Differences In Microenvironment Research Articles

Multiple Machine Learning Models, Molecular Subtyping and Singlecell Analysis Identify PANoptosis-related Core Genes and their Association with Subtypes in Crohn's Disease.

PANoptosis plays an important role in many inflammatory diseases. However, there are no reports on the association between PANoptosis and CD. This study used five machine learning algorithms - least absolute shrinkage and selection operator, support vector machine, random forest, decision tree and Gaussian mixture models - to construct CD's PANoptosis signature. Unsupervised hierarchical clustering analysis was used to identify PANoptosis-associated subgroups of CD. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were conducted to compare the PANoptosis-associated subgroups of CD among the potential biological mechanisms. Single sample GSEA was used to assess immune microenvironmental differences among the subgroups. The potential role of PANoptosis in CD was further explored using single-cell RNA-Seq (scRNA-Seq) for PANoptosis scoring, differential analysis, pseudotime analysis, cellular communication analysis and weighted gene co-expression network analysis (WGCNA) analysis. CD's PANoptosis signature consisted of seven genes: CEACAM6, CHP2, PIK3R1, CASP10, PSMB1, PSMB8 and UBC. The PANoptosis signature in multiple cohorts had a strong ability to recognise CD. The levels of immune cell infiltration and the vigour of the immune responses significantly varied between the two subpopulations of CD associated with PANoptosis. Multiple lines of evidence from the GO, KEGG, GSEA, GSVA, scRNA-Seq and WGCNA analyses suggested that I-kappaB kinase/NF- kappaB signalling, mitogen-activated protein kinase (MAPK), leukocyte activation and leukocyte migration were mechanisms closely associated with PANoptosis in CD. This study is the first to construct a PANoptosis signature with excellent efficacy in recognising CD. PANoptosis may mediate the process of CD through inflammatory and immune mechanisms, such as NF- kappaB, MAPK and leukocyte migration.

Potential pathways for traditional Chinese medicine intervention in diabetic kidney disease: macrophage polarization

Diabetic kidney disease(DKD) is a prevalent and severe microvascular complication of type 2 diabetes mellitus(T2DM). Chronic microinflammation is an important factor exacerbating renal tissue damage in DKD individuals. Macrophages play a crucial role in immune-inflammatory responses, and they can transiently and reversibly polarize into the pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype based on microenvironmental differences. The imbalance in M1/M2 macrophage polarization can exacerbate DKD progression by fostering inflammatory cytokine aggregation in the glomeruli and renal interstitium. Therefore, restoring the balance of macrophage is a pivotal avenue to ameliorate the chronic microinflammation state in DKD. Macrophage polarization is a complex and dynamic process. Various information molecules and cytokines involved in the polarization process play important roles in regulating phenotypes during the progression of DKD. They are closely related to various mechanisms such as metabolism, inflammation, fibrosis, and mitochondrial autophagy in DKD. By coordinating the inflammatory responses through polarization, they play a key role in regulating inflammation in metabolic-related diseases. The complex network of pathways involved in macrophage polarization corresponds well with the multi-pathway, multi-target treatment model of traditional Chinese medicine(TCM). Active ingredients and formulas of TCM can intervene in DKD by regulating macrophage polarization. Studies on relieving renal inflammation, repairing renal tissues, and promoting renal function recovery through macrophage polarization modulation are not uncommon. Therefore, based on exis-ting evidence, this study reviews TCM in targeting M1/M2 macrophage polarization balance to improve DKD, aiming to explore the potential of macrophage polarization in regulating DKD, which is expected to provide evidence support for the clinical diagnosis and treatment of DKD with TCM as well as the exploration of its biological mechanisms.

Effects of Different Shelling Methods on Data Variability during Field Screening for Reduced Aflatoxin Contamination in Maize.

Non-genetic variation limits the identification of novel maize germplasm with genetic markers for reduced Aspergillus flavus infection and aflatoxin contamination. Aflatoxin measurements can vary substantially within fields containing the same germplasm following inoculation with A. flavus. While some variation is expected due to microenvironmental differences, components of field screening methodologies may also contribute to variability in collected data. Therefore, the objective of this study is to test the effects of three different shelling methods (whole ear (WE), ear end removal (EER), and inoculation site-surrounding (ISS)) to obtain bulk samples from maize on aflatoxin measurements. Five ears per row of three inbred lines and two hybrids were inoculated with A. flavus, then shelled using the three different methods, and aflatoxin was quantified. Overall, EER and ISS resulted in reduced coefficients of variance (CVs) in comparison to WE for both inbred and hybrid maize lines, with two exceptions. Susceptible B73 showed increased CVs with both EER and ISS compared to WE, and resistant Mp719's EER CVs marginally increased compared to WE. While WE is the standard practice for most breeding programs due to its technical simplicity, EER and ISS may allow for finely phenotyping parental lines for further breeding applications.

