The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment.

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment.

In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories "cell adhesion" and the "extracellular matrix." The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the "spliceosome" and "actin binding." STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domain-containing receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme that promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blotting analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Further, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.

Characterization of the ERG-regulated Kinome in Prostate Cancer Identifies TNIK as a Potential Therapeutic Target.

Approximately 50% of prostate cancers harbor the TMPRSS2:ERG fusion, resulting in elevated expression of the ERG transcription factor. Despite the identification of this subclass of prostate cancers, no personalized therapeutic strategies have achieved clinical implementation. Kinases are attractive therapeutic targets as signaling networks are commonly perturbed in cancers. The impact of elevated ERG expression on kinase signaling networks in prostate cancer has not been investigated. Resolution of this issue may identify novel therapeutic approaches for ERG-positive prostate cancers. In this study, we used quantitative mass spectrometry-based kinomic profiling to identify ERG-mediated changes to cellular signaling networks. We identified 76 kinases that were differentially expressed and/or phosphorylated in DU145 cells engineered to express ERG. In particular, the Traf2 and Nck-interacting kinase (TNIK) was markedly upregulated and phosphorylated on multiple sites upon ERG overexpression. Importantly, TNIK has not previously been implicated in prostate cancer. To validate the clinical relevance of these findings, we characterized expression of TNIK and TNIK phosphorylated at serine 764 (pS764) in a localized prostate cancer patient cohort and showed that nuclear enrichment of TNIK (pS764) was significantly positively correlated with ERG expression. Moreover, TNIK protein levels were dependent upon ERG expression in VCaP cells and primary cells established from a prostate cancer patient-derived xenograft. Furthermore, reduction of TNIK expression and activity by silencing TNIK expression or using the TNIK inhibitor NCB-0846 reduced cell viability, colony formation and anchorage independent growth. Therefore, TNIK represents a novel and actionable therapeutic target for ERG-positive prostate cancers that could be exploited to develop new treatments for these patients.

Tissue engineered human prostate microtissues reveal key role of mast cell-derived tryptase in potentiating cancer-associated fibroblast (CAF)-induced morphometric transition in vitro.

The tumour microenvironment plays a vital role in the development of solid malignancies. Here we describe an in vitro human prostate cancer microtissue model that facilitates the incorporation and interrogation of key elements of the local prostatic tumour microenvironment. Primary patient-derived cancer-associated fibroblasts (CAFs) were cultured in three-dimensional (3D) melt electrowritten scaffolds where they deposited extensive extracellular matrix (ECM) and promoted significant changes in prostate epithelial morphology, when compared to matched non-malignant prostatic fibroblasts (NPFs). The addition of mast cells, a resident prostatic immune population that is expanded during early malignancy, enhanced the morphometric transition of benign epithelia via a tryptase-mediated mechanism. Our patient-specific 3D microtissues reveal a cascade of interactions between prostatic CAFs, their native ECM and mast cell-derived tryptase, rendering them important microenvironmental drivers of prostate cancer progression.

Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus.

Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration.