Lymphocytes In Tumor Microenvironment Research Articles

Abstract 2660: CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment

Abstract Background: Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only about 20% of patients with a variety of cancers. Literature has demonstrated that “hot tumor” which contains high T lymphocytes in tumor microenvironment exhibits a better response to immunotherapies than “cold tumor”. Therefore, we hypothesize that T lymphocyte-recruited factors play the roles determining the immunotherapeutic efficacy, including CXCL9, 10, and 11, which are located in human chromosome 4 with consistent co-expression and protein structure. Aims: The aim of this study is to investigate whether CXCL9/10 is critical in determining the anti-PD-(L)1 immunotherapeutic efficacy. Methods: GEPIA, cBioPortal, and Kaplan-Meier Plotter were used to investigate the gene expression, co-expression, and immunotherapeutic association for CD274 (PD-L1), ICAM1, and CXCL9/10 in lung, colon, and liver cancers. qPCR and flow cytometry were used to detect the gene expression in tumor cells and T lymphocytes, particularly comparing the differences between LL/2 and CT26 cells whereas LL/2 is immunotherapy-resistant and CT26 is immunotherapy-sensitive. CD8+ T cell transwell assay and qPCR were used to measure the function of CXCL10 on CD8+ T cell migration and activation, whereas IFNγ and IL-2 as the activation markers. Results: We found that CXCL9/10 overexpressed in a variety of tumors, including lung, colon, and liver tumors with a correlation with PD-L1. However, PD-L1 was not overexpressed in most tumors. High PD-L1 and CXCL10 are basically associated with better survival rates in tumor patients receiving immunotherapies. Correlation analysis revealed that IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression in lung cancer. Since literature has indicated that LL/2-derived syngeneic tumor exhibits immunotherapy-resistant and CT26 exhibits immunotherapy-sensitive, we further compared the gene expression between them. We found that IFNA, IFNG, CXCL10, and PD-L1 all expressed higher levels in CT26 cells than LL/2. Moreover, we found that T lymphocytes specifically expressed high CXCR3, a receptor of CXCL9/10, including CD4+ T and CD8+ T cells by flow cytometry. As expected, CD8+ T cells can be recruited in a transwell assay by IFNα and CXCL9. In addition, we found that IFNα induced CXCL10 expression in monocytes and CXCL10 increased activation markers IFNγ and IL-2 in CD4+ T and CD8+ T cells. Conclusions: This study revealed that CXCL9/10 expression is correlated with PD-L1 in tumors, resulting in better immunotherapeutic efficacy. The evidence demonstrated that CT26 intrinsically exhibited higher IFNs and CXCL9/10 compared to LL/2. Therefore, CXCL9/10 potentially recruits and activates CD8+ T cells to tumor microenvironment, transferring “cold tumor” to “hot tumor”. This study indicates that CXCL9/10 is critical for immunotherapeutic efficacy. Citation Format: Chun-Chia Cheng, Jungshan Chang, Zong-Lin Sie, Ai-Sheng Ho, Cheng-Liang Peng, Chun-Chao Chang. CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2660.

ScRNA+TCR+BCR-seq revealed the proportions and gene expression patterns of dual receptor T and B lymphocytes in NPC and NLH

While the relationship between single receptor lymphocytes and cancer has been deeply researched, the origin and biological roles of dual receptor lymphocytes in tumor microenvironment (TME) remain largely unknown. And since nasopharyngeal carcinoma (NPC) is a type of cancer closely associated with immune infiltration, studying the TME of NPC holds particular significance. Utilizing single-cell RNA sequencing combined with T cell receptor (TCR) and B cell receptor (BCR) sequencing (scRNA + TCR + BCR-seq), we analyzed data from 7 patients with NPC and 3 patients with nasopharyngeal lymphatic hyperplasia (NLH). In our research, it was firstly found that the presence of dual receptor lymphocytes in both the TME of NPC and the inflammatory environment of NLH. We also confirmed their clonal expansion, suggesting their potential involvement in the immune response. Subsequently, we further discovered the lineage and the pairing characteristics. It was found that the dual receptor lymphocytes in NPC and NLH mainly originate from memory cells, and the predominant pairing type for dual TCR was β+α1+α2 and dual BCR was heavy+κ+λ. By further analyzing their gene expression, we compared the function of dual receptor cells with single receptor cells in the context of both NPC and NLH. This groundbreaking research has enhanced our comprehension of the features of dual-receptor cells and has contributed to a better understanding of the TME in NPC. By comparing with NLH, it illuminates part of the alterations in the process of malignant transformation in NPC. These findings present the potential to acquire improved diagnostic markers and treatment modalities.

