Prostate Tumor Microenvironment Research Articles

The functions and mechanisms of RNA modification in prostate: Current status and future perspectives.

The increasing incidence and mortality of prostate cancer worldwide significantly impact the life span of male patients, emphasizing the urgency of understanding its pathogenic mechanism and associated molecular changes that regulate tumor progression for effective prevention and treatment. RNA modification, an important post-transcriptional regulatory process, profoundly influences tumor cell growth and metabolism, shaping cell fate. Over 170 RNA modification methods are known, with prominent research focusing on N6-methyladenosine, N7-methylguanosine, N1-methyladenosine, 5-methylcytidine, pseudouridine, and N4-acetylcytidine modifications. These alterations intricately regulate coding and non-coding RNA post-transcriptionally, affecting the stability of RNA and protein expression levels. This article delves into the latest advancements and challenges associated with various RNA modifications in prostate cancer tumor cells, tumor microenvironment, and core signaling molecule androgen receptors. It aims to provide new research targets and avenues for molecular diagnosis, treatment strategies, and improvement of the prognosis in prostate cancer.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [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 LB210.

Abstract CT023: Prostate cancer in situ autovaccination with the intratumoral viral mimic poly-ICLC: Making a cold tumor hot

Abstract PURPOSE To test the safety of sequential intratumoral plus systemic intramuscular injection of poly-ICLC, and its efficacy in priming antitumor immune responses in patients with prostate cancer (PCa). EXPERIMENTAL PROCEDURES This is an open-label dose-escalating phase 1 neoadjuvant clinical trial of poly-ICLC (NCT03262103) in 12 patients diagnosed with clinically localized PCa with plans to undergo radical prostatectomy (RP). Poly-ICLC was administered intratumorally (Artemis MRI-TRUS-guided) and intramuscularly (e.g., deltoid muscle). Comprehensive transcriptional profiling of tissues, phenotypic and transcriptional analysis of longitudinally collected peripheral blood, and analysis of the prostate tumor microenvironment (TME) were performed before and after poly-ICLC treatment in order to identify innate and adaptive antitumor immune responses within the tumor and in peripheral blood. This trial is the first to use human intratumoral immunotherapy instead of systemic immunomodulation for high-risk PCa patients. RESULTS Poly-ICLC was well tolerated (safe) in all 12 patients. There were no dose-limiting toxicities or TEAE (treatment-emergent adverse events) withdrawals during treatment. Eight of 10 evaluable patients (80%) had undetectable PSA (<0.1 ng/ml) at 1 year of follow-up post-RP. Eight of 12 evaluable patients (66.7%) and 7 of 10 patients (70%) with biopsy Gleason 8-10 had a lower Gleason score in the post-treatment RP specimen. Transcriptome profiling of paired biopsy and RP specimens showed significant upregulation of gene signatures associated with immune cell infiltration and TP53-associated metabolic genes, but downregulation of gene signatures associated with metastasis and DNA replication. Genes upregulated by poly-ICLC were associated with a good prognosis. Treatment with poly-ICLC increased systemic immune responses, as demonstrated by an increase in cytolytic signatures, T- and NK-cell signatures, and NK-cell subsets in the blood. Multiplex immunohistochemistry analysis revealed a significant increase in the densities of CD4- and CD8-T cells and B cells, as well as evidence of tertiary lymphoid structures (TLS) within the TME of post-treatment RP specimens compared to baseline. CONCLUSIONS Poly-ICLC treatment can reliably transform the cold TME of PCa into a hot, immune-enhanced ecosystem. The identified baseline and response biomarkers, tertiary lymphoid structures, potential clinical benefit, and immunologic correlates require validation in larger studies. Citation Format: Sujit S. Nair, Dimple Chakravarty, Sreekumar Balan, Alexander Hakansson, Manuel Duval, Elai Davicioni, Yang Liu, Swati Bhardwaj, Tin Htwe Thin, Monica Garcia-barros, Kenneth Haines, Majd Al Shaarani, Rachel Weil, Marcia Meseck, Parita Ratnani, Monali Fatterpekar, Ivan Jambor, Elena Gonzalez-Gugel, Adam Farkas, Vinayak Wagaskar, Kacie Schlussel, Cristina Pasat-karasik, Kamala Bhatt, Zachary Dovey, Adriana Pedraza, Akriti Gupta, Dara Lundon, Ante Peros, Sneha Parekh, Lily Davenport, Xiangfu Zhang, Raghav Gupta, Macy Robison, Cynthia Knauer, Ethan Ellis, Dmitry Rykunov, Boris Reva, Babu Padanilam, Matthew D. Galsky, Rachel Brody, Mani Menon, Andres Salazar, Nina Bhardwaj, Ashutosh K. Tewari. Prostate cancer in situ autovaccination with the intratumoral viral mimic poly-ICLC: Making a cold tumor hot [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 CT023.

