Inflammatory Tumor Microenvironment Research Articles

Non-Small Cell Lung Cancer Metastatic Without Oncogenic Alterations.

This overview provides a thorough review of current treatment approaches for first-line management of nononcogenic addicted non-small cell lung cancer. We also address pertinent clinical decision-making queries encountered in everyday practice, such as the optimal treatment strategy for PD-L1-high patients, predictive factors for response to immune checkpoint inhibitors (ICI) both in terms of patient and cancer characteristics, the potential benefits of dual checkpoint blockade, and the unresolved issue of safe discontinuation strategies for long-term responders. Around one in five patients falls into this latter category while the majority develop either primary or acquired resistance to ICI-based first-line therapy, necessitating effective subsequent lines of treatment. Docetaxel, with or without combination of antiangiogenic agents, serves as the backbone of treatment, although evidence in the post-ICI setting is limited. Given that an inflamed tumor microenvironment (TME) is crucial for ICI responses, targeting the TME in cases of acquired resistance alongside continued ICI administration appears rational, although clinical trials so far have failed to confirm this hypothesis. Antibody-drug conjugates have emerged as a promising treatment modality, offering the potential for reduced toxicity and improved efficacy by targeting specific cancer antigens. Moreover, several chemotherapy-free approaches are currently under investigation for treatment-naïve patients, including alternative ICI and drugs targeting epitopes on both cancer and immune cells.

Neuroinflammation in Glioblastoma: The Role of the Microenvironment in Tumour Progression.

Glioblastoma (GBM) stands as the most aggressive and lethal among the main types of primary brain tumors. It exhibits malignant growth, infiltrating the brain tissue, and displaying resistance toward treatment. GBM is a complex disease characterized by high degrees of heterogeneity. During tumour growth, microglia and astrocytes, among other cells, infiltrate the tumour microenvironment and contribute extensively to gliomagenesis. Tumour-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, are the most numerous nonneoplastic populations in the tumour microenvironment in GBM. The complex heterogeneous nature of GBM cells is facilitated by the local inflammatory tumour microenvironment, which mostly induces tumour aggressiveness and drug resistance. The immunosuppressive tumour microenvironment of GBM provides multiple pathways for tumour immune evasion, contributing to tumour progression. Additionally, TAMs and astrocytes can contribute to tumour progression through the release of cytokines and activation of signalling pathways. In this review, we summarize the role of the microenvironment in GBM progression, focusing on neuroinflammation. These recent advancements in research of the microenvironment hold the potential to offer a promising approach to the treatment of GBM in the coming times.

Celastrus orbiculatus Extract Inhibits Immune Inflammatory Thrombotic State of B-Lymphoma.

To investigate the inhibitory effect of Celastrus orbiculatus extracts (COE) on the proliferation of lymphoma cells and the immune regulation ability on inflammation and thrombophilia in vivo. The 38B9 lymphoma cells were treated with COE (160 µ g/mL) and CTX (25 µ mol/L). The apoptosis rate and cell cycle of each group were detected by flow cytometry. The secretion of inflammatory factors, including interleukin (IL)-6, IL-10, and tumor necrosis factor α (TNF-α), in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In vivo, BALB/c mice were subcutaneously injected with 38B9 lymphoma cells to establish lymphoma model. COE (3 mg·kg-1·d-1) and CTX (40 mg·kg-1·d-1) were administered to the model mice, respectively. The expression of plasma inflammatory factors (IL-6, IL-10 and TNF-α) and thrombus indexes, including D-dimer (D-D), von Willebrand factor (vWF) and tissue factor (TF), were detected by ELISA before tumor bearing (1 d), after tumor formation (14 d) and after intervention (21 d). PicoGreen dsDNA was used to detect the level of serum neutrophil extracellular traps (NETs). Flow cytometry was used to detect the expression of platelet activation marker calcium-dependent lectin-like receptor 2 (CLEC-2). The tumor growth and survival of mice were recorded. The 38B9 lymphoma cells were apoptotic after the intervention of COE and CTX. The ratio of G2-M phase cells decreased in COE intervented cells compared with the control cells (P<0.05), and S phase cells decreased in CTX intervented cells (P<0.05). Also, the secretion level of IL-6 was significantly reduced after COE or CTX intervention (P<0.05), and IL-10 was significantly increased (P<0.05). Furthermore, the tumor mass was reduced, and the median survival time was longer in COE and CTX intervented tumor-bearing mice than in non-intervented mice. The significantly lower levels of TNF-α, IL-6, NETs, TF, DD and CLEC-2, as well as higher IL-10 were observed in COE and CTX treatment mice in comparision with the control mice (P<0.05). COE has a mild and stable anti-tumor effect, which can reduce the secretion of inflammatory factors by lymphoma cells and regulate thrombophilic state caused by tumor inflammatory microenvironment.

Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

Regulatory effect of Yinchenhao decoction on bile acid metabolism to improve the inflammatory microenvironment of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is a malignant tumor with extremely high mortality. The tumor microenvironment is the "soil" of its occurrence and development, and the inflammatory microenvironment is an important part of the "soil". Bile acid is closely related to the occurrence of HCC. Bile acid metabolism disorder is not only directly involved in the occurrence and development of HCC but also affects the inflammatory microenvironment of HCC. Yinchenhao decoction, a traditional Chinese medicine formula, can regulate bile acid metabolism and may affect the inflammatory microenvironment of HCC. To determine the effect of Yinchenhao decoction on bile acid metabolism in mice with HCC and to explore the possible mechanism by which Yinchenhao decoction improves the inflammatory microenvironment of HCC by regulating bile acid metabolism, we established mice model of orthotopic transplantation of hepatocellular carcinoma. These mice were treated with three doses of Yinchenhao decoction, then liver samples were collected and tested. Yinchenhao decoction can regulate the disorder of bile acid metabolism in liver cancer mice. Besides, it can improve inflammatory reactions, reduce hepatocyte degeneration and necrosis, and even reduce liver weight and the liver index. Taurochenodeoxycholic acid, hyodeoxycholic acid, and taurohyodeoxycholic acid are important molecules in the regulation of the liver inflammatory microenvironment, laying a foundation for the regulation of the liver tumor inflammatory microenvironment based on bile acids. Yinchenhao decoction may improve the inflammatory microenvironment of mice with HCC by ameliorating hepatic bile acid metabolism.

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer

BackgroundIn prostate cancer (PCa), well-established biomarkers such as MSI status, TMB high, and PDL1 expression serve as reliable indicators for favorable responses to immunotherapy. Recent studies have suggested a potential association between CDK12 mutations and immunotherapy response; however, the precise mechanisms through which CDK12 mutation may influence immune response remain unclear. A plausible explanation for immune evasion in this subset of CDK12-mutated PCa may be reduced MHC expression.ResultsUsing genomic data of CDK12-mutated PCa from 48 primary and 10 metastatic public domain samples and a retrospective cohort of 53 low-intermediate risk primary PCa, we investigated how variation in the expression of the MHC genes affected associated downstream pathways. We classified the patients based on gene expression quartiles of MHC-related genes and categorized the tumors into “High” and “Low” expression levels. CDK12-mutated tumors with higher MHC-expressed pathways were associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. Consistent with an inflamed tumor microenvironment (TME) phenotype, digital cytometric analyses identified increased CD8 + T cells, B cells, γδ T cells, and M1 Macrophages in this group. In contrast, CDK12-mutated tumors with lower MHC expression exhibited features consistent with an immune cold TME phenotype and immunoediting. Significantly, low MHC expression was also associated with chromosome 6 loss of heterozygosity (LOH) affecting the entire HLA gene cluster. These LOH events were observed in both major clonal and minor subclonal populations of tumor cells. In our retrospective study of 53 primary PCa cases from this Institute, we found a 4% (2/53) prevalence of CDK12 mutations, with the confirmation of this defect in one tumor through Sanger sequencing. In keeping with our analysis of public domain data this tumor exhibited low MHC expression at the RNA level. More extensive studies will be required to determine whether reduced HLA expression is generally associated with primary tumors or is a specific feature of CDK12 mutated PCa.ConclusionsThese data show that analysis of CDK12 alteration, in the context of MHC expression levels, and LOH status may offer improved predictive value for outcomes in this potentially actionable genomic subgroup of PCa. In addition, these findings highlight the need to explore novel therapeutic strategies to enhance MHC expression in CDK12-defective PCa to improve immunotherapy responses.

