Components Of Tumor Microenvironment Research Articles

Hydrogen Therapy Reverses Cancer-Associated Fibroblasts Phenotypes and Remodels Stromal Microenvironment to Stimulate Systematic Anti-Tumor Immunity.

Tumor microenvironment (TME) plays an important role in the tumor progression. Among TME components, cancer-associated fibroblasts (CAFs) show multiple tumor-promoting effects and can induce tumor immune evasion and drug-resistance. Regulating CAFs can be a potential strategy to augment systemic anti-tumor immunity. Here, the study observes that hydrogen treatment can alleviate intracellular reactive oxygen species of CAFs and reshape CAFs' tumor-promoting and immune-suppressive phenotypes. Accordingly, a controllable and TME-responsive hydrogen therapy based on a CaCO3 nanoparticles-coated magnesium system (Mg-CaCO3) is developed. The hydrogen therapy by Mg-CaCO3 can not only directly kill tumor cells, but also inhibit pro-tumor and immune suppressive factors in CAFs, and thus augment immune activities of CD4+ T cells. As implanted in situ, Mg-CaCO3 can significantly suppress tumor growth, turn the "cold" primary tumor into "hot", and stimulate systematic anti-tumor immunity, which is confirmed by the bilateral tumor transplantation models of "cold tumor" (4T1 cells) and "hot tumor" (MC38 cells). This hydrogen therapy system reverses immune suppressive phenotypes of CAFs, thus providing a systematic anti-tumor immune stimulating strategy by remodeling tumor stromal microenvironment.

Clinical Significance of a Novel Vasculogenic Mimicry-Based Prognostic Model in Hepatocellular Carcinoma.

Vasculogenic mimicry, a novel neovascularization pattern of aggressive tumors, is associated with poor clinical outcomes. The aim of this research was to establish a new model, termed VC score, to predict the prognosis, Tumor Microenvironment (TME) components, and immunotherapeutic response in Hepatocellular Carcinoma (HCC). The expression data of the public databases were used to develop the prognostic model. Consensus clustering was performed to confirm the molecular subtypes with ideal clustering efficacy. The high- and low-risk groups were stratified utilizing the VC score. Various methodologies, including survival analysis, single-sample Gene Set Enrichment Analysis (ssGSEA), Tumor Immune Dysfunction and Exclusion scores (TIDE), Immunophenoscore (IPS), and nomogram, were utilized for verification of the model performance and to characterize the immune status of HCC tissues. GSEA was performed to mine functional pathway information. The survival and immune characteristics varied between the three molecular subtypes. A five-gene signature (TPX2, CDC20, CFHR4, SPP1, and NQO1) was verified to function as an independent predictive factor for the prognosis of patients with HCC. The high-risk group exhibited lower Overall Survival (OS) rates and higher mortality rates in comparison to the low-risk group. Patients in the low-risk group were predicted to benefit from immune checkpoint inhibitor therapy and exhibit increased sensitivity to immunotherapy. Enrichment analysis revealed that signaling pathways linked to the cell cycle and DNA replication processes exhibited enrichment in the high-risk group. The VC score holds the potential to establish individualized treatment plans and clinical management strategies for patients with HCC.

Analysis of tumor microenvironment composition in vestibular schwannomas: insights into NF2-associated and sporadic variations and their clinical correlations.

Vestibular schwannomas (VS), benign tumors stemming from the eighth cranial nerve's Schwann cells, are associated with Merlin gene mutations, inflammation, and the tumor microenvironment (TME), influencing tumor initiation, maintenance, and potential neural dysfunction. Understanding TME composition holds promise for systemic therapeutic interventions, particularly for NF2-related schwannomatosis. A retrospective analysis of paraffin-embedded tissue from 40 patients (2013-2020), evenly divided by neurofibromatosis type 2 status, with further stratification based on magnetic resonance imaging (MRI) progression and hearing function. Immunohistochemistry assessed TME components, including T-cell markers (CD4, CD8, CD25), NK cells (CD7), and macrophages (CD14, CD68, CD163, CCR2). Fiji software facilitated image analysis. T-cell markers (CD4, CD8, CD7) exhibited low expression in VS, with no significant NF2-associated vs. sporadic distinctions. Macrophage-related markers (CD14, CD68, CD163, CCR2) showed significantly higher expression (CD14: p = 0.0187, CD68: p < 0.0001, CD163: p = 0.0006, CCR2: p < 0.0001). CCR2 and CD163 significantly differed between NF2-associated and sporadic VS. iNOS, an M1-macrophage marker, was downregulated. CD25, a regulatory T-cell marker, correlated significantly with tumor growth dynamics (p = 0.016). Immune cells, notably monocytes and macrophages, crucially contribute to VS pathogenesis in both NF2-associated and sporadic cases. Significant differences in CCR2 and CD163 expression suggest distinct immune responses. Regulatory T-cells may serve as growth dynamic markers. These findings highlight immune cells as potential biomarkers and therapeutic targets for managing VS.

