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How prostate cancer cells and their precursors mediate changes in the tumor microenvironment (TME) to drive prostate cancer progression is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we perform extensive single-cell RNA-sequencing (scRNA-seq) and molecular pathology of the comparative biology between human prostate cancer and key stages in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues reveal that cancer cell-intrinsic activation of MYC signaling is a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Cell communication network and pathway analyses in GEMMs show that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogram the TME during carcinogenesis, leading to a convergence of cell state alterations in neighboring epithelial, immune, and fibroblast cell types that parallel key findings in human prostate cancer., (© 2024. The Author(s).)

α-particle emitters are emerging as a potent modality for disseminated cancer therapy because of their high linear energy transfer and localized absorbed dose profile. Despite great interest and pharmaceutical development, there is scant information on the distribution of these agents at the scale of the α-particle pathlength. We sought to determine the distribution of clinically approved [ 223 Ra]RaCl 2 in bone metastatic castration-resistant prostate cancer at this resolution, for the first time to our knowledge, to inform activity distribution and dose at the near-cell scale. Methods: Biopsy specimens and blood were collected from 7 patients 24 h after administration. 223 Ra activity in each sample was recorded, and the microstructure of biopsy specimens was analyzed by micro-CT. Quantitative autoradiography and histopathology were segmented and registered with an automated procedure. Activity distributions by tissue compartment and dosimetry calculations based on the MIRD formalism were performed. Results: We revealed the activity distribution differences across and within patient samples at the macro- and microscopic scales. Microdistribution analysis confirmed localized high-activity regions in a background of low-activity tissue. We evaluated heterogeneous α-particle emission distribution concentrated at bone-tissue interfaces and calculated spatially nonuniform absorbed-dose profiles. Conclusion: Primary patient data of radiopharmaceutical therapy distribution at the small scale revealed that 223 Ra uptake is nonuniform. Dose estimates present both opportunities and challenges to enhance patient outcomes and are a first step toward personalized treatment approaches and improved understanding of α-particle radiopharmaceutical therapies., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Prostate cancer (PCa) is the second most common cancer and constitutes about 14.7% of total cancer cases. PCa is highly prevalent and more aggressive in African-American (AA) men than in European-American (EA) men. PCa tends to be highly heterogeneous, and its complex biology is not fully understood. We use metabolomics to better understand the mechanisms behind PCa progression and disparities in its clinical outcome. Adenosine deaminase (ADA) is a key enzyme in the purine metabolic pathway; it was found to be upregulated in PCa and is associated with higher-grade PCa and poor disease-free survival. The inosine-to-adenosine ratio, which is a surrogate for ADA activity was high in PCa patient urine and higher in AA PCa compared to EA PCa. To understand the significance of high ADA in PCa, we established ADA overexpression models and performed various in vitro and in vivo studies. Our studies have revealed that an acute increase in ADA expression during later stages of tumor development enhances in vivo growth in multiple pre-clinical models. Further analysis revealed that mTOR signaling activation could be associated with this tumor growth. Chronic ADA overexpression shows alterations in the cells' adhesion machinery and a decrease in cells' ability to adhere to the extracellular matrix in vitro . Losing cell-matrix interaction is critical for metastatic dissemination which suggests that ADA could potentially be involved in promoting metastasis. This is supported by the association of higher ADA expression with higher-grade tumors and poor patient survival. Overall, our findings suggest that increased ADA expression may promote PCa progression, specifically tumor growth and metastatic dissemination., Competing Interests: None., (AJCEU Copyright © 2023.)

The biological influence of physicochemical parameters of "targeted" nanoparticles on their delivery to cancer tumors remains poorly understood. A comparative analysis of nanoparticle distributions in tumors following systemic delivery across several models can provide valuable insights. Methods: Bionized nanoferrite nanoparticles (iron oxide core coated with starch), either conjugated with a targeted anti-HER2 antibody (BH), or unconjugated (BP), were intravenously injected into athymic nude or NOD-scid gamma (NSG) female mice bearing one of five human breast cancer tumor xenografts growing in a mammary fat pad. Tumors were harvested 24 hours after nanoparticle injection, fixed, mounted, and stained. We performed detailed histopathology analysis by comparing spatial distributions of nanoparticles (Prussian blue) with various stromal cells (CD31, SMA, F4/80, CD11c, etc.) and the target antigen-expressing (HER2) tumor cells. Results: Only BH nanoparticles were retained in tumors and generally concentrated in the tumor periphery, with nanoparticle content diminishing towards the tumor interior. Nanoparticle distribution correlated strongly with specific stromal cells within each tumor type, which varied among tumor types and between mouse strains. Weak or no correlation between nanoparticle distribution and HER2 positive cells, or CD31 cells was observed. Conclusion: Antibody-labeled nanoparticles were retained across all tumors, irrespective of presence of the "target" antigen. Though presence of antibody on nanoparticles correlated with retention, non-cancerous host stromal cells were responsible for their retention in the tumor microenvironment. This study highlights gaps in our understanding of the complex biological interplay between disease and host immune biology, and the need to account for the influence of underlying aberrant tumor biology as factors determining nanoparticle fate in vivo., Competing Interests: Competing Interests: R.I. is an inventor listed on several nanoparticle patents. All patents are assigned to either The Johns Hopkins University or Aduro Biosciences, Inc. R.I. is a member of the Scientific Advisory Board of Imagion Biosystems. All other authors report no other competing interests., (© The author(s).)

Background: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood., Methods: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types., Results: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate., Conclusions: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes., (© 2022 Wiley Periodicals LLC.)

