Tumor Development Research Articles

Functional role of P2X7 purinergic receptor in cancer and cancer-related pain.

Numerous studies have revealed that the ATP-gated ion channel purinergic 2X7 receptor (P2X7R) plays an important role in tumor progression and the pathogenesis of cancer pain. P2X7R requires activation by extracellular ATP to perform its regulatory role functions. During tumor development or cancer-induced pain, ATP is released from tumor cells or other cells in the tumor microenvironment (such as tumor-associated immune cells), which activates P2X7R, opens ion channels on the cell membrane, affects intracellular molecular metabolism, and regulates the activity of tumor cells. Furthermore, peripheral organs and receptors can be damaged during tumor progression, and P2X7R expression in nerve cells (such as microglia) is significantly upregulated, enhancing sensory afferent information, sensitizing the central nervous system, and inducing or exacerbating pain. These findings reveal that the ATP-P2X7R signaling axis plays a key regulatory role in the pathogenesis of tumors and cancer pain and also has a therapeutic role. Accordingly, in this study, we explored the role of P2X7R in tumors and cancer pain, discussed the pharmacological properties of inhibiting P2X7R activity (such as the use of antagonists) or blocking its expression in the treatment of tumor and cancer pain, and provided an important evidence for the treatment of both in the future.

Role of TIM-1 in the development and treatment of tumours

T-cell immunoglobulin and mucin structural domain 1 (TIM-1, also known as hepatitis A virus cell receptor 1) is a co-stimulatory molecule that is expressed predominantly on the surface of T cells. TIM-1 promotes the activation and proliferation of T cells, cytokine secretion, and can also be overexpressed in various types of cancer. Upregulation of TIM-1 expression may be associated with the development and progression of cancer. After reviewing the literature, we propose that TIM-1 affects tumour development mainly through two pathways. In the Direct pathway: overexpression in tumours activates tumour-related signaling pathways, mediates the proliferation, apoptosis, invasion and metastasis, and directly affects tumour development directly. In the indirect pathway: In addition to changing the tumour microenvironment and influencing the growth of tumours, TIM-1 binds to ligands to encourage the activation, proliferation, and generation of cytokines by immune cells. This review examines how TIM-1 stimulates the development of tumours in direct and indirect ways, and how TIM-1 is exploited as a target for cancer therapy.

Advancement in Cancer Vasculogenesis Modeling through 3D Bioprinting Technology

Cancer vasculogenesis is a pivotal focus of cancer research and treatment given its critical role in tumor development, metastasis, and the formation of vasculogenic microenvironments. Traditional approaches to investigating cancer vasculogenesis face significant challenges in accurately modeling intricate microenvironments. Recent advancements in three-dimensional (3D) bioprinting technology present promising solutions to these challenges. This review provides an overview of cancer vasculogenesis and underscores the importance of precise modeling. It juxtaposes traditional techniques with 3D bioprinting technologies, elucidating the advantages of the latter in developing cancer vasculogenesis models. Furthermore, it explores applications in pathological investigations, preclinical medication screening for personalized treatment and cancer diagnostics, and envisages future prospects for 3D bioprinted cancer vasculogenesis models. Despite notable advancements, current 3D bioprinting techniques for cancer vasculogenesis modeling have several limitations. Nonetheless, by overcoming these challenges and with technological advances, 3D bioprinting exhibits immense potential for revolutionizing the understanding of cancer vasculogenesis and augmenting treatment modalities.

Programmed Cell Death-Related Gene Signature Associated with Prognosis and Immune Infiltration and the Roles of HMOX1 in the Proliferation and Apoptosis were Investigated in Uveal Melanoma.

