Modulation Of Immune Microenvironment Research Articles

Spatiotemporal modulation of immune microenvironment via composite hydrogel brakes for diabetic wound healing

Uncontrolled programmed polarization of macrophages plays an important role in chronic diabetic wound healing. Therefore, correcting programmed macrophage polarization while inducing new tissue remodelling through materials and preventing scar formation is desirable. Herein, a composite hydrogel brake that enables spatiotemporal regulation of the immune microenvironment was constructed to promote skin tissue regeneration. The system was based on thiolated hyaluronic acid to reshape the programmed polarization of macrophages, methacrylate gelatin as a mechanical support, and poly (L-lactic acid) nanoparticles loaded with TGF-β receptor antagonists to inhibit scar formation. Notably, the composite hydrogel acts as a double brake: in the early stage, self-induced M2-type polarization is achieved with the help of thiols; the TNF and NLRP signalling pathways are downregulated; and the immune microenvironment is regulated. During tissue remodeling, the long-term biological effect of TGF-β secretion by M2-type macrophages is interrupted by the slow release of antagonists, which inhibits excessive collagen deposition and prevents scar formation. The dual effect of restraint on reprogramming macrophage polarization and inhibiting scar formation was further confirmed in a diabetic rat skin defect model. Therefore, based on the immunological mechanism, a composite hydrogel brake was specifically designed to regulate the immune microenvironment, induce new tissue remodelling, and provide a promising clinical strategy for treating chronic diabetic wounds.

Abstract CT094: Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma

Abstract Colony-stimulating factor 1 receptor (CSF-1R) signaling is involved in regulating monocyte differentiation, and the recruitment and survival of tumor-associated macrophages (TAMs). BLZ945 is a small molecule, highly selective, orally bioavailable, potent kinase inhibitor of CSF-1R that has shown the ability to reduce the recruitment of TAMs in animal models and promote infiltration of effector T cells into the tumor in preclinical models. Given the potential effects of BLZ945 on effector T-cell activity, a combination of BLZ945 with the programmed death 1 (PD-1) inhibitor spartalizumab was investigated in 1) a dose escalation phase I clinical trial with advanced solid tumor patients; and 2) an expansion phase II clinical trial in adult patients with relapsed/refractory glioblastoma (GBM) after standard of care and other lines of therapy (NCT02829723). The percentages of non-classical and intermediate monocytes in the PBMC fraction were decreased after treatment at multiple doses and schedules of treatment tested in the escalation phase. Screening and on-treatment paired tumor biopsy samples from the escalation phase were analyzed by RNA sequencing. Downregulated genes have been noted as macrophage-specific genes, suggesting that treatment with BLZ945 is downregulating macrophages in the tumor microenvironment. There appeared to be a trend for a reduction in TAM gene signatures and increase in T-cell inflammation signatures upon treatment with single-agent BLZ945 or BLZ945 + spartalizumab. RNA-sequencing analysis from archival surgical resection GBM samples from the expansion phase, revealed a potential correlation between PDGFRA gene expression and best percent change in tumor size. This suggests that non-mesenchymal GBM, for which PDGFRA is part of the defining signature, are more sensitive to the treatment. DNA-sequencing of these archival samples also revealed mutations in p53 in the tumors with partial responses but no mutations in EGFR or TERT. Citation Format: Carlos Rodrigo Gil Del Alcazar, Virginia Xu, Chia-chi Lin, Marta Gil-Martin, Aung Naing, Liqiong Fan, Fang Yang, David Quinn, Jincheng Wu, Cornelia Quadt, Jennifer Mataraza. Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT094.

