Pro-tumor Cells Research Articles

Engineered Immunologic Niche Monitors Checkpoint Blockade Response and Probes Mechanisms of Resistance.

Antibodies to programmed cell death protein1 (anti-PD-1) have become a promising immunotherapy for triple negative breast cancer (TNBC), blocking PD-L1 signaling from pro-tumor cells through T cell PD-1 receptor binding. Nevertheless, only 10-20% of PD-L1+ metastatic TNBC patients who meet criteria benefit from ICB, and biomarkers to predict patient response have been elusive. We have previously developed an immunological niche, consisting of a microporous implant in the subcutaneous space, that supports tissue formation whose immune composition is consistent with that within vital organs. Herein, we investigated dynamic gene expression within this immunological niche to provide biomarkers of response to anti-PD-1. In a 4T1 model of metastatic TNBC, we observed sensitivity and resistance to anti-PD-1 based on primary tumor growth and survival. The niche was biopsied before, during, and after anti-PD-1 therapy, and analyzed for cell types and gene expression indicative of treatment refractivity. Myeloid cell-to-lymphocyte ratios were altered between ICB-sensitivity and resistance. Longitudinal analysis of gene expression implicated dynamic myeloid cell function that stratified sensitivity from resistance. A niche-derived gene signature predicted sensitivity or resistance prior to therapy. Analysis of the niche to monitor immunotherapy response presents a new opportunity to personalize care and investigate mechanisms underlying treatment resistance.

Machine learning‐based identification of a cell death‐related signature associated with prognosis and immune infiltration in glioma

AbstractAccumulating evidence suggests that a wide variety of cell deaths are deeply involved in cancer immunity. However, their roles in glioma have not been explored. We employed a logistic regression model with the shrinkage regularization operator (LASSO) Cox combined with seven machine learning algorithms to analyse the patterns of cell death (including cuproptosis, ferroptosis, pyroptosis, apoptosis and necrosis) in The Cancer Genome Atlas (TCGA) cohort. The performance of the nomogram was assessed through the use of receiver operating characteristic (ROC) curves and calibration curves. Cell‐type identification was estimated by using the cell‐type identification by estimating relative subsets of known RNA transcripts (CIBERSORT) and single sample gene set enrichment analysis methods. Hub genes associated with the prognostic model were screened through machine learning techniques. The expression pattern and clinical significance of MYD88 were investigated via immunohistochemistry (IHC). The cell death score represents an independent prognostic factor for poor outcomes in glioma patients and has a distinctly superior accuracy to that of 10 published signatures. The nomogram performed well in predicting outcomes according to time‐dependent ROC and calibration plots. In addition, a high‐risk score was significantly related to high expression of immune checkpoint molecules and dense infiltration of protumor cells, these findings were associated with a cell death‐based prognostic model. Upregulated MYD88 expression was associated with malignant phenotypes and undesirable prognoses according to the IHC. Furthermore, high MYD88 expression was associated with poor clinical outcomes and was positively related to CD163, PD‐L1 and vimentin expression in the in‐horse cohort. The cell death score provides a precise stratification and immune status for glioma. MYD88 was found to be an outstanding representative that might play an important role in glioma.

Abstract B014: Immuno-modulation of the tumor microenvironment of pancreatic adenocarcinoma following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT)