Uveal melanoma (UVM) is the most common primary ocular malignancy, with a wide range of symptoms and outcomes. The programmed cell death (PCD) plays an important role in tumor development, diagnosis, and prognosis. There is still no research on the relationship between PCD-related genes and UVM. A novel PCD-associated prognostic model is urgently needed to improve treatment strategies. We aim to screen PCD-related prognostic signature and investigate its proliferation ability and apoptosis in UVM cells. The clinical information and RNA-seq data of the UVM patients were collected from the TCGA cohort. All the patients were classified using consensus clustering by the selected PCD-related genes. After univariate Cox regression and PPI network analysis, the prognostic PCD-related genes were then submitted to the LASSO regression analysis to build a prognostic model. The level of immune infiltration of 8-PCD signature in high- and low-risk patients was analyzed using xCell. The prediction on chemotherapy and immunotherapy response in UVM patients was assessed by GDSC and TIDE algorithm. CCK-8, western blot and Annexin V-FITC/PI staining were used to explore the roles of HMOX1 in UVM cells. A total of 8-PCD signature was constructed and the risk score of the PCD signature was negatively correlated with the overall survival, indicating strong predictive ability and independent prognostic value. The risk score was positively correlated with CD8 Tcm, CD8 Tem and Th2 cells. Immune cells in high-risk group had poorer overall survival. The drug sensitivity demonstrated that cisplatin might impact the progression of UVM and better immunotherapy responsiveness in the high-risk group. Finally, Overespression HMOX1 (OE-HMOX1) decreased the cell viability and induced apoptosis in UVM cells. Recuse experiment results showed that ferrostatin-1 (fer-1) protected MP65 cells from apoptosis and necrosis caused by OE-HMOX1. The PCD signature may have a significant role in the tumor microenvironment, clinicopathological characteristics, prognosis and drug sensitivity. More importantly, HMOX1 depletion greatly induced tumor cell growth and inhibited cell apoptosis and fer-1 protected UVM cells from apoptosis and necrosis induced by OE-HMOX1. This work provides a foundation for effective therapeutic strategy in tumour treatment.

Characteristics of lipid metabolism after treatment of colon cancer mice with American ginseng vesicles.

Lipid molecules are present in tumours and play an important role in the anti-inflammatory response as well as in antiviral protection. Changes in the type and location of lipids in the intestine following exposure to environmental stressors play an important role in several disorders, including ulcerative colitis (UC), inflammatory bowel disease (IBD), and colorectal cancer. The aim of this work is to provide a new theoretical basis for tumour initiation and development by accurately measuring the spatial distribution of lipids and metabolites in intestinal tissue. Spatial metabolomics allows the detection of samples with minimal sample volume by label-free imaging of complex samples in their original state. The distribution of lipid molecules in tumours has not been reported, although the distribution of lipid molecules in intestinal tissue has been reported in the literature. The range of lipid profiles in colon cancer mouse tumour tissue was compiled using a spatial metabolomics: lipid extraction method. The changes in lipid distribution in two regions after oral administration of American Ginseng (Panax quinquefolius L.) vesicles were also compared. Tumour tissue samples were extracted with 80% methanol-20% formic acid in water. The resulting spatial metabolic profile allowed the identification of seven lipid classes in mouse tumours. The distribution of fibre tissue cells was 23.2% higher than tumour tissue cells, with the exception of the fatty acid (FA) species.

Molecular Pathways of Genistein Activity in Breast Cancer Cells

The most common malignancy in women is breast cancer. During the development of cancer, oncogenic transcription factors facilitate the overproduction of inflammatory cytokines and cell adhesion molecules. Antiapoptotic proteins are markedly upregulated in cancer cells, which promotes tumor development, metastasis, and cell survival. Promising findings have been found in studies on the cell cycle-mediated apoptosis pathway for medication development and treatment. Dietary phytoconstituents have been studied in great detail for their potential to prevent cancer by triggering the body’s defense mechanisms. The underlying mechanisms of action may be clarified by considering the role of polyphenols in important cancer signaling pathways. Phenolic acids, flavonoids, tannins, coumarins, lignans, lignins, naphthoquinones, anthraquinones, xanthones, and stilbenes are examples of natural chemicals that are being studied for potential anticancer drugs. These substances are also vital for signaling pathways. This review focuses on innovations in the study of polyphenol genistein’s effects on breast cancer cells and presents integrated chemical biology methods to harness mechanisms of action for important therapeutic advances.

Cancer associated fibroblast secreted miR-432-5p targets CHAC1 to inhibit ferroptosis and promote acquired chemoresistance in prostate cancer.