Abstract 1528: Flowcytometric based approach to investigate prostate tumor microenvironment

Abstract Prostate cancer (PC) and its tumor microenvironment (TME) are heterogenous and evolving along with the treatment patients are exposed to. Modelling of the TME represents an attractive approach to understand how PC evolves and spreads. Flow cytometry is a technique for quantitative measurement of single cell profiling based on the cell surface as well as intracellular markers. We designed a flowcytometric based approach to investigate the interplay between the PC cells and immune cells in a direct coculture. Cells are fluorescently tagged with a cell tracer and the analysis is performed separately on the adherent and non-adherent cells. To optimize the readout method, we investigate the interplay between Jurkat cells and PC cells (LNCaP) as well as immortalized prostate epithelial cell models modified to mimic early events in prostate carcinogenesis (RWPE-1 cells overexpressing androgen receptor, RWPE-1-AR, and having PTEN deletion, RWPE-1-AR-PTENdel).Jurkat (suspension culture) cocultured with LNCaP (adherent culture) exhibited 40% decrease in viability but only 30% of LNCaP dying cell in the floating population compared to LNcaP only which is 60%. When Jurkat were activated using Phorbol 12-myristate 13-acetate (PMA), we observed an increase in LNCaP cell death. Jurkat viability was not impacted in presence of RWPE-1 neither when exposed to RWPE1-AR-PTENdel. In summary our Flowcytometric-based approach will help to understand the interplay between immune cells and prostate cancer cells. Citation Format: karishma sajnani, Sini Eerola, Konsta Kukkonen, Hanna Rauhala, Tapio Visakorpi, Alfonso Urbanucci. Flowcytometric based approach to investigate prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1528.

Abstract 3417: Dissecting tumor microenvironment to predict clinical outcome in prostate cancer

Abstract Prostate cancer is one of the major causes of cancer death in males, and its prevalence is particularly high in developed countries such as the United States and Western Europe. However, death rates in developing and underdeveloped countries are greater, presumably, due to a lack of routine clinical check-ups, poor health awareness, inadequate screening resources, and clinical intervention. Much progress has already been made using cutting-edge technologies like whole genome sequencing, RNAseq, phosphoproteomics, and targeted transcriptomic Nanostring profiling, etc. However, understanding of the prostate tumor microenvironment as a whole in clinical samples remains very limited. Here we use the latest genomic, proteomics, and metabolomic tools not only to understand the biology of prostate cancers but also their correlation with patient demography and clinical outcome. We recruited 80 patients from both tertiary research hospitals and clinical centers in the eastern region of India. All of their demographic information is collected along with their clinical outcome. Pathological evaluation revealed only 50 patients with positive Prostate Carcinoma. We performed a detailed analysis using experimental data (from genomic, and proteomic) in a cohort of patients' routine biomarkers (PSA and PSMA), histopathology, and clinical outcome. Our data demonstrate no linear correlation of any of the individual markers or parameters like AR status, PSA, Prostate Specific Acid Phosphatase, Prostate-Specific Membrane Antigen (PSMA), Alpha-methylacyl-CoA racemase (AMACR), Mesothelin, PD-L1, and other potential genomic and proteomic alterations or any histopathological readouts. However, a clear understanding of the relationship between these multiparameter data, disease pathology, and clinical outcomes in a group of patients has been established. The heterogeneity of these individual data for tumor microenvironments will lead us to identify novel biomarkers for the clinical response of targeted drugs, accelerating personalized treatment strategies. Citation Format: Aritri Bhattacharjee, Manjusha Biswas, Amjad Husain, Asim Kr Das, Sourav Karmakar Karmakar, Tapan Mondal, Pinaki Roy, Manas Kr Mondal, Badal Kumar Sahu, Nabendu Murmu, Partho Nath, Chandra C. Ghosh, Pradip Kumar Majumder. Dissecting tumor microenvironment to predict clinical outcome in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3417.

Abstract 4150: Development of a three-dimensional spatialomics map of lethal prostate cancer driven by clinical imaging