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors. Unlike other tumors, the progress of systemic therapies has been very slow, mainly due to the peculiar and resistant tumor microenvironment (TME) of PDAC. The addition of ablative stereotactic body radiation therapy (SBRT) in a total neoadjuvant strategy is promising for the treatment of localized PDAC and is currently being explored in several clinical trials. However, if radiation therapy possesses the ability to modulate the TME, the impact of high-dose SBRT is still poorly known in PDAC. Here, we aim to characterize by immunohistochemistry (IHC) and RNA sequencing (RNAseq) analysis the immuno-modulations of the TME following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT). MATERIAL: Paraffin-embedded residual tumoral tissues of 50 localized PDAC resected between 2011 and 2020 were used: seventeen patients had surgery first, seventeen received an induction chemotherapy with FOLFIRINOX (FFX) only and sixteen with FFX followed by an iHD-SBRT designed to individually maximize the dose prescribed to the tumor and vessels interfaces up to Dmax(0.5cc)<53Gy in 5 fractions. RESULTS: We found that although collagen deposition increases significantly after iHD-SBRT (COL1: 83.27 vs 78.60 vs 68.04%, p<0.001 for iHD-SBRT, FFX and treatment naïve cohort respectively), the intra-tumoral lymphocyte infiltration is globally not altered, including for cytotoxic CD8+ lymphocytes. Only the IHC expression of CD4+ T helper population is significantly decreased after iHD-SBRT. Using gene set enrichment analysis (GSEA) of the KEGG pathway, the IL-17 signaling pathway related to the T17 cells is particularly significantly increased after iHD-SBRT compared to FFX only. After iHD-SBRT, the IHC expression levels of FOXP3+ cells and PD-L1 are significantly increased. No difference is observed for CD68+ expression however, cell type enrichment analysis from our RNA-seq data using xCell algorithm shows that the M2-polarized CD68+ cells are increased after iHD-SBRT compared to FFX alone. PD-1 expression is significantly decreased after iHD-SBRT. Furthermore, RNAseq data revealed that iHD-SBRT is associated with significant enrichment in basaloid cells (p<0.0024), a subtype of basal–like cells associated with immunomodulatory stroma and better clinical outcomes. GSEA canonical pathway analysis further revealed metabolism-related pathways such as oxidative phosphorylation and gluthathione metabolism significantly enriched in iHD-SBRT treated group, suggesting the potential of mitochondrial inhibitors as candidates for combination therapy. CONCLUSIONS: iHD-SBRT is able to durably immuno-modulate the TME of PDAC. If the overall T-cell infiltration is preserved, we have identified some increased pro-tumoral cells and pathways after iHD-SBRT that could improve the development of better-oriented combination trials involving high-dose SBRT. Citation Format: Christelle Bouchart, Oier Azurmendi Senar, Julie Navez, Laurine Verset, Anaïs Boisson, Matthieu Hein, Nicky D’Haene, Dirk Van Gestel, Luigi Moretti, Pieter Demetter, Jean-Baptiste Bachet, Karen Willard-Gallo, Rémy Nicolle, Tatjana Arsenijevic, Jean-Luc Van Laethem. Immuno-modulation of the tumor microenvironment of pancreatic adenocarcinoma following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B014.

C5aR1 blockade reshapes immunosuppressive tumor microenvironment and synergizes with immune checkpoint blockade therapy in high-grade serous ovarian cancer

ABSTRACT High-grade serous ovarian cancer (HGSC), with a modest response to immune checkpoint blockade (ICB) targeting PD-1/PD-L1 monotherapy, is densely infiltrated by M2-polarized tumor-associated macrophages (TAMs) and regulatory T (Treg) cells. The complement C5a/C5aR1 axis contributes to the programming of the immunosuppressive phenotype of TAMs in solid tumors and represents a promising immunomodulatory target for treating HGSCs. Here, we aimed to identify the relevance of C5aR1 in prognosis, immune microenvironment, and immunotherapy response in HGSCs. The expression and relationship of C5aR1 with tumor-infiltrating immune cells were assessed by immunohistochemistry and flow cytometry in the training cohort (n = 120) and fresh HGSC tissues (n = 36). Transcriptomic analyses of the xenografts delineated the mechanisms driving the immunomodulatory activity of PMX53, an orally bioavailable C5aR1 inhibitor. Therapeutic relevance was confirmed in ex vivo tumor cultures and The Cancer Genome Atlas (TCGA) datasets. C5aR1 expression independently predicted dismal prognosis and was linked to the immunoevasive subtype of HGSC, characterized by increased infiltration of pro-tumor cells (Treg cells, M2-polarized macrophages, and neutrophils) and impaired CD8+T functions. PMX53 antagonized subcutaneous tumor growth, modulated immunosuppressive mechanisms and synergized with aPD-1 in several tumor types. Single-cell RNA-seq analysis revealed predominant C5aR1 expression in TAMs, with an immunosuppressive-related expression signature in C5aR1+TAMs. Furthermore, the combination of C5aR1 and PD-L1 was associated with specific molecular characteristics and matched clinical response annotations. Therefore, the abundance of C5aR1 could predict an inferior prognosis in HGSCs, and incorporating PD-L1 may serve as a novel predictive biomarker to guide therapeutic options.

The immune checkpoint adenosine 2A receptor is associated with aggressive clinical outcomes and reflects an immunosuppressive tumor microenvironment in human breast cancer.

