Prostate Cancer Tumor Microenvironment Research Articles

Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

Abstract 3983: Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells

Abstract In the tumor microenvironment of prostate cancer (PCa), cancer cells frequently invade the surrounding stroma in the way of single cell migration (SCM) or collective cell migration (CCM). This phenomenon can be responsible for the heterogeneous formation of structural atypia such as Gleason pattern. Generally, PCa cells undergo a phenotypic change which is called the epithelial-to-mesenchymal transition (EMT), e.g., downregulation of epithelial cell-cell adhesion molecules such as E-cadherin and gain of mesenchymal morphology and motility. The surrounding stroma is mainly composed of fibroblasts and extracellular matrix (ECM). We hypothesize that direct contact-mediated interactions at the interface between PCa cells and fibroblasts/ECM can trigger numerous changes in PCa cells including morphology and motility. In this study, we investigated the roles of fibroblasts in the three-dimensional (3D) morphogenesis of PCa cells on a viscous substrate. To examine the effects of fibroblasts on the 3D morphogenesis of PCa cells on Matrigel, we mixed human PCa cell line LNCaP cells with commercially available normal human prostate stromal cells (PrSC) or nine primary cultures of prostate fibroblasts (pcPrFs and NPFs) isolated from patients with PCa and bladder cancer. The 3D morphogenesis of LNCaP cells mixed with fibroblasts on Matrigel was confirmed by E-cadherin immunohistochemical staining. All ten fibroblasts alone were aggregated on Matrigel, whereas LNCaP cells alone were not. LNCaP cells mixed with six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a deep crater-like hole on Matrigel. In the 3D structure, the upper and lower tiers were composed of LNCaP cells and fibroblasts, respectively. Of note, direct contact of four fibroblasts (NPF-M13, pcPrF-M7, pcPrF-M10, and pcPrF-M26) with LNCaP cells decreased the aggregation of fibroblasts on Matrigel. Five of six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M28, and pcPrF-M31) induced SCM of LNCaP cells, while only one fibroblasts (pcPrF-M24) induced CCM. In the scratch assay, the cell motility of CCM-inducing fibroblasts was slower than that of SCM-inducing fibroblasts. Expression pattern of primary cilia in SCM-inducing fibroblasts was changed by mixing with LNCaP cells, whereas that in CCM-inducing fibroblasts was not. PCa is quite interesting because of the multi-focal and heterologous progression of primary tumors. The heterogeneous stromal compartment in PCa tissues contains multiple populations of fibroblasts that are associated with morphology and motility of PCa cells. Here our results showed that direct contact of heterogeneous fibroblasts with PCa cells on Matrigel regulated the invasive phenotype of PCa cells such as SCM and CCM. Impairing the malignant phenotype of fibroblasts in PCa tissues, e.g., differentiation therapy targeting tumor stroma, may abrogate PCa cell invasion. Citation Format: Kenichiro Ishii, Yasuhisa Nakagawa, Taku Shirai, Masako Ichishi, Masaya Fujiwara, Daisuke Kato, Chise Matsuda, Yoshifumi Hirokawa, Yoshiki Sugimura, Masaki Inagaki, Masatoshi Watanabe. Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3983.

Enzyme-responsive polymeric micelles of cabazitaxel for prostate cancer targeted therapy