Prostate cancer (PCa) ranks as the sixth most serious male malignant disease globally. While docetaxel (DTX) chemotherapy is the standard treatment for advanced PCa patients with distant metastasis, some individuals exhibit insensitivity or resistance to DTX. Cancer-associated fibroblasts (CAFs) play a pivotal role as stromal cells within the tumor microenvironment, influencing tumor development, progression, and drug resistance through exosomes. Ferroptosis, a novel form of programmed cell death, is characterized by intracellular iron accumulation that triggers lipid peroxidation, ultimately leading to cell demise. To delve into the potential mechanisms of chemotherapy resistance in prostate cancer, our research delved into the impact of CAF-derived exosomes on ferroptosis. Our findings revealed that CAF exosomes hindered the buildup of lipid reactive oxygen species (ROS) in prostate cancer cells induced by erastin, as well as mitigated erastin-induced mitochondrial damage, thereby impeding iron-induced cell death in prostate cancer cells. Furthermore, miR-432-5p was identified to diminish glutathione (GSH) consumption by targeting CHAC1, consequently inhibiting ferroptosis in prostate cancer cells. Our study found that miR-432-5p, originating from cancer-associated fibroblast (CAF) exosomes, suppresses ferroptosis by targeting CHAC1, thereby increasing resistance to docetaxel (DTX) in PCa. This research introduces a novel approach to address resistance to DTX.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

An Aggregation-Induced Fluorescence Probe for Detection H2S and Its Application in Cell Imaging

Monitoring hydrogen sulfide (H2S) in living organisms is very important because H2S acts as a regulator in many physiological and pathological processes. Upregulation of endogenous H2S concentration has been shown to be closely related to the occurrence and development of tumors, atherosclerosis, neurodegenerative diseases and diabetes. Herin, a novel fluorescent probe HND with aggregation-induced emission was designed. Impressively, HND exhibited a high selectivity, fast response (1 min) and low detection limit (0.61 μM) for H2S in PBS buffer (10 mM, pH = 7.42). Moreover, the reaction mechanism between HND and H2S was conducted by Job’s plot, HR-MS, and DFT. In particular, HND was successfully employed to detect H2S in HeLa cells.

Gain-Type Aneuploidies Influence the Burden of Selective Long Non-Coding Transcripts in Colorectal Cancer

Chromosomal instability is a hallmark of colorectal carcinogenesis and produces an accumulation of different forms of aneuploidies or broad copy number aberrations. Colorectal cancer is characterized by gain-type broad copy number aberrations, specifically in Chr20, Chr8q, Chr13 and Chr7, but their roles and mechanisms in cancer progression are not fully understood. It has been suggested that broad copy number gains might contribute to tumor development through the so-called caricature transcriptomic effect. We intend to investigate the impact of broad copy number gains on long non-coding RNAs’ expression in colorectal cancer, given their well-known role in oncogenesis. The influence of such chromosomal aberrations on lncRNAs’ transcriptome profile was investigated by SNP and transcriptome arrays in our series of colorectal cancer samples and cell lines. The correlation between aneuploidies and transcriptomic profiles led us to obtain a class of Over-UpT lncRNAs, which are transcripts upregulated in CRC and further overexpressed in colon tumors bearing specific chromosomal aberrations. The identified lncRNAs can contribute to a wide interaction network to establish the cancer driving effect of gain-type aneuploidies.

Integrative analyses identified gap junction beta‐2 as a prognostic biomarker and therapeutic target for breast cancer

AbstractBackgroundIncreasing evidence has shown that connexins are involved in the regulation of tumor development, immune escape, and drug resistance. This study investigated the gene expression patterns, prognostic values, and potential mechanisms of connexins in breast cancer.MethodsWe conducted a comprehensive analysis of connexins using public gene and protein expression databases and clinical samples from our institution. Connexin mRNA expressions in breast cancer and matched normal tissues were compared, and multiomics studies were performed.ResultsGap junction beta‐2 mRNA was overexpressed in breast cancers of different pathological types and molecular subtypes, and its high expression was associated with poor prognosis. The tumor membrane of the gap junction beta‐2 mutated group was positive, and the corresponding protein was expressed. Somatic mutation and copy number variation of gap junction beta‐2 are rare in breast cancer. The gap junction beta‐2 transcription level in the p110α subunit of the phosphoinositide 3‐kinase mutant subgroup was higher than that in the wild‐type subgroup. Gap junction beta‐2 was associated with the phosphoinositide 3‐kinase‐Akt signaling pathway, extracellular matrix–receptor interaction, focal adhesion, and proteoglycans in cancer. Furthermore, gap junction beta‐2 overexpression may be associated with phosphoinositide 3‐kinase and histone deacetylase inhibitor resistance, and its expression level correlated with infiltrating CD8+ T cells, macrophages, neutrophils, and dendritic cells.ConclusionsGap junction beta‐2 may be a promising therapeutic target for targeted therapy and immunotherapy and may be used to predict breast cancer prognosis.