Cellular collusion: cracking the code of immunosuppression and chemo resistance in PDAC.

Despite the efforts, pancreatic ductal adenocarcinoma (PDAC) is still highly lethal. Therapeutic challenges reside in late diagnosis and establishment of peculiar tumor microenvironment (TME) supporting tumor outgrowth. This stromal landscape is highly heterogeneous between patients and even in the same patient. The organization of functional sub-TME with different cellular compositions provides evolutive advantages and sustains therapeutic resistance. Tumor progressively establishes a TME that can suit its own needs, including proliferation, stemness and invasion. Cancer-associated fibroblasts and immune cells, the main non-neoplastic cellular TME components, follow soluble factors-mediated neoplastic instructions and synergize to promote chemoresistance and immune surveillance destruction. Unveiling heterotypic stromal-neoplastic interactions is thus pivotal to breaking this synergism and promoting the reprogramming of the TME toward an anti-tumor milieu, improving thus the efficacy of conventional and immune-based therapies. We underscore recent advances in the characterization of immune and fibroblast stromal components supporting or dampening pancreatic cancer progression, as well as novel multi-omic technologies improving the current knowledge of PDAC biology. Finally, we put into context how the clinic will translate the acquired knowledge to design new-generation clinical trials with the final aim of improving the outcome of PDAC patients.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [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 PO4-24-03.

Abstract PO2-07-06: Multimodal learning predictor of HER2-positive breast cancer therapy response in the randomized PREDIX HER2 trial

Abstract Background: The PREDIX HER2 trial, compared six courses of docetaxel, trastuzumab, and pertuzumab (DTP) vs. trastuzumab emtansine (T-DM1) as neoadjuvant treatment for HER2-positive breast cancer (BC). Similar rates of pathologic complete response (pCR) were seen. Methods: Clinicopathological, shallow whole-genome sequencing (CUTseq, n=176), whole exome sequencing (WES, n=192), and RNA-sequencing (RNA-seq, n=187) data were generated using fresh-frozen baseline core biopsies. Potential tumor intrinsic resistance factors and microenvironment components were quantified by multi-omics analysis, including BC-specific somatic mutations and copy number alterations (CNA), COSMIC mutational signatures, CNA-based chromosomal instability signatures (CIN), subclone percentage, PAM50 subtype, GGI/PIK3CA score, HER2DX score, immune profiles (Danaher signature score, TIDE score and immune repertoires). We assessed the association of biomarkers with pCR in each treatment arm using logistic regression adjusting for hormone receptor (HR) status, and evaluated their predictive value by adding the interaction term (biomarker x treatment arm). In addition, a machine learning (ML) analysis was conducted from different classifiers, comprising unimodal ML-based models from clinical, RNA and DNA information, respectively. Model performance was assessed using the mean and standard deviation (mean ± std) of the area under receiver operator characteristic curve (AUC), positive predictive value (PPV) and negative predictive value (NPV) using a nested stratified cross-validation (CV) schema of 200 outer shuffle splits and 100 inner 5-fold CV to mitigate potential risk of overfitting. Results: In DTP arm, patients with higher ERBB2 copy ratio (ORadj=1.98, p=0.004) or mRNA (ORadj=3.08, p&amp;lt; 0.001) or HER2-enriched subtype (PAM50) (ORadj=1.78, p=0.02) had higher pCR rates, while ESR1 gene expression (ORadj=0.59, p=0.07) predicted treatment resistance despite adjustment for HR status. Conversely, response to T-DM1 was less likely to depend on ERBB2 profiles and only PAM50 HER2 enriched subtype (ORadj=1.53, p=0.1) showed higher pCR rate (52% vs. 25%) than other subtypes. Both ESR1 (ORadj=0.4, p=0.008) and PGR (ORadj=0.5, p=0.03) gene expression were independent predictors of T-DM1 resistance. Pre-treatment immune exclusion metrics could predict resistance to DTP (endothelial cell, ORadj=0.67, p=0.07) and T-DM1 (neutrophils, ORadj=0.54, p=0.02; mast cells, ORadj=0.57, p=0.02; cancer-associated fibroblasts, ORadj=0.67, p=0.09)), respectively. Predefined metrics such as PIK3CA signature score (ORadj =1.67, p=0.04) and Taxane response score (ORadj =1.64, p=0.03) were positively related to pCR in DTP arm. Genome instability, involving CIN CX2 signature (impaired homologous recombination) (ORadj=1.71, p=0.05), COSMIC signature6 (ORadj =1.53, p=0.07) and signature13 (ORadj =1.57, p=0.05), predicted benefit from DTP. The biomarker-treatment interaction tests were significant for HER2DX (pinteraction=0.004) and COSMIC signature15 (defective DNA mismatch repair) (pinteraction=0.007): lower HERDX score (ORadj =0.73, p=0.14) or higher COSMIC signature15 score (ORadj =1.51, p=0.1) could identify patients benefiting from T-DM1, while being resistant to DTP (HERDX: ORadj =1.46, p=0.13; signature15: ORadj =0.70, p=0.1). In the ML models, clinical information yielded an AUC=0.62±0.07, PPV=0.64±0.12 and NPV=0.64±0.06; for DNA data, AUC was equal to 0.70±0.08, PPV=0.72±0.09 and NPV=0.71±0.07; an adaptive boosting ensemble learning on RNA reported slightly increased pCR prediction performance (AUC=0.72±0.06, PPV=0.64±0.06, NPV=0.80±0.09). Conclusion: This study demonstrates that antibody–drug conjugates and standard treatment harbor strikingly distinctive biomarkers across tumor ecosystem. Further external validation and integrated ML model comprising all unimodal models are ongoing. Citation Format: Kang Wang, Yajing Zhu, Ioannis Zerdes, Emmanouil Sifakis, Georgios Manikis, Dimitrios Salgkamis, Nikolaos Tsiknakis, Luuk Harbers, Nicola Crosetto, Jonas Bergh, Alexios Matikas, Thomas Hatschek, Theodoros Foukakis. Multimodal learning predictor of HER2-positive breast cancer therapy response in the randomized PREDIX HER2 trial [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 PO2-07-06.