Abstract PO5-06-09: Metastatic triple negative breast cancer has distinct tumor immune landscape

Abstract Background: Triple negative breast cancer (TNBC) is considered the most immunogenic breast cancer subtype due to higher levels of tumor infiltrating immune cells (TILS), elevated tumor mutational burden and PD-L1 expression, providing a rationale for immunotherapy. Here we aimed to investigate the differences in the immune microenvironment of primary vs. metastatic sites in TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes, was performed on 147 real-world FFPE breast cancer samples (32 primary, 115 metastatic). 37 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. PD-L1 (CPS positive ≥1% and CPS positive ≥10%) was determined using immunohistochemistry (22C3). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong) and cell proliferation (CP, poor/moderate/high) were determined by RNA-sequencing. Demographic and clinicopathologic variables were collected. Statistical comparisons of biomarkers between groups were calculated using the Wilcoxon Rank-Sum test for continuous variables and the proportions test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Cox proportional hazards analysis (p≤0.05 for significance). Results: Triple negative status was associated with current use of alcohol [p=0.03]. Black patients were also more likely to have TNBC, constituting 24% of TNBC patients while only making up 14% of the total cohort [p=0.04]. Conversely, white patients were less likely to have TNBC, constituting 73% of TNBC patients while making up 84% of the total cohort [p=0.04]. Comparing TNBC and non-TNBC cases, TNBC cases were observed to have significantly higher TIGS [p=0.014] and PD-L1 expression [p=4.4 × 10−7]. Additionally, TNBC cases trended towards exhibiting greater cell proliferation [p=0.069]. These differences were found to be driven primarily by metastatic tumors, among which TNBC tumors showed significantly higher TIGS [p=0.015], cell proliferation [p=0.021], and PD-L1 expression [p=2.9 × 10−7] than non-TNBC tumors, while none of these significant associations were observed among primary tumors. Triple negative status was significantly associated with PD-L1 expression (assessed by IHC), with a majority of PD-L1 positive cases determined to be triple negative [70% of CPS≥1% cases, p=5 × 10-5; 59% of CPS≥10% cases, p=6 × 10−4]. Several immune-related genes and immune checkpoint molecules were over-expressed in triple negative breast cancer, including CD8, GZMB, PRF1, CCL5, IFNG, TIGIT and CXCL10. A number of genes were also significantly overexpressed in metastatic tumors: KRT5 [p=0.02], TFRC [p=0.04], ABCF1 [p=0.04], FCRLA [p=0.05], LAMP3 [p=0.05], and underexpressed in metastatic tumors: ENTPD1 [p=0.05], IGSF6 [p=0.03], and ITGA1 [p=0.03]. Conclusions: Our comprehensive biomarker analyses showed that metastatic TNBC has a more inflamed tumor microenvironment and higher checkpoint target expression compared to non-TNBC. Further analysis of immune expression by site-specific metastases and correlation with immunotherapy outcomes is warranted to guide clinicians in selection of the ideal metastatic site to biopsy for therapeutic decision making. However, in a collective TNBC context, these data support the use of immunotherapy in metastatic TNBC. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Metastatic triple negative breast cancer has distinct tumor immune landscape [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-09.

Abstract PO1-14-06: Integrated analysis reveals the impact of obesity on triple-negative breast cancer

Abstract Background: Obesity and overweight status, which has been growing rapidly over the past few decades, is considered as a risk factor for many types of cancers including breast cancer. Despite the multi-omics profile of triple-negative breast cancer (TNBC) has been comprehensively characterized, the impact of obesity on molecular features of TNBC is not fully appreciated. Methods: We applied an integrative analysis on clinicopathological data and molecular data (including genomic, transcriptomic, proteomic and metabolomic profiling) using the multi-omics database of TNBC (N = 465) from Fudan University Shanghai Cancer Center (FUSCC) for associations with patient body mass index (BMI). Patients were categorized into overweight/obese (OW/OB, BMI ≥ 24 kg/m2) and normal (NL, BMI &amp;lt; 24 kg/m2) group according to the Chinese criteria of BMI. The clinical and molecular differences between OW/OB and NL patients were systematically explored. We also constructed high-fat diet (HFD)-induced obese mouse tumor models and used single-cell RNA sequencing to investigate the impact of obesity on the tumor microenvironment. Furthermore, we analyzed the efficacy of anti-PD-1 immunotherapy on TNBC tumors in both obese and normal mice. Results: OW/OB patients exhibited higher proportion of metabolic syndrome, more adipose tissue in the breast and worse survival than NL patients. Among most frequently mutated genes, OBSCN showed statistically significantly less mutated in the OW/OB group (3.2% vs 9.6%), while TP53 (68.3% vs 76.9%) and PIK3CA (21.4% vs 14.1%) had tendency to be different. In terms of copy number alterations, we found OW/OB patients had a higher amplified or gained frequency of 13q14.11 (FOXO1) and a lower frequency of deletion or loss of chromosomal region 7p22.1 (FOXK1). We further dissect the expression profile of TNBC. Differentially expressed gene analysis and pathway enrichment analysis demonstrated that immune and metabolic pathways were the major distinction between OW/OB and NL tumors. OW/OB tumors were characterized with elevated inflammation of tumor microenvironment, as well as higher expression of immune checkpoints. Moreover, analyses focusing on metabolic heterogeneity using transcriptomic, proteomic and metabolomic data revealed upregulation of lipid metabolism and reactive oxygen species pathway in OW/OB group. In addition, our in vivo experiments demonstrated that TNBC in the obese mice displayed faster growth rates. Flow cytometry analysis and single-cell RNA sequencing showed that higher proportion of immunosuppressive myeloid cells and exhausted CD8+ T cells and upregulation of lipid metabolism in HFD group. Applying anti-PD-1 immunotherapy in both obese and normal mice displayed that tumors in the obese mice showed more sensitive to anti-PD-1 immunotherapy. Conclusion: Our study systematically revealed that obesity might play a significant role in the molecular heterogeneity of TNBC and showed distinct sensitivities to immunotherapy, which should be taken in account in the field of precision medicine. Keywords: triple-negative breast cancer, obesity, immune, tumor microenvironment, metabolism Citation Format: Yue Gong, Peng Ji, Huai-liang Wu, Li-Hua He, Ming-Liang Jin, Xin Hu, Yi-Zhou Jiang, Zhiming Shao. Integrated analysis reveals the impact of obesity on triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-06.