Abstract Prostate adenocarcinoma (PCa) is the most common malignancy and the second leading cause of cancer-related mortality in United States men. Following treatment for localized PCa, up to 50% of these men can experience disease recurrence, with subsequent metastasis and death. This indicates that there is suboptimal identification of lethal PCa at the time of diagnosis, despite advancements in imaging, genomics, and biomarkers. To improve the noninvasive diagnosis of lethal PCa at initial diagnosis, we have developed a novel magnetic resonance imaging (MRI) method, diffusion basis spectrum imaging (DBSI), that noninvasively measures cancer, stromal, and immune cell contributions to the prostate tumor microenvironment (TME). To validate DBSI for PCa, we have applied novel technologies to matched prostatectomy specimens that include (i) fully automated tissue section processing with serial 2-photon tomographic (STP) imaging of the sections to generate 3D volumetric structural maps of the tumor tissue and (ii) multiplex MALDI mass spectrometry imaging of untargeted glycans and extracellular matrix (ECM) peptides and of proteins targeted with novel photocleavable-mass-tagged antibodies (MALDI-IHC) to spatially resolve hundreds of biomolecules from the TME. To establish the feasibility of this approach, we recruited a patient with an unusually aggressive prostate cancer consisting of Gleason 5+4 acinar adenocarcinoma histology with intraductal growth that was locally invasive and lymph node positive at prostatectomy (stage T3bN1). This patient also had the unusual finding of low activity on prostate specific membrane antigen positron emission tomography (PSMA-PET). Following gross sectioning of the prostate with MRI-guided 3D printed molds to co-register clinical DBSI with histology, a 5-mm thick tissue cross-section containing tumor and normal prostate was imaged with high resolution ex vivo DBSI followed by 3D structural mapping with STP imaging. Selected slides in the 3D stack were subject to multiplexed MALDI imaging to identify relationships between cellular and molecular heterogeneity with tumor structure identified with DBSI and STP imaging. We discovered unique structural differences in the prostate tumor compared with the surrounding stroma based upon collagen and glycan modifications to the ECM that were recapitulated with second harmonic maps of collagen from STP imaging as well as stromal maps generated from DBSI imaging. Additional novel protein and glycan ECM components were expressed in this tumor known to be associated with poor patient outcomes. Together, these data represent the first evidence successfully linking noninvasive in vivo clinical imaging and de novo spatialomics technologies with potential to improve PCa diagnostics. This advanced imaging workflow can be applied to any tissue type, underscoring its relevance to translational cancer research. Citation Format: Joseph E. Ippolito, David H. Ballard, Karla Bergeron, Ritesh Chidambaram, Anusha Elumalai, Tyler J. Fraum, Huaping Jing, Peter Kamelin, Eric H. Kim, Catherine Kita, Aleksandra Klim, Anthony Knesis, Mark Lim, José Marcio Luna, Hani Nakhoul, Timothy Ragan, Kenneth J. Rothschild, Sheng-Kwei Song, Kainen Utt, Cody Weimholt, Gargey Yagnik, Elijah Yew, Peggi M. Angel, Richard R. Drake. Development of a three-dimensional spatialomics map of lethal prostate cancer driven by clinical imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4150.

Recent Trends in Nanocarrier-Based Drug Delivery System for Prostate Cancer.

Prostate cancer remains a significant global health concern, requiring innovative approaches for improved therapeutic outcomes. In recent years, nanoparticle-based drug delivery systems have emerged as promising strategies to address the limitations of conventional cancer chemotherapy. The key trends include utilizing nanoparticles for enhancing drug delivery to prostate cancer cells. Nanoparticles have some advantages such as improved drug solubility, prolonged circulation time, and targeted delivery of drugs. Encapsulation of chemotherapeutic agents within nanoparticles allows for controlled release kinetics, reducing systemic toxicity while maintaining therapeutic efficacy. Additionally, site-specific accumulation within the prostate tumor microenvironment is made possible by the functionalization of nanocarrier with targeted ligands, improving therapeutic effectiveness. This article highlights the basics of prostate cancer, statistics of prostate cancer, mechanism of multidrug resistance, targeting approach, and different types of nanocarrier used for the treatment of prostate cancer. It also includes the applications of nanocarriers for the treatment of prostate cancer and clinical trial studies to validate the safety and efficacy of the innovative drug delivery systems. The article focused on developing nanocarrier-based drug delivery systems, with the goal of translating these advancements into clinical applications in the future.

Agent-based modeling of the prostate tumor microenvironment uncovers spatial tumor growth constraints and immunomodulatory properties

Inhibiting androgen receptor (AR) signaling through androgen deprivation therapy (ADT) reduces prostate cancer (PCa) growth in virtually all patients, but response may be temporary, in which case resistance develops, ultimately leading to lethal castration-resistant prostate cancer (CRPC). The tumor microenvironment (TME) plays an important role in the development and progression of PCa. In addition to tumor cells, TME-resident macrophages and fibroblasts express AR and are therefore also affected by ADT. However, the interplay of different TME cell types in the development of CRPC remains largely unexplored. To understand the complex stochastic nature of cell-cell interactions, we created a PCa-specific agent-based model (PCABM) based on in vitro cell proliferation data. PCa cells, fibroblasts, “pro-inflammatory” M1-like and “pro-tumor” M2-like polarized macrophages are modeled as agents from a simple set of validated base assumptions. PCABM allows us to simulate the effect of ADT on the interplay between various prostate TME cell types. The resulting in vitro growth patterns mimic human PCa. Our PCABM can effectively model hormonal perturbations by ADT, in which PCABM suggests that CRPC arises in clusters of resistant cells, as is observed in multifocal PCa. In addition, fibroblasts compete for cellular space in the TME while simultaneously creating niches for tumor cells to proliferate in. Finally, PCABM predicts that ADT has immunomodulatory effects on macrophages that may enhance tumor survival. Taken together, these results suggest that AR plays a critical role in the cellular interplay and stochastic interactions in the TME that influence tumor cell behavior and CRPC development.