Cabazitaxel, a novel tubulin inhibitor with poor affinity for P-glycoprotein, is a second-generation taxane holding great promise for the treatment of metastatic castration-resistant prostate cancer. However, its poor solubility and lack of target-ability limit its therapeutic applications. Herein, we develop a biodegradable, enzyme-responsive, and targeted polymeric micelle for cabazitaxel. The micelle is formed from two amphiphilic block copolymers. The first block copolymer consists of PEG, an enzyme-responsive peptide, and cholesterol; whereas the second block copolymer consists of a targeting ligand, PEG and cholesterol. The enzyme-responsive peptide is cleavable in the presence of matrixmetaloproteinase-2 (MMP-2), which is overexpressed in the tumor microenvironment of prostate cancer. The micelle showed a very low critical micelle concentration (CMC), high drug loading, and high entrapment efficiency. Release of cabazitaxel from the micelle is dependent on the cleavage of the enzyme-responsive peptide. Moreover, the micelle showed dramatically higher cellular uptake in prostate cancer cells compared to free cabazitaxel. Importantly, the ligand-coupled polymeric micelle demonstrated better inhibition of tumor growth in mice bearing prostate cancer xenografts compared to unmodified micelle and free cabazitaxel. Taken together, these findings suggest that the enzyme-responsive cabazitaxel micelle is a potent and promising drug delivery system for advanced prostate cancer therapy. Statement of SignificanceHerein, we develop a biodegradable, enzyme-responsive, and actively targeted polymer micelle for cabazitaxel, which is a novel tubulin inhibitor with poor affinity for P-glycoprotein. Despite cabazitaxel’s great promise for metastatic castration-resistant prostate cancer, its poor solubility, lack of target-ability, and high systemic toxicity limit its therapeutic applications, and therefore a targeted delivery system is highly needed for cabazitaxel. Our results demonstrate the importance of active targeting in targeted prostate cancer therapy. Encapsulating cabazitaxel in the micelle increases its activity and is expected to reduce its systemic toxicity, which is a major hurdle in its clinical applications. Moreover, the polymeric micelle may servers as a promising nanoscale platform for the targeted delivery of other chemotherapeutic agents to prostate cancer.

Clinical application of immune checkpoints in targeted immunotherapy of prostate cancer.

Immunotherapy is considered as an effective method for cancer treatment owing to the induction of specific and long-lasting anti-cancer effects. Immunotherapeutic strategies have shown significant success in human malignancies, particularly in prostate cancer (PCa), a major global health issue regarding its high metastatic rates. In fact, the first cancer vaccine approved by FDA was Provenge, which has been successfully used for treatment of PCa. Despite the remarkable success of cancer immunotherapy in PCa, many of the developed immunotherapy methods show poor therapeutic outcomes. Immunosuppression in tumor microenvironment (TME) induced by non-functional T cells (CD4+ and CD8+), tolerogenic dendritic cells (DCs), and regulatory T cells, has been reported to be the main obstacle to the effectiveness of anti-tumor immune responses induced by an immunotherapy method. The present review particularly focuses on the latest findings of the immune checkpoints (ICPs), including CTLA-4, PD-1, PD-L1, LAG-3, OX40, B7-H3, 4-1BB, VISTA, TIM-3, and ICOS; these checkpoints are able to have immune modulatory effects on the TME of PCa. This paper further discusses different approaches in ICPs targeting therapy and summarizes the latest advances in the clinical application of ICP-targeted therapy as monotherapy or in combination with other cancer therapy modalities in PCa.

Modulation of CXC-motif chemokine receptor 7, but not 4, expression is related to migration of the human prostate cancer cell LNCaP: regulation by androgen and inflammatory stimuli.

To elucidate the regulation, function of the chemokine CXC-motif ligand 12 (CXCL12) and its receptors (CXCR) 4 and 7 in prostate cancer tumor microenvironment. In-silico-analysis of expression in prostate cancer tissues. In-vitro comparison, testing of regulation in human prostate cancer cells LNCaP, DU145, and PC3. Dihydrotestosterone (DHT) treatments (0-10nM) were for 0-48h. The inflammatory agent Flagellin treatment (20ng/ml) was for 2h. Migration assays were performed for 24h using 10ng/ml CXCL12. Real-time PCR, western analysis, and migration assays were used to determine mRNA, protein, and functional changes, respectively. Malignant prostate cancer tissues exhibit higher CXCR4/7 mRNA ratio, and higher CXCR7 mRNA levels were detected in the androgen-responsive LNCaP cells. Putative androgen-responsive elements were identified in CXCR4, 7 gene, and exposure to DHT, flagellin increased CXCR4 mRNA but decreased CXCR7 mRNA levels in LNCaP cells. Androgen receptor siRNA significantly attenuated the effects of DHT on CXCR4, 7 mRNA in LNCaP cells. However, DHT and flagellin only decrease CXCR7 protein and additively increased migration of LNCaP cells towards CXCL12. Down regulation of CXCR7 protein by DHT and flagellin increased migration, supporting CXCR7 as decoy receptor counteracting CXCL12/CXCR4-mediated migration in prostate cancer cells.