Taurine Rescues Cancer-induced Atrophy in Human Skeletal Muscle Cells via Ameliorating the Inflammatory Tumor Microenvironment.

Cancer cachexia is a wasting syndrome that has a devastating impact on the prognosis of patients with cancer. It is well-documented that pro-inflammatory cytokines are involved in the progression of this disorder. Therefore, this study was conducted to investigate the protective effect of taurine, an essential nonprotein amino acid with great anti-inflammatory properties, in attenuating muscle atrophy induced by cancer. Conditioned media (CM) derived from T24 human bladder carcinoma cells with or without 5 mM taurine were incubated with human skeletal muscle cells (HSkMCs) and their differentiation was examined. The intracellular reactive oxygen species (ROS), morphology, and the catabolic pathway were monitored. T24-derived CM with high levels of TNF-α and IL-6 caused aberrant ROS accumulation and formation of atrophic myotubes by HSkMCs. In T24 cancer cells, taurine significantly inhibited the production of TNF-α and IL-6. In HSkMCs, taurine increased ROS clearance during differentiation and preserved the myotube differentiation ability impaired by the inflammatory tumor microenvironment. In addition, taurine ameliorated myotube atrophy by regulating the Akt/FoxO1/MuRF1 and MAFbx signaling pathways. Taurine rescues cancer-induced atrophy in human skeletal muscle cells by ameliorating the inflammatory tumor microenvironment. Taurine supplementation may be a promising approach for intervening with the progression of cancer cachexia.

DNA methylation regulator-based molecular subtyping and tumor microenvironment characterization in hepatocellular carcinoma.

Although recent studies have reported the regulation of the immune response in hepatocellular carcinoma (HCC) through DNA methylation, the comprehensive impact methylation modifications on tumor microenvironment characteristics and immunotherapy efficacy has not been fully elucidated. In this research, we conducted a comprehensive assessment of the patterns of DNA methylation regulators and the profiles of the tumor microenvironment (TME) in HCC, focusing on 21 specific DNA methylation regulators. We subsequently developed a unique scoring system, a DNA methylation score (DMscore), to assess the individual DNA methylation modifications among the three distinct methylation patterns for differentially expressed genes (DEGs). Three distinct methylation modification patterns were identified with distinct TME infiltration characteristics. We demonstrated that the DMscore could predict patient subtype, TME infiltration, and patient prognosis. A low DMscore, characterized by an elevated tumor mutation burden (TMB), hepatitis B virus (HBV)/hepatitis C virus (HCV) infection, and immune activation, indicates an inflamed tumor microenvironment phenotype with a 5-year survival rate of 7.8%. Moreover, a low DMscore appeared to increase the efficacy of immunotherapy in the anti-CTLA-4/PD-1/PD-L1 cohort. In brief, this research has enhanced our understanding of the correlation between modifications in DNA methylation patterns and the profile of the tumor microenvironment in individuals diagnosed with HCC. The DMscore may serve as an alternative biomarker for survival and efficacy of immunotherapy in patients with HCC.