The growing interest and clinical translation of alpha particle (α) therapies brings with it new challenges to assess target cell engagement and to monitor therapeutic effect. Noninvasive imaging has great potential to guide α-treatment and to harness the potential of these agents in the complex environment of disseminated disease. Poly(ADP) ribose polymerase 1 (PARP-1) is among the most abundantly expressed DNA repair enzymes with key roles in multiple repair pathways-such as those induced by irradiation. Here, we used a third-generation PARP1-specific radiotracer, [ 18 F]-PARPZ, to delineate castrate resistant prostate cancer xenografts. Following treatment with the clinically applied [ 225 Ac]-PSMA-617, positron emission tomography was performed and correlative autoradiography and histology acquired. [ 18 F]-PARPZ was able to distinguish treated from control (saline) xenografts by increased uptake. Kinetic analysis of tracer accumulation also suggests that the localization of the agent to sites of increased PARP-1 expression is a consequence of DNA damage response. Together, these data support expanded investigation of [ 18 F]-PARPZ to facilitate clinical translation in the ⍺-therapy space., (© 2022. The Author(s).)

Digital autoradiography (DAR) is a powerful tool to quantitatively determine the distribution of a radiopharmaceutical within a tissue section and is widely used in drug discovery and development. However, the low image resolution and significant background noise can result in poor correlation, even errors, between radiotracer distribution, anatomic structure, and molecular expression profiles. Differing from conventional optical systems, the point-spread function in DAR is determined by properties of radioisotope decay, phosphor, and digitizer. Calibration of an experimental point-spread function a priori is difficult, prone to error, and impractical. We have developed a content-adaptive restoration algorithm to address these problems. Methods: We model the DAR imaging process using a mixed Poisson-gaussian model and blindly restore the image by a penalized maximum-likelihood expectation-maximization algorithm (PG-PEM). PG-PEM implements a patch-based estimation algorithm with density-based spatial clustering of applications with noise to estimate noise parameters and uses L2 and Hessian Frebonius norms as regularization functions to improve performance. Results: First, PG-PEM outperformed other restoration algorithms at the denoising task ( P < 0.01). Next, we implemented PG-PEM on preclinical DAR images ( 18 F-FDG, treated mouse tumor and heart; 18 F-NaF, treated mouse femur) and clinical DAR images (bone biopsy sections from 223 RaCl 2 -treated castration-resistant prostate cancer patients). DAR images restored by PG-PEM of all samples achieved a significantly higher effective resolution and contrast-to-noise ratio and a lower SD of background ( P < 0.0001). Additionally, by comparing the registration results between the clinical DAR images and the segmented bone masks from the corresponding histologic images, we found that the radiopharmaceutical distribution was significantly improved ( P < 0.0001). Conclusion: PG-PEM is able to increase resolution and contrast while robustly accounting for DAR noise and demonstrates the capacity to be widely implemented to improve preclinical and clinical DAR imaging of radiopharmaceutical distribution., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-RasG12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid-Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Introduction: Percutaneous fetoscopic surgery is hampered by an increased risk of preterm prelabor rupture of membranes (PPROM). Recent surgical techniques have shown that suturing the chorioamniotic membranes following laparotomy and uterine exteriorization is associated with a lower risk of PPROM compared to percutaneous in utero surgery. This study presents the ChorioAnchor, a novel resorbable device that percutaneously anchors the chorioamniotic membranes to the uterine wall., Methods: Human factors testing and peel tests were used to simulate the worst-case in-use loading conditions, establishing the device strength requirements. Tensile testing was used to measure the time-zero strength of the device. Porcine cadaver testing was used to examine ultrasound visibility and acute handling characteristics. Short-term host response was examined through an acute 7-day implantation study in a rabbit model., Results: With a time-zero tensile strength of 47 N, the ChorioAnchor exceeded the established 4 N strength requirement. Both the ChorioAnchor and delivery device were seen to be clearly visible under ultrasound imaging. Short-term host response to the device was well within the range expected for this type of device., Conclusion: The ChorioAnchor meets its engineering requirements in the early stages of implantation. Future studies will examine the kinetics of degradation of the device in vitro and in vivo., (The Author(s). Published by S. Karger AG, Basel.)

PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer., (©2020 American Association for Cancer Research.)

Background: Liver metastasis is not uncommon in men with metastatic castration-resistant prostate cancer (mCRPC), estimated at ~20% to 60% of advanced late-stage patients. Liver and other visceral metastases are associated with worse overall survival. Recent evidence suggests the frequency of visceral metastases may be increasing for reasons that are unclear but may be related to selective pressures induced by modern therapies, including second-generation antiandrogen receptor signaling inhibitors such as enzalutamide and abiraterone. Consequently, robust models to study the pathobiology of prostate cancer liver metastases and their response to therapy are urgently needed., Methods: Hemi-spleen injection of human (LN95, PC3, VCaP, and MDA-PCa-2b) or syngeneic (Myc-CaP) prostate cancer cells (1 × 10 6 ) was performed to seed liver metastases via the splenic vessels. Plasma levels of prostate-specific antigen (PSA) were monitored longitudinally in human androgen receptor-positive (AR+) models. Immunohistochemical staining of AR and HoxB13 was performed to document the prostatic origin of hepatic lesions., Results: LN95, PC3, and Myc-CaP produced distinct liver micrometastases that progressed to macrometastases by ~2 to 4 weeks postinoculation, while inoculation of MDA-PCa-2b and VCaP only produced occasional micrometastases and seeding of individual cells adjacent to blood vessels, respectively, at the time points analyzed. All lesions are characterized by positive staining for nuclear AR and/or the prostate-specific differentiation marker HoxB13 depending on the model. Circulating PSA levels are strongly correlated with overall tumor burden in mice seeded with LN95. Histologic micrometastases and low levels of circulating PSA are detected in mice seeded with MDA-PCa-2b at ~60 days postinoculation, but no circulating PSA was detected in animals inoculated with VCaP up to ~75 days despite the presence of rare AR+ cells in the liver., Conclusion: The studies reported herein establish intrasplenic injection as a robust model of mCRPC liver metastasis. In addition, circulating PSA was validated as a noninvasive biomarker to longitudinally monitor overall tumor burden when using PSA+ models. Therefore, this model can be used to interrogate the pathophysiology of prostate cancer liver metastases, the microenvironmental factors permissive to such growth, immunologic variables, and the response of hepatic lesions to therapy., (© 2020 Wiley Periodicals LLC.)

Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion, and metastasis, but how stromal lipids influence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment. IMPLICATIONS: This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content, and inflammatory response., (©2020 American Association for Cancer Research.)