The crosstalk between the immune system and cancer cells has aroused considerable interest over the past decades. To escape immune surveillance cancer cells evolve various strategies orchestrating tumor microenvironment. The discovery of the inhibitory immune checkpoints was a major breakthrough due to their crucial contribution to immune evasion. The A2AR receptor represents one of the most essential pathways within the TME. It is involved in several processes such as hypoxia, tumor progression, and chemoresistance. However, its clinical and immunological significance in human breast cancer remains elusive. The mRNA expression and protein analysis were performed by RT-qPCR and immunohistochemistry. The log-rank (Mantel-Cox) test was used to estimate Kaplan-Meier analysis for overall survival. Using large-scale microarray data (METABRIC), digital cytometry was conducted to estimate cell abundance. Analysis was performed using RStudio software (7.8 + 2023.03.0) with EPIC, CIBERSORT, and ImmuneCellAI algorithms. Tumor purity, stromal and immune scores were calculated using the ESTIMATE computational method. Finally, analysis of gene set enrichment (GSEA) and the TISCH2 scRNA-seq database were carried out. Gene and protein analysis showed that A2AR was overexpressed in breast tumors and was significantly associated with high grade, elevated Ki-67, aggressive molecular and histological subtypes, as well as poor survival. On tumor infiltrating immune cells, A2AR was found to correlate positively with PD-1 and negatively with CTLA-4. On the other hand, our findings disclosed more profuse infiltration of protumoral cells such as M0 and M2 macrophages, Tregs, endothelial and exhausted CD8+ T cells within A2ARhigh tumors. According to the Single-Cell database, A2AR is expressed in malignant, stromal and immune cells. Moreover, it is related to tumor purity, stromal and immune scores. Our results also revealed that CD8+T cells from A2ARhigh patients exhibited an exhausted functional profile. Finally, GSEA analysis highlighted the association of A2AR with biological mechanisms involved in tumor escape and progression. The present study is the first to elucidate the clinical and immunological relevance of A2AR in breast cancer patients. In light of these findings, A2AR could be deemed a promising therapeutic target to overcome immune evasion prevailing within the TME of breast cancer patients.

A key driver to promote HCC: Cellular crosstalk in tumor microenvironment.

Liver cancer is the third greatest cause of cancer-related mortality, which of the major pathological type is hepatocellular carcinoma (HCC) accounting for more than 90%. HCC is characterized by high mortality and is predisposed to metastasis and relapse, leading to a low five-year survival rate and poor clinical prognosis. Numerous crosstalk among tumor parenchymal cells, anti-tumor cells, stroma cells, and immunosuppressive cells contributes to the immunosuppressive tumor microenvironment (TME), in which the function and frequency of anti-tumor cells are reduced with that of associated pro-tumor cells increasing, accordingly resulting in tumor malignant progression. Indeed, sorting out and understanding the signaling pathways and molecular mechanisms of cellular crosstalk in TME is crucial to discover more key targets and specific biomarkers, so that develop more efficient methods for early diagnosis and individualized treatment of liver cancer. This piece of writing offers insight into the recent advances in HCC-TME and reviews various mechanisms that promote HCC malignant progression from the perspective of mutual crosstalk among different types of cells in TME, aiming to assist in identifying the possible research directions and methods in the future for discovering new targets that could prevent HCC malignant progression.