Targeting the prostate tumor microenvironment by plant-derived natural products

Prostate cancer is among the most common malignancies for men, with limited therapy options for last stages of the tumor. There are some different options for treatment and control of prostate tumor growth. However, targeting some specific molecules and cells within tumors has been attracted interests in recent years. The tumor microenvironment (TME) has an important role in the initiation of various malignancies, which can also expand the progression of tumor and facilitate invasion of malignant cells. By regulating immune responses and distinct changes in the metabolism of cells in the tumor, TME has substantial effects in the resistance of cancer cells to therapy. TME in various solid cancers like prostate cancer includes various cells, including cancer cells, supportive stromal cells, immunosuppressive cells, and anticancer inflammatory cells. Natural products including herbal-derived agents and also other natural compounds have been well studied for their anti-tumor potentials. These compounds may modulate various signaling pathways involved in TME, such as immune responses, the metabolism of cells, epigenetics, angiogenesis, and extracellular matrix (ECM). This paper provides a review of the current knowledge of prostate TME and complex interactions in this environment. Additionally, the potential use of natural products and also nanoparticles loaded with natural products as therapeutic adjuvants on different cells and therapeutic targets within prostate TME will be discussed.

Digital analysis of the prostate tumor microenvironment with high-order chromogenic multiplexing

As our understanding of the tumor microenvironment grows, the pathology field is increasingly utilizing multianalyte diagnostic assays to understand important characteristics of tumor growth. In clinical settings, brightfield chromogenic assays represent the gold-standard and have developed significant trust as the first-line diagnostic method. However, conventional brightfield tests have been limited to low-order assays that are visually interrogated. We have developed a hybrid method of brightfield chromogenic multiplexing that overcomes these limitations and enables high-order multiplex assays. However, how compatible high-order brightfield multiplexed images are with advanced analytical algorithms has not been extensively evaluated. In the present study, we address this gap by developing a novel 6-marker prostate cancer assay that targets diverse aspects of the tumor microenvironment such as prostate-specific biomarkers (PSMA and p504s), immune biomarkers (CD8 and PD-L1), a prognostic biomarker (Ki-67), as well as an adjunctive diagnostic biomarker (basal cell cocktail) and apply the assay to 143 differentially graded adenocarcinoma prostate tissues. The tissues were then imaged on our spectroscopic multiplexing imaging platform and mined for proteomic and spatial features that were correlated with cancer presence and disease grade. Extracted features were used to train a UMAP model that differentiated healthy from cancerous tissue with an accuracy of 89% and identified clusters of cells based on cancer grade. For spatial analysis, cell-to-cell distances were calculated for all biomarkers and differences between healthy and adenocarcinoma tissues were studied. We report that p504s positive cells were at least 2× closer to cells expressing PD-L1, CD8, Ki-67, and basal cell in adenocarcinoma tissues relative to the healthy control tissues. These findings offer a powerful insight to understand the fingerprint of the prostate tumor microenvironment and indicate that high-order chromogenic multiplexing is compatible with digital analysis. Thus, the presented chromogenic multiplexing system combines the clinical applicability of brightfield assays with the emerging diagnostic power of high-order multiplexing in a digital pathology friendly format that is well-suited for translational studies to better understand mechanisms of tumor development and growth.

Androgen receptor contributes to repairing DNA damage induced by inflammation and oxidative stress in prostate cancer.