Abstract 5924: RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide, and the third most common cause of cancer-related deaths. For the 40% to 50% of HCC patients who are candidates for systemic therapies, the landscape has evolved significantly in the last 10 years, with most recently, immunotherapies such as combination of atezolizumab and bevacizumab emerging as the current first-line systemic therapy for patients with intermediate or advanced disease. However, there are no predictive biomarkers for patients with HCC to enable selection of those most likely to benefit from these therapies and this remains a disease with high unmet medical need. Activation of the WNT pathway via mutational events or indirect pathway activation is implicated in many cancers including up to 50% of HCC patient tumors. High expression of beta-catenin, encoded by the CTNNB1 gene, has been shown to correlate with decreased survival and more rapid disease progression in HCC patients. In addition, WNT/beta-catenin pathway activation has been shown to result in immune exclusion and resistance to immunotherapy in HCC. ALN-BCAT comprises a chemically modified siRNA encapsulated in a lipid nanoparticle formulation which generates robust and highly specific reductions of CTNNB1 mRNA, downstream biomarkers and tumor cell proliferation in in vitro model systems. Anti-tumor activity has been demonstrated in multiple in vivo orthotopic HCC mouse models, in both early and late treatment settings as measured by liver to body weight ratio. Reductions in tumor burden were orthogonally confirmed by histology analysis and shown to be associated with changes in CTNNB1-related biomarkers. Finally, ALN-BCAT treatment results in changes in the tumor microenvironment in a mouse syngeneic HCC model, with increases in CD4+ and CD8+ T cells and reductions in M2 macrophages, consistent with a shift to a more immune stimulatory anti-tumor microenvironment. Here, we demonstrate that ALN-BCAT provides a novel approach to the inhibition of the WNT pathway as a therapeutic approach for treatment of HCC patients and WNT pathway activated tumors. Citation Format: Tulin Dadali, Spencer Miller, Gaurav Saawant, Stephen Abbott, Mikyung Yu, Svetlana Morskaya, Brandon Lehrich, Satdarshan Monga, Gloria Lau, Martin Maier, Wendy Broom. RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models [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 5924.

Lentinan-loaded GelMA hydrogel accelerates diabetic wound healing through enhanced angiogenesis and immune microenvironment modulation

Diabetic wound healing is a substantial clinical challenge, characterized by delayed angiogenesis and unresolved inflammation. Lentinan, a polysaccharide extracted from shiitake mushrooms, has the potential to regulate both macrophage polarization and angiogenesis, though this aspect remains inadequately explored. To facilitate lentinan's clinical utility, we have developed a GelMA hydrogel encapsulated with lentinan (10 μM), offering a controlled release mechanism for sustained lentinan delivery at the wound site. Application of the lentinan-encapsulated delivery system topically significantly expedites wound closure compared to control groups. Furthermore, histological examination demonstrates enhanced neovascularization and reduced inflammation in lentinan-treated wounds, as evidenced by increased M2 macrophage infiltration. Moreover, our results indicated that lentinan-induced AMPK activation promotes DAF16 expression, enhancing the resistance of macrophages and HUVECs to oxidative stress in high-glucose environments, thereby promoting M2 macrophage polarization and angiogenesis. All these findings underscore lentinan's capacity to modulate macrophage polarization and angiogenesis via the AMPK/DAF16 pathway, ultimately facilitating the healing of diabetic wounds.

Efficacy of combined treatment with pirfenidone and PD-L1 inhibitor in mice bearing ectopic bladder cancer xenograft

To assess the efficacy of pirfenidone combined with PD-L1 inhibitor for treatment of bladder cancer in a mouse model and its effect on tumor immune microenvironment modulation. Forty C57BL/6 mouse models bearing ectopic human bladder cancer xenografts were randomized into control group, PD-L1 inhibitor group, pirfenidone group and combined treatment group (n=10). After successful modeling, PD-L1 inhibitor treatment was administered via intraperitoneal injection at 12.5 mg/kg every 3 days, and oral pirfenidone (500 mg/kg) was given on a daily basis. The survival rate of the mice and tumor growth rate were compared among the 4 groups. The expressions of CD3, CD8, CD45, E-cadherin and N-cadherin in the tumor tissues were detected with immunohistochemistry after the 21-day treatment, and bone marrow-derived suppressor cells (MDSCs) were observed with immunofluorescence staining; serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), creatinine (CRE) and lactate dehydrogenase (LDH-L) were analyzed using an automated biochemical analyzer. Treatment with PD-L1 inhibitor and pirfenidone alone both significantly decreased tumor growth rate and tumor volume at 21 days (P < 0.05), but the combined treatment produced an obviously stronger inhibitory effect (P < 0.05). PD-L1 inhibitor and pirfenidone alone significantly increased E- cadherin expression and decreased N-cadherin expression in the tumor tissue (P < 0.05). The two treatments both significantly increased the percentage of CD3+, CD8 and CD45+ T cells and decreased the percentage of Ly-6G+CD11b+MDSCs in the tumor tissue, and these changes were more obvious in the combined treatment group (P < 0.05). No significant differences were found in serum ALT, AST, BUN, CRE or LDH-L levels among the 4 groups (P>0.05). Combined treatment with pirfenidone and PD-L1 inhibitor significantly inhibits the progression of bladder cancer in mice possibly by regulating tumor immune microenvironment and inhibiting epithelial-mesenchymal transition of the tumor cells.