Abstract PO4-25-03: Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in triple negative breast cancer

Abstract Background: KEYNOTE-522 has resulted in FDA approval of the immune checkpoint blocker pembrolizumab with neoadjuvant chemotherapy for patients with high-risk triple negative breast cancer (TNBC), given the remarkable improvement in pCR rate to 65% along with improvement in event free survival, while with chemotherapy alone, the pCR rate is 40-50%. Unfortunately, use of pembrolizumab is associated with several immune related adverse events (irAE), some of which can be life-threatening and debilitating, including cardiomyositis, encephalitis, and adrenal insufficiency, among others. Hence, there is an unmet need to identify biomarkers in the tumor microenvironment which could predict patients who may attain pCR with chemotherapy alone and be spared the side effects from the added PD-1 inhibition. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes by RNA-seq, was performed on 1:2 matched FFPE tumor samples from 23 stage 1-3 TNBC patients (2 stage 1, 6 stage 2, 15 stage 3). All patients were female with an average age of 48 years (range: 25-79 years). 14 patients were treated with neoadjuvant chemotherapy alone (NAC) and 9 were treated with neoadjuvant chemotherapy combined with the checkpoint inhibitor pembrolizumab (NAC+I). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong), cell proliferation (CP, poor/moderate/high), and cancer testis antigen expression burden (CTAB) were determined by RNA-sequencing. Statistical comparisons of quantitative biomarkers between groups were calculated using the Wilcoxon Rank-Sum test, overrepresentation analysis of categorical variables between groups was calculated by proportions test, and survival differences were quantified by Cox proportional hazards analysis. Pathological responses were documented as pathological complete response (pCR) vs. non-pCR. Results: Across the entire cohort, patients with a BMI < 30 had significantly higher PD-L1 expression (assessed by RNA) than those with a BMI ³ 30 [p=0.047]. While not a statistically significant association, the NAC+I group tended to have a higher pCR rate compared to the NAC group [44.4% vs. 28.6%, p=0.66]. Across the entire cohort, tumors with high CTA expression, designated as CTAB high, had a higher pCR rate than CTAB low tumors [54.5% vs 16.7%, p=0.089]. No significant difference in pCR rate between TIGS or CP groups was detected for the NAC group, but in the NAC+I group, highly proliferative tumors had a lower pCR rate than moderately proliferative tumors [0% vs. 80%, p=0.048]. The pCR and non-pCR groups for each treatment type also exhibited distinct gene expression profiles. Among the NAC group, underexpression of FOXP3 and overexpression of MAPK14 and CD44 were associated with pCR [p≤0.031], while underexpression of IFNB1, MAPK14, and CD44 was associated with non-pCR [p≤0.029]. Among the NAC+I group, overexpression of CXCR4, TNFR and CCL20 was associated with non-pCR [p≤0.046]. Interestingly, SDHA was significantly overexpressed in the non-pCR subset of patients in the NAC group and significantly underexpressed in the non-pCR subset of patients in the NAC+I group [p≤0.031]. Conclusions: The development of biomarkers of treatment response is essential to the integration of immunotherapy with chemotherapy as a combined cancer treatment. Our study profiled the immune context of both NAC and NAC+I and identified several key microenvironmental differences underlying divergent treatment response in both groups. A comprehensive understanding of these factors could potentially predict pCR to chemotherapy alone, enabling the avoidance of the unnecessary treatment of these tumors with immunotherapy. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Pawel Kalinski, 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. Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in 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 PO4-25-03.

Abstract PO2-13-04: Single cell characterization of senescent CD8+ T cell promotes immunotherapy resistance in early-stage triple-negative breast cancer

Abstract Background: Immune checkpoint blockade (ICB) has been explored in triple-negative breast cancer (TNBC) and has been proven to improve patient survival in advanced TNBCs (IMpassion130) or increase the pathological complete response rate in early TNBCs (KEYNOTE522). However, ICB could only benefit a subset of patients, highlighting the urgent need to optimize patient selection. Unlike in advanced TNBCs, a predictive biomarker for ICB in early TNBCs is still lacking. The main reason might be that previous studies neglected the heterogeneity of tumor ecosystems between early and advanced TNBCs. There is an urgent need to understand the unique ecosystem of early-stage TNBC and develop biomarkers for precision immunotherapy. Objective: This study aims to characterize the micro-loop associated with the immune efficacy of early TNBC, so as to explain the mechanisms behind the differences in early and late efficacy markers, identify efficacy markers in early TNBC immunotherapy, and search for clinical targeting strategies to sensitize TNBC for neoadjuvant therapy. Methods: We conducted an in-depth analysis of 32 single-cell transcriptomes, comprising 26 early-stage TNBC enrolled in prospective clinical trials (NCT04418154 and NCT04613674), receiving neoadjuvant chemotherapy alone (n=8) or combined with anti-PD1 (n=18), and 6 advanced TNBC. We analyzed the microenvironmental differences between early and advanced tumors, and between different treatment response groups in early stages, and identified a subpopulation of cells specific to early stages and associated with immunotherapeutic efficacy. We used mIHC, flow-through, and validated the presence of this cell in clinical samples. We also explored its predictive performance on four independent cohorts of TNBCs with ICB treatment (GSE194040, NCT04129996, GSE169246, GSE124821). Furthermore, to understand the impact of ISG+ T cells on immunotherapy efficacy and explore targeting strategies, we employed a multi-faceted approach to assess their tumor-killing ability, response to Anti-PD1 treatment, and sensitization effect of drugs targeting ISG+ T cells. ISG+ T cells were sorted using flow cytometry for further experiment. In vitro, we established a co-culture model of primary TNBC patients-derived organoid (PDO) and tumor infiltrated CD8+T cells and evaluated the tumor cell viability as well as T cell cytotoxicity. In vivo, we established TS/A tumor cell bearing BalB/c mouse and E0771 tumor cell-bearing C57BL mouse and measured tumor size and tumor microenvironment by flow cytometry. Results: We obtained 236,017 high-quality cells and identified cell subsets across 32 samples with different stages, treatments and responses. We then uncovered an enriched subset of interferon-induced (ISG+) CD8+ T cells specific to early TNBC, serving as a predictive factor for ICB resistance. Mechanistically, the upregulation of the IFN pathway in early-stage TNBC triggers a transition in ISG+ CD8+ T cells, resulting in cellular senescence, diminished cytotoxicity, and impaired response to ICB. Encouragingly, we made a noteworthy discovery that Nicotinamide mononucleotide, an anti-aging drug, effectively restores the functionality of ISG+ CD8+ T cells, amplifying immunotherapy sensitivity. Conclusions: This study provided a comprehensive single-cell transcriptome atlas of early-stage TNBC and depicted stage-related ISG+CD8+ T cell for predicting ICB response in early TNBC. We emphasize the critical role of T cell dysfunction prompted by cellular senescence in driving ICB resistance during the early stages of TNBC and propose an approach for sensitizing immunotherapy with aging drugs. Citation Format: Tong Fu, Yi-Yu Chen, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. Single cell characterization of senescent CD8+ T cell promotes immunotherapy resistance in early-stage 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 PO2-13-04.