Androgen deprivation therapy remains the first-line therapy option for prostate cancer, mostly resulting in the transition of the disease to a castration-resistant state. The lack of androgen signaling during therapy affects various cellular processes, which sometimes paradoxically contributes to cancer progression. As androgen receptor (AR) signaling is known to contribute to oxidative stress regulation, loss of AR may also affect DNA damage level and the response mechanism in oxidant and inflammatory conditions of the prostate tumor microenvironment. Therefore, this study aimed to investigate the role of AR and AR-regulated tumor suppressor NKX3.1 upon oxidative stress-induced DNA damage response (DDR) in the inflammatory tumor microenvironment of the prostate. Intracellular reactive oxygen species (ROS) level was induced by either inflammatory conditioned media obtained from lipopolysaccharide-induced macrophages or oxidants and measured by dichlorodihydrofluorescein diacetate. In addition to this, DNA damage was subsequently quantified by counting gH2AX foci using an immunofluorescence-based Aklides platform. Altered expression of proteins function in DDR detected by western blotting. Cellular levels of ROS and ROS-induced DNA double-strand break damage were analyzed in the absence and presence of AR signaling upon treatment of prostate cancer cells by either oxidants or inflammatory microenvironment exposure. The results showed that AR suppresses intracellular ROS and contributes to DNA damage recognition under oxidant conditions. Besides, increased DNA damage due to loss of NKX3.1 under inflammatory conditions was alleviated by its overexpression. Moreover, the activation of the DDR mediators caused by AR and NKX3.1 activation in androgen-responsive and castration-resistant prostate cancer cells indicated that the androgen receptor function is essential both in controlling oxidative stress and in activating the ROS-induced DDR. Taken together, it is concluded that the regulatory function of androgen receptor signaling has a vital function in the balance between antioxidant response and DDR activation.

Castration promotes the browning of the prostate tumor microenvironment

BackgroundAdipose tissue has gained attention due to its potential paracrine role. Periprostatic adipose tissue surrounds the prostate and the prostatic urethra, and it is an essential player in prostate cancer progression. Since obesity is directly related to human tumor progression, and adipose tissue depots are one of the significant components of the tumor microenvironment, the molecular mediators of the communication between adipocytes and epithelial cells are in the spotlight. Although periprostatic white adipose tissue contributes to prostate cancer progression, brown adipose tissue (BAT), which has beneficial effects in metabolic pathologies, has been scarcely investigated concerning cancer progression. Given that adipose tissue is a target of androgen signaling, the actual role of androgen removal on the periprostatic adipose tissue was the aim of this work.MethodsSurgical castration of the transgenic adenocarcinoma of the mouse prostate (TRAMP) was employed. By histology examination and software analysis, WAT and BAT tissue was quantified. 3T3-like adipocytes were used to study the role of Casodex® in modifying adipocyte differentiation and to investigate the function of the secretome of adipocytes on the proliferation of androgen-dependent and independent prostate cancer cells. Finally, the role of cell communication was assayed by TRAMP-C1 xenograft implanted in the presence of 3T3-like adipocytes.ResultsAndrogen removal increases brown/beige adipose tissue in the fat immediately surrounding the prostate glands of TRAMP mice, concomitant with an adjustment of the metabolism. Castration increases body temperature, respiratory exchange rate, and energy expenditure. Also, in vitro, it is described that blocking androgen signaling by Casodex® increases the uncoupling protein 1 (UCP1) marker in 3T3-like adipocytes. Finally, the effect of brown/beige adipocyte secretome was studied on the proliferation of prostate cancer cells in vivo and in vitro. The secretome of brown/beige adipocytes reduces the proliferation of prostate cancer cells mediated partly by the secretion of extracellular vesicles.ConclusionsConsequently, we concluded that hampering androgen signaling plays a crucial role in the browning of the periprostatic adipose tissue. Also, the presence of brown adipocytes exhibits the opposite effect to that of white adipocytes in vitro regulating processes that govern the mechanisms of cell proliferation of prostate cancer cells. And finally, promoting the browning of adipose tissue in the periprostatic adipose tissue might be a way to handle prostate cancer cell progression.EECkevEGf-HMwkJP3j4bZsVideo

Spatial whole transcriptome profiling of primary tumor from patients with metastatic prostate cancer.

Prostate cancer (PCa) is a highly heterogeneous disease in terms of its molecular makeup and clinical prognosis. The prostate tumor microenvironment (TME) is hypothesized to play an important role in driving disease aggressiveness, but precise mechanisms remain elusive. In our study, we used spatial transcriptomics to explore for the first time the spatial gene expression heterogeneity within primary prostate tumors from patients with metastatic disease. In total, we analyzed 5459 tissue spots from three PCa patients comprising castration-resistant (CRPC) and neuroendocrine (NEPC) disease stages. Within CRPC, we identified a T cell cluster whose activity might be impaired by nearby regulatory T cells, potentially mediating the aggressive disease phenotype. Moreover, we identified Hallmark signatures of epithelial-mesenchymal transition in a cancer-associated fibroblast (CAF) cluster, indicating the aggressive characteristic of the primary TME leading to metastatic dissemination. Within NEPC, we identified active immune-stroma cross-talk exemplified by significant ligand-receptor interactions between CAFs and M2 macrophages. Further, we identified a malignant cell population that was associated with the down-regulation of an immune-related gene signature. Lower expression of this signature was associated with higher levels of genomic instability in advanced PCa patients (SU2C cohort, n = 99) and poor recurrence free survival in early-stage PCa patients (TCGA cohort, n = 395), suggesting prognostic biomarker potential. Taken together, our study reveals the importance of whole transcriptome profiling at spatial resolution for biomarker discovery and for advancing our understanding of tumor biology.