Comprehensive pan-cancer analysis unveils the significant prognostic value and potential role in immune microenvironment modulation of TRIB3

TRIB3, a pseudokinase, was previously studied within only some specific cancer types, leaving its comprehensive functions in pan-cancer contexts largely unexplored. Here, we performed an integrated analysis of TRIB3 expression, prognosis, genetic alterations, functional enrichment and tumor immune-related characteristics in 33 cancer types. Our results showed that TRIB3 exhibits high expression levels across 24 different cancer types and correlates closely with unfavorable prognoses. Meanwhile, TRIB3 shows mutations in a wide spectrum of 22 distinct cancer types, with the predominant mutation types being missense mutations and gene amplifications, and significant changes in DNA methylation levels in 14 types of cancer. We further discovered that TRIB3 expression is significantly associated with cancer immune-related genome mutations, such as tumor mutational burden (TMB), microsatellite instability (MSI) and DNA mismatch repair (MMR), and infiltration of immunosuppressive cells, such as CD4+ Th2 cells and myeloid-derived suppressor cells (MDSCs), into the tumor microenvironment. These results indicated that the expression of TRIB3 might reshape the tumor immune microenvironment (TIME) and lead to immunosuppressive "cold" tumors. In addition, our results confirmed that the loss of function of TRIB3 inhibits cell proliferation, promotes apoptosis, and leads to significant enrichment of "hot" tumor-related immune pathways, at least in breast cancer cells, which further supports the important role of TRIB3 in cancer prognosis and TIME regulation. Together, this pan-cancer investigation provided a comprehensive understanding of the critical role of TRIB3 in human cancers, and suggested that TRIB3 might be a promising prognostic biomarker and a potential target for cancer immunotherapy.

First person – Wei-Lun Hsu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models &amp; Mechanisms, helping researchers promote themselves alongside their papers. Wei-Lun Hsu is first author on ‘ High-fat diet induces C-reactive protein secretion, promoting lung adenocarcinoma via immune microenvironment modulation’, published in DMM. Wei-Lun is a PhD student in the lab of Kang-Yi Su at National Taiwan University, Taipei, Taiwan, investigating possible treatments for obesity-related diseases and obesity.

High-fat diet induces C-reactive protein secretion, promoting lung adenocarcinoma via immune microenvironment modulation

ABSTRACT To understand the effects of a high-fat diet (HFD) on lung cancer progression and biomarkers, we here used an inducible mutant epidermal growth factor receptor (EGFR)-driven lung cancer transgenic mouse model fed a regular diet (RD) or HFD. The HFD lung cancer (LC-HFD) group exhibited significant tumor formation and deterioration, such as higher EGFR activity and proliferation marker expression, compared with the RD lung cancer (LC-RD) group. Transcriptomic analysis of the lung tissues revealed that the significantly changed genes in the LC-HFD group were highly enriched in immune-related signaling pathways, suggesting that an HFD alters the immune microenvironment to promote tumor growth. Cytokine and adipokine arrays combined with a comprehensive analysis using meta-database software indicated upregulation of C-reactive protein (CRP) in the LC-HFD group, which presented with increased lung cancer proliferation and metastasis; this was confirmed experimentally. Our results imply that an HFD can turn the tumor growth environment into an immune-related pro-tumorigenic microenvironment and demonstrate that CRP has a role in promoting lung cancer development in this microenvironment.

Engineering of copper sulfide mediated by phototherapy performance.