The C1q and gC1qR axis as a novel checkpoint inhibitor in cancer.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

DNA nanodevice as a multi-module co-delivery platform for combination cancer immunotherapy

A major challenge in combining cancer immunotherapy is the efficient delivery of multiple types of immunological stimulators to elicit a robust anti-tumor immune response and reprogram the immunosuppressive tumor microenvironment (TME). Here, we developed a DNA nanodevice that was generated by precisely assembling three types of immunological stimulators. The doxorubicin (Dox) component induced immunogenic cell death (ICD) in tumor cells and enhanced phagocytosis of antigen-presenting cells (APCs). Exogenous double-stranded DNA (dsDNA) could act as a molecular adjuvant to activate the stimulator of interferon genes (STING) signaling in APCs by engulfing dying tumor cells. Interleukin (IL)-12 and small hairpin programmed cell death-ligand 1 (shPD-L1) transcription templates were designed to regulate TME. Additionally, for targeted drug delivery, multiple cyclo[Arg-Gly-Asp-(d-Phe)-Cys] (cRGD) peptide units on DNA origami were employed. The incorporation of disulfide bonds allowed the release of multiple modules in response to intracellular glutathione (GSH) in tumors. The nanodevice promoted the infiltration of CD8+ and CD4+ cells into the tumor and generated a highly inflamed TME, thereby enhancing the effectiveness of cancer immunotherapy. Our research results indicate that the nanodevice we constructed can effectively inhibit tumor growth and prevent lung metastasis without obvious systemic toxicity, providing a promising strategy for cancer combination treatment.

Inflammatory and subtype-dependent serum protein signatures predict survival beyond the ctDNA in aggressive B cell lymphomas.

Biological heterogeneity of large B cell lymphomas (LBCLs) is poorly captured by current prognostic tools, hampering optimal treatment decisions. We dissected the levels of 1,463 serum proteins in a uniformly treated trial cohort of 109 patients with high-risk primary LBCL (ClinicalTrials.gov: NCT01325194) and correlated the profiles with molecular data from tumor tissue and circulating tumor DNA (ctDNA) together with clinical data. We discovered clinically and biologically relevant associations beyond established clinical estimates and ctDNA. We identified an inflamed serum protein profile, which reflected host response to lymphoma, associated with inflamed and exhausted tumor microenvironment features and high ctDNA burden, and translated to poor outcome. We composed an inflammation score based on the identified inflammatory proteins and used the score to predict survival in an independent LBCL trial cohort (ClinicalTrials.gov: NCT03293173). Furthermore, joint analyses with ctDNA uncovered multiple serum proteins that correlate with tumor burden. We found that SERPINA9, TACI, and TARC complement minimally invasive subtype profiling and that TACI and TARC can be used to evaluate treatment response in a subtype-dependent manner in the liquid biopsy. Altogether, we discovered distinct serum protein landscapes that dissect the heterogeneity of LBCLs and provide agile, minimally invasive tools for precision oncology. This research was funded by grants from the Research Council of Finland, Finnish Cancer Organizations, Sigrid Juselius Foundation, University of Helsinki, iCAN Digital Precision Cancer Medicine Flagship, Orion Research Foundation sr, and Helsinki University Hospital.

The senescence journey in cancer immunoediting.

Cancer progression is continuously controlled by the immune system which can identify and destroy nascent tumor cells or inhibit metastatic spreading. However, the immune system and its deregulated activity in the tumor microenvironment can also promote tumor progression favoring the outgrowth of cancers capable of escaping immune control, in a process termed cancer immunoediting. This process, which has been classified into three phases, i.e. "elimination", "equilibrium" and "escape", is influenced by several cancer- and microenvironment-dependent factors. Senescence is a cellular program primed by cells in response to different pathophysiological stimuli, which is based on long-lasting cell cycle arrest and the secretion of numerous bioactive and inflammatory molecules. Because of this, cellular senescence is a potent immunomodulatory factor promptly recruiting immune cells and actively promoting tissue remodeling. In the context of cancer, these functions can lead to both cancer immunosurveillance and immunosuppression. In this review, the authors will discuss the role of senescence in cancer immunoediting, highlighting its context- and timing-dependent effects on the different three phases, describing how senescent cells promote immune cell recruitment for cancer cell elimination or sustain tumor microenvironment inflammation for immune escape. A potential contribution of senescent cells in cancer dormancy, as a mechanism of therapy resistance and cancer relapse, will be discussed with the final objective to unravel the immunotherapeutic implications of senescence modulation in cancer.