Expression of IDO1 in Tumor Microenvironment Significantly Predicts the Risk of Recurrence/Distant Metastasis for Patients With Esophageal Squamous Cell Carcinoma

In this study, we aimed to explore immune markers predicting locoregional recurrence/distant metastasis (R/M) for patients with esophageal squamous cell carcinoma (ESCC) post-surgical intervention by using a novel high-throughput spatial tool to quantify multiple immune proteins expressed in ESCC and lymphocytes in tumor microenvironment (TME-L). First, formalin-fixed paraffin-embedded tissues from surgical patients with ESCC (n = 94) were constructed on a microarray, which was then divided into discovery (n = 36) and validation cohorts (n = 58). Using a newly developed GeoMx digital spatial profiling tool, 31 immune proteins in paired ESCC and TME-L, morphologically segmented by PANCK and CD45, respectively, from the discovery cohort were quantified, releasing 2,232 variables. Next, the correlation matrix was analyzed using the Corrplot package in R Studio, resulting in 6 closely correlated clusters. The Least Absolute Shrinkage and Selection Operator regression scoring model predictive of R/M risk with superior specificity was successfully established based on the 3 following hierarchically clustered immune proteins: ARG1 in ESCC/PANCK+, STING, and IDO1 in TME-L/CD45+. Moreover, the expression of IDO1 in TME-L, rather than in ESCC, significantly predicted the R/M risk score with an area under curve of 0.9598. In addition, its correlation with R/M status was further validated by dual immunohistochemistry staining of IDO1 and CD45 in discovery and validation cohorts. Above all, our findings not only provide a more accurate scoring approach based on quantitative immune proteins for the prediction of R/M risk, but also propose that IDO1 in TME-L potentially plays a driving role in mediating R/M in ESCC.