Copper sulfide based phototherapy, including photothermal therapy and photodynamic therapy, is an emerging minimally invasive treatment of tumor, which the light was converted to heat or reactive oxygen to kill the tumor cells. Compared with conventional chemotherapy and radiation therapy, Cu2-x S based phototherapy is more efficient and has fewer side effects. However, considering the dose-dependent toxicity of Cu2-x S, the performance of Cu2-x S based phototherapy still cannot meet the requirement of the clinical application to now. To overcome this limitation, engineering of Cu2-x S to improve the phototherapy performance by increasing light absorption has attracted extensive attention. For better guidance of Cu2-x S engineering, we outline the currently engineering method being explored, including (1) structural engineering, (2) compositional engineering, (3) functional engineering, and (4) performance engineering. Also, the relationship between the engineering method and phototherapy performance was discussed in this review. In addition, the further development of Cu2-x S based phototherapy is prospected, including smart materials based phototherapy, phototherapy induced immune microenvironment modulation et al. This review will provide new ideas and opportunities for engineering of Cu2-x S with better phototherapy performance. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Comprehensive bioinformatics analysis reveals the oncogenic role of FoxM1 and its impact on prognosis, immune microenvironment, and drug sensitivity in osteosarcoma.

Osteosarcoma, a highly malignant bone tumor primarily affecting adolescents, presents a significant challenge in cancer therapy due to its resistance to chemotherapy. This study explores the multifaceted impact of the transcription factor FoxM1 on osteosarcoma, shedding light on its pivotal role in tumor progression, immune microenvironment modulation, and drug response. Utilizing publicly available datasets from the Gene Expression Omnibus (GEO) and Therapeutically Applicable Research To Generate Effective Treatments (TARGET) databases, we conducted an in-depth bioinformatics analysis. Our findings illuminate the far-reaching implications of FoxM1 in osteosarcoma, emphasizing its significance as a potential therapeutic target. Differential expression analysis and Gene Set Enrichment Analysis (GSEA) revealed FoxM1's influence on critical pathways related to apoptosis, cell cycle regulation, and DNA repair. Notably, FoxM1 expression correlated with poor clinical outcomes in osteosarcoma patients, highlighting its prognostic relevance. Additionally, FoxM1 was found to modulate the immune microenvironment within tumor tissues, impacting immune cell infiltration, immunomodulators, immune checkpoints, and chemokines. Furthermore, a prognostic model based on FoxM1-coexpressed genes demonstrated its effectiveness in predicting patient survival. Drug sensitivity analysis indicated FoxM1's association with drug response, potentially guiding personalized treatment approaches. Hub gene screening identified RAB23 as a key target regulated by FoxM1, with RAB23 shown to influence osteosarcoma cell growth. This study also confirmed FoxM1's overexpression in osteosarcoma tissues compared to normal tissues, and its association with clinicopathological characteristics, including clinical stage, pathological type, and lung metastasis. In conclusion, FoxM1 emerges as a central player in the pathogenesis of osteosarcoma, impacting gene expression, immune responses, and therapeutic outcomes. This comprehensive analysis deepens our understanding of FoxM1's role in osteosarcoma and offers potential avenues for improved diagnosis and treatment.

Bifidobacterium adolescentis induces Decorin+ macrophages via TLR2 to suppress colorectal carcinogenesis

BackgroundThe interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis and its possible mechanism on the manipulation of Decorin+ macrophages in colorectal cancer.MethodsThe relative abundance of B. adolescentis in tumor or para-tumor tissue of CRC patients was analyzed. The role of B. adolescentis was explored in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the myeloid cells subsets in TME. The expression level of TLR2/YAP axis and its downstream Decorin in macrophages were tested by Western blot and qRT-PCR. Knockdown of Decorin in Raw264.7 was performed to investigate the effect of Decorin+ macrophages on subcutaneous tumor formation. Multi-immunofluorescence assay examined the number of Decorin+ macrophages on the CRC tissue.ResultsWe found that the abundance of B. adolescentis was significantly reduced in tumor tissue of CRC patients. Supplementation with B. adolescentis suppressed AOM/DSS-induced tumorigenesis in mice. ScRNA-seq and animal experiment revealed that B. adolescentis increased Decorin+ macrophages. Mechanically, Decorin was activated by TLR2/YAP axis in macrophages. The abundance of B. adolescentis was correlated with the number of Decorin+ macrophages and the expression level of TLR2 in tumor tissue of CRC patients.ConclusionsThese results highlight that B. adolescentis induced Decorin+ macrophages and provide a novel therapeutic target for probiotic-based modulation of immune microenvironment in CRC.