New Pathway for Cisplatin Prodrug to Utilize Metabolic Substrate Preference to Overcome Cancer Intrinsic Resistance.

Tumor cells adapt to diverse survival strategies defying our pursuit of multimodal cancer therapy. Prostate cancer (PCa) is an example that is resistant to one of the most potent chemotherapeutics, cisplatin. PCa cells survive and proliferate using fatty acid oxidation (FAO), and the dependence on fat utilization increases as the disease progresses toward a resistant form. Using a pool of patient biopsies, we validated the expression of a key enzyme carnitine palmitoyltransferase 1 A (CPT1A) needed for fat metabolism. We then discovered that a cisplatin prodrug, Platin-L, can inhibit the FAO of PCa cells by interacting with CPT1A. Synthesizing additional cisplatin-based prodrugs, we documented that the presence of an available carboxylic acid group near the long chain fatty acid linker on the Pt(IV) center is crucial for CPT1A binding. As a result of fat metabolism disruption by Platin-L, PCa cells transition to an adaptive glucose-dependent chemosensitive state. Potential clinical translation of Platin-L will require a delivery vehicle to direct it to the prostate tumor microenvironment. Thus, we incorporated Platin-L in a biodegradable prostate tumor-targeted orally administrable nanoformulation and demonstrated its safety and efficacy. The distinctive FAO inhibitory property of Platin-L can be of potential clinical relevance as it offers the use of cisplatin for otherwise resistant cancer.

Collagen VI deposition mediates stromal T cell trapping through inhibition of T cell motility in the prostate tumor microenvironment

The tumor extracellular matrix (ECM) is a barrier to anti-tumor immunity in solid tumors by disrupting T cell-tumor cell interaction underlying the need for elucidating mechanisms by which specific ECM proteins impact T cell motility and activity within the desmoplastic stroma of solid tumors. Here, we show that Collagen VI (Col VI) deposition correlates with stromal T cell density in human prostate cancer specimens. Furthermore, motility of CD4+ T cells is completely ablated on purified Col VI surfaces when compared with Fibronectin and Collagen I. Importantly, T cells adhered to Col VI surfaces displayed reduced cell spreading and fibrillar actin, indicating a reduction in traction force generation accompanied by a decrease in integrin β1 clustering. We found that CD4+ T cells largely lack expression of integrin α1 in the prostate tumor microenvironment and that blockade of α1β1 integrin heterodimers inhibited CD8+ T cell motility on prostate fibroblast-derived matrix, while re-expression of ITGA1 improved motility. Taken together, we show that the Col VI-rich microenvironment in prostate cancer reduces the motility of CD4+ T cells lacking integrin α1, leading to their accumulation in the stroma, thus putatively inhibiting anti-tumor T cell responses.

Abstract A078: Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer

Abstract The advent of immunotherapies such as immune checkpoint blockade (ICB) has greatly expanded the therapeutic options available to patients with treatment-refractory solid tumors. However, patients with castration-resistant prostate cancer (CRPC) generally remain unresponsive to ICB therapy due to an immunologically “cold” prostate tumor microenvironment (TME) with limited accessibility to cytotoxic lymphocytes such as Natural Killer (NK) and T cells that can mediate potent anti-tumor immunity. During the course of tumor evolution, CRPCs acquire many genomic alterations and copy number changes that can influence therapy outcomes. Yet the impact of such genetic alterations on the immune TME and response to ICB remains largely unknown, in part due to the lack of models to functionally study them in an immune competent setting. To this end, we previously developed a novel electroporation-based genetically engineered mouse model (EPO-GEMM) to rapidly generate genetically defined prostate tumors de novo within an intact prostate TME (Leibold*, Ruscetti* et al., Cancer Discovery 2020). Here, we applied the EPO-GEMM approach to characterize the immune landscapes of distinct genetic subtypes of CRPC and subsequently identify and target tumor intrinsic mediators of immune suppression for prostate cancer immunotherapy. Using transposon systems to overexpress the MYC oncogene and CRISPR/Cas9 vectors to inactivate tumor suppressor genes (TSGs) commonly altered in CRPC (Pten, P53, Rb1, Apc), we generated a suite of genetically distinct prostate cancer-bearing EPO-GEMMs. Histological analysis revealed genotype-specific immune infiltrates, with MYC-driven tumors generally lacking cytotoxic CD8+ T cells and harboring increased numbers of regulatory T cells (Tregs). A similar correlation was also observed between MYC levels and cytotoxic lymphocyte suppression in CRPC patient samples. In particular, tumors with compound MYC overexpression and p53 loss (MP) displayed drastic NK cell suppression. Cytokine and RNA-seq profiling of primary EPO-GEMM CRPCs, tumor-derived cell lines, and isogenic Myc-CaP cells engineered with TSG losses revealed that MP alterations led to not only inhibition of inflammatory cytokine and interferon signaling and antigen presentation/processing gene expression, but also induction of VEGF signaling and increased angiogenesis in the prostate TME. Treatment of MP tumors with a VEGFR2 antibody to block VEGF signaling significantly reduced tumor growth and increased NK and CD8+T cell infiltration and activation, indicating a phenotypic switch from a traditionally “cold” TME. Our results demonstrate that the genetic configuration of prostate cancer shapes its surrounding TME. Additionally, targeting VEGF signaling may be a mechanism to overcome ICB resistance in prostate cancer. More broadly, we believe that by understanding the tumor intrinsic mechanisms driving immune suppression, we can identify rational immunotherapy combinations for CRPC based on the genetic fingerprint of a patient’s tumor for precision medicine. Citation Format: Katherine C. Murphy, Kelly DeMarco, Yvette Lopez-Diaz, Lin Zhou, Marcus Ruscetti. Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer [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 A078.