Abstract 5459: Inflammatory gene-based subtyping of osteosarcoma patients reveals the association of inflammation in tumor microenvironment with better survival

Abstract Introduction:Inflammation has a significant impact on the tumor microenvironment (TME) of osteosarcoma (OS). Nevertheless, due to the high heterogenecity and variable inflammatory characteristics among OS patients, it is still unclear which inflammatory factors or immune/stroma cell types are crucially associated with OS prognosis. Therefore, it is essential to explore the immune infiltration pattern and the related core inflammatory genes in OS. Method:We combined bulk RNA-seq data of 88 osteosarcoma (OS) patients from the TARGET database and single cell RNA-seq data in GSE152048 from the NCBI Gene Expression Omnibus. Firstly, we calculated the inflammatory gene expression matrix of bulk RNA-seq data by combining 80 inflammatory gene sets (3427 genes in total) from the MSigDB using the gene set variation analysis (GSVA). Secondly, consensus clustering was executed to evaluate the stability of clustering for the above matrix. Furthermore, the overall survival and differential expression analyses were used to screen for core inflammatory genes. Immune infiltration patterns among groups were profiled with CIBERSORT. Finally, we employed the scRNA-seq series to evaluate cell-level information to identify the survival-associated cell subpopulations using the Seurat package. The t-SNE was used for dimensionality reduction and cluster identification. Results:88 osteosarcoma (OS) patients were clustered into two distinct groups according to the consensus clustering of our formed GSVA inflammatory gene expression matrix. Group 1 (43 patients) exhibited evident inflammation features, while Group 2 (45 patients) displayed limited inflammation features. Patients from Group 1 had significantly better overall survival than those from Group 2. The difference in immune infiltration pattern of two groups were examined using bulk RNA-seq data. Group 1 showed a higher population of macrophages and CD8+ T cells compared to Group 2, implying high inflammatory features might lead to a better immune activation of TME in patients. To explore the factors and cell types that contributes to restricted immune infiltration and activation in OS TME, scRNA-seq data of 11 OS patient tissues were analyzed. Consistently with bulk RNA seq analysis, the 338 up-regulated genes from Group 1 were mainly expressed in immune cells including macrophages and myeloid cells. Interestingly, the 64 up-regulated genes from Group 2 were found nearly uniformly expressed in two type of non-immune cells: myoblasts and chondroblastic cells. Conclusion:Inflammatory gene-based subtyping of osteosarcoma (OS) patients reveals that the inflammation in TME led by myeloid-derived immune cells including macrophage is associated with better prognosis. Furthermore, myoblasts and chondroblastic cells might contribute to the limited inflammation response in OS TME. Citation Format: Liu Yang, Yalan Sheng, Shuangying Qiao, Desheng Hu, Debajyoti Chowdhury, Hiu Fung Yip, Zheng Chen, Yun He, Aiping Lu, Fangfei Li. Inflammatory gene-based subtyping of osteosarcoma patients reveals the association of inflammation in tumor microenvironment with better survival [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 5459.

Abstract 169: Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas

Abstract Background: Although there is raising evidence that the density and the level of immune checkpoint expression of immune cell subpopulations that are in direct contact to the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patient’s outcome, a comprehensive assessment of intraepithelial immune cell subpopulations and their spatial interplay is lacking. Material and methods: To comprehensively assess the density, level of immune checkpoint expression, and spatial interplay of 48 immune checkpoint expressing intraepithelial leukocyte subpopulations in 46 carcinoma entities, 4740 tumor samples in a tissue microarray format were stained with 21 antibodies using our BLEACH&amp;STAIN mfIHC approach. A deep learning-based framework comprising 18 different convolutional neuronal networks (U-Net and DeepLabv3+) were used for image analysis. Results: The mean intraepithelial immune cell density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ Tregs, CD20+ B-cells, M1/M2 macrophages and CD11c+ dendritic cells varied markedly between tumor entities and individual tumors. For example, 88 (±90) cells/mm2 were found in tubular breast cancer, 661 (±729) cells/mm2 in colorectal cancer, and 1280 (±1334) to 2325 (±2131) cells/mm2 in squamous cell cancers from various origins. The abundance of T-cell subpopulations, M2 macrophages, and dendritic cells was highly concordant (range r=0.49 to 0.92) across all tumor entities, while the density of M1 macrophages was unrelated (r=0.1, p&amp;lt;0.001). Unsupervised hierarchical clustering across all tumor entities revealed a cluster A of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells, thus a highly inflamed tumor microenvironment that was characterized by the strongest interaction between intraepithelial CD8+ T-cell and CD4+ T-helper cells, M2 macrophages, as well as dendritic cells (DC, p&amp;lt;0.002 each) along with the highest expression level of TIM3, PD-1, CLTA-4 and PD-L1. In contrast, a cluster C of 2245 patients with the lowest intraepithelial immune cell infiltration was characterized by a significantly increased M1 proportion (p&amp;lt;0.001) and an enriched proportion of regulatory T-cells (p&amp;lt;0.001). Across all analyzed tumor entities, the intraepithelial highly inflamed cluster A was significantly linked to low pT (p&amp;lt;0.001). Conclusion: The data from this study provide a comprehensive characterization of intraepithelial immune cells across 46 different human carcinomas and identify an inflamed phenotype defined by strong interactions of intraepithelial CD8+ cytotoxic T-cells, CD4+ T-cells, dendritic cells, and M2 macrophages along with highest levels of TIM3, PD-1, PD-L1, and CTLA-4 expression that is linked to a favorable tumor phenotype. Citation Format: Zhihao Huang, Tim Mandelkow, Jan H. Müller, Andreas H. Marx, Till Krech, Guido Sauter, Julia Ebner, Maximilian Lennartz, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Elena Bady, Niclas C. Blessin. Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas [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 169.