Background: C-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML., Methods: First, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student's t-tests were used and values of p<0.05 were considered significant. Survival was calculated using Mantel-Cox log-rank test., Results: In vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (p<0.0001) and had superior expansion compared with CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CLL-1 CAR T coexpressing transgenic IL15 initially expanded better than CD28z-CD8 CAR T without IL15 (p<0.0001), but produced severe acute toxicity associated with high level production of human tumor necrosis factor α (TNFα), IL15 and IL2. Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by two strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody, while excessive T cell expansion could be arrested by activation of an inducible caspase nine safety switch by administration of dimerizing drug. Both strategies successfully prolonged tumor-free survival., Conclusion: Combinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells., Competing Interests: Competing interests: HEH is a cofounder of Allovir and Marker Therapeutics, has served on advisory boards for Tessa Therapeutics, Gilead Biosciences, Novartis, Kiadis and PACT Pharma and received research support from Kuur Therapuetics and Tessa Therapuetics. MKB is a cofounder of Allovir and Marker Therapeutics, reports personal fees from Tessa Therapeutics and Allogene., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Dose optimization and pharmacokinetic evaluation of α-particle emitting radium-223 dichloride ( 223 RaCl 2 ) by planar γ-camera or single photon emission computed tomography (SPECT) imaging are hampered by the low photon abundance and injected activities. In this study, we demonstrate SPECT of 223 Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223 Ra were imaged using a dedicated small animal SPECT by detecting the low-energy photon emissions from 223 Ra. Localization of the therapeutic agent was verified by whole-body and whole-limb autoradiography and its radiobiological effect confirmed by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction at reasonable administered activities and acquisition times. Line sources of 223 Ra in both air and in a water scattering phantom gave a line spread function with a full-width-at-half-maximum of 1.45 mm. Early and late-phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling was correlated with DNA damage from the α particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223 RaCl 2 , a recently approved α-particle-emitting radionuclide. This approach allows quantitative assessment of 223 Ra distribution and may assist radiation-dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning.

Prostate cancer is a candidate for immunotherapy because cancer cells express tissue-specific proteins that can be therapeutic targets. However, immune checkpoint inhibitors and active immunization have performed poorly in clinical trials. We developed a novel virus-like particle (VLP) vaccine composed of bovine papillomavirus L1 protein engineered to display surface docking sites. We decorated VLPs with peptides encoding T cell epitopes from two prostate cancer-associated tumor antigens, prostate stem cell antigen (PSCA), and prostatic acid phosphatase (PAP-1 and PAP-2), and a neo-antigen, stimulator of prostatic adenocarcinoma-specific T cells (SPAS-1). The VLP vaccines induced a mean frequency of antigen-specific IFN-γ secreting CD8 + T cells of 2.9% to PSCA, 9.5% to SPAS-1, 0.03% to PAP-1, and 0.03% to PAP-2 in tumor-bearing TRAMP mice. We treated TRAMP mice at 19-20 weeks of age, when mice have advanced stages of carcinogenesis, with either VLP vaccine, anti-PD1 antibody, or combination immunotherapy. The VLP vaccine alone or in combination with anti-PD1 antibody significantly reduced tumor burden, while anti-PD1 antibody had a modest non-significant therapeutic effect. All treatments significantly increased CD3 + and CD8 + T cell infiltration into tumor tissue compared to control mice, and combination therapy resulted in significantly greater CD3 + and CD8 + T cell infiltration than monotherapy. Reduction in tumor burden in vaccine-treated mice was inversely correlated with CD8 + T cell numbers in tumor tissue. No other immunotherapy has shown efficacy in this animal model of advanced prostate cancer, making bovine papillomavirus VLPs an attractive vaccine technology to test in patients with metastatic prostate cancer.

The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8 + T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

We report the establishment of B6CaP, an allograft tumor line from a Hi-Myc transgenic mouse that had been backcrossed onto C57BL/6J background. This tumor line grows subcutaneously in wildtype C57BL/6J immunocompetent mice, expresses AR, and has a luminal cytokeratin profile. When digested into single cells and injected via intracardiac injection, B6CaP produces metastatic widespread metastases including frequent bone lesions. Metastatic lesions occur most often in the femur, spine, and skull, and have a mixed osteolytic/osteoblastic phenotype. B6CaP allografts are androgen dependent, and regress after castration. However, castration resistant tumors regrow after 4-6 months and can be maintained as androgen-independent clones. This is the first example of a prostate-derived tumor line that shows frequent metastasis to bone and grows in an immunocompetent host, making this model useful for studying mechanisms of bone metastasis and tumor immune response., Competing Interests: CONFLICTS OF INTEREST The authors claim no conflict of interest.

Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8 + T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression., (©2019 American Association for Cancer Research.)

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is highly overexpressed in primary and metastatic prostate cancer (PCa). This has led to the development of radiopharmaceuticals for targeted imaging and therapy under current clinical evaluation. Despite this progress, the exact biological role of the protein in prostate cancer development and progression has not been fully elucidated. This is in part because the human PSMA and mouse PSMA (mPSMA) have different patterns of anatomical expression which confound study in the most widely utilized model organisms. Most notably, mPSMA is not expressed in the healthy murine prostate. Here, we reveal that mPSMA is highly upregulated in the prostate adenocarcinoma of the spontaneous Hi-Myc mouse model, a highly accurate and well characterized mouse model of prostate cancer development. Antibody detection and molecular imaging tools are used to confirm that mPSMA is expressed from early prostatic intraepithelial neoplasia (PIN) through adenocarcinoma., (© 2019 The Authors. The Prostate Published by Wiley Periodicals, Inc.)