Lack of Genetic Variation in Low Heterozygosity and Clonal Animals Creates Lack of Physiological Variation

Data variability frequently complicates reproducibility and interpretation of experimental results. Such variability arises from numerous sources such as differences in procedures or not accounting for key biological factors (e.g. sex, biological rhythms, prandial state). Making the situation more problematic, variation in physiological performance is often viewed as highly labile, easily and rapidly influenced by environmental stressors, development, etc., making it diffcult to pin down a source for variation. Undeniably, however, unknown (or ignored) genetic variation among and within strains/lines can also be a significant source of data variability in published physiological measurements, though surprisingly this has not been extensively investigated as a specific source of physiological variation. We hypothesized that variation in physiological performance is correlated with the intrinsic degree of genetic variability of the subject animal. To test this hypothesis, we employed two animal models: 1) Inbred lines ( e.g., NHGRI-1) derived from wild type strains of the zebrafish ( Danio rerio), with an estimated 15% of the genetic heterozygosity of wild type AB zebrafish, and 2) the parthenogenetically reproducing marbled crayfish ( Procambarus virginalis), all specimens of which are genetically identical clones. For these two animal models, we measured both physiological variables (e.g. heart rate, stroke volume, cardiac output, oxygen consumption) and morphological variables (e.g. yolk-chorion ratio, body mass, embryo mass, total length, condition factor, specific growth rate) during development. We subjected the two animal models to environmental stress in the form of both temperature and hypoxia to stimulate physiological responses that could be compared and contrasted among populations. From these data we then calculated the resultant coeffcients of variation for measured variables for wild type and low/zero heterozygosity populations and/or species. In zebrafish, both the wildtype AB and NHRGI-1 lines showed similar developmental trajectories characterized by similar mean values for physiological and morphological variables. Additionally, similar mean values for physiological and morphological variables were recorded in the face of temperature and hypoxia challenge. Yet, importantly, the coeffcient of variation for each measured variable was significantly lower in NHGRI-1 than AB larvae for >90% of the assessed endpoints. In the clonal crayfish, genetically identical early stage marbled crayfish reared in different temperatures or oxygen levels show major acclimation responses, but generally showed less morphological and physiological variation about the mean than sexually-reproducing species crayfish with inherently much greater genetic variation, as evident from comparisons of calculated coeffcients of variation. A key question regarding the clonal crayfish is how can there be any morphological or physiological variation between individuals? We suggest that variability that persists may arise from microenvironmental differences during rearing (e.g. egg position during incubation on the mother’s pleon) and/or stochastic differences in gene expression (e.g. due to random epimutations) in this clonal species. In conclusion, genetic diversity clearly contributes to physiological variability. For future experiments, low heterozygosity lines and/or clonal species may be useful for decreasing inter-individual variation, thus aiding interpretation of results and enhancing reproducibility. In any event, scientific documentation of physiological studies should include as much information on (genetic) background of the experimental animals as possible. NSF IOS-2103499. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Carbon and nutrient cycling responses to repeated application of biochar and NPK fertilizers depend on microenvironmental differences among hierarchical aggregate fractions

The conversion of bambara groundnut seed residues into biochar facilitated the bioavailability and retention of more nutrients (C, N, P, K and Mg) after repeated application of biochar. However, the mechanisms of microbially mediated biochar-C degradation and nutrient cycling responses to repeated biochar application, particularly in different hierarchical soil aggregates, are largely unknown. A 20-day incubation experiment was conducted on Ultisols from a 4-year cucumber field trial in Nsukka, Nigeria. The effects of repeated application of bambara seed residue biochar with or without NPK fertilizer on soil aggregate associated electrical conductivity (EC), basal respiration, microbial biomass, and soil enzyme activities were determined. The results showed that the concentration of organic matter in the bulk soils increased with biochar and NPK+biochar treatments. These treatments also increased the soil EC, cumulative CO2 respiration, microbial biomass C and N concentrations, and the activities of tyrosine-aminopeptidase and sulfatase enzymes, compared to the biochar treatment and the control soil. The NPK+biochar treatment contributed 35 % more to β-cellobiosidase activity, but the biochar treatment resulted in 85 % reduction in N-Acetyl-β-glucosaminidase activity, indicating microbial N mining. The NPK and NPK+biochar treatments accounted for a higher percentage of N-, C and N-, and S- cycle enzyme activities, although their composition was relatively higher with the latter treatment. The overall soil biochemical responses were significantly higher in the micro-aggregates (< 0.25 mm) than in the macro-aggregates (≤ 4.75–0.25 mm); least of all in the small macro-aggregates (0.25–1.00 mm). Therefore, repeated application of biochar to N-deficient soils generally does not result in positive soil biochemical responses. However, repeated application of biochar together with NPK fertilizer modulates N limitation and optimizes microbial nutrient cycling processes, especially in micro-aggregates.