Abstract 4333: Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer

Abstract Background: Recently, anti-PD-L1 in combination with cytotoxic chemotherapy has shown significant survival benefit in a randomized phase 3 trial for unresectable or metastatic biliary tract cancer (BTC). However, no biomarker including PD-L1 expression has been established to predict clinical outcomes, and there is an unmet need for a novel predictive biomarker for anti-PD-1 or PD-L1 therapy. Here, we assessed TILs using artificial intelligence (AI)-powered spatial analysis in advanced BTC treated with anti-PD-1 beyond 1st line treatment. Methods: An AI-powered whole-slide image (WSI) analyzer (Lunit SCOPE IO, Lunit, Seoul, Korea) was used to segment tumor epithelium and stroma, and identification of intratumor TIL (iTIL) and stromal TIL (sTIL). H&E stained WSI from pre-treatment samples was acquired from Asan Medical Center (n =166), and a total of 154 samples (92.8%) after quality control were used for the final analysis. Immune phenotypes (IP) were defined as follow: inflamed as high iTIL and sTIL; immune-excluded as low iTIL and high sTIL; immune-desert as low TIL overall. Among them, 20 patients were available for multi-color flow cytometry analysis (FACS) using peripheral blood mononuclear cells, collected at baseline, C1D8, and C2D1. Results: All patients (n=154) were treated with anti-PD-1 (pembrolizumab or nivolumab) monotherapy, and 72 of 154 patients (46.8%) were treated as 2nd line. Gemcitabine plus cisplatin (GemCis) was used prior to anti-PD-1 as first-line therapy in all patients. Overall, 15 (9.7%) patients showed inflamed IP. With median follow-up duration of 15.4 months, the inflamed IP group showed better overall survival (17.2 vs. 6.6 months, P=0.03), and progression-free survival (PFS; 4.5 vs. 2.6 months, P=0.09) along with higher PFS rate at 12 months (33.3% vs. 11.5%, P=0.035), and overall response rate (26. 7% vs. 8.6%, P=0.053) than other phenotype groups. There was no significant difference in median PFS with GemCis among IP groups (P=0.74). In the FACS available subgroup, inflamed IP showed higher baseline central memory T (Tcm)+effector memory T (Tem)/Tnaive ratio than other IPs. With the administration of anti-PD-1, Tcm+Tem/Tnaive ratio was increased, while the proportion of PD1+CD8+T, CD39+CD8+T, CD103+CD8+T and Treg were decreased in the inflamed IP group than other phenotype groups. Conclusions: Immune phenotypes classified by AI-powered spatial TIL analysis was effective to predict the clinical outcomes of patients with advanced BTC treated with anti-PD-1 therapy. Citation Format: Yeong Hak Bang, Kyunghye Bang, Jin Ho Shin, Hyunseok Yoon, Kyu-Pyo Kim, Inkeun Park, Jae Ho Jeong, Heung-Moon Chang, Baek-Yeol Ryoo, Chiyoon Oum, Seulki Kim, Yoojoo Lim, Gahee Park, Chan-Young Ock, Changhoon Yoo. Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4333.

Whole-slide image analysis identifies a high content of Hodgkin Reed-Sternberg cells and a low content of T lymphocytes in tumor microenvironment as predictors of adverse outcome in patients with classic Hodgkin lymphoma treated with ABVD.

Classic Hodgkin lymphoma (cHL) constitutes the most frequent lymphoma in young adults. Its histopathology is unique as a scattered tumor population, termed Hodgkin Reed-Sternberg (HRS) cells is diluted in a prominent tumor microenvironment (TME) composed of T lymphocytes, B lymphocytes, macrophages, neutrophils, eosinophils and histiocytes. Traditionally, the identification of prognostic biomarkers in the cHL TME has required visual inspection and manual counting by pathologists. The advent of whole-slide imaging (WSI) and digital image analysis methods could significantly contribute to improve this essential objective in cHL research, as a 10-20% of patients are still refractory or relapsed after conventional chemotherapy. In this work, we have digitized a total of 255 diagnostic cHL slides and quantified the proportion of HRS cells (CD30), B cells (CD20) and T cells (CD3) by digital image analysis. Data obtained where then correlated with the overall survival (OS) and progression free survival (PFS) of cHL patients. Quantification of HRS cells, B cells and T cells reflects the biological heterogeneity of the different cHL histological subtypes analyzed. A percentage of 2.00% of HRS cells statistically significantly discriminated between patients achieving a complete metabolic response (CMR) and refractory or relapsed (R/R) patients both for the OS (P=0.001) and PFS (P=0.005). Furthermore, patients with a percentage of T cells below the 26.70% in the TME showed a statistically significantly shorter OS (P=0.019) and PFS (P=0.041) in comparison with patients above this threshold. A subgroup of patients with a low content of T cells and high content of HRS cells exhibited a special aggressive clinical course. Currently, there is the need to implement quantitative and easy scalable methods to enhance clinical translation, as the cHL TME plays a central role in the clinical course of the disease. The results of this study could contribute to the identification of prognostic biomarkers specifically looking at the cHL TME and their inclusion in future clinical trials.

CD40L-armed oncolytic herpes simplex virus suppresses pancreatic ductal adenocarcinoma by facilitating the tumor microenvironment favorable to cytotoxic T cell response in the syngeneic mouse model

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Despite the promising outcome of immune checkpoint inhibitors and agonist antibody therapies in different malignancies, PDAC exhibits high...