Pigeons ( Columba livia ) are used in biomedical research for studies of vision, cognition, neuronal pathways, and spatial orientation. Because there are few commercial laboratory sources, research pigeons are typically acquired from local fancier breeders or bred onsite. For acquired pigeons, the health and vaccine status is often unknown. A juvenile pigeon, born onsite and living in an enclosed outdoor loft, presented with small, bleeding, wart-like lesions on the medial aspects of digits 1 and 4. Topical treatment was initiated. Within a week, 4 fledglings were reported for small, dark papular lesions on the face, head, neck, and beak, and shortly thereafter, 2 additional juvenile pigeons developed similar lesions. The fledglings were euthanized, and histologic examination revealed numerous intralesional eosinophilic cytoplasmic viral inclusions (Bollinger bodies) confirming a diagnosis of poxvirus infection, likely pigeon pox. Although usually self-limiting, pigeon pox can cause moderate to severe lesions in fledgling and juvenile birds. Vaccination with a modified live poxvirus labeled for chickens was used to create herd immunity to pigeon poxvirus. Since vaccination of our entire flock and implementation of more stringent health protocols, all lesions have resolved, and no new lesions have been noted.

Purpose: Androgen receptor ( AR ) gene alterations, including ligand-binding domain mutations and copy number (CN) gain, have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration-resistant prostate cancer (mCRPC). The goal of this study was to validate AR gene alterations detected in cell-free DNA (cfDNA) as markers of enzalutamide and abiraterone resistance in patients with mCRPC., Methods: Patients with mCRPC (N = 62) were prospectively enrolled between 2014 and 2018. Blood was collected before therapies-enzalutamide (n = 25), abiraterone (n = 35), or enzalutamide and abiraterone (n = 2) -and at disease progression. We used deep next-generation sequencing to analyze cfDNA for sequence variants and CN status in AR and 45 additional cancer-associated genes. Primary end points were prostate-specific antigen response, progression-free survival (PFS), and overall survival (OS)., Results: Elevated tumor-specific cfDNA (circulating tumor DNA) was associated with a worse prostate-specific antigen response (hazard ratio [HR], 3.17; 95% CI, 1.11 to 9.05; P = .031), PFS (HR, 1.76; 95% CI, 1.03 to 3.01; P = .039), and OS (HR, 2.92; 95% CI, 1.40 to 6.11; P = .004). AR ligand-binding domain missense mutations (HR, 2.51; 95% CI, 1.15 to 5.72; P = .020) were associated with a shorter PFS in multivariable models. AR CN gain was associated with a shorter PFS; however, significance was lost in multivariable modeling. Genetic alterations in tumor protein p53 (HR, 2.70; 95% CI, 1.27 to 5.72; P = .009) and phosphoinositide 3-kinase pathway defects (HR, 2.62; 95% CI, 1.12 to 6.10; P = .026) were associated with a worse OS in multivariable models., Conclusion: These findings support the conclusion that high circulating tumor DNA burden is associated with worse outcomes to enzalutamide and abiraterone in men with mCRPC. Tumor protein p53 loss and phosphoinositide 3-kinase pathway defects were associated with worse OS in men with mCRPC. AR status associations with outcomes were not robust, and additional validation is needed., Competing Interests: AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or po.ascopubs.org/site/ifc.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

Introduction: Radium-223 dichloride is the first alpha-particle emitting therapeutic agent approved by FDA and EMA for bone metastatic castration-resistant prostate cancer. We studied its age-dependent biodistribution in mice, and compared it with [ 99m Tc]Tc-MDP and [ 18 F]NaF aiming to identify a potential imaging surrogate to predict [ 223 Ra]RaCl 2 whole-body localization., Methods: Male C57Bl/6 mice dosed with [ 223 Ra]RaCl 2 were sacrificed at different time points to explore [ 223 Ra]RaCl 2 whole-body distribution. In another experiment, mice at different ages were dosed with [ 223 Ra]RaCl 2 to evaluate the aging impact. Finally, [ 99m Tc]Tc-MDP and [ 18 F]NaF were administered to mice, and we compared their biodistributions with [ 223 Ra]RaCl 2 . Detailed micro-localization of each tracer was visualized using autoradiography and histochemical staining., Results: [ 223 Ra]RaCl 2 uptake in bone was rapid and stable. We observed persistent localization at bone epiphyses, as well as the red pulp of the spleen, while its uptake in most soft tissues cleared within 24 h. [ 223 Ra]RaCl 2 distribution in soft tissues is similar in all age groups tested, while bone activity significantly decreased with aging. Although the diagnostic tracers cleared much faster from soft tissues than the therapeutic radionuclide, [ 99m Tc]Tc-MDP and [ 18 F]NaF both co-localized with [ 223 Ra]RaCl 2 in the skeletal compartment., Conclusions: Radium-223 localization to the bone is dependent on age-varying factors, which implies that radium-223 dosimetry should take patient age into account. [ 99m Tc]Tc-MDP shows a different biodistribution from [ 223 Ra]RaCl 2 , both in soft tissues and in bone. [ 18 F]NaF presents a high similarity with [ 223 Ra]RaCl 2 in skeletal uptake, which validates the potential of [ 18 F]NaF as an imaging surrogate to predict radium-223 radiotherapeutic distribution in bone., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Prostate cancer remains the second leading cause of cancer related death in men. Immune check point blocking antibodies have revolutionized treatment of multiple solid tumors, but results in prostate cancer remain marginal. Previous reports have suggested that local therapies, in particular cryoablation might increase tumor immunogenicity. In this work, we examine potential synergism between tumor cryoabalation and check point blocking antibodies., Methods: FVB/NJ mice were injected subcutaneously into each flank with either 1 × 10 6 or 0.2 × 10 6 isogenic hormone sensitive Myc-Cap cells to establish synchronous grafts. Mice were treated with four intraperitoneal injections of anti-PD-1 (10 mg/kg), anti-CTLA-4 (1 mg/kg), or isotype control antibody with or without adjuvant cryoablation of the larger tumor graft and with or without neo-adjuvant androgen deprivation with degarelix (ADT). Mouse survival and growth rates of tumor grafts were measured. The immune dependency of observed oncological effects was evaluated by T cell depletion experiments., Results: Treatment with anti-CTLA-4 antibody and cryoablation delayed the growth of the distant tumor by 14.8 days (p = 0.0006) and decreased the mortality rate by factor of 4 (p = 0.0003) when compared to cryoablation alone. This synergy was found to be dependent on CD3 +  and CD8 +  cells. Combining PD-1 blockade with cryoablation did not show a benefit over use of either treatment alone. Addition of ADT to anti-PD1 therapy and cryoablation doubled the time to accelerated growth in the untreated tumors (p = 0.0021) and extended survival when compared to cryoablation combined with ADT in 25% of the mice. Effects of combining anti-PD1 with ADT and cryoablation on mouse survival were obviated by T cell depletion., Conclusion: Trimodal therapy consisting of androgen deprivation, cryoablation and PD-1 blockade, as well as the combination of cryoablation and low dose anti-CTLA-4 blockade showed that local therapies with cryoablation could be considered to augment the effects of checkpoint blockade in prostate cancer.