Abstract 2748: The role of TMPRSS2_ERG fusions in modulating tumor microenvironment in prostate cancer

Abstract The control region of TMPRSS2 fuses with ETS transcription factor family members, particularly ERG. This occurs in ~20-50% of human prostate cancers depending on the ethnicity. TMPRSS2-ERG (T2E) fusions, however, do not display a notable phenotype in genetically engineered mouse models (GEMM) recapitulating the fusion expression and T2E also has no prognostic significance in humans, despite its high incidence. Wnt signaling has been implicated in prostate cancer. Here, we report a discovery of a role for T2E fusion that impacts the prostate stroma in pre-clinical models. The composition of stromal cells within the microenvironment in T2E mice was studied by single cell RNAseq analysis. We identified stromal cell clusters in T2E mice that differed from wild type mice in the expression of Pdgfrβ, Pdgfrα and Col1a1. Interestingly, these clusters showed an increase of expression of Wnt receptors such as Lgr5 and Fzd7 as well as Wnt ligands such as Sfrp2, Wnt2 and Wnt6. T2E GEMMs also exhibited upregulation of the Wnt-secretion regulator porcupine (PORCN-Protein-serine O-palmitoleoyltransferase) in the stroma adjacent to the T2E-expression prostate epithelial cells. Furthermore, lineage-tracing with the Wnt reporter and epithelial stem cell marker Lgr5 showed that Wnt-active cells were increased in prostatic intraepithelial neoplasia (PIN) lesions in T2E;Pten+/- mice. Strikingly, Lgr5+ cells were also found in the prostate stroma surrounding these PINs. Since, TMPRSS2/ERG fusions represent an early event in prostate tumorigenesis, here we provide a mechanism whereby induction of Wnt signaling in the stroma by T2E-expressing prostate epithelial cells increases the stem cell compartments in both epithelial and stromal cells. These data suggest that aberrant ERG-expressing prostate epithelial cells activate upregulation of Wnt signaling in stromal cells, which provides a possible route for enhancing prostate carcinogenesis. Citation Format: Hubert Pakula, Caroline F. Ribeiro, Giuseppe N. Fanelli, Rory Kirchner, Sudeepa Syamala, Basudev Chowdhury, Giorgia Zadra, Paolo Chetta, Zhe Li, Massimo Loda. The role of TMPRSS2_ERG fusions in modulating tumor microenvironment in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2748.

Abstract 1913: Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment

Abstract Cancer is a heterogeneous collection of neoplastic cells, surrounded by non-malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross-talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can inhibit cancer growth and proliferation during the initial stages of carcinogenesis; however, cancer cells can evade these inhibitory signals, and reshape the TME, making it more amenable to their own growth. This gives rise to cancer-associated stromal cells, including cancer-associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial-mesenchymal transition. Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan (HSPG) that has both pro-mitogenic and pro-metastatic activity. GPC1’s role in regulating fibroblasts and their transformation into CAFs has not been investigated. We studied the ability of prostate cancer cells to alter the TME in vitro by exposing human bone fibroblasts (HS-5 cells) to tumor conditioned media (TCM) isolated from a human neuroendocrine (DU-145) and bone (PC-3) metastatic prostate cancer cell lines and assessed changes in cell morphology and cytotoxicity (using MTT). Exposure of HS-5 cells to TCM from DU-145 and PC-3 cells altered cellular morphology to a more mesenchymal phenotype similar to that seen in active myofibroblasts. TCM also decreased MTT staining in a time-dependent manner. We also determined the effect of GPC1 knockdown (KD) in HS-5 cells on cell morphology, migration and cytotoxicity. Interestingly, we saw phenotypic switching of GPC1 KD HS-5 cells to a myofibroblast like morphology akin to that when treated with TCM. There was no change in cytotoxicity in GPC1 KD HS-5 cells as compared to control cells. However, GPC1 KD enhanced the migration of HS-5 cells in wound healing assay. Migration was also increased in GPC1 KD HS-5 cells upon treatment with TCM. GPC1 KD also increased HS-5 cell contractility significantly. Collectively, these data support the hypothesis that GPC1 plays an inhibitory role in the TME of prostate cancer by regulating the activation of fibroblasts. Citation Format: Sukhneeraj P. Kaur, Hee K. Lee, Robert D. Arnold, Brian S. Cummings. Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1913.