Role of m6A modifications in immune evasion and immunotherapy.

RNA modification has garnered increasing attention in recent years due to its pivotal role in tumorigenesis and immune surveillance. N6-methyladenosine (m6A) modification is the most prevalent RNA modification, which can affect the expression of RNA by methylating adenylate at the sixth N position to regulate the occurrence and development of tumors. Dysregulation of m6A affects the activation of cancer-promoting pathways, destroys immune cell function, maintains immunosuppressive microenvironment, and promotes tumor cell growth. In this review, we delve into the latest insights into how abnormalities in m6A modification in both tumor and immune cells orchestrate immune evasion through the activation of signaling pathways. Furthermore, we explore how dysregulated m6A modification in tumor cells influences immune cells, thereby regulating tumor immune evasion via interactions within the tumor microenvironment (TME). Lastly, we highlight recent discoveries regarding specific inhibitors of m6A modulators and the encapsulation of m6A-targeting nanomaterials for cancer therapy, discussing their potential applications in immunotherapy.

PPARγ Antagonists Exhibit Antitumor Effects by Regulating Ferroptosis and Disulfidptosis

Oral squamous cell carcinoma (OSCC) stands as a prevalent subtype of head and neck squamous cell carcinoma, leading to disease recurrence and low survival rates. PPARγ, a ligand-dependent nuclear transcription factor, holds significance in tumor development. However, the role of PPARγ in the development of OSCC has not been fully elucidated. Through transcriptome sequencing analysis, we discovered a notable enrichment of ferroptosis-related molecules upon treatment with PPARγ antagonist. We subsequently confirmed the occurrence of ferroptosis through transmission electron microscopy, iron detection, etc. Notably, ferroptosis inhibitors could not completely rescue the cell death caused by PPARγ inhibitors, and the rescue effect was the greatest when disulfidptosis and ferroptosis inhibitors coexisted. We confirmed that the disulfidptosis phenotype indeed existed. Mechanistically, through qPCR and Western blotting, we observed that the inhibition of PPARγ resulted in the upregulation of heme oxygenase 1 (HMOX1), thereby promoting ferroptosis, while solute carrier family 7 member 11 (SLC7A11) was also upregulated to promote disulfidptosis in OSCC. Finally, a flow cytometry analysis of flight and multiplex immunohistochemical staining was used to characterize the immune status of PPARγ antagonist-treated OSCC tissues in a mouse tongue orthotopic transplantation tumor model, and the results showed that the inhibition of PPARγ led to ferroptosis and disulfidptosis, promoted the aggregation of cDCs and CD8+ T cells, and inhibited the progression of OSCC. Overall, our findings reveal that PPARγ plays a key role in regulating cell death in OSCC and that targeting PPARγ may be a potential therapeutic approach for OSCC.

Inflammatory myofibroblastic tumor of the liver after adrenal neuroblastoma surgery: a case report

A boy aged 55 months was diagnosed with stage IV Neuroblastoma (NB) of the right adrenal gland 2 years ago. Preoperative chemotherapy was given and he was then treated with retroperitoneal tumor resection and lymph node dissection. After surgery, the children were transferred to the Hemato-Oncology Department for chemotherapy according to the high-risk group NB, with outpatient follow-up every 6 months. In the second postoperative year, abdominal computed tomography (CT) scan revealed a rounded hypodense area in the upper part of the right posterior lobe of the liver, with marked inhomogeneous enhancement in the venous phase after enhancement, which was surgically resected, and postoperative pathology confirmed inflammatory myofibroblastic tumor (IMT) of liver. The patient was not given any special treatment after surgery. In this study, whole transcriptome sequencing was performed on the postoperative specimen of adrenal NB and the specimen of IMT of liver. This unusual case emphasizes the need for close monitoring of second tumor development in NB survivors even in the absence of known predisposing factors.