The microenvironment of prostate cancer often includes abundant tumor-associated macrophages (TAMs), with their acquisition of an M2 phenotype correlating with local aggressiveness and metastasis. Tumor-derived M-CSF contributes to TAM M2 polarization, and M-CSF receptor inhibition slows prostate cancer growth in model systems. As additional cytokines can direct TAM M2 polarization, targeting downstream transcription factors could avoid resistance. Klf4 and C/EBPβ each contribute to monocyte development, and reduced expression of macrophage Klf4 or C/EBPβ favors their adoption of a pro-inflammatory M1 state. We find that a Hi-Myc C57BL/6 prostate cancer line grows more slowly in syngeneic Klf4(f/f);Lys-Cre compared with Klf4(f/f) mice when inoculated subcutaneously, but grows equally rapidly in C/EBPβ(f/f);Lys-Cre and C/EBPβ(f/f) hosts. In the absence of myeloid Klf4, TAMs have reduced expression of surface mannose receptor and Fizz1 mRNA, both M2 markers. Global gene expression analysis further revealed activation of pro-inflammatory, pro-atherosclerotic pathways. Analysis of tumor-infiltrating lymphocytes (TILs) demonstrated markedly increased activated CD8 T cell numbers, and CD8 T cell depletion obviated the inhibitory effect of myeloid Klf4 deletion on prostate cancer growth. These findings suggest that reducing expression or activity of the Klf4 transcription factor in tumor myeloid cells may contribute to prostate cancer therapy.

Urinary complications resulting from benign prostatic hyperplasia and bladder outlet obstruction continue to be a serious health problem. Novel animal model systems and imaging approaches are needed to understand the mechanisms of disease initiation, and to develop novel therapies for benign prostatic hyperplasia. Long-term administration of both estradiol and testosterone in mice can result in prostatic enlargement and recapitulate several clinical components of lower urinary tract symptoms. Herein, we use longitudinal magnetic resonance imaging and histological analyses to quantify changes in prostatic volume, urethral volume, and genitourinary vascularization over time in response to estradiol-induced prostatic enlargement. Our data demonstrate significant prostatic enlargement by 12 weeks after treatment, with no detectable immune infiltration by macrophages or T- or B-cell populations. Importantly, the percentage of cell death, as measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling, was significantly decreased in the prostatic epithelium of treated animals as compared to controls. We found no significant change in prostate cell proliferation in treated mice when compared to controls. These studies highlight the utility of magnetic resonance imaging to quantify changes in prostatic and urethral volumes over time. In conjunction with histological analyses, this approach has the high potential to enable mechanistic studies of initiation and progression of clinically relevant lower urinary tract symptoms. In addition, this model is tractable for investigation and testing of therapeutic interventions to ameliorate or potentially reverse prostatic enlargement., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Due to inadequate screening methods and the common coexistence of limited functional liver reserves, curative treatment options are limited. Liver transplantation is the only curative treatment modality for early HCC. There are multidisciplinary treatment options like ablative treatments, radiation and systemic therapy available for more advanced patients or those that are inoperable. Treatment resistance and progression is inevitable for these HCC patients. Newer therapeutics need to be explored for better management of HCC. HCC is a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. Here we explored the therapeutic potential of the anti-angiogenic, anti-lymphangiogenic, and directly anti-tumorigenic biomimetic collagen IV-derived peptide developed by our group. Human HCC cell lines HuH7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration upon treatment with the peptide. Consistent with previously described multimodal inhibitory properties, the peptide was found to inhibit both c-Met and IGF1R signaling in HepG2 cells and blocked HepG2 conditioned media stimulation of microvascular endothelial cell (MEC) tube formation. Furthermore, the peptide treatment of mouse HepG2 tumor xenografts significantly inhibited growth relative to untreated controls. The peptide was also found to improve the survival of autochthonous Myc-induced HCC in a transgenic mouse model. Mechanistically, we found that the peptide treatment reduced microvascular density in the autochthonous liver tumors with increased apoptosis. This study shows the promising therapeutic potential of our biomimetic peptide in the treatment of HCC., Competing Interests: CONFLICTS OF INTEREST ASP is a co-founder and serves as the CSO, JJG is a co-founder and serves as the CTO and NBP is the Senior Director of R&D of AsclepiX Therapeutics, LLC. The terms of these arrangements are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasive phenotypes that becomes dysregulated in primary and metastatic prostate cancer.

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Mucoadhesive particles (MAP) have been widely explored for pulmonary drug delivery because of their perceived benefits in improving particle residence in the lungs. However, retention of particles adhesively trapped in airway mucus may be limited by physiologic mucus clearance mechanisms. In contrast, particles that avoid mucoadhesion and have diameters smaller than mucus mesh spacings rapidly penetrate mucus layers [mucus-penetrating particles (MPP)], which we hypothesized would provide prolonged lung retention compared to MAP. We compared in vivo behaviors of variously sized, polystyrene-based MAP and MPP in the lungs following inhalation. MAP, regardless of particle size, were aggregated and poorly distributed throughout the airways, leading to rapid clearance from the lungs. Conversely, MPP as large as 300 nm exhibited uniform distribution and markedly enhanced retention compared to size-matched MAP. On the basis of these findings, we formulated biodegradable MPP (b-MPP) with an average diameter of <300 nm and examined their behavior following inhalation relative to similarly sized biodegradable MAP (b-MAP). Although b-MPP diffused rapidly through human airway mucus ex vivo, b-MAP did not. Rapid b-MPP movements in mucus ex vivo correlated to a more uniform distribution within the airways and enhanced lung retention time as compared to b-MAP. Furthermore, inhalation of b-MPP loaded with dexamethasone sodium phosphate (DP) significantly reduced inflammation in a mouse model of acute lung inflammation compared to both carrier-free DP and DP-loaded MAP. These studies provide a careful head-to-head comparison of MAP versus MPP following inhalation and challenge a long-standing dogma that favored the use of MAP for pulmonary drug delivery.