Exosome-derived miR-27a produced by PSC-27 cells contributes to prostate cancer chemoresistance through p53

Prostate cancer (PCa) is a major malignancy in men. Hitherto that date, surgical or chemical castration is the standard treatment to PCa. Nevertheless, there are still many patients with initial treatment progress to metastatic castration-resistant prostate cancer (mCRPC). There are many effective chemotherapeutic drugs for mCRPC, but the tumors will be resistant to these chemotherapeutic drugs, which is an urgent problem to be solved. Specifically, tumor therapy resistance driven by the pathologically active host stroma has gradually becoming the spotlight of oncotherapy in recent years. The exosome-derived miR-27a plays an important role in PCa cell chemoresistance. However, the functions of miR-27a on PCa developing chemoresistance remain unknown. In the present study, we aimed to construct potential regulatory networks of exosomal miR-27a in PCa chemoresistance. The expression of miR-27a was significantly increased by treatment with cisplatin, doxorubicin (DOX) and docetaxel in PCa tissues. We next co-cultured PCa cells (PC3 cells) with primary prostate fibroblasts (PSC27 cells) to explore the mechanisms of tumor therapy resistance. Further studies delineate that exosome-derived miR-27a produced by PSC-27 cells improved chemoresistance by restraining the expression of P53 gene. Our studies provide a new direction for exploring the effects of PCa tumor microenvironment of chemoresistance.

NT5E expression and the immune landscape of prostate cancer (PC): An analysis from The Cancer Genome Atlas database.

e16591 Background: Immune checkpoint inhibitors targeting PD-1/L1 and CTLA-4 pathway have shown modest activity in patients with advanced PC. Additional immunosuppressive mechanisms in the PC tumor microenvironment need to be investigated. Increased CD73 (encoded by NT5E) expression results in generation of immunosuppressive adenosine in the tumor microenvironment and has been associated with metastasis and poor survival in PC. Utilizing the TCGA dataset, we investigated the association of NT5E expression with the immune landscape of PC. Methods: RNA-seq data for 331 PC tumor samples and 51 normal adjacent tissue (NAT) samples was downloaded and log2 transformed. Patients were split into low, intermediate, and high expression groups based on NT5E expression (≤ -1, -1 to 1 and ≥1 standard deviation from the overall mean) in tumor and NAT. A tumor inflammation signature (TIS) reflecting an inflamed tumor phenotype was calculated based on the averaged tumor expression of 18 previously validated genes (Ayers et al, 2017). Abundance of infiltrating immune cell subsets was estimated based on expression of previously identified 782 immune metagenes (Charoentong et al, 2017). Immune cell abundance scores and TIS were compared between NT5E expression groups using the Mann-Whitney U test and the Bonferroni correction was used to control for false discovery rate. Results: NT5E expression in NAT was not associated with the TIS or expression of immune cell marker genes. In contrast, NT5E expression in tumor tissue correlated positively with TIS (P < 0.001). Compared to tumors with low NT5E expression, those in high NT5E expression group had higher expression of central memory CD4+, effector memory CD8+, type 1 helper, NK and regulatory T (Treg) cell markers. Conclusions: In our analysis, NT5E expression correlated with markers of inflamed tumor phenotype in PC. Although NT5E expression was associated with higher CD8+and CD4+ T cells, concurrent increase in Tregs could inhibit the infiltrating lymphocytes and promote tumor growth. Our findings indicate a possible role for the adenosine pathway as a mediator of immunosuppression in PC and a potential therapeutic target. AT and EL: Equal contribution

Preparation and Evaluation of ZD2 Peptide 64Cu-DOTA Conjugate as a Positron Emission Tomography Probe for Detection and Characterization of Prostate Cancer.

Positron emission tomography (PET) is a sensitive modality for cancer molecular imaging. We aim to develop a PET probe for sensitive detection and risk stratification of prostate cancer by targeting an abundant microenvironment oncoprotein, extradomain-B fibronectin (EDB-FN). The probe consists of a small ZD2 peptide specific to EDB-FN and a 64Cu-DOTA chelate. The probe was synthesized using standard solid-phase peptide chemistry and chelated to 64Cu prior to imaging. PET images were acquired at 4 and 22 h after intravenously injecting a 200 μCi probe into mice bearing human PC3 and LNCaP tumors, which represent highly aggressive and slow-growing prostate tumors, respectively. At 4 and 22 h postinjection, tumors could be clearly identified in the PET images. A significant higher signal was observed in PC3 tumors than in LNCaP tumors at 22 h (p = 0.01). Probe accumulation was also higher in PC3 tumors at 24 h. These data demonstrated that PET molecular imaging of EDB-FN in the tumor microenvironment of prostate cancer allows efficient differentiation of PC3 and LNCaP tumors in vivo. The ZD2 peptide-targeted PET probe shows potential in the detection and characterization of high-risk prostate cancer to improve the clinical management of prostate cancer.