Identification and verification of eight cancer-associated fibroblasts related genes as a prognostic signature for head and neck squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) can exert their immunosuppressive effects by secreting various effectors that are involved in the regulation of tumor-infiltrating immune cells as well as other immune components in the tumor immune microenvironment (TIME), thereby promoting tumorigenesis, progression, metastasis, and drug resistance. Although a large number of studies suggest that CAFs play a key regulatory role in the development of head and neck squamous cell carcinoma (HNSCC), there are limited studies on the relevance of CAFs to the prognosis of HNSCC. In this study, we identified a prognostic signature containing eight CAF-related genes for HNSCC by univariate Cox analysis, lasso regression, stepwise regression, and multivariate Cox analysis. Our validation in primary cultures of CAFs from human HNSCC and four human HNSCC cell lines confirmed that these eight genes are indeed characteristic markers of CAFs. Immune cell infiltration differences analysis between high-risk and low-risk groups according to the eight CAF-related genes signature hinted at CAFs regulatory roles in the TIME, further revealing its potential role on prognosis. The signature of the eight CAF-related genes was validated in different independent validation cohorts and all showed that it was a valid marker for prognosis. The significantly higher overall survival (OS) in the low-risk group compared to the high-risk group was confirmed by Kaplan-Meier (K-M) analysis, suggesting that the signature of CAF-related genes can be used as a non-invasive predictive tool for HNSCC prognosis. The low-risk group had significantly higher levels of tumor-killing immune cell infiltration, as confirmed by CIBERSORT analysis, such as CD8+ T cells, follicular helper T cells, and Dendritic cells (DCs) in the low-risk group. In contrast, the level of infiltration of pro-tumor cells such as M0 macrophages and activated Mast cells (MCs) was lower. It is crucial to delve into the complex mechanisms between CAFs and immune cells to find potential regulatory targets and may provide new evidence for subsequently targeted immunotherapy. These results suggest that the signature of the eight CAF-related genes is a powerful indicator for the assessment of the TIME of HNSCC. It may provide a new and reliable potential indicator for clinicians to predict the prognosis of HNSCC, which may be used to guide treatment and clinical decision-making in HNSCC patients. Meanwhile, CAF-related genes are expected to become tumor biomarkers and effective targets for HNSCC.

Targeting of non-apoptotic cancer cell death mechanisms by quercetin: Implications in cancer therapy.

The ultimate goal of cancer treatment is to kill cancer cells, based on the use of various therapeutic agents, such as chemotherapy, radiotherapy, or targeted therapy drugs. Most drugs exert their therapeutic effects on cancer by targeting apoptosis. However, alterations in apoptosis-related molecules and thus assisting cells to evade death, eventually lead to tumor cell resistance to therapeutic drugs. The increased incidence of non-apoptotic cell death modes such as induced autophagy, mitotic catastrophe, senescence, and necrosis is beneficial to overcoming multidrug resistance mediated by apoptosis resistance in tumor cells. Therefore, investigating the function and mechanism of drug-induced non-apoptotic cell death modes has positive implications for the development of new anti-cancer drugs and therapeutic strategies. Phytochemicals show strong potential as an alternative or complementary medicine for alleviating various types of cancer. Quercetin is a flavonoid compound widely found in the daily diet that demonstrates a significant role in inhibiting numerous human cancers. In addition to direct pro-tumor cell apoptosis, both in vivo and in vitro experiments have shown that quercetin exerts anti-tumor properties by triggering diverse non-apoptotic cell death modes. This review summarized the current status of research on the molecular mechanisms and targets through which quercetin-mediated non-apoptotic mode of cancer cell death, including autophagic cell death, senescence, mitotic catastrophe, ferroptosis, necroptosis, etc.

Neuronal CaMKK2 promotes immunosuppression and checkpoint blockade resistance in glioblastoma

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME) and is refractory to immune checkpoint blockade (ICB). Here, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a driver of ICB resistance. CaMKK2 is highly expressed in pro-tumor cells and is associated with worsened survival in patients with GBM. Host CaMKK2, specifically, reduces survival and promotes ICB resistance. Multimodal profiling of the TME reveals that CaMKK2 is associated with several ICB resistance-associated immune phenotypes. CaMKK2 promotes exhaustion in CD8+ T cells and reduces the expansion of effector CD4+ T cells, additionally limiting their tumor penetrance. CaMKK2 also maintains myeloid cells in a disease-associated microglia-like phenotype. Lastly, neuronal CaMKK2 is required for maintaining the ICB resistance-associated myeloid phenotype, is deleterious to survival, and promotes ICB resistance. Our findings reveal CaMKK2 as a contributor to ICB resistance and identify neurons as a driver of immunotherapeutic resistance in GBM.