Background: B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer., Methods: Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson's correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease., Results: B7-H3 mRNA expression was positively associated with higher Gleason score (P<0.001), tumor stage (P<0.001), and castrate resistant metastatic disease (P<0.0001). High B7-H3 expression correlated with the development of metastasis and prostate cancer specific mortality, but this was not significant on multi-variable analysis. B7-H3 expression correlated with ERG-positive disease (r=0.99) and AR expression (r=0.36). ChIP revealed an AR-binding site upstream of B7-H3, and the presence of androgens decreased B7-H3 expression in LNCaP suggesting potential direct AR regulation. Gene set enrichment analysis demonstrated an association of B7-H3 with androgen signaling as well as immune regulatory pathways., Conclusions: Higher B7-H3 expression correlates with Gleason grade, prostate cancer stage and poor oncologic outcomes in prostatectomy cohorts. B7-H3 expression appears to be related to androgen signaling as well as the immune reactome.

Prostate cancer is one of the most common cancers in men in the United States. Comprehensive understanding of the biology contributing to prostate cancer will have important clinical implications. Animal models have greatly impacted our knowledge of disease and will continue to be a valuable resource for future studies. Herein, we describe a detailed protocol for the orthotopic engraftment of a murine prostate cancer cell line (Myc-CaP) into the anterior prostate of an immune competent mouse.

Objective: The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer., Methods: We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR., Results: ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3., Conclusions: Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed., Experimental Design: Germline ASPN D-repeat-length was retrospectively analyzed in 1,600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 noncancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes, including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer., Results: Variation at the ASPN D locus was differentially associated with poorer oncologic outcomes. ASPN D14 [HR, 1.72; 95% confidence interval (CI), 1.05-2.81, P = 0.032] and heterozygosity for ASPN D13/14 (HR, 1.86; 95% CI, 1.03-3.35, P = 0.040) were significantly associated with metastatic recurrence, while homozygosity for the ASPN D13 variant was significantly associated with a reduced risk of metastatic recurrence (HR, 0.44; 95% CI, 0.21-0.94, P = 0.035) in multivariable analyses. Orthotopic xenografts established biologic roles for ASPN D14 and ASPN D13 variants in metastatic prostate cancer progression that were consistent with patient-based data., Conclusions: We observed associations between ASPN D variants and oncologic outcomes, including metastasis. Our data suggest that ASPN expressed in the tumor microenvironment is a heritable modulator of metastatic progression., (©2015 American Association for Cancer Research.)

Prostate cancer is a leading cause of cancer death in men due to the subset of cancers that progress to metastasis. Prostate cancers are thought to be hardwired to androgen receptor (AR) signaling, but AR-regulated changes in the prostate that facilitate metastasis remain poorly understood. We previously noted a marked reduction in secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) expression during invasive phases of androgen-induced prostate growth, suggesting that this may be a novel invasive program governed by AR. Herein, we show that SPARCL1 loss occurs concurrently with AR amplification or overexpression in patient-based data. Mechanistically, we demonstrate that SPARCL1 expression is directly suppressed by androgen-induced AR activation and binding at the SPARCL1 locus via an epigenetic mechanism, and these events can be pharmacologically attenuated with either AR antagonists or HDAC inhibitors. We establish using the Hi-Myc model of prostate cancer that in Hi-Myc/Sparcl1(-/-) mice, SPARCL1 functions to suppress cancer formation. Moreover, metastatic progression of Myc-CaP orthotopic allografts is restricted by SPARCL1 in the tumor microenvironment. Specifically, we show that SPARCL1 both tethers to collagen in the extracellular matrix (ECM) and binds to the cell's cytoskeleton. SPARCL1 directly inhibits the assembly of focal adhesions, thereby constraining the transmission of cell traction forces. Our findings establish a new insight into AR-regulated prostate epithelial movement and provide a novel framework whereby SPARCL1 in the ECM microenvironment restricts tumor progression by regulating the initiation of the network of physical forces that may be required for metastatic invasion of prostate cancer., (©2015 American Association for Cancer Research.)

Dietary carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and chronic inflammation have each been implicated as etiologic agents in prostate cancer. We hypothesized that bacterial prostatitis would accelerate PhIP-induced preinvasive lesions in the rat prostate. Male Fischer 344 rats were assigned into 4 groups: Control (untreated), PhIP (200 ppm in the diet for 20 weeks), Escherichia coli (E. coli, prostatic inoculation in week 10), or PhIP + E. coli. Study animals were monitored for a total of 52 weeks and were euthanized as necessary based on strict criteria for health status and tumor burden. Animals treated with E. coli initially developed acute and chronic inflammation in all lobes of the prostate, whereas inflammation was observed predominantly in the ventral lobe at time of death. PhIP + E. coli-treated animals exhibited a marked decrease in survival compared with PhIP-alone-treated animals as a result of an increase in the number of invasive cancers that developed at multiple sites, including the skin, small intestine, and Zymbal's gland. Despite their earlier mortality, PhIP + E. coli-treated animals developed an increased average number of precancerous lesions within the prostate compared with PhIP-treated animals, with a significantly increased Ki-67 index. Multiplexed serum cytokine analysis indicated an increase in the level of circulating IL6 and IL12 in PhIP + E. coli-treated animals. Elevated serum IL6 levels correlated with the development of precancerous lesions within the prostate. These results suggest that bacterial infections and dietary carcinogens, two conceivably preventable cancer risk factors, may synergistically promote tumorigenesis., (©2015 American Association for Cancer Research.)