Lysyl oxidase-responsive anchoring nanoparticles for modulation of the tumor immune microenvironment

In the tumor microenvironment, lysyl oxidase (LOX) is known to play a key role in stabilizing the tumor extracellular matrix. Here, we designed LOX-responsive nanoparticles to interact with the collagen matrix of the tumor microenvironment. Collagen-coated and imiquimod-loaded polydopamine nanoparticles (CPN/IQ) could form crosslinked structures with the collagen matrix via LOX. In vitro, anchoring of CPN/IQ nanoparticles was observed with LOX-secreting CT26 cells, but this was blocked by a LOX inhibitor. In CT26 tumor-bearing mice, co-administration of nanoparticles plus the LOX inhibitor did not significantly alter the antitumor efficacy among nanoparticles. In the absence of the LOX inhibitor, however, a single administration of CPN/IQ could provide sustained responsiveness to near-infrared irradiation and ablation of primary tumors. In the primary tumor microenvironment, CPN/IQ lowered the Treg cell population but increased the cytotoxic CD3+CD8+ T cell population. In splenic dendritic cells, CPN/IQ treatment significantly increased the CD11c+CD86+ and CD11c+CD80+ cell populations. In a CT26 distant tumor-rechallenge model, CPN/IQ treatment increased the cytotoxic CD3+CD8+ T cell population and provided 100% survival of mice until 64 days. This study indicates the feasibility of tumor immune microenvironment modulation using LOX-responsive size-transforming nanoparticles. Although we tested the concept in a CT26 cell-derived tumor model, the concept of LOX-responsive collagen matrix- anchoring nanoparticles may be broadly applied to other tumor tissues with LOX-rich tumor microenvironments.

Role of Interleukins in Inflammation-Mediated Tumor Immune Microenvironment Modulation in Colorectal Cancer Pathogenesis.

Tumor cells invade and spread through a procedure termed as epithelial-to-mesenchymal cell transition (EMT). EMT is triggered by any alterations in the genes that encode the extracellular matrix (ECM) proteins, the enzymes that break down the ECM, and the activation of the genes that causes the epithelial cell to change into a mesenchymal type. The transcription factors NF-κB, Smads, STAT3, Snail, Zeb, and Twist are activated by inflammatory cytokines, for instance, Tumor Necrosis Factor, Tumor Growth Factors, Interleukin-1, Interleukin-8, and Interleukin-6, which promotes EMT. The current piece of work has been reviewed from the literature works published in last 10 years on the role interleukins in inflammation-mediated tumor immune microenvironment modulation in colorectal cancer pathogenesis utilizing the databases like Google Scholar, PubMed, Science Direct. Recent studies have demonstrated that pathological situations, such as epithelial malignancies, exhibit EMT characteristics, such as the downregulation of epithelial markers and the overexpression of mesenchymal markers. Several growing evidence have also proved its existence in the human colon during the carcinogenesis of colorectal cancer. Most often, persistent inflammation is thought to be one factor contributing to the initiation of human cancers, such as colorectal cancer (CRC). Therefore, according to epidemiologic and clinical research, people with ulcerative colitis and Crohn's disease have a greater probability of developing CRC. A substantial amount of data points to the involvement of the NF-κB system, SMAD/STAT3 signaling cascade, microRNAs, and the Ras-mitogen-activated protein kinase/Snail/Slug in the epithelial-to-mesenchymal transition-mediated development of colorectal malignancies. As a result, EMT is reported to play an active task in the pathogenesis of colorectal cancer, and therapeutic interventions targeting the inflammation-mediated EMT might serve as a novel strategy for treating CRC. The illustration depicts the relationship between interleukins and their receptors as a driver of CRC development and the potential therapeutic targets.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels T cell-related prognostic risk model and tumor immune microenvironment modulation in triple-negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive and fatal malignancy. The current success of tumor immunotherapy has focused attention on intermediate T-cell subsets and the tumor microenvironment, which are essential for activation of the anti-tumor response. Therefore, both areas require further research to accelerate progress in developing tailored immunotherapeutic approaches for patients with TNBC. We obtained scRNA-seq data of TNBC from the GEO database. A multiplex strategy was used to analyze and identify the T-cell heterogeneity of TNBC. By combining the METABRIC and GEO databases, a prognostic risk model for T-cell marker genes was constructed and validated. In addition, the immune-infiltrating cells of TNBC was analyzed using CIBERSORT, and the association between the risk model and response to immunotherapy was investigated. Based on scRNA-seq data, 25,932cells were identified for multiple analyzes. T cells were studied with a focus on 2 subtypes, including CD8+ and CD4+. There were also communication relationships between T cells and multiple cell types. The results of the enrichment analysis showed that the T-cell marker genes were focused in pathways related to the immune system. In addition, OPTN, TMEM176A, PKM and HES1 deserve attention as prognostic markers in TNBC. The immune infiltration results showed that the high-risk group had significant immune cell infiltration and immunosuppression status. This study provides a resource for understanding T-cell heterogeneity and the associated prognostic risk model for TNBC. The results show that the model helps predict prognosis and response to treatment in breast cancer.