Abstract LB362: Systematic development and characterization of orthotopic animal tumor models: A proactive approach to mimic patients' tumor microenvironments for drug discovery

Abstract Preclinical in vivo tumor models are crucial in translational research and the pharmaceutical industry, particularly for evaluating novel anti-cancer agents. While animal tumor models are widely used in both drug development and academic research, the majority of investigations have relied on subcutaneous (SC) transplantable tumor models. These models often fail to consider the orthotopic organ origin or the unique microenvironmental differences, leading to significant limitations. Despite many publications highlighting the advantages of orthotopic tumor models (ORTH) over SC models, there is a lack of systematic analysis and comparative characterization of these formats. In this study, we have systematically developed a comprehensive ORTH tumor model platform comprising over 80 orthotopic tumor models and covering more than 25 tumor types. ORTH tumor models have been enhanced for non-invasive monitoring and increased sensitivity of tumor growth and metastasis detection through labeling with multimodality imaging markers such as GFP, RFP, and bioluminescence or imaging with micro-CT or ultrasound. We optimized and streamlined technical procedures, throughput, and reproducibility of ORTH model development, facilitating the use of these models to assess anticancer agents in a large-scale format. To mimic the clinical situation, our characterization of these models includes growth profiles, metastatic features, and survival status. Additionally, ORTH models were compared with SC models regarding the efficacy of standard-of-care (SOC) drugs through side-by-side comparison studies. As expected, inconsistent responses to SOC therapeutics were observed in some models when comparing ORTH versus SC tumors. For models yielding inconsistent results, molecular analysis and model selection recommendations will be discussed. To date, this is the first study describing the generation of a considerably large orthotopic tumor model collection and conducting a comprehensive side-by-side characterization of ORTH and SC models. This proactive approach aims to improve the mimicry of patients' tumor microenvironments and enhance the clinical relevance and potentially increase the efficiency of translational research processes. Citation Format: WenQing Yang, Xusheng Yuan, Guangliang Yang, Xiaojun Tong, Weiqun Ding, Qiuxia Zhang, Feng Lu, Zhijian Yang. Systematic development and characterization of orthotopic animal tumor models: A proactive approach to mimic patients' tumor microenvironments for drug discovery [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 LB362.

Abstract B082: A pro-tumorigenic, aged microenvironment may aid in worsened disease progression in an HGSOC model

Abstract Over 70% of HGSOC patients are diagnosed after 55 and the average patient succumbs to disease at 70 years old. Despite this, most ovarian cancer research, and cancer studies generally, utilize young mice that are mimetic of human teenagers and/or do not investigate age as a variable despite it being the top cancer risk factor. An improved understanding of how age affects HGSOC progression may enable development of alternative treatment strategies aimed at intercepting metastasis and preventing cancer cells from establishing in the peritoneum. Our preliminary studies show tumor burden and disease severity are significantly higher in aged versus young animals. We developed an epithelial cell line derived from C57Bl/6 oviducts and used CRISPR-Cas9 genome editing to develop a syngeneic tumorigenic line harboring Trp53, Pten, and Brca2 deletions. After intraperitoneal injection into 6-week and 14-month-old immunocompetent C57Bl/6 mice, we observed a stark increase in tumor burden in aged mice. Furthermore, older mice died at a faster rate; all aged mice succumbed to disease within 1-month of inoculation compared to young mice surviving up to 3 months. Total RNA-sequencing of tumors harvested from aged and young mice is ongoing. Characterization of the tumor-naïve peritoneal compartment revealed the aged microenvironment to be highly inflammatory. When assessed via cytokine array, peritoneal fluid of aged mice had higher concentrations of inflammatory markers including soluble TNF receptor-1 (sTNFR-1), L-selectin, and VCAM-1. Flow cytometry of peritoneal lavage indicated an over 2-fold increase in the number CD45+ cells present in the peritoneal cavity, coinciding with cytokine array results. Within this population, there was nearly complete depletion of macrophage and neutrophil populations and a significant increase in B cells and CD4+ and CD8+ T cells. The percentage of PD-1+ CD8+ cells also increased. In addition to increased signs of inflammation, a significantly higher concentration of insulin-like growth factor-1 (IGF-I) and higher concentrations of the associated IGF binding proteins -3 and -6 were found in peritoneal fluid of aged mice. In addition, during tissue collection, organs of the aged mice were found to be much larger than young mice, a common effect of elevated IGF-I; aged omentum weighed 2-4 times that of young. Notably, IGF-I is known to inhibit apoptosis and promote proliferation and increased IGF-I is correlated with increased risk of breast, prostate, and lung cancer. A drastic difference in disease severity occurs when identical cancer cells are injected into 6-week and 14-month-old mice. To identify possible microenvironmental differences in the two populations, a broad panel of cytokines was assessed, and immune populations were quantified in pre-cancerous mice. Overall, multiple pro-inflammatory markers and the number of lymphocytes were upregulated in the aged peritoneal cavity, and markers of immune fatigue were present. Further work is aimed at identifying immune cell state via further cytometry and sequencing. Citation Format: Katherine A. Cummins, Ronny I. Drapkin, Matthew Knarr. A pro-tumorigenic, aged microenvironment may aid in worsened disease progression in an HGSOC model [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B082.

Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Development of a Novel CD8+ T Cell-Associated Signature for Prognostic Assessment in Hepatocellular Carcinoma.

The aim of this study was to analyze the clinical significance and prognostic value of CD8+ T cell-related regulatory genes in hepatocellular carcinoma (HCC). This was a retrospective study. We combined TCGA-LIHC and single-cell RNA sequencing data for Lasso-Cox regression analysis to screen for CD8+ T cell-associated genes to construct a novel signature. The expression of the signature genes was detected at cellular and tissue levels using qRT-PCR, immunohistochemistry, and tissue microarrays. The CIBERSORT algorithm was then used to assess the immune microenvironmental differences between the different risk groups and a drug sensitivity analysis was performed to screen for potential HCC therapeutic agents. An 8-gene CD8 + T cell-associated signature (FABP5, GZMH, ANXA2, KLRB1, CD7, IL7R, BATF, and RGS2) was constructed. Survival analysis showed that high-risk patients had a poorer prognosis in all cohorts. Tumor immune microenvironment analysis revealed 22 immune cell types that differed significantly between patients in different risk groups, with patients in the low-risk group having an immune system that was more active in terms of immune function. Patients in the high-risk group were more prone to immune escape and had a poorer response to immunotherapy, and AZD7762 was screened as the most sensitive drug in the high-risk group. Finally, preliminary experiments have shown that BATF has a promoting effect on the proliferation, migration and invasion of HuH-7 cells. The CD8+ T-cell-associated signature is expected to be a tool for optimizing individual patient decision-making and monitoring protocols, and to provide new ideas for treatment and prognostic assessment of HCC.

Simulation of transvascular transport of nanoparticles in tumor microenvironments for drug delivery applications

Nanomedicine is a promising approach for tumor therapy but penetration is challenged by complex tumor microenvironments. The purpose of this study is to design nanoparticles and analyze their transport in two abnormal microenvironments through a 2-D simulation. Employing a Computational Fluid Dynamics (CFD) approach, tumor vascular-interstitial models were initially simulated, and the impact of nanoparticles on the velocity profile and pressure gradient within the tumor microenvironment was observed. Through meticulous mesh analysis, it was determined that optimal outcomes were achieved using a quadrilateral meshing method for pancreatic tumor and a quad/tri meshing method for hepatic tumor. Results showed an increase in vessel diameter correlated with elevated blood flow velocity, reaching a maximum of 1.40 × 10^−3 m/s with an expanding cell gap. The simulation results for pressure distribution show that as vessel diameter increases, the velocity of nanoparticles in blood increases and decreases the pressure of blood. Intriguingly, distinct fluid flow patterns in pancreatic and hepatic tumors, emphasize how microenvironmental differences, specifically cell pore size, profoundly impact therapeutic agent transport, with implications for drug delivery strategies in cancer therapy. These simulation-based insights enable researchers to anticipate nanofluid behavior in realistic settings. Future work, incorporating immune cells, will enhance the understanding of nanoparticle efficiency in cancer therapy.

Identification of pyroptosis-related subtypes and comprehensive analysis of characteristics of the tumor microenvironment infiltration in clear cell renal cell carcinoma

Pyroptosis is a kind of programmed cell death triggered by the inflammasome. Growing evidence has revealed the crucial utility of pyroptosis in tumors. However, the potential mechanism of pyroptosis in clear cell renal cell carcinoma (ccRCC) is still unclear. In this research, we systematically analyze the genetic and transcriptional alterations of pyroptosis-related genes (PRGs) in ccRCC, identify pyroptosis-related subtypes, analyze the clinical and microenvironmental differences among different subtypes, develop a corresponding prognostic model to predict the prognosis of patients, and interpret the effect of pyroptosis on ccRCC microenvironment. This study provides a new perspective for a comprehensive understanding of the role of pyroptosis in ccRCC and its impact on the immune microenvironment, and a reliable scoring system was established to predict patients’ prognosis.

Abstract PO-025: The effect of tissue of origin on oncogenesis in HPV-related malignancies: Comparative analysis of molecular and immune signatures in HPV-positive head and neck cancer and cervical cancer

