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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, anatomical structure, and molecular expression profiles. Differing from conventional optical systems, the point spread function (PSF) in DAR is determined by properties of radioisotope decay, phosphor and digitizer. Calibration of an experimental PSF 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 Clus- tering of Applications with Noise" to estimate noise parameters, and utilizes L2 and Hessian Frobenius (HF) norms as regularization functions to improve performance. Results: First, PG-PEM outperformed other restoration algorithms at the denoising task (p

Background: Radium-223 dichloride ([223Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability.

Background Penis transplantation represents an exciting new avenue for restoration of male urogenitalia. However, little is known about the specific immunological features of penile transplants, limiting their application in complex urogenital reconstruction. To properly study this emerging form of transplantation, adequate preclinical models are a necessity. The purpose of this study is to establish a clinical and histological rejection classification of urogenital tissue transplants using a new rat heterotopic penile transplant model that includes preputial skin. Methods Syngeneic and allogeneic heterotopic penile transplantations were performed on Lewis and Brown Norway rats using a new model designed by our group. Grafts were clinically and histologically monitored at postoperative days (POD) 3-30. Results Six syngeneic and 25 allogeneic transplants were performed. All syngeneic and tacrolimus-treated grafts survived until endpoint. Allogeneic graft rejection is shown to follow a 4-stage clinical progression with all untreated allografts developing epidermal sloughing at POD7 and full rejecting between POD14 and POD16. Histological samples were used to develop a specific 4-grade rejection classification analogous to the 2007 Banff Criteria for skin-containing allografts. Conclusions Graft skin and urethral lining tissue are first rejection targets followed by tunica albuginea and corpora cavernosa in a distal to proximal pattern. We established a robust and reproducible murine model to study the immunobiology of male genital tissue in the context of transplantation and developed a novel 4-grade clinical and histological rejection scale based on graft skin and urethral lining as the main targets of rejection.

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 × 106 ) 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.

// Mustafa A. Barbhuiya 1, * , Adam C. Mirando 2, * , Brian W. Simons 3 , Ghali Lemtiri-Chlieh 1 , Jordan J. Green 2 , Aleksander S. Popel 2 , Niranjan B. Pandey 2 and Phuoc T. Tran 1, 3 1 Department of Radiation Oncology and Molecular and Radiation Sciences, Sidney Kimmel Comprehensive Cancer Centre, Johns Hopkins School of Medicine, Baltimore, MD, USA 2 Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3 Department of Medical Oncology, Sidney Kimmel Comprehensive Cancer Centre and Department of Urology, The Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA * These authors have contributed equally to this work Correspondence to: Phuoc T. Tran, email: tranp@jhmi.edu , tranpt21@sbcglobal.net Niranjan B. Pandey, email: npandey4@jhmi.edu , npandey@asclepix.com Aleksander S. Popel, email: apopel@jhu.edu Keywords: hepatocellular carcinoma; angiogenesis; c-Met; HGF; IGF1R Received: April 07, 2017 Accepted: August 26, 2017 Published: September 21, 2017 ABSTRACT 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.

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.

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.

Purpose: Enhancing immune responses in triple negative breast cancers (TNBCs) remains a challenge. Our study aimed to determine whether magnetic iron oxide nanoparticle (MION) hyperthermia (HT) can enhance abscopal effects with radiotherapy (RT) and immune checkpoint inhibitors (IT) in a metastatic TNBC model. Methods: One week after implanting 4T1-luc cells into the mammary glands of BALB/c mice, tumors were treated with RT (3 × 8Gy)±local HT, mild (HTM, 43°C/20 min) or partially ablative (HTAbl, 45°C/5min plus 43°C/15min),±IT with anti-PD-1 and anti-CTLA-4 antibodies (both 4 × 10mg/kg, i.p.). Tumor growth was measured daily. Two weeks after treatment, lungs and livers were harvested for histopathology evaluation of metastases. Results: Compared to untreated controls, all treatment groups demonstrated a decreased tumor volume; however, when compared against surgical resection, only RT + HTM+IT, RT + HTAbl+IT and RT + HTAbl had similar or smaller tumors. These cohorts showed more infiltration of CD3+ T-lymphocytes into the primary tumor. Tumor growth effects were partially reversed with T-cell depletion. Combinations that proved most effective for primary tumors generated modest reductions in numbers of lung metastases. Conversely, numbers of lung metastases showed potential to increase following HT+ IT treatment, particularly when compared to RT. Compared to untreated controls, there was no improvement in survival with any treatment. Conclusions: Single-fraction MION HT added to RT + IT improved local tumor control and recruitment of CD3+ T-lymphocytes, with only a modest effect to reduce lung metastases and no improvement in overall survival. HT + IT showed potential to increase metastatic dissemination to lungs., GRAPHICAL ABSTRACT