Abstract 1986: Differential T cell response within the tumor microenvironment observed across different indications

Abstract Cytotoxic T-cells (CTLs) in the Tumor immune Microenvironment (TiME) play an important role in mounting anti-tumor immune response. Immunotherapeutic (I/O) agents like checkpoint inhibitor aim to prevent anergy whilst T cell agonists co-stimulate CTLs to reinvigorate them and thus leading to tumor cytotoxicity. In this study we investigate the abundance and functionality of the intratumoral CTLs post stimulation, across different tumor indications, to understand the differential response across samples and indications to immune cell stimulation. These include Head and Neck Squamous cell carcinoma (HNSCC), Renal Cell Carcinoma (RCC), stomach adenocarcinoma (ca-Stomach) and Triple Negative Breast Cancer (TNBC). Using the Farcast࣪ TiME tumor histo-culture platform, that preserves both tumor and immune components in culture, we stimulated tumor explants with anti-CD3 (10ng/ml) + IL2 (100units/ml) for T-cell activation. Supernatants were collected pre-treatment and at 24-hour intervals for evaluation of cytokine release. At termination the explants were either fixed in formalin to generate paraffin blocks or dissociated into single cells for flowcytometry. 3-5 samples from each tumor indication were included in this study. Flowcytometry based immunophenotyping indicated presence of 48.6% ±5.4 live CD3+ T-cells and 20.6% ± 3.1 live CD3+CD8+ CTLs across all indications in the absence of any stimulation. HNSCC, RCC and ca-Stomach had similar proportion of CTLs, while TNBC had much lower proportions of T cell and CTLs. HNSCC and TNBC had a very low proportion of functional Granzyme-B+ CTLs (0.29%±0.08) whereas ca-Stomach and RCC had relatively higher proportion of these cells (6.9%±2.5). The proliferating Ki67+ CTLs sub-population was low across all indications (0.37%±0.2). Upon stimulation there was a significant increase (>2.5-fold, p<0.05) in the proliferating CTLs sub-population across all indications though the cytokine release pattern varied. HNSCC showed a consistent increase (7.4-fold) in functional CTLs in all samples accompanied by a significant increase in INFɣ, Granzyme B and Perforin secretion. RCC and TNBC showed minimal change in functional CTLs but showed a significant increase in secretion of these cytokines. In ca-Stomach there was an increase in INFɣ secretion, but not Granzyme B and Perforin. In summary, while CTLs were present in TiME, the proportions of activated and proliferating sub-populations differed across indications. The presence of active CTLs did not always translate to tumor cytotoxicity. This could be due to other pro-tumor cells and factors that offset the anti-tumor activity of T cells. The TiME platform thus provides a unique platform to study complex interactions between tumor and intratumoral immune cell populations that could be further probed to understand differential response in patients to immunotherapy mediated tumor cytotoxicity. Citation Format: Nandini P. Basak, Vasanth K, Kowshik Jaganathan, Gowri Shankar K, Manimaran A, Rajashekar M, MS Ganesh, Amritha Prabha, Prakash BV, C Jaya Prakash, Sindhu Govindan, Ritu Malhotra, Pradeep Kar, Oliyarasi M, Anirudh Suri, Rachita Rao, Satish Sankaran. Differential T cell response within the tumor microenvironment observed across different indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1986.

BAG3 induces α-SMA expression in human fibroblasts and its over-expression correlates with poorer survival in fibrotic cancer patients.

Hypoxia and angiogenesis in solid tumors are often strictly linked to the development of fibrotic tissues, a detrimental event that compromises the antitumor immunity. As a consequence, tumor aggressiveness and poor patient prognosis relate to higher incidence of tissue fibrosis and stromal stiffness. The molecular pathways through which normal fibroblasts are converted in cancer‐associated fibroblasts (CAFs) have a central role in the onset of fibrosis in tumor stroma, thus emerging as a strategic target of novel therapeutic approaches for cancer disease. Several studies addressed the role of BAG3 in sustaining growth and survival of cancer cell and also shed light on the different mechanisms in which the intracellular protein is involved. More recently, new pieces of evidence revealed a pivotal role of extracellular BAG3 in pro‐tumor cell signaling in the tumor microenvironment, as well as its involvement in the development of fibrosis in tumor tissues. Here we report further data showing the presence of the BAG3 receptor (Interferon‐induced transmembrane protein [IFITM]‐2) on the plasma membrane of normal dermal fibroblasts and the activity of BAG3 as a factor able to induce the expression of α‐smooth muscle actin and the phosphorylation of AKT and focal adhesion kinase, that sustain CAF functions in tumor microenvironment. Furthermore, in agreement with these findings, bag3 gene expression has been analyzed by high throughput RNA sequencing databases from patients‐derived xenografts. A strong correlation between bag3 gene expression and patients' survival was found in several types of fibrotic tumors. The results obtained provide encouraging data that identify BAG3 as a promising therapeutic target to counteract fibrosis in tumors.