Abstract 1478: Broad downregulation of normal-specific super-enhancer associated genes in lung adenocarcinoma

Abstract Background Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and a highly heterogeneous disease whose mechanism is not fully uncovered. Recently, Super-enhancers (SEs), tissue-specific clusters of enhancer elements, were found to drive cell identity genes and play an important role in tumorigenesis. However, current studies focus on SE in lung tumors but largely neglect those specific in normal lung tissues, which also provide critical data for understanding transformation of normal cells into cancer cells. For the first time, this study characterized normal-specific SE-associated genes (SEGs) in LUAD by dysregulation in tumor, prognostic significance and tumor microenvironment (TME) interactions via a comprehensive analysis of 22 bulk and single-cell transcriptomic datasets. Methods We analyzed TCGA datasets of 10 cancer types, and additional datasets comprising 4 datasets of LUAD tumor and normal paired samples, 5 datasets of LUAD tumors with survival information, and 3 single-cell transcriptomic datasets. For each dataset, standard methods were used for data preprocessing and normalization according to its generation platforms (e.g., microarray, bulk RNA-seq and scRNA-seq). For downstream analyses, paired t-test and linear model were used to identify dysregulated SEGs; Cox Proportional Hazards Regression was used to evaluate their prognostic significance and LASSO was used to select features for building multivariate prognostic models; bulk data deconvolution and correlation analysis were applied to assess the association of TME cell composition and SEG expression; Gene set enrichment analysis was applied for functional analysis. Results We found normal-lung SEGs were significantly disproportionally downregulated in LUAD tumors (Chi-squared test, p &amp;lt; 0.01), and associated with poor prognosis of patients. The patterns were consistently found in discovery (TCGA) and independent validation datasets. Interestingly, the effect appears specific to lung as such downregulation of normal-lung SEGs was also found in lung squamous cell carcinoma but not observed in most of other 8 cancer types. The downregulated SEGs were significantly involved in cell differentiation, apoptosis, proliferation, and stem cell pluripotency, and were enriched in tumor suppressors. Also, their downregulation correlate with reduced endothelial cells and lower immune activity with reduced B and CD8 T cells and inhibited immune target genes. Conclusions Our study characterized the genes associated to normal-specific SEs in LUAD, shedding sheds light on their role in tumorigenesis of lung cancers. Their broad downregulation indicates a significant effect of SE aberrancy in the transformation of normal cell into cancer cells with a modulation of immune microenvironment; further functional studies are needed to elucidate the underlying mechanism. Citation Format: Guoshuai Cai, Yihan Liu, Xiang Cui, Christopher Amos, Feifei Xiao. Broad downregulation of normal-specific super-enhancer associated genes in lung adenocarcinoma [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 1478.

MUC1 Expression Affects the Immunoflogosis in Renal Cell Carcinoma Microenvironment through Complement System Activation and Immune Infiltrate Modulation

Mucin1 (MUC1), a glycoprotein associated with an aggressive cancer phenotype and chemoresistance, is aberrantly overexpressed in a subset of clear cell renal cell carcinoma (ccRCC). Recent studies suggest that MUC1 plays a role in modulating cancer cell metabolism, but its role in regulating immunoflogosis in the tumor microenvironment remains poorly understood. In a previous study, we showed that pentraxin-3 (PTX3) can affect the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system (C1q) and releasing proangiogenic factors (C3a, C5a). In this scenario, we evaluated the PTX3 expression and analyzed the potential role of complement system activation on tumor site and immune microenvironment modulation, stratifying samples in tumors with high (MUC1H) versus tumors with low MUC1 expression (MUC1L). We found that PTX3 tissue expression was significantly higher in MUC1H ccRCC. In addition, C1q deposition and the expressions of CD59, C3aR, and C5aR were extensively present in MUC1H ccRCC tissue samples and colocalized with PTX3. Finally, MUC1 expression was associated with an increased number of infiltrating mast cells, M2-macrophage, and IDO1+ cells, and a reduced number of CD8+ T cells. Taken together, our results suggest that expression of MUC1 can modulate the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system and regulating the immune infiltrate, promoting an immune-silent microenvironment.