A 25-yr-old Diana monkey (Cercopithecus diana) with a 1.5-yr history of chronic colitis and diarrhea was found to have disseminated granulomatous disease with intralesional acid fast bacilli. Bacilli were identified as Mycobacterium genavense by polymerase chain reaction, sequencing of the 16S-23S ribosomal RNA intergenic spacer (ITS) gene, and mycolic acid analysis by high-performance liquid chromatography. Mycobacterium genavense is a common cause of mycobacteriosis in free-ranging and captive birds. In addition, recognition of opportunistic infection in human immunodeficiency virus-positive patients is increasing. Disease manifestations of M. genavense are similar to Mycobacterium avium complex (MAC) and include fever, wasting, and diarrhea with disseminated disease. Similar clinical signs and lesions were observed in this monkey. Mycobacterium genavense should be considered as a differential for disseminated mycobacterial disease in nonhuman primates as this agent can mimic MAC and related mycobacteria.

Inflammation is associated with several diseases of the prostate including benign enlargement and cancer, but a causal relationship has not been established. Our objective was to characterize the prostate inflammatory microenvironment after infection with a human prostate-derived bacterial strain and to determine the effect of inflammation on prostate cancer progression. To this end, we mimicked typical human prostate infection with retrograde urethral instillation of CP1, a human prostatic isolate of Escherichia coli. CP1 bacteria were tropic for the accessory sex glands and induced acute inflammation in the prostate and seminal vesicles, with chronic inflammation lasting at least 1 year. Compared to controls, infection induced both acute and chronic inflammation with epithelial hyperplasia, stromal hyperplasia, and inflammatory cell infiltrates. In areas of inflammation, epithelial proliferation and hyperplasia often persist, despite decreased expression of androgen receptor (AR). Inflammatory cells in the prostates of CP1-infected mice were characterized at 8 weeks post-infection by flow cytometry, which showed an increase in macrophages and lymphocytes, particularly Th17 cells. Inflammation was additionally assessed in the context of carcinogenesis. Multiplex cytokine profiles of inflamed prostates showed that distinct inflammatory cytokines were expressed during prostate inflammation and cancer, with a subset of cytokines synergistically increased during concurrent inflammation and cancer. Furthermore, CP1 infection in the Hi-Myc mouse model of prostate cancer accelerated the development of invasive prostate adenocarcinoma, with 70% more mice developing cancer by 4.5 months of age. This study provides direct evidence that prostate inflammation accelerates prostate cancer progression and gives insight into the microenvironment changes induced by inflammation that may accelerate tumour initiation or progression., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background and Study Aims: Endoscopic submucosal dissection (ESD) is a technically challenging procedure. A novel gel can facilitate ESD due to its submucosal dissecting properties. This prospective porcine survival study evaluated clinical and histologic parameters of hybrid ESD using the gel., Patients and Methods: Gastric submucosal lesions were created in six pigs and hybrid ESD was performed. Healing was assessed weekly until necropsy at Day 28., Results: En bloc resection was achieved in all lesions (mean size 40.7 mm). The mean total procedure time was 13.5 minutes and the mean resection time was 5.5 minutes. The mean total histologic injury score was 4. At necropsy, four ulcers had healed completely and two were < 6 mm in size., Conclusion: Hybrid ESD of large gastric lesions in a porcine model can be facilitated by the novel gel, dramatically reducing procedure and resection times by eliminating the need for time-consuming submucosal dissection. The novel gel is safe and easy to use, and has the potential to simplify ESD. Further prospective human studies are needed to validate these findings., (© Georg Thieme Verlag KG Stuttgart · New York.)

Local delivery of chemotherapeutics in the cervicovaginal tract using nanoparticles may reduce adverse side effects associated with systemic chemotherapy, while improving outcomes for early-stage cervical cancer. It is hypothesized here that drug-loaded nanoparticles that rapidly penetrate cervicovaginal mucus (CVM) lining the female reproductive tract will more effectively deliver their payload to underlying diseased tissues in a uniform and sustained manner compared with nanoparticles that do not efficiently penetrate CVM. Paclitaxel-loaded nanoparticles are developed, composed entirely of polymers used in FDA-approved products, which rapidly penetrate human CVM and provide sustained drug release with minimal burst effect. A mouse model is further employed with aggressive cervical tumors established in the cervicovaginal tract to compare paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (conventional particles, or CP) and similar particles coated with Pluronic F127 (mucus-penetrating particles, or MPP). CP are mucoadhesive and, thus, aggregated in mucus, while MPP achieve more uniform distribution and close proximity to cervical tumors. Paclitaxel-MPP suppress tumor growth more effectively and prolong median survival of mice compared with unencapsulated paclitaxel or paclitaxel-CP. Histopathological studies demonstrate minimal toxicity to the cervicovaginal epithelia, suggesting paclitaxel-MPP may be safe for intravaginal use. These results demonstrate the in vivo advantages of polymer-based MPP for treatment of tumors localized to a mucosal surface., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: By regulating cell fate, proliferation, and survival, Notch pathway signaling provides critical input into differentiation, organization, and function of multiple tissues. Notch signaling is also becoming an increasingly recognized feature in malignancy, including prostate cancer, where it may play oncogenic or tumor suppressive roles., Methods: Based on an electronic literature search from 2000 to 2013 we identified, summarized, and integrated published research on Notch signaling dynamics in prostate homeostasis and prostate cancer., Results: In benign prostate, Notch controls the differentiation state and architecture of the gland. In prostate cancer, similar features correlate with lethal potential and may be influenced by Notch. Increased Notch1 can confer a survival advantage on prostate cancer cells, and levels of Notch family members, such as Jagged2, Notch3, and Hes6 increase with higher cancer grade. However, Notch signaling can also antagonize growth and survival of both benign and malignant prostate cells, possibly through antagonistic effects of the Notch target HEY1 on androgen receptor function., Discussion: Notch signaling can dramatically influence prostate development and disease. Determining the cellular contexts where Notch promotes or suppresses prostate growth could open opportunities for diagnostic and therapeutic interventions., (© 2014 Wiley Periodicals, Inc.)