Infiltration and Polarization of Tumor-associated Macrophages Predict Prognosis and Therapeutic Benefit in Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive and heterogeneous malignancy. Tumor-associated macrophages (TAMs) are key infiltrating cell populations in the inflammatory microenvironment of malignant tumors including MIBC. It intrigues us to explore the clinical significance and immunoregulatory role of TAMs infiltration and polarization in MIBC. A total of 141 patients with MIBC from Zhongshan Hospital and 391 patients with MIBC from The Cancer Genome Atlas (TCGA) database were included in this study. Moreover, 195 patients who received anti-PD-L1 therapy from the IMvigor210 trial were enrolled. Patients were categorized into three subtypes considering the infiltration level and polarization status of TAMs, denoted as TAMlow (Subtype I), TAMhigh&M2/M1low (Subtype II), and TAMhigh&M2/M1high (Subtype III). Subtype III suffered inferior prognosis, and Subtype II could benefit more from adjuvant chemotherapy (ACT). Subtype III was featured with increased pro-tumor cells and immunosuppressive cytokines, while Subtype II possessed more immunogenic cells infiltration with activated and tumoricidal properties. Subtype II and Subtype III presented basal/squamous-like characterization and showed additional prognostic merit beyond molecular classification. Subtype I exhibited elevated level of FGFR3 signature, while Subtype II had EGFR signaling activation and immunotherapeutic indication. Additionally, Subtype II patients were indeed highly sensitive to PD-L1 blockade therapy in IMvigor210 trial. The infiltration and polarization status of TAMs shaped distinct immune microenvironment with predictive significance for survival outcome, ACT benefit, and PD-L1 blockade therapy sensitivity in MIBC. Immune classification based on TAMs polarization and infiltration might provide tools to tailor chemotherapy and immunotherapy.

Stromal infiltrating mast cells identify immunoevasive subtype high-grade serous ovarian cancer with poor prognosis and inferior immunotherapeutic response

ABSTRACT Tumor infiltrating mast cells (TIMs), with pro- or anti-tumorigenic role in different types of malignancies, have been implicated in resistance to anti-PD1 therapy. Here, we aimed to identify the relevance of TIMs with the prognosis, immune contexture, and immunotherapy in high-grade serous ovarian cancer (HGSOC). Tissue microarrays containing 197 HGSOC patients were assessed by immunohistochemistry (IHC) for detecting the expression of mast cell tryptase and other immune markers. Kaplan-Meier curve, log-rank test, and Cox regression model were applied to perform survival analysis. Single-cell RNA-seq analysis and flow cytometric analysis were selected to characterize TIMs. Furthermore, short-term HGSOC organoids were employed to validate the effect of TIMs on anti-PD1 therapy. Abundance of stromal TIMs (sTIMs) predicted dismal prognosis and linked to immunoevasive subtype of HGSOC, characterized by increased infiltration of pro-tumor cells (Treg cells, M2-polarized macrophages, and neutrophils) and impaired anti-tumor immune functions. Intensive inter-cell interactions between TIMs and other immune cells were identified, suggesting potential cross-talks to foster an immunosuppressive microenvironment. Organoids derived from sTIMs-low patients were associated with increased response to anti-PD-1 treatment other than the presence of high sTIMs infiltration. A nomogram, constructed by combining FIGO stage, sTIMs, and PD-L1, with an area under the curve (AUC) for predicting 5-year overall survival of 0.771 was better than that of FIGO staging system of 0.619. sTIMs/PD-L1-based classifier has potential clinical application in predicting prognosis of patients with HGSOC. sTIMs-high tumors correlate with immunosuppressive tumor microenvironment (TME) and possess potential insensitivity to immunotherapy.

Stromal LAG-3+cells infiltration defines poor prognosis subtype muscle-invasive bladder cancer with immunoevasive contexture