Comprehensive Analysis Identified Glycosyltransferase Signature to Predict Glioma Prognosis and TAM Phenotype.

Glycosylation is the most common posttranslational modification of proteins. Glycosyltransferase gene differential expression dictates the glycosylation model and is epigenetically regulating glioma progression and immunity. This study is aimed at identifying the glycosyltransferase gene signature to predict the prognosis and immune characteristics of glioma. The glycosyltransferase gene signature of glioma was identified in the TCGA database and validated in the CGGA database. Glioma patients were then divided into high- and low-risk groups based on risk scores to compare survival differences and predictive capacity. Subsequently, validation of glycosyltransferase gene signature merits by comparing with other signatures and utility in clinical judgment. The immune cell infiltration, immune pathways, and immune checkpoint expression level were also analyzed and compared in the high- and low-risk groups. Finally, the signature and its gene function were tested in our cohort and in vitro experiments. Eight glycosyltransferase genes were identified to establish the glycosyltransferase signature to predict the prognosis of glioma patients. The survival time was shorter in the high-risk group compared to the low-risk group based on glycosyltransferase signature and was confirmed in an independent external cohort. The glycosyltransferase signature displayed outstanding predictive capacity than other signatures in the TCGA and CGGA database cohorts. Furthermore, patients in the high-risk group were positively correlated with TAM infiltration, immune checkpoint expression level, and protumor immune pathways in TCGA cohorts. Validation of clinical tissue specimens revealed that the high-risk group was closely associated with infiltration of M2 TAMs. High-risk genes in the signature promote glioma proliferation, invasion, and macrophage recruitment in an in vitro validation of U87 and U251 cell lines. This carefully constructed that glycosyltransferase signature can predict the prognosis and immune profile of gliomas and help us evaluate subsequent macrophage-targeted therapies as well as other immune microenvironment modulation therapeutic strategies.

Novel cellular senescence-related risk model identified as the prognostic biomarkers for lung squamous cell carcinoma.

Lung cancer is one of the top causes of cancer-related death worldwide. Cellular senescence is a characteristic of cell cycle arrest that plays a role in carcinogenesis and immune microenvironment modulation. Despite this, the clinical and immune cell infiltration features of senescence in lung squamous cell carcinoma (LUSC) are unknown. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to get RNA-seq data and clinical information for LUSC. The least absolute shrinkage and selection operator (LASSO)-Cox regression, receiver operating characteristic (ROC), and Kaplan-Meier analysis were used to evaluate a risk model for predicting overall survival based on six differentially expressed genes. The tumor microenvironment (TME) and immunotherapy response were also studied. To discriminate LUSC into high- and low-risk subgroups, a risk model comprised of six cellular senescence-related genes (CDKN1A, CEBPB, MDH1, SIX1, SNAI1, and SOX5) was developed. The model could stratify patients into high-risk and low-risk groups, according to ROC and Kaplan-Meier analysis. In the TCGA-LUSC and GSE73403 cohorts, the high-risk group had a worse prognosis (P<0.05), and was associated with immune cell inactivation and being insensitive to immunotherapy in IMvigor210. We discovered a new LUSC classification based on six cellular senescence-related genes, which will aid in identifying patients who will benefit from anti-PD-1 treatment. Targeting senescence-related genes appears to be another option for improving clinical therapy for LUSC.