Abstract 5351: Tumor PKC-Delta induces immune exclusion in non-small cell lung cancer

Abstract The recruitment of a sufficient number of immune cells to induce an inflamed tumor microenvironment (TME) is a prerequisite for effective response to cancer immunotherapy. The immunological phenotypes in the TME of EGFR-mutated lung cancer were characterized as non-inflamed, for which immunotherapy is largely ineffective. To orchestrate host immunity against lung tumors, we proposed that PKCδ, a gatekeeper of immune homeostasis with kinase activity, is responsible for the un-inflamed phenotype in EGFR-mutated lung tumors. It promotes tumor progression by stimulating extracellular matrix (ECM) and PD-L1 expression which leads to immune exclusion and assists cancer cell escape from T cell surveillance. Ablation of PKCδ enhances the intratumoral penetration of T cells and suppresses the growth of tumors. Furthermore, blocking PKCδ significantly sensitizes the tumor to immune checkpoint blockade (ICB) therapy (αPD-1) in vitro and in vivo models. These findings revealed that PKCδ is a critical switch to induce inflamed tumors and consequently enhances the efficacy of ICB therapy in EGFR-mutated lung cancer. This opens a new avenue for applying immunotherapy against recalcitrant tumors. This work was funded by the FDCT project grant to Dr. Xing-Xing Fan (Grant no. 0058/2020/A2). Citation Format: Xing Xing Fan. Tumor PKC-Delta induces immune exclusion in non-small cell lung cancer [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 5351.

Abstract 5490: Spatial profiling of inflamed tumor microenvironment of clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) presents a distinctive tumor microenvironment (TME) characterized by extensive vascularization and immune infiltration. This unique TME makes ccRCC an attractive target for anti-angiogenesis and immune checkpoint blockade (ICB). To better understand the complexity of ccRCC and the associated cell types and states involved in disease progression, we conducted high-throughput single-cell immunofluorescence imaging on tissue sections from 437 localized tumors that were surgically resected. These samples included regions from the tumor center, tumor edge, and adjacent benign areas (TMA cores, n=1859). Our primary focus was on tumors with elevated immune cell enrichment (CD45high), as these tumors are known to be aggressive and may respond well to immunotherapy. Our study reveals the intricate interactions between tumor, vascular, stromal, and immune cells within ccRCC. We find that CD45high tumors, upon losing epithelial marker expression, acquire tumor cell PD-L1 and mesenchymal markers such as Fibroblast activation protein (FAP). This shift is indicative of the epithelial-mesenchymal transition (EMT) process. These CD45high and EMThigh tumors are enriched with changes in TME, including elevated FAP+ cancer-associated fibroblasts (CAFs) and subsets of immune suppressive T cells and macrophages. We report that ccRCC patients with high tumor PD-L1 expression or abundant FAP+ CAFs are associated with earlier metastasis and, in sunitinib-treated patients, shorter progression-free survival. This study illuminates the intricate connection between immune cell infiltration, stromal biology, and EMT in ccRCC, offering a deeper understanding of the disease's complex biology and potentially guiding future therapeutic approaches and strategies. Citation Format: Teijo Pellinen, Lassi Luomala, Kalle Mattila, Jenni Säilä, Annabrita Hemmes, Katja Valimaki, Oscar Bruck, Lassi Paavolainen, Harry Nisen, Petrus Järvinen, Olli Kallioniemi, Paula Vainio, Panu Jaakkola, Tuomas Mirtti. Spatial profiling of inflamed tumor microenvironment of clear cell renal cell carcinoma [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 5490.