Abstract Background: Despite sharing a common causal agent and viral oncogenes, HPV-positive head and neck cancer (HPV+ HNC) and cervical cancer (HPV+ CC) have markedly distinct natural histories and therapy responses. Here we sought to elucidate how the tissue of origin and oncogenic etiology drive molecular and microenvironmental differences between these two malignancies. Methods: Somatic mutation and RNA expression data for HPV+ HNC, HPV+ CC and HPV-negative head and neck cancer (HPV- HNC) were obtained from The Cancer Genome Atlas (TCGA). Mutation data were used to define repertoires of somatic mutations and mutational signatures. Single-sample gene set enrichment analysis (ssGSEA) was used to identify enriched immune signatures. Immune cell infiltration levels were estimated using xCell. Comparisons were primarily made between HPV+ HNC and HPV+ CC samples, with comparisons between HPV+ HNC vs. HPV- HNC tumors serving as a control to distinguish molecular/immune signatures driven by tissue of origin versus viral etiology. Results: Exome and RNA-sequencing data were available for 72 HPV+ HNC, 139 HPV+ CC, and 415 HPV- HNC tumors, and 70 HPV+ HNC, 139 HPV+ CC, and 413 HPV- HNC cases, respectively. Recurrently altered genes in head and neck and cervical cancer were not statistically significantly different between HPV+ HNC and HPV+ CC, although as previously reported HPV+ HNC and HPV- HNC differed by the higher frequency of TP53, CDKN2A, FAT1, and NOTCH1 mutations in the later. An evaluation of mutational signatures revealed that although both HPV+ HNC and CC had evidence of APOBEC mutagenesis (Signatures 2 and 13), Signature 2 and 13 exposures were both significantly higher in HPV+ CC relative to HPV+ HNC. APOBEC enrichment scores confirmed that HPV+ CC had higher APOBEC signal than HPV+ HNC despite both being virally driven. While both cancers were commonly affected by mutagenesis from aging and showed no differences in Signature 1, HPV+ HNC exhibited a higher correlation with Signature 5. Immune de-convolution evaluating 36 cell populations identified 13 cell types with different frequencies between two cancers (q &amp;lt; 0.1). The abundance of B-cell lineages, CD4+ naïve T-cells, and Tregs significantly differed in HPV+ HNC and HPV+ CC (p&amp;lt;0.001). These findings led us to investigate expression signatures of tertiary lymphoid structures, which we identified as markedly higher in HPV+ HNC versus HPV+ CC or HPV- HNC (p &amp;lt; 0.001, for both comparisons). Activated T-cells, however, did not appear to differ between HPV+ HNC or CC as determined by the previously defined cytolytic score. Conclusion: We find that while tissue of origin seems not to influence recurrently mutated oncogenes in HPV-related malignancies, it has significant effects on phenotypic consequences in these tumors including influencing both mutational signature exposure and the tumor microenvironment. These findings may inform further investigations into the unique molecular and microenvironmental characteristics of these virally driven tumors. Citation Format: Reith R. Sarkar, Xin Pei, Andrea Gazzo, Yingjie Zhu, Nancy Lee, Jorge Reis-Filho, Dmitriy Zamarin, Britta Weigelt, Nadeem Riaz. The effect of tissue of origin on oncogenesis in HPV-related malignancies: Comparative analysis of molecular and immune signatures in HPV-positive head and neck cancer and cervical cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-025.

P12.05.B THE ROLE OF ONCO-METABOLITE IN THE IDH MUTANT GLIOBLASTOMA MICROENVIRONMENT

Abstract BACKGROUND Recently, we found that the reactive astrocytes in the IDH wild type glioblastoma contribute to anti-tumor immunity and support pro-oncogenic signaling. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients due to an onco-metabolite R-2-hydroxyglutarate using Next generation sequencing and human ex-vivo slice model. MATERIAL AND METHODS We purified and transcriptionally profiled astrocytes from 9 patients with confirmed IDH1-R132H mutation, by means of RNA-sequencing and the data were analyzed using the established pipelines. We also used spatial transcriptomics to evidently show the spatial distribution of astrocytes in IDH-mutated/wildtype glioma samples. We validated our findings using human organotypic slice model inoculated with IDH-mutant cell line or treated with oncometabolite 2-hydroxy glutarate. Additionally, LC-MS was further used to give us a chart of neurotransmitters due to altered microenvironment. RESULTS Our results from RNA sequencing showed a transcriptional transformation of reactive astrocytes within the microenvironment of IDH-mutated tumors compared to wildtype glioma by means of RNA-sequencing of purified astrocytes. And, using our established human neocortical GBM model inoculated with IDH mutant tumor and R-2HG treatment, we showed that we were able to activate inflammatory transcriptional programs in astrocytes, mediated by the presence of microglia. Further, by spatially mapping the transcriptomic profiles of purified microglia, we were able to confirm that microglia also demonstrate inflammatory activation in IDH mutated glioma. This inflammatory astrocyte transformation is associated with a loss of neurotransmitter homeostasis (disrupted levels of glutamate) in the treated sections, as has been previously reported in IDH mutated tumors. Additionally, R-H2G increased neuronal spiking rate in, pointing to potential excitotoxicity. CONCLUSION Our work provides a crucial contribution towards understanding the role of R-2HG in the IDH mutant glioma microenvironment and sheds light on the significant microenvironmental differences to IDH wild-type glioma.

A study of waterside microenvironmental factors and their effects on summer outdoor thermal comfort in a Cfa-climate campus

Outdoor thermal comfort (OTC) studies explore outdoor subjects’ responses to their thermal environment, usually evaluated using the neutral temperature (NT). This study investigated the influences of microenvironmental factors around a waterbody on thermal perceptions, using questionnaires and meteorological measurements at the Central Lake of Southwest University of Science and Technology (SWUST) in Mianyang. Microenvironmental factors included sky view factor (SVF) and distance from the lake (DFL). It was found that people felt most comfortable in the shade of trees although some volunteers voted artificial canopy as their preferred thermal adaptation element. In addition, a linear regression yielded an NT of 28.44 °C in Mianyang during the summer of 2022. There were NT variations among different measurement sites (e.g., on the east shore, it was 28.18 °C on the waterside, 27.11 °C away from the lake, and 25.53 °C far from the lake; while it was 27.57 °C under the tree crown, 25.11 °C on the lawn, and 29.13 °C in the square). This variation may be due to human adaptation towards microenvironmental factors and their effects on microclimate. The variation in thermal responses owing to microenvironmental differences (different NTs at various types of sites) might be a novel finding in the field of OTC. This study provides important directions for microenvironment design in the future for OTC improvement.