CD169 Expression on Lymph Node Macrophages Predicts in Patients With Gastric Cancer.

The induction of an anti-cancer immune responses is potentially associated with the efficacy of anti-cancer therapy. Recent studies have indicated that sinus macrophages in regional lymph nodes are involved in anti-cancer immune responses in the cancer microenvironment. In the present study, we investigated the correlation between lymphocyte infiltration in cancer tissues and macrophage activation in regional lymph nodes. We retrospectively identified 294 patients with gastric cancer who underwent surgery from 2008 to 2012. Using immunohistochemistry, we evaluated CD169-expression on CD68-positive macrophages, and the density of CD8-postive lymphocytes in tumor microenvironment. We statistically examined the correlation between CD169 and CD8 expression, and performed Cox regression analysis of potential prognostic factors, including CD169 and CD8 expression, for cancer-specific survival (CSS) in patients with total and advanced gastric cancer. CD169 overexpression in lymph node sinus macrophages (LySMs) was positively correlated to the density of CD8-positive lymphocytes in primary cancer tissues (R = 0.367, p < 0.001). A high density of CD8-positive T lymphocytes in the primary site and a high level of CD169 expression in LySMs were independently associated with greater CSS in patients with total and advanced gastric cancer (p < 0.05 for all). The expression on CD169 in LySMs is a predictor of a favorable clinical course in patients with gastric cancer, and might be useful for evaluating anti-cancer immune responses.

813 Melanoma exosomes mediates the dysfunction of interferon signaling pathway in lymphocytes in tumor microenvironment

813 Melanoma exosomes mediates the dysfunction of interferon signaling pathway in lymphocytes in tumor microenvironment

Phenotypic Analysis of Tumor Tissue-Infiltrating Lymphocytes in Tumor Microenvironment of Bladder Cancer and Upper Urinary Tract Carcinoma.

There are no previous reports directly evaluating immunologic conditions in tumor microenvironment including both bladder cancer (BCa) and upper urinary tract carcinoma (UTUC). In this study, we aimed to clarify the difference of immunity status and its clinical significance depending on the tumor site in urothelial carcinoma. Tumor tissue-infiltrating lymphocytes were extracted from 70 urothelial cancer patients who underwent surgical resection (52 cases of BCa and 18 cases of UTUC). The immunologic classification was established by unsupervised clustering analysis according to the expression ratio of 9 extracellular surface markers measured by flow cytometry, and we examined the relationship between immunologic classification and clinical importance such as pathologic status and prognosis (progression-free survival and cancer-specific survival). The immunologic condition was classified into 2 groups. Group 1 (n= 41) comprised the CD4 T-cell-dominant group and group 2 (n=29) the immunologically activated group. This immunologic classification was significantly correlated with tumor grade (P= .020) but not tumor location in multivariate analysis. In invasive BCa patients (n= 33), progression-free survival and cancer-specific survival of group 2 were significantly worse than those of group 1 (P= .021 and P=.022, respectively), while there was no significant difference between groups 1 and 2 in patients with invasive UTUC (n= 17). Although there was no difference in the local immunologic condition of urothelial carcinoma between BCa and UTUC, its significance as a prognostic predictor might vary depending on tumor site.

Expression of PD-1 on CD4+ Tumor-Infiltrating Lymphocytes in Tumor Microenvironment Associated with Pathological Characteristics of Breast Cancer.