Targeting the autophagy-miRNA axis in prostate cancer: toward novel diagnostic and therapeutic strategies.

Since prostate cancer is one of the leading causes of cancer-related death, a better understanding of the molecular pathways guiding its development is imperative. A key factor in prostate cancer is autophagy, a cellular mechanism that affects both cell survival and death. Autophagy is essential in maintaining cellular homeostasis. Autophagy is a physiological mechanism wherein redundant or malfunctioning cellular constituents are broken down and recycled. It is essential for preserving cellular homeostasis and is implicated in several physiological and pathological conditions, including cancer. Autophagy has been linked to metastasis, tumor development, and treatment resistance in prostate cancer. The deregulation of miRNAs related to autophagy appears to be a crucial element in the etiology of prostate cancer. These miRNAs influence the destiny of cancer cells by finely regulating autophagic mechanisms. Numerous investigations have emphasized the dual function of specific miRNAs in prostate cancer, which alter autophagy-related pathways to function as either tumor suppressors or oncogenes. Notably, miRNAs have been linked to the control of autophagy and the proliferation, apoptosis, and migration of prostate cancer cells. To create customized therapy approaches, it is imperative to comprehend the dynamic interplay between autophagy and miRNAs in prostate cancer. The identification of key miRNAs provides potential diagnostic and prognostic markers. Unraveling the complex network of lncRNAs, like PCA3, also expands the repertoire of molecular targets for therapeutic interventions. This review explores the intricate interplay between autophagy and miRNAs in prostate cancer, focusing on their regulatory roles in cellular processes ranging from survival to programmed cell death.

NAT10-mediated upregulation of GAS5 facilitates immune cell infiltration in non-small cell lung cancer via the MYBBP1A-p53/IRF1/type I interferon signaling axis

Interactions of tumor cells with immune cells in the tumor microenvironment play an important role during malignancy progression. We previously identified that GAS5 inhibited tumor development by suppressing proliferation of tumor cells in non-small cell lung cancer (NSCLC). Herein, we discovered a tumor-suppressing role for tumor cell-derived GAS5 in regulating tumor microenvironment. GAS5 positively coordinated with the infiltration of macrophages and T cells in NSCLC clinically, and overexpression of GAS5 promoted macrophages and T cells recruitment both in vitro and in vivo. Mechanistically, GAS5 stabilized p53 by directly binding to MYBBP1A and facilitating MYBBP1A-p53 interaction, and enhanced p53-mediated transcription of IRF1, which activated type I interferon signaling and increased the production of downstream CXCL10 and CCL5. We also found that activation of type I interferon signaling was associated with better immunotherapy efficacy in NSCLC. Furthermore, the stability of GAS5 was regulated by NAT10, the key enzyme responsible for N4-acetylcytidine (ac4C) modification, which bound to GAS5 and mediated its ac4C modification. Collectively, tumor cell-derived GAS5 could activate type I interferon signaling via the MYBBP1A-p53/IRF1 axis, promoting immune cell infiltration and potentially correlating with immunotherapy efficacy, which suppressed NSCLC progression. Our results suggested GAS5 as a promising predictive marker and potential therapeutic target for combination therapy in NSCLC.A schematic diagram demonstrating the regulatory effect of GAS5 on immune cell infiltration by activating type I interferon signaling via MYBBP1A-p53/IRF1 axis in non-small cell lung cancer. IFN, interferon.

Unraveling Immune Heterogeneity across Pan-Cancer and Deep Insights in Lung Adenocarcinoma based on Alternative Splicing.