Abstract PO3-24-09: May ESR1 mutant breast cancer cells influence fibroblast phenotype?

Abstract For almost 50 years, it has been postulated that carcinogenesis relies on the intrinsic genetic abnormalities of tumor cells, but in the last decades, it has been reported that their functional interactions with the tumor microenvironment (TME) might affect therapy response and contribute to the disease progression. Based on these observations, the aim of the present study was to investigate the functional interactions between one of the essential components of TME, the fibroblasts, and breast cancer (BC) cells, expressing mutations in the hormone-binding domain (HBD) of the gene encoding for the estrogen receptor alpha (ESR1). Thus, we employed estrogen receptor-positive MCF-7 BC cells CRISPR engineered to express the Y537S-HBD mutation. Y537S is the most commonly detected mutation harbored in HBD-ESR1 and is a major contributor to endocrine therapy resistance and poor clinical outcome in BC. We collected conditioned media (CM) from BC cells and fibroblasts isolated from primary human tissues (normal fibroblasts, NFs, and cancer-associated fibroblasts, CAFs) in order to assess in vitro co-culture systems that can mimic the complex in vivo TME. It was interesting to observe that CAFs displayed a significant increase in their proliferation and migration when exposed to CM of mutant BC cells with respect to the parental ones. These latter findings fit with the immunofluorescence analysis showing a more evident formation of actin stress fibers. However, proteomic analysis demonstrated, in the same circumstances, more significant changes in NFs than in CAFs. Among the up-regulated proteins revealed by NFs upon Y537S-CM exposure, most of them overlap the up-regulated proteins featuring the intrinsic phenotype of CAFs, mainly involved in the cellular ultrastructural organization (i.e. cytoskeleton, focal adhesion, vesicle, and organelle) as evidenced by Metacore functional analysis. On the other side, we demonstrated that mutant clones showed more aggressive behavior in terms of proliferation, growth, migration, and invasion, compared to the parental counterpart upon exposure to the CM-derived from NFs and CAFs. This led us to further investigate the intrinsic properties of the mutated clones through their genomic and proteomic profiles that evidenced, by using Metacore software, an enrichment of insulin-like growth factor 1 receptor (IGF1R) pathway. The relevance of the latter datum was confirmed by the higher content of the IGF-1 ligand found in the secretome of mutant cells. Thus, the short autocrine loop maintaining IGF1/IGF1R signaling activation in mutant clones might reasonably play an important role in mediating the autocrine and paracrine effects between mutant cells and fibroblasts. For instance, the pharmacological blockade of IGF1R signaling interfered with the reciprocal interactions between fibroblasts and BC cells that sustained tumor growth and progression as evidenced in both “in vitro” and “in vivo” experiments. In conclusion, our study addressed the IGF1R pathway as a potential druggable target helpful to disconnect mutant breast cancer cell-fibroblast crosstalk that potentiates breast tumor growth and progression. Citation Format: Luca Gelsomino, Amanda Caruso, Rocco Malivindi, Adel Elisabetta Leonetti, Emine Tasan, Salvatore Panza, Giuseppina Daniela Naimo, Ines Barone, Cinzia Giordano, Daniela Bonofiglio, Loredana Mauro, Guowei Gu, Suzanne Fuqua, Stefania Catalano, Sebastiano Andò. May ESR1 mutant breast cancer cells influence fibroblast phenotype? [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 PO3-24-09.