BackgroundLymphocyte activation gene 3 (LAG-3) is a promising immune checkpoint therapeutic target being evaluated in clinical trials. We assessed the LAG-3+ cells distribution, its association with clinical outcomes and immune contexture and its role in the landscape of muscle-invasive bladder cancer (MIBC) treatment.Methods141 patients with MIBC from Zhongshan Hospital were included for survival and adjuvant chemotherapy (ACT) benefit analyses. 32 fresh resected samples of MIBC were collected to detect CD8+ T cells functional state. The molecular classification analyses were based on 391 patients with MIBC from The Cancer Genome Atlas. Immunohistochemistry and flow cytometry were performed to characterize various immune cells infiltration.ResultsIn Kaplan-Meier analyses and Cox regression models, stromal LAG-3+ cells enrichment was consistently associated with inferior overall survival and disease-free survival, and indicated suboptimal responsiveness to ACT. Patents with high stromal LAG-3+ cells possessed increased protumor cells, immunosuppressive cytokines and immune checkpoint expression. The phenotypic analyses of CD8+ T cells correlated its dysfunctional state with LAG-3+ cells. Besides, LAG-3 mRNA level was linked to luminal and basal subtypes of MIBC. LAG-3-high tumors exhibited limited FGFR3 mutation and signaling signature, and displayed activated immunotherapeutic and EGFR-associated pathway.ConclusionsStromal LAG-3+ cells abundance indicated an immunoevasive contexture with dysfunctional CD8+ T cells, and represented an independent predictor for adverse survival outcome and ACT resistance in MIBC. LAG-3 expression could potentially be a novel biomarker for FGFR3-targeted and EGFR-targeted therapies and immunotherapy. The crucial role of LAG-3+ cells in the therapeutic landscape of MIBC needs further validation retrospectively and prospectively.

Intratumoral IL22-producing cells define immunoevasive subtype muscle-invasive bladder cancer with poor prognosis and superior nivolumab responses.

Our previous researches have identified immunoevasive subtype muscle-invasive bladder cancer (MIBC) characterized with immune cells infiltration patterns. Our study explored the clinical significance, immunoregulatory role and therapeutic value of intratumoral IL22-producing cells in MIBC. Two hundred and fifty-nine formalin-fixed paraffin-embedded MIBC samples and 83 freshly resected MIBC tissues and 391 TCGA MIBC samples were retrospectively evaluated. Immunohistochemistry and flow cytometry were applied to identify immune cell infiltration and functional status. In vitro intervention studies were to test the therapeutic and predictive potential of IL22+ cells. Our data revealed patients with high IL22+ cells infiltration suffered poor overall survival and recurrence-free survival in both training and validation cohorts. Only pT2 patients of combined cohort with low IL22+ cells infiltration gained survival benefits from adjuvant chemotherapy (ACT) significantly. Besides, immune contexture featured with increased pro-tumor cells and immunosuppressive cytokines was identified in patients with high IL22+ cells density. The expression pattern of exhausted and effector markers in CD8+ T cells from high IL22+ cells subgroup indicated their dysfunctional status. Importantly, nivolumab showed tumor-killing efficacy in tumors with high IL22+ cells infiltration, and immunosuppressive contexture with CD8+ T cells exhaustion was abrogated in tumors treated with anti-IL22 antibody. In summary, IL22+ cells infiltration determined immunosuppressive contexture with CD8+ T cell dysfunction. Tumor-infiltrating IL22+ cells could be used as an independent marker to predict prognosis and ACT responses. IL22+ cells infiltration possessed the potential to be a favorable predictor for nivolumab application and IL22 blockade could be a novel therapeutic strategy in MIBC.

Abstract 4130: Anti-cancer efficacy of systemic and Intratumoral YS-ON-001 as monotherapy or in combination with checkpoint inhibitor in syngeneic cancer models