Unraveling the heterogeneity of intercellular crosstalk in intrahepatic and extrahepatic cholangiocarcinoma using single-cell and spatial transcriptome profiling.

50 Background: Extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) are two types of malignant tumors that arise from the bile duct epithelium at different locations. Currently, effective treatment options for these tumors are limited, and their microenvironmental differences remain poorly understood. Methods: We collected retrospective clinicopathological data of ECC (N=64) and ICC (N=153) patients from our hospital and integrated public database information to analyze the clinical characteristics, survival, and prognosis of ECC and ICC. We performed single-cell sequencing (scRNA-seq) and spatial transcriptomic techniques on three ECC and three ICC tumor tissues. Verification of scRNA-seq results was done using immunohistochemistry, flow cytometry, and polychromatic immunofluorescence. Additionally, we analyzed RNA and ATAC sequencing data for exhausted CD8+ T cells TEX obtained from ICC and ECC tissue samples. We used InferCNV to infer copy number variations (CNVs) of epithelial cells from ICC and ECC. Results: Our results showed that ECC and ICC differ in their clinical characteristics such as age, gender, bilirubin, CEA, and outcome. ECC had higher copy number amplification variation and exhibited inflammatory signaling and ongoing immunoediting, reflected in higher immune checkpoint expression such as PDCD1, CTLA4, and LAG3, and tumor infiltration with highly differentiated dysfunctional PDCD1+ LAG3+ CXCL13+ exhausted CD8+ T cells (TEX) and precursors of exhausted T (TPEX). RNA velocity analysis revealed different developmental trajectories of T cells in ECC and ICC, predominantly in naive/stem-like and memory T cells. We also identified a trefoil factor 3 (TFF3)-positive epithelial subpopulation in ECC. TFF3+ epithelial cells had closer interaction with CTHRC1+ fibroblasts, which induced more CD8+ T cells to TEX and TPEX. TFF3+ cells exhibited higher CNV scores and higher PD-L1 and PD-L2 expression, and co-localized with TEX and TPEX, providing one explanation for the immunosuppressive environment in ECC and suggesting a potential response to immune checkpoint therapy (ICT). Conclusions: Our study provides a comprehensive characterization of the cellular heterogeneity landscape of ECC and ICC, which can guide the optimization of personalized cancer immunotherapy. We identified potential biomarkers and targets for immunotherapies, and our findings shed light on the underlying mechanisms of tumor immune evasion and provide a foundation for further research in this area.

LncRNAs associated with vascular mimicry establish a novel molecular subtype and prognostic model for pancreatic cancer.

Vascular mimicry (VM) epitomizes an innovative tumor angiogenesis pathway, potentially serving as an alternate conduit under the assumption of traditional tumor angiogenesis pathway inhibition. The role of VM in pancreatic cancer (PC), however, remains unexplored. Using differential analysis and Spearman correlation, we identified key long non-coding RNAs (lncRNAs) signatures in PC from the collected set of VM-associated genes in the literature. We identified optimal clusters using the non-negative matrix decomposition (NMF) algorithm, and then compared clinicopathological features and prognostic differences between clusters. We also assessed tumor microenvironmental (TME) differences between clusters using multiple algorithms. Using univariate Cox regression analyses as well as lasso regression, we constructed and validated new lncRNA prognostic risk models for PC. We used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze model-enriched functions and pathways. Nomograms were then developed to predict patient survival in association with clinicopathological factors. In addition, single-cell RNA-sequencing (scRNA-seq) analysis was used to analyze the expression patterns of VM-related genes and lncRNAs in the PC of TME. Finally, we used the Connectivity Map (cMap) database to predict local anaesthetics that could modify the VM of PC. In this study, we developed a novel three-cluster molecular subtype using the identified VM-associated lncRNA signatures of PC. The different subtypes have significantly different clinical characteristics and prognostic value, and also show differential treatment response and TME. Following an in-depth analysis, we constructed and validated a novel prognostic risk model for PC based on the VM-associated lncRNA signatures. Enrichment analysis suggested that high riskscores were significantly associated with functions and pathways, including extracellular matrix remodeling, et al. In addition, we predicted eight local anaesthetics that could modulate VM in PC. Finally, we discovered differential expression of VM-related genes and lncRNAs across various cell types within pancreatic cancer. VM has a critical role in PC. This study pioneers the development of a VM-based molecular subtype that demonstrates substantial differentiation in PC populations. Furthermore, we highlighted the significance of VM within the immune microenvironment of PC. Moreover, VM might contribute to PC tumorigenesis through its mediation of mesenchymal remodeling and endothelial transdifferentiation-related pathways, which offers a new perspective on its role in PC.

Abstract B030: Spatially-resolved cell-cell interactions between tumor microenvironment and immunologically “hot” and “cold” locally advanced prostate tumors

