signal amplification was used to improve signal and specificity, as well as to reduce background. The Vectra multispectral imaging instrument was used for image acquisition and InForm software was used to quantitate the density (number of cells per square millimeter) of specific cell types in the tumor and in the surrounding stroma. Results: Multispectral imaging successfully captured and quantified multiple immune cell types in all tissues (images to be shown at time of presentation). CD8+ and CD4+ T cell densities as well as PD-L1 expression in the tumor and surrounding stroma are shown. Summary of immune cell infiltrate Within Tumor Within Stroma SampleCD8 densityCD4 densityPDL1 H-scoreCD8 densityCD4 densityPDL1 H-score1050631301207342523059189238312260022111331104305083833106345020808206131751011891591110700417754621858511431739186812198591617448342570196 For patients not achieving a pathologic complete response (pCR), the density of both the CD8 (p=.03) and CD4 (p=.05) infiltrates in the stroma were significantly greater than in the tumor. For patients achieving a pCR, there was no significant difference in the densities of stromal and intratumoral CD8 (p=.11) or CD4 (p=.75) infiltrates suggesting that T cell infiltration into the tumor from the stroma is critical. Conclusion: Multispectral imaging allows different immune cell phenotypes to be visualized and quantified simultaneously in the same tissue section enabling further study of the relationships and distribution of these cells within the tumor and tumor microenvironment, and their spatial distribution and proximity to the tumor cells. This technology will enable improved understanding of the immune infiltrate in breast tumors thereby facilitating the rational design and use of immunotherapeutic agents in combination with standard systemic therapies. Citation Format: Elizabeth A Mittendorf, Chichung Wang, Peng Yau, Kristin Roman, Yun Wu, Gheath Alatrash, Clifford Hoyt. Multispectral imaging allows visualization and quantification of multiple immunologic cell types in breast tumor tissues [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-07.