The Importance of Time to Prostate-Specific Antigen (PSA) Nadir after Primary Androgen Deprivation Therapy in Hormone-Naïve Prostate Cancer Patients.

Prostate-specific antigen (PSA) is currently the most useful biomarker for detection of prostate cancer (PCa). The ability to measure serum PSA levels has affected all aspects of PCa management over the past two decades. The standard initial systemic therapy for advanced PCa is androgen-deprivation therapy (ADT). Although PCa patients with metastatic disease initially respond well to ADT, they often progress to castration-resistant prostate cancer (CRPC), which has a high mortality rate. We have demonstrated that time to PSA nadir (TTN) after primary ADT is an important early predictor of overall survival and progression-free survival for advanced PCa patients. In in vivo experiments, we demonstrated that the presence of fibroblasts in the PCa tumor microenvironment can prolong the period for serum PSA decline after ADT, and enhance the efficacy of ADT. Clarification of the mechanisms that affect TTN after ADT could be useful to guide selection of optimal PCa treatment strategies. In this review, we discuss recent in vitro and in vivo findings concerning the involvement of stromal–epithelial interactions in the biological mechanism of TTN after ADT to support the novel concept of “tumor regulating fibroblasts”.

The progress of study on the targeted immunotherapy based on XAGE-1b antigen in prostate cancer

Prostate cancer is one of the most common malignancies in men, which threats to men’s healthseriously. Its development is a complex process of multi-gene interaction and interaction, the immune response plays an important role in the whole process. With the development of immunology and tumor microenvironmentinprostate cancer, the targeted immunotherapy isconsidered the most promising treatment for prostate cancer. XAGE-1b antigen is a prostate cancer-associated antigen with strong immunogenicity. A large number of studies have shown that it is closely related to the occurrence and progress of prostate cancer, many researchers believe that XAGE-1b antigen isa potential target forimmunotherapy in advanced prostate cancer. Inthis review, we perform a review of the research progress on the targeted immunotherapy based on XAGE-1b antigen in prostate cancer. Key words: XAGE-1b; Prostate Cancer; Cancer-testis antigen; Targeted immunotherapy

Tumor-infiltrating mesenchymal stem cells: Drivers of the immunosuppressive tumor microenvironment in prostate cancer?

Prostate cancer is characterized by T-cell exclusion, which is consistent with their poor responses to immunotherapy. In addition, T-cells restricted to the adjacent stroma and benign areas are characterized by anergic and immunosuppressive phenotypes. In order for immunotherapies to produce robust anti-tumor responses in prostate cancer, this exclusion barrier and immunosuppressive microenvironment must first be overcome. We have previously identified mesenchymal stem cells (MSCs) in primary and metastatic human prostate cancer tissue. An Opal Multiplex immunofluorescence assay based on CD73, CD90, and CD105 staining was used to identify triple-labeled MSCs in human prostate cancer tissue. T-cell suppression assays and flow cytometry were used to demonstrate the immunosuppressive potential of primary MSCs expanded from human bone marrow and prostate cancer tissue from independent donors. Endogenous MSCs were confirmed to be present at sites of human prostate cancer. These prostate cancer-infiltrating MSCs suppress T-cell proliferation in a dose-dependent manner similar to their bone marrow-derived counterparts. Also similar to bone marrow-derived MSCs, prostate cancer-infiltrating MSCs upregulate expression of PD-L1 and PD-L2 on their cell surface in the presence of IFNγ and TNFα. Prostate cancer-infiltrating MSCs suppress T-cell proliferation similar to canonical bone marrow-derived MSCs, which have well-documented immunosuppressive properties with numerous effects on both innate and adaptive immune system function. Thus, we hypothesize that selective depletion of MSCs infiltrating sites of prostate cancer should restore immunologic recognition and elimination of malignant cells via broad re-activation of cytotoxic pro-inflammatory pathways.