Prostate cancer metastasis and health disparities: a systematic review.

Prostate cancer (PCa), one of the most prevalent malignancies affecting men, significantly contributes to increased mortality rates worldwide. While the causative death is due to advanced metastatic disease, this occurrence disproportionately impacts men of African descent compared to men of European descent. In this review, we describe potential mechanisms underlying PCa metastases disparities and current treatments for metastatic disease among these populations, differences in treatment outcomes, and survival rates, in hopes of highlighting a need to address disparities in PCa metastases. We reviewed existing literature using databases such as PubMed, Google Scholar, and Science Direct using the following keywords: "prostate cancer metastases", "metastatic prostate cancer disparity", "metastatic prostate cancer diagnosis and treatment", "prostate cancer genetic differences and mechanisms", "genetic differences and prostate tumor microenvironment", and "men of African descent and access to clinical treatments". The inclusion criteria for literature usage were original research articles and review articles. Studies indicate unique genetic signatures and molecular mechanisms such as Epithelial-Mesenchymal Transition (EMT), inflammation, and growth hormone signaling involved in metastatic PCa disparities. Clinical studies also demonstrate differences in treatment outcomes that are race-specific, for example, patients of African descent have a better response to enzalutamide and immunotherapy yet have less access to these drugs as compared to patients of European descent. Growing evidence suggests a connection between a patient's genetic profile, the prostate tumor microenvironment, and social determinants of health that contribute to the aggressiveness of metastatic disease and treatment outcomes. With several potential pathways highlighted, the limitations in current diagnostic and therapeutic applications that target disparity in PCa metastases warrant rigorous research attention.

Anoikis in phenotypic reprogramming of the prostate tumor microenvironment.

Prostate cancer is one of the most common malignancies in males wherein 1 in 8 men are diagnosed with this disease in their lifetime. The urgency to find novel therapeutic interventions is associated with high treatment resistance and mortality rates associated with castration-resistant prostate cancer. Anoikis is an apoptotic phenomenon for normal epithelial or endothelial cells that have lost their attachment to the extracellular matrix (ECM). Tumor cells that lose their connection to the ECM can die via apoptosis or survive via anoikis resistance and thus escaping to distant organs for metastatic progression. This review discusses the recent advances made in our understanding of the signaling effectors of anoikis in prostate cancer and the approaches to translate these mechanistic insights into therapeutic benefits for reducing lethal disease outcomes (by overcoming anoikis resistance). The prostate tumor microenvironment is a highly dynamic landscape wherein the balance between androgen signaling, cell lineage changes, epithelial-mesenchymal transition (EMT), extracellular matrix interactions, actin cytoskeleton remodeling as well as metabolic changes, confer anoikis resistance and metastatic spread. Thus, these mechanisms also offer unique molecular treatment signatures, exploitation of which can prime prostate tumors to anoikis induction with a high translational significance.