Alternative splicing (AS) participates in tumor development and tumor microenvironment formation. However, the landscape of immune infiltrating AS events (IIASE) in pan-cancer and mechanisms of AS in lung adenocarcinoma (LUAD) have not been comprehensively characterized. We systematically profiled the IIASE landscape of pan-cancer using data from The Cancer Genome Atlas (TCGA), analyzing both commonalities and specific characteristics among different cancer types. We found that AS events tend to occur specifically in one cancer type rather than in multiple cancer types. AS events were used to classify 512 LUAD samples into two subtypes by unsupervised clustering: aberrant splicing subtype (ABS) and immune infiltrating subtype (IIS). The two subtypes showed significant differences in clinicopathology, prognosis, transcriptomics, genomics and immune microenvironment. We constructed a classification signature comprising 10 genes involved in 14 AS events using Logistic regression. The robustness of the signature was validated in three independent datasets using survival analysis. To explore AS mechanisms in LUAD, we constructed subtype-specific co-expression networks using Pearson correlation analysis. AS event of AKT3 regulated by splicing factor ENOX1 was associated with poor prognosis in LUAD. Overall, we outline AS events associated with immune infiltration in pan-cancer and this study provides insights into AS mechanisms in LUAD patient classification.

Abstract PR004: Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment

Abstract Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are recommended treatment with Bacillus Calmette-Guérin (BCG). The therapeutic effect of BCG is highly dependent on the host immune system and the tumor microenvironment (TME). The cellular composition and the functional status of the TME have been shown to play crucial roles for treatment efficacy. However, much is still unknown regarding the cellular interactions and mechanisms of BCG. Using spatial proteomics and transcriptomics, we investigated the immune cell landscape in a cohort of BCG treated patients. Materials and methods: A total of 183 tumors from 111 patients with NMIBC treated with BCG were included on a tissue microarray (TMA). The TMA included paired pre- and post-BCG tumors. We analyzed the tumor tissue using Imaging Mass Cytometry (IMC; StandardBioTools) for simultaneous detection of 35 immune and tumor-related proteins at single cell resolution. Ilastik was used to generate probability maps and DeepCell Mesmer was used for cell segmentation. Markers were quantified for the mean intensity per cell. GeoMX Digital Spatial Profiling (DSP) was used for spatial proteomics and transcriptomics profiling of tumor and stromal areas, targeting 63 protein targets and whole transcriptome amplification, respectively. Results: We identified a total of 364,504 cells in the tumor tissue using IMC. After computing a neighborhood graph using the log normalised and z-scaled signal and embedding it using UMAP, leiden clustering was performed to detect communities of unique cell types by interrogating the presence of phenotypic markers. We identified major lineages such as macrophages, CD8 T cells, dendritic cells (DCs), NK cells and neutrophils in the TME of analyzed tumors. Specifically, we observed more CD8 T cells after BCG (p=0.015), especially in females (p=0.048). Females also had higher levels of DCs and macrophages after treatment compared to males (p=0.024 and p=0.048). Patients who progressed to MIBC after BCG had higher numbers of CD8 T cells and NK cells prior to BCG (p=0.039 and p=0.021). Prolonged exposure to interferons has been associated with immune evasion and tumor cell proliferation. In concordance, we found high pretreatment protein expression of IFNα and IFNγ in tumor regions from patients with a pronounced signature of CD8 T cell exhaustion after BCG treatment using GeoMX DSP proteomic analysis. Data analysis is currently ongoing, and additional results will be presented at the conference. Conclusion: The composition and functional status of the TME is associated with clinical and biological features such as immune cell abundance, CD8 T cell exhaustion, sex and progression. A greater understanding of the TME may help identify patients unresponsive to BCG earlier and improve the understanding of biological differences in tumor development and aggressiveness. This may ultimately improve patient outcomes. Citation Format: Trine Strandgaard, Tine Ginnerup Andreasen, Tessa Jane Divita, Liina Salminen, Sean Houghton, Kasper Thorsen, Iver Nordentoft, Iyinyeoluwa Okulate, Alexander Schmitz, Søren Riis Paludan, John Sfakianos, Amir Horowitz, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR004.