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.

VDR is a potential prognostic biomarker and positively correlated with immune infiltration: a comprehensive pan-cancer analysis with experimental verification.

The vitamin D receptor (VDR) is a transcription factor that mediates a variety of biological functions of 1,25-dihydroxyvitamin D3. Although there is growing evidence of cytological and animal studies supporting the suppressive role of VDR in cancers, the conclusion is still controversial in human cancers and no systematic pan-cancer analysis of VDR is available. We explored the relationships between VDR expression and prognosis, immune infiltration, tumor microenvironment, or gene set enrichment analysis (GSEA) in 33 types of human cancers based on multiple public databases and R software. Meanwhile, the expression and role of VDR were experimentally validated in papillary thyroid cancer (PTC). VDR expression decreased in 8 types and increased in 12 types of cancer compared with normal tissues. Increased expression of VDR was associated with either good or poor prognosis in 13 cancer types. VDR expression was positively correlated with the infiltration of cancer-associated fibroblasts, macrophages, or neutrophils in 20, 12, and 10 cancer types respectively and this correlation was experimentally validated in PTC. Increased VDR expression was associated with increased percentage of stromal or immune components in tumor microenvironment (TME) in 24 cancer types. VDR positively and negatively correlated genes were enriched in immune cell function and energy metabolism pathways, respectively, in the top 9 highly lethal tumors. Additionally, VDR expression was increased in PTC and inhibited cell proliferation and migration. In conclusion, VDR is a potential prognostic biomarker and positively correlated with immune infiltration as well as stromal or immune components in TME in multiple human cancers.

Formyl peptide enhances cancer immunotherapy by activating antitumoral neutrophils, and T cells

Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.

A critical review on exploring tumor Microenvironment's impacts on radioresistance

Malignant tumors comprise cancer cells and their surrounding tumor microenvironment (TME). This environment includes cellular and non-cellular components that collectively form a sophisticated network contributing to cancer cell survival, progression, immune evasion, and resistance to treatment. Recent findings have shown that TME components act as a protective "fortress," effectively shielding cancer cells from the impact of radiation. This review provides a comprehensive overview of our current knowledge of how TME reinforces the resistance of cancer cells to radiotherapy. Furthermore, it outlines the growing body of evidence from preclinical and clinical studies regarding the influence of TME in fostering radioresistance. Additionally, it explores the potential of targeting various TME components to amplify responses to radiation in clinical settings, and delves into the associated challenges and future perspectives in this rapidly evolving field.

Increased Multiplexity in Optical Tissue Clearing-Based Three-Dimensional Immunofluorescence Microscopy of the Tumor Microenvironment by Light-Emitting Diode Photobleaching

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 μm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.

Identifying Diffuse Glioma Subtypes Based on Pathway Enrichment Evaluation.