African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. This adhesion decrease is facilitated by a decrease in cell adhesion protein integrin β1 (ITGB1). Elevated ADA enzyme activity decreases cAMP which mediates this adhesion decrease by disrupting the Epac-Rap1 pathway. It is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes this decrease in cAMP levels. These molecular findings suggest that ADA-mediated adhesion decrease could facilitate metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels (ADA enzyme activity) in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i) Biochemical Recurrence (ii) Time to attaining castration resistance in castration sensitive patients (iii) Time to metastasis (iv) Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Brian Simons, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR17.

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 and prostate adenocarcinoma in 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.

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 × 106 or 0.2 × 106 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.

INTRODUCTION AND OBJECTIVES:MicroRNA 21 (miR-21) is overexpressed in virtually all types of cancers including prostate cancer (PCa). We have previously reported that the androgen receptor (AR) indu...

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.

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 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.

Introduction: C-type lectin 1 (CLL-1, CD371) is highly expressed on the malignant cells from many patients with AML, and CAR T cells directed to this antigen can selectively target both leukemic progenitor cells (LSC) as well as AML blasts whilst sparing normal tissues. We previously showed (1) that such CAR-Ts can recognize and eliminate both AML blasts and primitive AML colony-forming cells in a low tumor-burden model. We have now modified the structure of the CLL-1 CAR and added transgenic expression of IL15 to enhance performance sufficiently for activity even against more extensive disease. Material and Methods: We assessed the phenotype and cytolytic ability of T cells transduced with 5 CLL-1 CAR constructs, varying in their spacer, transmembrane and costimulatory sequences (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3), and compared these with the effects of our published construct (4-1BBz-CD8)(1). We used flow cytometry to determine the effects of each construct on T cell phenotype and differentiation, and sequential (recursive) co-culture assays with tumor-cell targets to determine the durability of the anti-tumor activity. The most active constructs (CD28z-CD8 and 4-1BBz-CD8) were then evaluated in NOD.SCID IL-2Rg-/- (NSGS) mice engrafted with 1.5x10ˆ6 FFLuc-modified HL 60 AML cells, which received 2x10ˆ6 CLL-1 CAR T cells on day 3. To determine if we could further potentiate the in vivo expansion, persistence and anti-tumor activity of the CLL-1 CAR-T cells, we used a second retroviral vector to co-express transgenic IL15, measuring the effects in vitro and in vivo. Mice engrafted with 1.5x10ˆ6 tumor cells and received 2.5x10ˆ6 CLL-1 CAR T cells on week 3 in patient derived xenograft (PDX) model. We determined antitumor activity by bioluminescence imaging and weekly bleeding and measured serum cytokines by multiplex analysis (Luminex, TX). After euthanasia, we examined formalin-fixed/paraffin embedded sections. Results: Modified CLL-1 CAR constructs were expressed by 70-80% of cells irrespective of CAR sequence, but CD28z-CD8 CAR T cell expansion was significantly higher than CAR T cells with 4-1BBz endodomains (p Conclusion: Addition of transgenic IL15 to CLL-1-CD28z-CD8 CAR augmented activity against AML in a range of cell line and PDX models, and toxicity associated with exuberant CART expansion could be prevented by cytokine blockade and/or an inducible safety switch. References: 1. Tashiro H, et al. Mol Ther. 2017 2.Straathof KC et al. Blood. 2005 Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees.