Abstract Background: YS-ON-001 is a complex containing synthetic double-strand RNA, a toll-like receptor 3 (TLR3) agonist. TLR3 agonist stimulates antigen-presenting cells, increases T, NK, NKT cells and decreases regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), reprogrames macrophages from protumorigenic to anti-tumor. YS-ON-001 is being evaluated for systemic and intratumoral use alone or combined with checkpoint inhibitor to treat cancer. Method: The anti-tumor efficacy of systemically or intratumorally (it) injected YS-ON-001 alone or in combination with PD-1 antagonist was assessed in several syngeneic tumor models including pancreatic cancer, hepatocellular carcinoma. Tumors were implanted into left and/or right flanks while only one tumor was injected with YS-ON-001. YS-ON-001 was administered by IM injection every other day or it injection twice a week. Murine PD-1 antibody was infused intraperitoneally (ip) once a week. In addition to body weight, tumor volume was monitored on both flanks. Surviving mice with unnoticeable tumor were rechallenged. Flow cytometry was used to analyze immune cells from the tumors including T, NK, NKT cells, macrophages 1 and 2, Tregs, and MDSCs. Results: Within tumor microenvironment YS-ON-001 IM monotherapy resulted in increased infiltration of CD4+ T cells and CD8+ T cells, NK cells and NKT cells, reduced Tregs and MDSCs, and increased the M1/M2 ratio, leading to significant tumor growth inhibition in more than 10 cancer models. Combination of YS-ON-001 im with anti-PD1 ip resulted in greater anti-tumor efficacy compared to anti-PD1 monotherapy in both 4T1 breast cancer and LLC2 lung cancer models. Rejecting tumor rechallenge in surviving mice from the initial challenge treated with IM YS-ON-001suggested long-term anti-tumor immunity. Monotherapy of YS-ON-001 it or anti-PD-1 ip significantly suppresses tumor growth not only at the primary injection site, but also at uninjected site (p<0.05). Combination of YS-ON-001 it with anti-PD-1 ip significantly enhanced suppressive effect of anti-PD1 at both primary injected and distant uninjected sites. Conclusions: these experiments demonstrated systemic or intratumoral delivery of YS-ON-001 can modulates immune responses in tumor microenvironment by increasing anti-tumor cells in T, NK, NKt cells and decreasing pro-tumor cells in Treg, MDSC, leading to significant tumor growth inhibition. Combination of YS-on-001, IM or It with PD-1 antibodies significantly enhanced the anti-tumor efficacy of PD-1 antibodies. These results provide a strong rationale and support clinical development as standalone therapy or in combination with other modalities such checkpoint inhibitors. Citation Format: Zhongkai Shi, Yuan Liu, Yi Zhang. Anti-cancer efficacy of systemic and Intratumoral YS-ON-001 as monotherapy or in combination with checkpoint inhibitor in syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4130.

Oncogenic Notch Triggers Neoplastic Tumorigenesis in a Transition-Zone-like Tissue Microenvironment

During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. Here, we show that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.

The Initial Stage of Tumorigenesis in Drosophila Epithelial Tissues.

Cancer development originates in a single mutant cell transformed from a normal cell, including further evolution of pro-tumor cells through additional mutations into malignant cancer tissues. Data from recent studies, however, suggest that most pro-tumor cells do not develop into tumors but remain dormant within or are prophylactically eliminated from tissues unless bestowed with additional driver mutations. Drosophila melanogaster has provided very efficient model systems, such as imaginal discs and ovarian follicular epithelia, to study the initial stage of tumorigenesis. This review will focus on the behaviors of emerging pro-tumor cells surrounded by normal cells and situations where they initiate tumor development.

Nanoparticles That Reshape the Tumor Milieu Create a Therapeutic Window for Effective T-cell Therapy in Solid Malignancies.

A major obstacle to the success rate of chimeric antigen receptor (CAR-) T-cell therapy against solid tumors is the microenvironment antagonistic to T cells that solid tumors create. Conventional checkpoint blockade can silence lymphocyte antisurvival pathways activated by tumors, but because they are systemic, these treatments disrupt immune homeostasis and induce autoimmune side effects. Thus, new technologies are required to remodel the tumor milieu without causing systemic toxicities. Here, we demonstrate that targeted nanocarriers that deliver a combination of immune-modulatory agents can remove protumor cell populations and simultaneously stimulate antitumor effector cells. We administered repeated infusions of lipid nanoparticles coated with the tumor-targeting peptide iRGD and loaded with a combination of a PI3K inhibitor to inhibit immune-suppressive tumor cells and an α-GalCer agonist of therapeutic T cells to synergistically sway the tumor microenvironment of solid tumors from suppressive to stimulatory. This treatment created a therapeutic window of 2 weeks, enabling tumor-specific CAR-T cells to home to the lesion, undergo robust expansion, and trigger tumor regression. CAR-T cells administered outside this therapeutic window had no curative effect. The lipid nanoparticles we used are easy to manufacture in substantial amounts, and we demonstrate that repeated infusions of them are safe. Our technology may therefore provide a practical and low-cost strategy to potentiate many cancer immunotherapies used to treat solid tumors, including T-cell therapy, vaccines, and BITE platforms.Significance: A new nanotechnology approach can promote T-cell therapy for solid tumors. Cancer Res; 78(13); 3718-30. ©2018 AACR.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia.

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.