Abstract 1538: Impact of C-reactive protein on the efficacy of immune checkpoint inhibitors in non-small cell lung cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) therapy has achieved remarkable success in the treatment of non-small cell lung cancer (NSCLC). Clinical studies have suggested a correlation between C-reactive protein (CRP) levels and treatment efficacy, but it remains unclear whether CRP can directly impact the efficacy. Therefore, we investigated the imapct of CRP on the efficacy of ICIs in NSCLC. Methods: The tumor growth model was established in CRP knockout and wildtype mice by injecting LLC cells to evaluate the effect of CRP on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse tumors were detected by flow cytometry. The impact of CRP on macrophage polarization was evaluated using flow cytometry and IF, while its impact on T cell chemokines CCL5, CXCL9, and CXCL10 was analyzed by ELISA and qRT-PCR. mCRP and α-PD-1 were implemented in C57/BL6 mice injected with LLC cells to evaluate the effect of mCRP on tumor immunotherapy. The frequencies of T cells in mouse tumors were detected by flow cytometry. Expression of mCRP and infiltration of immune cells in human NSCLC tissues were analyzed using IHC. Results: We found that tumor progression was enhanced and the proportion of tumor-infiltrated M1 macrophages was decreased in CRP knockout mice. Using human PBMC-derived macrophage and RAW264.7 cell lines, we demonstrated that the monomeric form of CRP, known as mCRP, induced M1 polarization of macrophages, leading to increased secretion of CCL5, CXCL9, and CXCL10. In tumor bearing mice, mCRP treatment enhanced anti-tumor immunity and suppressed tumor progression by increasing CD4+ and CD8+ T cell recruitment. Surprisingly, the combined treatment of mCRP and α-PD-1 induced more significant tumor regression than α-PD-1 treatment alone. Additionally, high level of mCRP in NSCLC tissues is correlated with a more inflamed tumor microenvironment phenotype. Conclusion: mCRP can enhance the efficacy of immune checkpoint inhibitors in NSCLC by activating anti-tumor immunity. Citation Format: Wenyuan Li, Hui Guo. Impact of C-reactive protein on the efficacy of immune checkpoint inhibitors in non-small cell lung cancer [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 1538.

Abstract 2691: Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancer cases, and is among the most aggressive and lethal malignancies worldwide. PDAC is driven by genetic alterations in the pancreatic epithelium (e.g. KRAS, TRP53) coupled with a dysregulated innate immune response, the latter leading to an inflammatory tumor microenvironment enriched in innate immune cells (e.g. macrophages). However, immune-based treatment regimens for PDAC patients have primarily focused on immunotherapy with adaptive immune checkpoint inhibitors (e.g. PD-1) which disappointingly have yielded minimal clinical benefit. Thus, there is an unmet clinical need to identity disease-associated innate immune regulators as therapeutic targets in PDAC. Innate immune responses depend on a series of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs) that are expressed in immune and non-immune cells. PRRs are classified into several structurally and functionally conserved subfamilies, including Toll-like receptors (TLRs), Absent in melanoma 2 (AIM2)-like receptors (ALRs), and Nod-like receptors (NLRs). A subset of NLRs and AIM2 are also well documented for their formation of multiprotein complexes called ‘inflammasomes’ - comprising the adaptor ASC and Caspase-1 - which direct the maturation and release of bioactive pro-inflammatory cytokines, IL-18 and IL-1β. However, the role of these critical innate immune regulators in PDAC has been underexplored, and is virtually unknown. By coupling endoscopic ultrasound-guided fine needle aspiration (EUS FNA) to capture tumor biopsies from all stages of PDAC, with whole transcriptome profiling of PDAC patient primary tumors, we reveal specific enrichment of the innate immune TLR2 and ASC inflammasome molecular pathways. Augmented TLR2 and ASC expression was prognostic for low survival, and a “TLR2 activation” signature was also predictive for chemoresistance. Antibody-mediated anti-TLR2 therapy suppressed tumor growth in human PDAC tumor xenografts (patient and cell line) in both immunocompromised and ‘humanized’ mice. With respect to ASC, using the genetically engineered KPC PDAC mouse model, we show that Caspase-1 activation, a read-out for inflammasome activation, is upregulated in pancreatic tumors. Moreover, genetic ablation of ASC in KPC mice markedly suppressed the development of pancreatic tumors, which was associated with significant reductions in levels of both Caspase-1 activation and inflammation. Transcriptome profiling further identified unique downstream molecular networks associated with a range of oncogenic cellular processes. Taken together, our results support innate immune TLR2- and ASC inflammasome-based therapeutic targeting in PDAC, with further clinical utility that ASC inflammasome and TLR2 activation is prognostic, with TLR2 also predictive for chemoresponsiveness. Citation Format: Bassam Kashgari, Louise McLeod, Linden J. Gearing, Daniel Croagh, Brendan J. Jenkins. Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer [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 2691.