FLASH Modulation of Immune Microenvironment in Diffuse Midline Glioma (DMG) at Single-Cell Resolution

<h3>Purpose/Objective(s)</h3> Diffuse Midline Glioma with H3K27M mutation (DMG) is a universally fatal tumor primarily treated with radiotherapy (RT) and typically unresponsive to immune-based therapies. FLASH is a novel RT technique associated with decreased toxicity and effective tumor control in glioma models as well as immune-sparing properties in other cancer models. However, the effect of FLASH on the DMG tumor immune microenvironment (TIME) has not yet been explored. <h3>Materials/Methods</h3> Using a syngeneic murine brainstem DMG, we performed single-cell RNA sequencing on CD45+ cells isolated from tumors 4 days after posterior fossa irradiation (15Gy x 1, 9MeV), utilizing either ultra-high dose rate (90Gy/sec, FLASH) or conventional (2Gy/min, CONV) RT, and compared to unirradiated tumor (SHAM). For differential gene expression analysis, we compared CONV to SHAM (CvS) and FLASH to SHAM (FvS). We also performed single cell gene set enrichment scores (ssGSEA) for evaluation of immune signatures from gene ontology (GO); ssGSEA scores were deemed significant by a p<0.05 using Mann-Whitney U test. We validated our most robust immune markers with dual immunofluorescence. <h3>Results</h3> Unsupervised clustering of 23,165 CD45+ cells revealed 16 immune clusters, including subtypes of resident microglia (MG, 78.2% of total cells), lymphocytes (6.4%), macrophages (MPs, 5.2%), monocytes (1.8%), and dendritic cells (DCs, 1.7%). MG consisted of four distinct subtypes representing a spectrum of homeostatic to activated cellular states. In the most activated MG subtype, both CONV and FLASH showed an upregulation of interferon (IFN) genes such as Isg15 (Fold Change (FC) 1.03 CvS, 0.94 FvS) and Ifit3 (FC 0.77 CvS, 0.66 FvS) when compared to SHAM. Furthermore, ssGSEA of MG found IFN scores increased in both FvS (p < 0.0001) and CvS (p < 0.0001) while TGFbR signaling score was increased in FvS (p < 0.0001) and decreased in CvS (p < 0.0001). In non-resident myeloid clusters, the most differential response was found in MPs and DCs with robust upregulation of IFN genes for CvS (Isg15 FC 2.30 MPs, 2.67 DCs; Ifit3 FC 2.02 MPs, 2.06 DCs), while this response was not seen with FvS. This was confirmed with ssGSEA with increased IFN scores in both MPs (CvS p<0.0001, p=0.04) and DCs (CvS p<0.0001, FvS p=0.69). FLASH showed an increase in anti-inflammatory MP markers such as Mrc1, Cd163, and Maf (FC 1.40, 1.11, 1.19) and an upregulated myeloid-derived suppressor cell signature in monocytes (FvS, p < 0.0001), not seen in CvS (p = 0.09). <h3>Conclusion</h3> Our work is the first to map CONV and FLASH immune alterations with single-cell resolution in the DMG TIME. FLASH induces IFN differentially across microglia and non-resident myeloid subsets. Further exploration is warranted to understand how FLASH impacts tumor growth through differential cytokine signaling, inflammatory responses and immune checkpoint expression within the DMG TIME.

CXCL8 Up-Regulated LSECtin through AKT Signal and Correlates with the Immune Microenvironment Modulation in Colon Cancer.

The role of CXCL8 and LSECtin in colon cancer liver metastasis and immune checkpoint inhibitors (ICIs) treatment effect were widely recognized. However, the regulatory role of CXCL8 on LSECtin is still unclear. The expression of CXCL8 or LSECtin was analyzed by TCGA database, and verified by GES110225 and clinical samples. The relationship between the expression of CXCL8 or LSECtin and immune cells infiltration, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO) items, stromal score, Estimation of STromal and Immune cells in MAlignant Tumours (ESTIMAT) immune score, tumor mutation burden (TMB), mismatch repair gene and immune checkpoints expression were analyzed by Spearman. The effects of CXCL8 on LSECtin expression, proliferation, and invasion ability were clarified by recombinant CXCL8 or CXCL8 interfering RNA. In colon cancer, the expression of CXCL8 was higher, but LSECtin was lower than that in normal mucosa. The expression of CXCL8 or LSECtin was significantly positively correlated with immune cells infiltration, stromal score, ESTIMATE immune score, TMB, and immune checkpoints expression. The expression of LSECtin was closely related to the cytokine-cytokine receptor interaction pathway and response of chemokine function, such as CXCL8/CXCR1/2 pathway. There was a significant positive correlation between the expression of CXCL8 and LSECtin in colon cancer. CXCL8 up-regulated LSECtin through AKT signal and promoted the proliferation and invasion ability of colon cancer. CXCL8 up-regulated LSECtin by activating AKT signal and correlated with the immune microenvironment modulation in colon cancer.