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

What’s known on the subject? and What does the study add? Sexual function is often impaired after radical prostatectomy (RP) resulting in reduced sexual activity and sexual bother. The main focus in the literature concerning sexual adverse effects has been on erectile dysfunction and impairment of sexual function rather than the actual sexual bother it causes, although the sexual bother is most important to the individual patient’s quality of life. The relation between these measures, and in particular pre-operative prediction of post-operative sexual bother, has only been studied limitedly and with varying results. Some studies have found good mental health, low levels of pre-operative sexual bother, and higher education to be associated with absence of post-operative sexual bother, but another study could not identify any pre-operative predictors of a post-operative sexual bother. Severe sexual bother after RP were reported by 64% to 95% of the patients three years after operation, and the prevalence was associated with the level of pre-treatment sexual bother and pre-operative nerve-preservation. On the other hand, others have reported that only 43% of men have sexual bother 2 years after RP. However, none of these studies stratified patients according to their pre-operative sexual activity and most of them were American. It has been shown that American findings concerning sexual bother may not always be valid for non-American patients due to differing sex role expectations thus warranting the need for more non-American studies. This study has shown that two thirds of patients experienced sexual bother one year after RP. We have identified patients with increased risk of experiencing overall sexual bother post-operatively: those who report pre-operative sexual bother, those who are sexually active before RP, and those who display neurotic personality-traits. Another important finding was that the proportion of patients who experienced bother relevant to having impaired post-operative SF was significantly higher among pre-operatively sexually active patients than those who had been inactive. This study adds knowledge that patients’ pre-operative sexual activity, sexual bother and personality should be taken into account to be able to give individualized information about the risk of getting sexual bother after RP. OBJECTIVES • To evaluate the cytotoxicity of dimeric naphthoquinones (BiQs) in prostate cancer cells. • To assess the interaction of dimeric naphthoquinones with common therapies including radiation and docetaxel. MATERIALS AND METHODS • The cytotoxicity of 12 different dimeric naphthoquinones was assessed in androgen-independent (PC-3, DU-145) and androgen-responsive (LNCaP, 22RV1) prostate cancer cell lines and in prostate epithelial cells (PrECs). • BiQ2 and BiQ11 were selected for determination of dose response, effects on colony formation and initial exploration into mechanism of action. • Synergistic effects with radiation and docetaxel were explored using colony-forming and MTT assays. RESULTS • At concentrations of 15µM, BiQ2, BiQ3, BiQ11, BiQ12, and BiQ15 demonstrated cytotoxicity in all prostate cancer cell lines. • Treatment with BiQs limited the ability of prostate cancer cells to form colonies in clonogenic assays. • Exposure of prostate cancer to BiQs increased cellular reactive oxygen species (ROS), decreased ATP production, and promoted apoptosis. • BiQ cytotoxicity was independent of NADP(H):quinone oxidoreductase 1 (NQO1) activity in PrECs, PC-3 and 22RV1, but not DU-145 cells. • Exposure of prostate cancer cells to radiation before treatment with BiQs increased their activity allowing for inhibitory effects well below the IC50s of these compounds in PrECs. • Co-administration of BiQs with docetaxel had minimal additive effects. CONCLUSIONS • Dimeric naphthoquinones represent a new class of compounds with prostate cancer cytotoxicity and synergistic effects with radiation. The cytotoxic effect of these agents is probably contributed to by the accumulation of ROS and mitochondrial dysfunction. • Further studies are warranted to better characterize this class of potential chemo-therapeutics.