Integrative metabolic and transcriptomic profiling of prostate cancer tissue containing reactive stroma

Reactive stroma is a tissue feature commonly observed in the tumor microenvironment of prostate cancer and has previously been associated with more aggressive tumors. The aim of this study was to detect differentially expressed genes and metabolites according to reactive stroma content measured on the exact same prostate cancer tissue sample. Reactive stroma was evaluated using histopathology from 108 fresh frozen prostate cancer samples gathered from 43 patients after prostatectomy (Biobank1). A subset of the samples was analyzed both for metabolic (n = 85) and transcriptomic alterations (n = 78) using high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) and RNA microarray, respectively. Recurrence-free survival was assessed in patients with clinical follow-up of minimum five years (n = 38) using biochemical recurrence (BCR) as endpoint. Multivariate metabolomics and gene expression analysis compared low (≤15%) against high reactive stroma content (≥16%). High reactive stroma content was associated with BCR in prostate cancer patients even when accounting for the influence of Grade Group (Cox hazard proportional analysis, p = 0.013). In samples with high reactive stroma content, metabolites and genes linked to immune functions and extracellular matrix (ECM) remodeling were significantly upregulated. Future validation of these findings is important to reveal novel biomarkers and drug targets connected to immune mechanisms and ECM in prostate cancer. The fact that high reactive stroma grading is connected to BCR adds further support for the clinical integration of this histopathological evaluation.

Immunotherapy for Prostate Cancer.

Immunotherapy with agents that block immune checkpoints is a mainstay of therapy for several common tumor types; so far, prostate cancer is not among those treated using this method. The observed lack of activity in prostate cancer is not due to a lack of testing; several agents have been evaluated both alone and in combination. Although several combination strategies show some promise, it appears likely that a greater understanding of the prostate cancer tumor microenvironment and baseline immune response will be required to optimize future treatment strategies.

2025 Targeting immunosuppressive myeloid cells to enhance cancer immunotherapy

OBJECTIVES/SPECIFIC AIMS: Prostate cancer (PCa) is the most common noncutaneous malignancy in men in the United States. A significant fraction of advanced PCa treated with androgen deprivation therapy experience relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). The PCa tumor microenvironment is comprised of a complex mixture of epithelial and stroma cell types engaged in multifaceted heterotypic interactions functioning to maintain tumor growth and immune evasion. We recently uncovered the important role played by myeloid-derived suppressor cells (MDSCs) to mediate tumor immune evasion in aggressive PCa (Wang, Lu et al., Cancer Discovery, 2016). Immune checkpoint blockade (ICB) has elicited durable therapeutic responses across a number of cancer types. However, the impact of ICB on mCRPC has been disappointing, which may signal the need to combine mechanistically-distinct ICB agents and/or override immunosuppression in the tumor microenvironment. Our objective is to determine if robust immunotherapy responses in mCRPC may be elicited by the combined actions of ICB agents together with targeted agents that neutralize MDSCs yet preserve T cell function. METHODS/STUDY POPULATION: We created a novel embryonic stem cell-based chimeric mouse model of mCRPC engineered with signature mutations to study the response to single and combination immunotherapy. The efficacy studies were followed with detailed mechanistic investigation and clinical validation. RESULTS/ANTICIPATED RESULTS: Consonant with early stage clinical trials experience, anti-CTLA4 or anti-PD1 monotherapy failed to impact disease progression. Similarly, modest anti-tumor activity was observed with combination ICB as well as monotherapy with targeted agents including Cabozantinib (tyrosine kinase inhibitor), Dactolisib (PI3K/mTOR inhibitor), and Dasatinib (tyrosine kinase inhibitor). In contrast, mCRPC primary and metastatic disease showed robust responses to dual ICB treatment together with either Cabozantinib or Dactolisib, but not with Dasatinib which impaired T cell infiltration in the tumor. Detailed intratumoral immune profiling with mass cytometry (CyTOF) showed that combined ICB and Cabozantinib or Dactolisib was associated with significant depletion of MDSCs. Cabozantinib and Dactolisib blocked the PI3K signaling activity in MDSCs and reduced their immunosupppresive activity. Mechanistically, the combination efficacy was due to the upregulation of IL-1RA and suppression of MDSC-promoting cytokines secreted by mCRPC cells. DISCUSSION/SIGNIFICANCE OF IMPACT: We demonstrated that an antibody cocktail targeting CTLA4 and PD1 was insufficient to generate effective anti-tumor response, but combination of ICB with targeted therapy that inactivates PI3K signaling displayed superior synergistic efficacy through impairing MDSCs in the tumor microenvironment. These observations illuminate a clinical path hypothesis for combining ICB with MDSC-targeted therapies in the treatment of mCRPC. Importantly, conclusions from the study on PCa may have implications in combination immunotherapy for aggressive breast cancer which is also largely resistant to immune checkpoint blockade and replete with immunosuppressive myeloid cells.