Objective This study aimed to investigate the correlation of CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes with pathological characteristics in breast cancer patients. Methods A cross-sectional study consecutively recruited 133 patients with invasive ductal breast cancer. The expression of CD4, programmed cell death protein 1 (PD-1), CK7, CK20, E-cadherin, or Ki-67 was detected by immunohistochemistry. The associations between CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes and pathological characteristics were evaluated. Results Elderly patients intended to have a lower level of CD4+/PD-1− tumor-infiltrating lymphocytes (p < 0.05). Patients with positive E-cadherin expression had higher median cell counts of CD4+/PD-1− tumor-infiltrating lymphocytes than patients with negative E-cadherin expression (30/HPF versus 10/HPF, p < 0.05). Counts of CD4+/PD-1+ tumor-infiltrating lymphocytes had a significant correlation with Ki-67 index that the correlation coefficient was 0.29 (p = 0.001). Positive CK20 expression was related to a higher level of CD4+/PD-1− tumor-infiltrating lymphocytes than negative CK20 expression (73/HPF versus 30/HPF, p < 0.05). Conclusion CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes showed diverse association with pathological features of breast cancer. CD4+/PD-1+ tumor-infiltrating lymphocytes had a significant relationship with Ki-67 expression whereas CD4+/PD-1− tumor-infiltrating lymphocytes had a significant relationship with E-cadherin expression. Further studies are warranted to explore the immunomodulatory effects of phenotypes of CD4+ T cell subsets in breast cancer.

SAT-145 - Fgl2 contributes to hepatocellular carcinoma via suppressing the inflammatory phenotypes and functions of dentritic cells and cytotoxic lymphocytes in tumor microenvironment

SAT-145 - Fgl2 contributes to hepatocellular carcinoma via suppressing the inflammatory phenotypes and functions of dentritic cells and cytotoxic lymphocytes in tumor microenvironment

Hypofractionated radiotherapy and tumor immunity — new concepts and new combination

Modern immunology has established that tumor immune escape is associated with hidden or missing tumor-specific antigens and tumor-associated antigens, as well as immune suppressors that are released from tumor cells to inhibit the immune cytotoxicity and antigen-presenting cells (APCs). The changes in tumor microenvironment have an impact on tumor immunity and treatment outcomes. The immune effects finally depend on activation and inhibition of T cell receptors and other co-regulated receptors (CD28, CD80/CD86, and CTLA-4) in spite of the existence of APCs and cytotoxic T lymphocytes in tumor microenvironment. Recent studies have revealed that radiotherapy induced not only DNA damage but also immunogenesis in tumor cells. Both conventionally fractionated radiotherapy and hypofractionated radiotherapy can induce immunogenesis in tumor cells. Immunogenic regulation makes many tumor antigens expressed in cells exposed to irradiation, which induces immune recognition and cytotoxicity; cell content (DNA, HMGB1, etc.) released from dead immunogenic cells can trigger immune effects and in situ tumor vaccination, which further induce an abscopal effect of radiotherapy. A lot of anti-tumor immunotherapy fails to achieve satisfactory treatment outcomes. Therefore, how to combine radiotherapy, especially stereotactic body radiotherapy, with anti-tumor immunotherapy has recently become a new challenge for researchers. Key words: Neoplasms/radiotherapy; Neoplasms/immunotherapy

Hereditary cancer syndromes: utilizing DNA repair deficiency as therapeutic target.

Human cells have numerous repair mechanisms to counteract various insults incurred on the DNA. Any mutation in these repair mechanisms can lead to accumulation of DNA errors and carcinogenesis. This review aims to discuss the therapeutic options in the two most common DNA repair deficient cancer syndromes, namely Lynch syndrome (hereditary non-polyposis colorectal cancer) and breast cancer susceptibility gene (BRCA) associated ovarian and breast cancer. Deficiency in DNA repair mechanisms renders these tumors with increased sensitivity to platinum agents. There has been increasing amount of information on the utility of the defects in DNA repair as targets for cancer therapy in these syndromes. Novel therapies like poly (ADP-ribose) polymerase (PARP) inhibitors are one of such example where the induction of double stranded breaks in DNA leads to tumoricidal effect in patients with homologous DNA repair deficiency. Interestingly, patients with DNA repair deficiencies tend to have a more favorable prognosis than sporadic malignancies. In microsatellite high colorectal cancer patients, this has been attributed to increased recruitment of CD8+ T lymphocytes in tumor microenvironment. However, these tumors are able to limit the host immune response by activation of immune checkpoints that seem like attractive targets of therapy in the future.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy.

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.