Abstract Background: Immune-checkpoint inhibitors (ICI) such as anti-PD-L1 have shown promise in solid tumors with high mutational burden and immune infiltration such as melanoma. However, ICI have shown have only shown mild success in prostate cancer. One likely explanation is that majority of prostate cancer have low mutational burden leading to poor immune infiltration. However, exceptions to the immune infiltration have been observed in a subset of locally advanced prostate cancer specimens. We hypothesized that specific immune subsets, particular myeloid progenitor cells, drive microenvironmental differences between hot and cold tumors. Methods: A total of four formalin-fixed paraffin-embedded tissue punch samples were obtained from biobank at UCLA and were sectioned at 5µm thickness. The sections were deparaffinized and de-crosslinked per manufacture’s instructions, followed by spatial transcriptome profiling using the Visium (10x Genomics) platform. Libraries were constructed from RNA and sequenced on Illumina platform. The reads were processed using Spaceranger. Spot level read counts were first filtered by gene detection rate, total gene count and mitochondrial content. Spots passing QC were normalized and clustered based on the top 2000 most highly variable genes. Cell-cell communication was assessed using the LIANA ligand-receptor analysis framework. To identify regions of inflammation, cell type deconvolution was performed using SpatialDWLS (dampened weighted least squares) and gene-set enrichment was performed using ssgsea from GSVA package. All analysis and statistics were perform using R version 4.1. Results: The number of spots passed QC ranged from approximately 3400 – 4000. Clustering by gene expression resulted in 14 – 23 clusters that showed high concordance with expert pathologist annotation of tumor and tumor subtype mapping to intraductal carcinoma and invasive carcinoma. Cell-type deconvolution by SpatialDWLS found that different tissue regions harbored approximately 1.5-4.5% of immune cells. The myeloid fraction was 0.8%- 3% and T-cells of total immune cells, representing 0.4% - 0.9% of the total cell population. These regions with myeloid cell enrichment were associated with elevated interferon alpha and gamma response, as well as IL2-STAT5 signaling in geographically proximal regions. Conclusion: The presence of myeloid cells in the microenvironments of immunologically “hot” tumors may represent a major factor contributing to immunosuppression in tumors predicted to respond to ICI. Ongoing efforts profiling “hot” tumors will assess the feasibility of modulating myeloid cells in order to recruit cytotoxic T cells. Citation Format: Anson Ku, Rong Rong Huang, Huihui Ye, Adam Sowalsky. Spatially-resolved cell-cell interactions between tumor microenvironment and immunologically “hot” and “cold” locally advanced prostate tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B030.

Abstract 5788: Single-cell and spatial transcriptomic mapping of human renal cell carcinoma brain metastases uncovers actionable immune-resistance targets

Abstract Introduction: The discovery of immune checkpoint inhibitors has revolutionized metastatic renal cell carcinoma (RCC) treatment. However, in patients with RCC brain metastases, response rates are low and survival outcomes poor. To understand the tumor microenvironmental differences between primary kidney tumors, extracranial metastases, and brain metastases, we developed a detailed single-cell atlas of RCC brain metastases along with their matched extracranial and primary tumors. Methods: We performed single-nucleus RNA-seq on 27 samples (nearly 200,000 cells) from RCC patients; samples included 14 brain metastases, 8 matched primary kidney tumors, and 5 matched extracranial metastases. We performed multiplex IHC to validate selected transcriptomic findings. We used Nanostring CosMx 960-plex RNA spatial molecular imaging technique on selected samples to validate cellular interactions in a spatial context. Results: We established a multi-tissue single-cell atlas of RCC brain metastases by identifying 9 major and 37 minor malignant, immune, and stromal cell clusters. Brain metastases had higher neuronal and glial cells interacting with immune and tumor cells. Brain metastasis tumor cells were also transcriptomically reprogrammed to adapt to the brain microenvironment through enrichment of MYC targets, MTORC1 signaling, epithelial-mesenchymal transition, fatty-acid metabolism, oxidative phosphorylation, and reactive oxygen species pathways. Moreover, cell-to-cell communication and downstream target gene expression analyses showed that brain metastasis tumor cells expressed ligands and receptors that induce tumor cell proliferation in both autocrine and paracrine fashions. Among T-cell populations, we found fewer proliferating cytotoxic T lymphocytes in the brain than in other sites. Moreover, T cells in brain metastases expressed higher levels of several targetable inhibitory checkpoints than did extracranial metastases. In addition, we found that naïve/memory T cells in brain metastases were a favorable prognostic marker for overall survival after craniotomy. Our characterization of myeloid cell populations across the 3 disease sites found fewer dendritic cells and monocytes in the brain compared to other sites. Macrophages in brain metastases more highly expressed an M2 immunosuppressive gene signature than did those in primary RCC tumors. Conclusion: Our findings from the largest single-cell atlas of RCC brain metastases with matched primary and extracranial metastases suggest several unique targetable, immunosuppressive biological mechanisms in the brain microenvironment. These results provide a foundation for a deeper understanding of RCC brain metastasis biology and can serve as a resource for the scientific community to further explore therapeutically targetable tumor and immune-related mechanisms. Citation Format: Elshad Hasanov, Truong Nguyen Anh Lam, Jerome Lin, Patrick K. Reville, Merve Hasanov, Anna K. Casasent, David Shih, Sahin Hanalioglu, Mehmet Asim Bilen, Omar Alhalabi, Berrin Babaoglu, Baylar Baylarov, Adeboye O. Osunkoya, Lisa M. Norberg, Joy Gumin, Tuan M. Tran, Jianzhuo Li, Anh G. Hoang, Haidee D. Chancoco, Brittany C. Parker Kerrigan, Erika J. Thompson, Betty YS Kim, Dima Suki, Melike Mut, Figen Soylemezoglu, Giannicola Genovese, Kadir C. Akdemir, Hussain A. Tawbi, Nizar M. Tannir, Florencia McAllister, Michael A. Davies, Padmanee Sharma, Jason Huse, Frederick Lang, Nicholas Navin, Eric Jonasch. Single-cell and spatial transcriptomic mapping of human renal cell carcinoma brain metastases uncovers actionable immune-resistance targets [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 5788.