Gliomas are highly heterogeneous in molecular, histology, and microenvironment. However, a classification of gliomas by integrating different tumor microenvironment (TME) components remains unexplored.Based on the enrichment scores of 17 pathways involved in immune, stromal, DNA repair, and nervous system signatures in diffuse gliomas, we performed consensus clustering to uncover novel subtypes of gliomas. Consistently in three glioma datasets (TCGA-glioma, CGGA325, and CGGA301), we identified three subtypes: Stromal-enriched (Str-G), Nerve-enriched (Ner-G), and mixed (Mix-G). Ner-G was charactered by low immune infiltration levels, stromal contents, tumor mutation burden, copy number alterations, DNA repair activity, cell proliferation, epithelial-mesenchymal transformation, stemness, intratumor heterogeneity, androgen receptorexpression and EGFR, PTEN, NF1 and MUC16 mutation rates, while high enrichment of neurons and nervous system pathways, and high tumor purity, estrogen receptor expression, IDH1 and CIC mutation rates, temozolomide response rate and overall and disease-free survival rates. In contrast, Str-G displayed contrastive characteristics to Ner-G. Our analysis indicates that the heterogeneity between glioma cells and neurons is lower than that between glioma cells and immune and stromal cells. Furthermore, the abundance of neurons is positively associated with clinical outcomes in gliomas, while the enrichment of immune and stromal cells has a negative association with them. Our classification method provides new insights into the tumor biology of gliomas, as well as clinical implications for the precise management of this disease.

Abstract P37: Establishment of Gastric Organoids Biobank and Its Usage

Abstract Gastric cancer is highly heterogeneous. To analyze their biology, patient-derived organoids (PDOs) are useful because they maintain intra-tumor heterogeneity (ITH) as well as inter-patient heterogeneity. Recently, four groups reported the establishment of gastric PDOs. However, it is still not easy because of the need to overcome a common problem: “contamination” of the culture with “non-cancer cell”-derived organoids from the specimen. These non-cancer organoids often overgrow and became dominant. Furthermore, PDOs have another limitation. They contain only epithelial cells and no other types of cells, making it difficult to study the tumor microenvironment (TME). Since October 2021, we have been working on establishing our own gastric PDO biobank. 63 gastric PDOs have been successfully established from 24 patients. Cancer organoids are enriched by manual selection under a microscopy. Our biobank contains 35 gastric cancer PDOs, 16 of which were confirmed as cancer using WES and WTS. Profiling for the remaining PDOs is ongoing. 12 fibroblasts and 26 cancer associated fibroblasts (CAFs) have also been successfully established. To overcome the difficulties of TME research with PDOs, co-culture platform for PDOs and fibroblasts has been established. This co-culture platform can provide new insights into the functions of CAFs, one of the major components of TME. In this poster, I will describe the progress of our gastric PDO biobank establishment and the prospects for my projects using it. Citation Format: Takeshi Hagihara, Kie Kyon Huang, Raghav Sundar, Tomoyuki Uchihara, Clara Shi Ya Ng, Su Ting Tay, Angie Lay Keng Tan, Jimmy Bok Yan So, Patrick Tan. Establishment of Gastric Organoids Biobank and Its Usage [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P37.

Cancer-associated fibroblasts: a versatile mediator in tumor progression, metastasis, and targeted therapy.

Tumor microenvironment (TME) has been demonstrated to play a significant role in tumor initiation, progression, and metastasis. Cancer-associated fibroblasts (CAFs) are the major component of TME and exhibit heterogeneous properties in their communication with tumor cells. This heterogeneity of CAFs can be attributed to various origins, including quiescent fibroblasts, mesenchymal stem cells (MSCs), adipocytes, pericytes, endothelial cells, and mesothelial cells. Moreover, single-cell RNA sequencing has identified diverse phenotypes of CAFs, with myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) being the most acknowledged, alongside newly discovered subtypes like antigen-presenting CAFs (apCAFs). Due to these heterogeneities, CAFs exert multiple functions in tumorigenesis, cancer stemness, angiogenesis, immunosuppression, metabolism, and metastasis. As a result, targeted therapies aimed at the TME, particularly focusing on CAFs, are rapidly developing, fueling the promising future of advanced tumor-targeted therapy.

Targeted Therapy for Highly Desmoplastic and Immunosuppressive Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a very poor prognosis. Despite advancements in treatment strategies, PDAC remains recalcitrant to therapies because patients are often diagnosed at an advanced stage. The advanced stage of PDAC is characterized by metastasis, which typically renders it unresectable by surgery or untreatable by chemotherapy. The tumor microenvironment (TME) of PDAC comprises highly proliferative myofibroblast-like cells and hosts the intense deposition of a extracellular matrix component that forms dense fibrous connective tissue, a process called the desmoplastic reaction. In desmoplastic TMEs, the incessant aberration of signaling pathways contributes to immunosuppression by suppressing antitumor immunity. This feature offers a protective barrier that impedes the targeted delivery of drugs. In addition, the efficacy of immunotherapy is compromised because of the immune cold TME of PDAC. Targeted therapy approaches towards stromal and immunosuppressive TMEs are challenging. In this review, we discuss cellular and non-cellular TME components that contain actionable targets for drug development. We also highlight findings from preclinical studies and provide updates about the efficacies of new investigational drugs in clinical trials.