Cell-type specific expression of oncogenic and tumor suppressive microRNAs in the human prostate and prostate cancer

MiR-1 and miR-143 are frequently reduced in human prostate cancer (PCa), while miR-141 and miR-21 are frequently elevated. Consequently, these miRNAs have been studied as cell-autonomous tumor suppressors and oncogenes. However, the cell-type specificity of these miRNAs is not well defined in prostate tissue. Through two different microdissection techniques, and droplet digital RT-PCR, we quantified these miRNAs in the stroma and epithelium of radical prostatectomy specimens. In contrast to their purported roles as cell-autonomous tumor suppressors, we found miR-1 and miR-143 expression to be predominantly stromal. Conversely, miR-141 was predominantly epithelial. miR-21 was detected in both stroma and epithelium. Strikingly, the levels of miR-1 and miR-143 were significantly reduced in tumor-associated stroma, but not tumor epithelium. Gene expression analyses in human cell lines, tissues, and prostate-derived stromal cultures support the cell-type selective expression of miR-1, miR-141, and miR-143. Analyses of the PCa Genome Atlas (TCGA-PRAD) showed a strong positive correlation between stromal markers and miR-1 and miR-143, and a strong negative correlation between stromal markers and miR-141. In these tumors, loss of miR-1 and gain of miR-21 was highly associated with biochemical recurrence. These data shed new light on stromal and epithelial miRNA expression in the PCa tumor microenvironment.

Immune mediators in the tumor microenvironment of prostate cancer

Immune mediators in the tumor microenvironment of prostate cancer

Prostate cancer immunotherapy: the path forward.

To provide an overview of current strategies being investigated in the development of immunotherapy in prostate cancer. Development of immunotherapy in prostate cancer actually began in 2010 with FDA approval of sipuleucel-T. Given that immune checkpoint inhibitor trials have either been negative at the phase III level or underwhelming in smaller studies, it is likely that combination strategies will be required to further maximize the impact immune-based therapies on the clinical course of the disease. Emerging data suggests the presence of multiple checkpoint inhibitors in the prostate cancer tumor microenvironment highlighting the need for combination immunotherapy platforms that would potentially include androgen deprivation, chemotherapy, or radiation. Preclinical and clinical data support immune-based combinations in prostate cancer and several trials are underway to better define the future of immunotherapy in prostate cancer.

Pathology-Driven Comprehensive Proteomic Profiling of the Prostate Cancer Tumor Microenvironment

Prostate cancer is the second most common cancer in men worldwide. Gleason grading is an important predictor of prostate cancer outcomes and is influential in determining patient treatment options. Clinical decisions based on a Gleason score of 7 are difficult as the prognosis for individuals diagnosed with Gleason 4+3 cancer is much worse than for those diagnosed with Gleason 3+4 cancer. Laser capture microdissection (LCM) is a highly precise method to isolate specific cell populations or discrete microregions from tissues. This report undertook a detailed molecular characterization of the tumor microenvironment in prostate cancer to define the proteome in the epithelial and stromal regions from tumor foci of Gleason grades 3 and 4. Tissue regions of interest were isolated from several Gleason 3+3 and Gleason 4+4 tumors using telepathology to leverage specialized pathology expertise to support LCM. Over 2,000 proteins were identified following liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of all regions of interest. Statistical analysis revealed significant differences in protein expression (>100 proteins) between Gleason 3 and Gleason 4 regions-in both stromal and epithelial compartments. A subset of these proteins has had prior strong association with prostate cancer, thereby providing evidence for the authenticity of the approach. Finally, validation of these proteins by immunohistochemistry has been obtained using an independent cohort of prostate cancer tumor specimens.Implications: This unbiased strategy provides a strong foundation for the development of biomarker protein panels with significant diagnostic and prognostic potential. Mol Cancer Res; 15(3); 281-93. ©2017 AACR.