Intraperitoneal (IP) chemotherapy is more effective than systemic chemotherapy for treating advanced ovarian cancer, but is typically associated with severe complications due to high dose, frequent administration schedule, and use of non-biocompatible excipients/delivery vehicles. Here, we developed paclitaxel (PTX)-loaded microspheres composed of di-block copolymers of poly(ethylene glycol) and poly(sebacic acid) (PEG-PSA) for safe and sustained IP chemotherapy. PEG-PSA microspheres provided efficient loading (~ 13% w/w) and prolonged release (~ 13 days) of PTX. In a murine ovarian cancer model, a single dose of IP PTX/PEG-PSA particles effectively suppressed tumor growth for more than 40 days and extended the median survival time to 75 days compared to treatments with Taxol(®) (47 days) or IP placebo particles (34 days). IP PTX/PEG-PSA was well tolerated, with only minimal to mild inflammation. Our findings support PTX/PEG-PSA microspheres as a promising drug delivery platform for IP therapy of ovarian cancer, and potentially other metastatic peritoneal cancers.

ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2(-/-) mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2(-/-) mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2(-/-) animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.

Animal models, particularly mouse models, play a central role in the study of the etiology, prevention, and treatment of human prostate cancer. While tissue culture models are extremely useful in understanding the biology of prostate cancer, they cannot recapitulate the complex cellular interactions within the tumor microenvironment that play a key role in cancer initiation and progression. The National Cancer Institute (NCI) Mouse Models of Human Cancers Consortium convened a group of human and veterinary pathologists to review the current animal models of prostate cancer and make recommendations about the pathologic analysis of these models. More than 40 different models with 439 samples were reviewed, including genetically engineered mouse models, xenograft, rat, and canine models. Numerous relevant models have been developed over the past 15 years, and each approach has strengths and weaknesses. Analysis of multiple genetically engineered models has shown that reactive stroma formation is present in all the models developing invasive carcinomas. In addition, numerous models with multiple genetic alterations display aggressive phenotypes characterized by sarcomatoid carcinomas and metastases, which is presumably a histologic manifestation of epithelial-mesenchymal transition. The significant progress in development of improved models of prostate cancer has already accelerated our understanding of the complex biology of prostate cancer and promises to enhance development of new approaches to prevention, detection, and treatment of this common malignancy.

Circulating tumor cells (CTCs) have received intense scientific scrutiny because they travel in the bloodstream and are therefore well situated to mediate hematogenous metastasis. However, the potential of CTCs to actually form new tumors has not been tested. Popular methods of isolating CTCs are biased towards larger, more differentiated, non-viable cells, creating a barrier to testing their tumor forming potential. Without relying on cell size or the expression of differentiation markers, our objective was to isolate viable prostate CTCs from mice and humans and assay their ability to initiate new tumors. Therefore, blood was collected from transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and from human patients with metastatic castration-resistant prostate cancer (PCa). Gradient density centrifugation or red cell lysis was used to remove erythrocytes, and then leukocytes were depleted by magnetic separation using CD45 immunoaffinity beads. CTCs fractions from TRAMP mice and PCa patients were verified by immunocytochemical staining for cytokeratin 8 and EpCAM, and inoculated into immunodeficient mice. TRAMP tumor growth was monitored by palpation. Human tumor growth formation was monitored up to 8 months by ultrasensitive PSA assays performed on mouse serum. We found viable tumor cells present in the bloodstream that were successfully isolated from mice without relying on cell surface markers. Two out of nine immunodeficient mice inoculated with TRAMP CTCs developed massive liver metastases. CTCs were identified in blood from PCa patients but did not form tumors. In conclusion, viable CTCs can be isolated without relying on epithelial surface markers or size fractionation. TRAMP CTCs were tumorigenic, so CTCs isolated in this way contain viable tumor-initiating cells. Only two of nine hosts grew TRAMP tumors and none of the human CTCs formed tumors, which suggests that most CTCs have relatively low tumor-forming potential. Future studies should identify and target the highly tumorigenic cells.

Androgens initiate a complex network of signals within the UGS that trigger prostate lineage commitment and bud formation. Given its contributions to organogenesis in other systems, we investigated a role for canonical Wnt signaling in prostate development. We developed a new method to achieve complete deletion of beta-catenin, the transcriptional coactivator required for canonical Wnt signaling, in early prostate development. Beta-catenin deletion abrogated canonical Wnt signaling and yielded prostate rudiments that exhibited dramatically decreased budding and failed to adopt prostatic identity. This requirement for canonical Wnt signaling was limited to a brief critical period during the initial molecular phase of prostate identity specification. Deletion of beta-catenin in the adult prostate did not significantly affect organ homeostasis. Collectively, these data establish that beta-catenin and Wnt signaling play key roles in prostate lineage specification and bud outgrowth., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Prostate cancer is the second leading cause of cancer death among United States men. However, disease aggressiveness is varied, with low-grade disease often being indolent and high-grade cancer accounting for the greatest density of deaths. Outcomes are also disparate among men with high-grade prostate cancer, with upwards of 65% having disease recurrence even after primary treatment. Identification of men at risk for recurrence and elucidation of the molecular processes that drive their disease is paramount, as these men are the most likely to benefit from multimodal therapy. We previously showed that androgen-induced expression profiles in prostate development are reactivated in aggressive prostate cancers. Herein, we report the down-regulation of one such gene, Sparcl1, a secreted protein, acidic and rich in cysteine (SPARC) family matricellular protein, during invasive phases of prostate development and regeneration. We further demonstrate a parallel process in prostate cancer, with decreased expression of SPARCL1 in high-grade/metastatic prostate cancer. Mechanistically, we demonstrate that SPARCL1 loss increases the migratory and invasive properties of prostate cancer cells through Ras homolog gene family, member C (RHOC), a known mediator of metastatic progression. By using models incorporating clinicopathologic parameters to predict prostate cancer recurrence after treatment, we show that SPARCL1 loss is a significant, independent prognostic marker of disease progression. Thus, SPARCL1 is a potent regulator of cell migration/invasion and its loss is independently associated with prostate cancer recurrence.