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at advanced stages and associated with early distant metastasis and poor survival. Besides clinical factors, the tumor microenvironment (TME) emerged as a crucial determinant of patient survival and therapy response in many tumors, including PDAC. Thus, the presence of tumor-infiltrating lymphocytes and the formation of tertiary lymphoid structures (TLS) is associated with longer survival in PDAC. Although neoadjuvant therapy (NeoTx) has improved the management of locally advanced tumors, detailed insight into its effect on various TME components is limited. While a remodeling towards a proinflammatory state was reported for PDAC-infiltrating T cells, the effect of NeoTx on B cell subsets, including plasma cells, and TLS formation is widely unclear. We thus investigated the frequency, composition, and spatial distribution of PDAC-infiltrating B cells in primary resected (PR) versus neoadjuvant-treated patients using a novel multiplex immunohistochemistry panel. The NeoTx group displayed significantly lower frequencies of pan B cells, GC B cells, plasmablasts, and plasma cells, accompanied by a reduced abundance of TLS. This finding was supported by bulk RNA-sequencing analysis of an independent fresh frozen tissue cohort, which revealed that major B cell pathways were downregulated in the NeoTx group. We further observed that plasma cells frequently formed aggregates that localized close to TLS and that TLS+ patients displayed significantly higher plasma cell frequencies compared to TLS- patients in the PR group. Additionally, high densities of CD20+ intratumoral B cells were significantly associated with longer overall survival in the PR group. While CD20+ B cells held no prognostic value for NeoTx patients, an increased frequency of proliferating CD20+Ki67+ B cells emerged as an independent prognostic factor for longer survival in the NeoTx group. These results indicate that NeoTx differentially affects PDAC-infiltrating immune cells and may have detrimental effects on the existing B cell landscape and the formation of TLS. Gaining further insight into the underlying molecular mechanisms is crucial to overcome the intrinsic immunotherapy resistance of PDAC and develop novel strategies to improve the long-term outcome of PDAC patients.

Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Pathway-based stratification of gliomas uncovers four subtypes with different TME characteristics and prognosis.

Increasing evidences have found that the interactions between hypoxia, immune response and metabolism status in tumour microenvironment (TME) have clinical importance of predicting clinical outcomes and therapeutic efficacy. This study aimed to develop a reliable molecular stratification based on these key components of TME. The TCGA data set (training cohort) and two independent cohorts from CGGA database (validation cohort) were enrolled in this study. First, the enrichment score of 277 TME-related signalling pathways was calculated by gene set variation analysis (GSVA). Then, consensus clustering identified four stable and reproducible subtypes (AFM, CSS, HIS and GLU) based on TME-related signalling pathways, which were characterized by differences in hypoxia and immune responses, metabolism status, somatic alterations and clinical outcomes. Among the four subtypes, HIS subtype had features of immunosuppression, oxygen deprivation and active energy metabolism, resulting in a worst prognosis. Thus, for better clinical application of this acquired stratification, we constructed a risk signature by using the LASSO regression model to identify patients in HIS subtype accurately. We found that the risk signature could accurately screen out the patients in HIS subtype and had important reference value for individualized treatment of glioma patients. In brief, the definition of the TME-related subtypes was a valuable tool for risk stratification in gliomas. It might serve as a reliable prognostic classifier and provide rational design of individualized treatment, and follow-up scheduling for patients with gliomas.

Roles and interactions of tumor microenvironment components in medulloblastoma with implications for novel therapeutics.

Medulloblastomas, the most common malignant pediatric brain tumors, can be classified into the wingless, sonic hedgehog (SHH), group 3, and group 4 subgroups. Among them, the SHH subgroup with the TP53 mutation and group 3 generally present with the worst patient outcomes due to their high rates of recurrence and metastasis. A novel and effective treatment for refractory medulloblastomas is urgently needed. To date, the tumor microenvironment (TME) has been shown to influence tumor growth, recurrence, and metastasis through immunosuppression, angiogenesis, and chronic inflammation. Treatments targeting TME components have emerged as promising approaches to the treatment of solid tumors. In this review, we summarize progress in research on medulloblastoma microenvironment components and their interactions. We also discuss challenges and future research directions for TME-targeting medulloblastoma therapy.