Abstract A016: NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype

Abstract Introduction: Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a key role in protecting cells from oxidative stress. NRF2 is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1), which facilitates NRF2 ubiquitination and proteosomal degradation under homeostatic conditions. NRF2 activating mutations occur at a frequency of 5.9% in muscle-invasive bladder cancer (MIBC). However, the role of NRF2 in MIBC has not been fully understood. The aim of this study is to clarify how NRF2 affect to the normal epithelial cells and the tumor cells in bladder. Methods: We used genetically engineered mice (GEM) with a Cre inducible Nrf2 activating mutation (LSL-Nrf2-E79Q). To evaluate how Nrf2 affects the tumorigenesis process in vivo, the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) was administered to Upk3a-CreERT2; LSL-Nrf2-E79Q/+ (UN) mice. We also established normal urothelial organoids and BBN tumor derived organoids from wild-type (WT) and Krt5-CreERT2; LSL-Nrf2-E79Q/+ (K5N) mice for ex vivo study. To induce overexpression of NRF2, we utilized KI696 (KEAP1-NRF2 inhibitor) as a pharmacological agent and performed NAD/NADH assay to quantify the reductive stress. Additionally, we performed single cell RNA sequencing (scRNA-seq) with WT, UN and K5N mice bladder to identify the effect of Nrf2E79Q on the normal epithelial cells. Results: In BBN induced tumor model in vivo, UN mice did not differ in time to tumor development from the control WT mice. Furthermore, UN tumors had lost the post-recombination allele of LSL-Nrf2-E79Q in genomic PCR, meaning cells expressing Nrf2E79Q are selected against during the process. In ex vivo study, KI696 induced reductive stress in both WT normal urothelial organoids and BBN tumor derived organoids, but inhibited cell proliferation only in normal urothelial organoids. In K5N organoids, Nrf2E79Q activation enhanced relative mRNA expression of a number of basal related genes. In addition to that, vehicle organoids in differentiation media showed phenotypic differentiation, whereas those treated with tamoxifen did not demonstrate phenotypic signs of differentiation suggesting Nrf2 activation promotes a basal-like phenotype. Furthermore, we annotated three clusters in mice bladder epithelial cells as luminal, intermediate and basal using scRNA-seq and looked at their proportion among WT, UN and K5N mice. We found that the percentage of luminal cells was decreased and intermediate and basal cells increased in both UN and K5N mice compared to WT mice. This indicated that Nrf2E79Q promotes disadvantage in luminal cells in UN mice, whereas it contributes to the increase in intermediate and basal cells in K5N mice. Conclusion: These results suggested that Nrf2E79Q promotes a fitness disadvantage in normal mice urothelium. Furthermore, Nrf2E79Q inhibits differentiation of K5N organoids and drives a basal-like phenotype in GEM models. Further investigation is needed to clarify the mechanism of NRF2 activating mutation contributing the tumorigenesis in bladder. Citation Format: Akihiro Hamada, Yuki Kita, Di Wu, Mi Zhou, Sean Clark-Garvey, Andrew Gdowski, Michael Sturdivant, Wolfgang Beckabir, Elliott Drew Toomer, Emily Kounlavong, Lucia Lim, Kathryn Gessner, Jeffrey Damrauer, William Kim. NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A016.

Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids.

Cervical cancer, primarily squamous cell carcinoma, is the most prevalent gynecologic malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment. This limitation compromises the accurate representation of tumor heterogeneity. We collected para-cancerous cervical tissues from patients diagnosed with cervical squamous cell carcinoma (CSCC) and prepared uterine cervix extracellular matrix (UCEM) hydrogels. Proteomic analysis of UCEM identified several tissue-specific signaling pathways including human papillomavirus, phosphatidylinositol 3-kinase-AKT, and extracellular matrix receptor. Secreted proteins like FLNA, MYH9, HSPA8, and EEF1A1 were present, indicating UCEM successfully maintained cervical proteins. UCEM provided a tailored microenvironment for CSCC organoids, enabling formation and growth while preserving tumorigenic potential. RNA sequencing showed UCEM-organoids exhibited greater similarity to native CSCC and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways including virus protein-cytokine, nuclear factor κB, tumor necrosis factor, and oncogenes EGR1, FPR1, and IFI6. Moreover, UCEM-organoids developed chemotherapy resistance. Our research provides insights into advanced organoid technology through native matrix hydrogels.