Abstract 2876: Characterization of P2X4 purinergic receptors in the prostate tumor microenvironment

Abstract The 5-year survival rate for patients with metastatic prostate cancer (PCa) in the United States is 30.6%. Therefore, more effective treatment approaches are urgently required. The P2X4 purinergic receptor is well studied for its role in inflammation. Our previous studies demonstrated that the P2X4 receptor is highly expressed in PCa compared to benign regions and is associated with poor prognosis. We observed intense P2X4 expression on most macrophages and some neutrophils in the primary tumor microenvironment (TME). Our study also showed that P2X4 knock-down in mouse PCa Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in mice when compared to control cells. Allograft tumors were heavily infiltrated by P2X4-positive host immune cells. However, which components of the prostate TME express P2X4 receptors and the functional consequence of that expression is not well defined. Therefore, the objective of this study is to characterize P2X4 receptor expression and function in the prostate TME and metastatic niche. A P2X4 antagonist has completed Phase 1 clinical trials for the treatment of neuropathic pain. As such, deciphering a role for P2X4 in the prostate TME promises novel cancer therapeutic approaches against aggressive PCa. We used RNA in situ hybridization and immunohistochemistry to assess P2X4 expression on immune cells in human PCa tissues and Myc-CaP allograft primary and metastatic tumor tissues. To determine P2X4 function in macrophages in vitro, we used THP1 human monocytic cells differentiated to M0 macrophages and polarized to the M1 and M2 subtypes. M1 and M2 cells were treated with P2X4 agonist, CTP or antagonist, 5-BDBD. RNA was extracted from cells to examine the expression of macrophage subtype-specific genes, and cytokine levels in cell media were measured by ELISA. Statistical analysis was done using GraphPad Prism. Dual staining revealed abundant CD163+ M2 macrophages that express P2X4 in human PCa tissues. In Myc-CaP allograft primary and metastatic tumor tissues, we observed abundant F4/80+ macrophages and few Ly-6G+ neutrophils. Staining patterns suggest that P2X4+ immune cells likely include both cell types. CTP treatment of THP1 cell-derived M2 macrophages promoted anti-inflammatory IL-10 secretion while 5-BDBD treatment induced IL-6 secretion in M1 cells. M1 marker CD80, and M2 markers CD163 and CD206 mRNA expression were comparable across treatment groups, suggesting that treatment does not likely influence macrophage polarization. In conclusion, our study indicates that tumor-associated macrophages express P2X4 receptors both in primary and metastatic tumors. Our in vitro studies suggest a role for P2X4 receptors in macrophage cytokine secretion. Future studies will include functional assays to determine the role of P2X4 receptors on macrophages in the context of PCa. Citation Format: Vahinipriya Manoharan, Angelo M. De Marzo, Janielle P. Maynard. Characterization of P2X4 purinergic receptors in the prostate tumor microenvironment [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 2876.

Abstract 5139: Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment

Abstract Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of malignant cells, thereby affecting tumorigenesis and tumor progression. While immune checkpoint inhibitors and agonism of costimulatory molecules can act to stimulate leukocytes, the lack of adequate recruitment of effector cells into TME often results in suboptimal immune responses. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous chemoattractant that is widely expressed by many tissues and recruits innate leukocytes through its receptor, CMKLR1 which is expressed on NK cells, some dendritic cells, and macrophages. Previous studies show that RARRES2 is downregulated across multiple tumor types including prostate cancer. Here, using a syngeneic transgenic adenocarcinoma mouse prostate (TRAMP-C1) model, we show that forced overexpression of RARRES2 by tumor cells results in significant tumor suppression and recruitment of NK and CD8+ T-cells within the TME. Male C57/BL/6 mice were inoculated with either RARRES2-overexpressing, vector control (VC) or mixed (50:50; VC: RARRES2) TRAMP-C1 cells. Chemerin overexpression in TRAMP-C1 cells did not alter their phenotypic behavior in vitro, but significantly suppressed tumor growth in vivo in mice. Furthermore, we observed a significantly higher influx of NK (CD3-CD19-NK1.1+) and CD8+ T-cells (CD3+CD8+) in tumor-infiltrating leukocytes (TILs) from mice with mixed tumors compared with VC tumors. Depletion of NK cells, CD4+, or CD8+ T cells showed that NK cells and CD8+ T-cells, but not CD4+ T-cells, were required for chemerin-dependent suppression of TRAMP-C1 tumor growth. We found significant increases in splenic NK and CD8 T cells, and a concomitant significant decrease in splenic G-MDSC in mice with chemerin-expressing tumors compared to controls, suggesting chemerin expression within the TME has an effect on systemic immune responses. Treatment of control tumors with anti-PD1 checkpoint inhibition did not result in significant tumor suppression, while anti-PD1 treatment of chemerin-expressing tumors significantly reduced tumor growth, doubling the complete response rate in these unresponsive tumors. Thus, for the first time we have demonstrated that increasing chemerin expression within the prostate TME can suppress growth by augmenting recruitment of NK and CD8+ T-cells and has the potential to be combined with checkpoint inhibitors in order to improve response rates in non-responsive tumors. Overall, these data suggest that a tumor-targeted chemerin therapeutic may have potential to treat human prostate cancers. Citation Format: Muhammad A. Saeed, Kevin Kim, Russell K. Pachynski. Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment. [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 5139.