BACKGROUND AND STUDY AIMS: Pancreatitis complicates 1 % - 22 % of endoscopic retrograde cholangiopancreatography procedures. The study aims were to develop a reproducible animal model of post-ERCP pancreatitis (PEP), and investigate the impact of endoscopic technique on severity of PEP. PATIENTS AND METHODS: ERCP was carried out in six male hound dogs. Pancreatitis was induced by one of three escalating methods: 1) pancreatic acinarization with 20 - 30 mL of contrast; 2) acinarization + ductal balloon occlusion + sphincterotomy; 3) acinarization + intraductal synthetic bile injection + ductal balloon occlusion + sphincterotomy. Dogs 5 and 6 received a pancreatic stent. Necropsy was performed on postoperative day 5. All pancreatic specimens were graded by two blinded pathologists according to a validated scoring system. All dogs were compared with three control dogs. RESULTS: Dogs 1 - 4 developed clinical pancreatitis and hyperamylasemia (11 736 vs. 722 U/L, P = 0.02). Total injury scores were significantly elevated compared with controls (6.85 vs. 1.06, P = 0.004). There was significant increase in acinar cell necrosis (0.86 vs. 0.06, P = < 0.001), and all other categories (except fibrosis) demonstrated elevated injury scores . Dogs 5 and 6 developed clinical pancreatitis without significant hyperamylasemia; total injury scores were elevated compared with controls (4.83 vs. 1.06, P = 0.01), but lower than in Dogs 1 - 4 (4.83 vs. 6.85, P = 0.25). There was escalating severity of pancreatic injury from Dogs 1 to 4 correlating with the method of endoscopic injury used. CONCLUSION: Severity of PEP is directly proportional to invasiveness of endoscopic intervention. Pancreatic acinarization, even without balloon occlusion and sphincterotomy, can be used as a reliable animal model for future studies investigating therapy and prevention of disease.

Metastatic castration resistant prostate cancer (CRPC) continues to result in over 26,000 deaths per year in the United States. For 70 years, therapy for advanced prostate cancer has relied on AR-directed therapies. Unfortunately, even with the second generation androgen deprivation compounds, treated cancers commonly progress to castration resistance and lethality. This progression to castration resistance remains the central therapeutic roadblock for advanced prostate cancer. Importantly, in the vast majority of CRPC cases, the tumor still depends on AR signaling, but this signaling becomes androgen independent. The most common mechanisms, including mutation, protein truncation, and alternative splicing, affect the C-terminal domain, suggesting that antagonism of AR function via the N-terminal domain may be a promising target for CRPC therapy. Several groups have shown that CDKs 1, 5 and 9 activate AR by phosphorylation of the N-terminal domain. Here, we show that the multi-CDK inhibitor roniciclib (BAY1000394) inhibits activation of AR in a panel of prostate cancer cell lines, including several cell lines which exhibit androgen-independent AR signaling via AR-V7 alternative splicing. In vivo, roniciclib inhibited growth of prostate cancer xenografts. Since AR inhibition in prostate cancer has been shown to induce compensatory PI3K signaling, we also treated xenografts with a combination of roniciclib and the PI3K inhibitor copanlisib (BAY 80-6946). This combination was especially effective, and was well tolerated by the mice. Our results suggest that a combination of a multi-CDK inhibitor and a PI3K inhibitor may be promising for CRPC therapy. Citation Format: Brian W. Simons, Maria Ybanez, Emmanuel S. Antonarakis, Jun Luo, Barry D. Nelkin. Multi-CDK inhibition efficiently suppresses AR function and cell growth in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2017-2171

Hepatocellular carcinoma (HCC) related mortality ranks second worldwide and stands one step behind lung cancer. Due to the asymptomatic nature of the cancer, early diagnosis is a major problem. Currently only liver transplantation is curative for early stage hepatocellular cancer. There are other multidisciplinary treatment options like radiation and chemotherapy available for advanced patients. However, chemotherapy resistance is a common problem in the treatment of liver cancer. Newer therapeutics are needed for better management of advanced HCC. Angiogenesis and lymphangiogenesis are processes that are vital for tumorigenesis and metastasis. HCC is known to be a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. We explored the therapeutic potential of the anti-angiogenic and anti-lymphangiogenic, biomimetic peptide SP2043 developed by our group in HCC. Hepatocellular carcinoma cell lines HuH-7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration following treatment. Furthermore, SP2043 was found to impair microvascular endothelial cell (MEC) tube formation induced by HepG2 tumor conditioned media and to significantly inhibit HepG2 tumor xenograft growth compared to untreated controls. The peptide drug was also found to improve the survival of autochthonous Myc induced HCC in a transgenic mouse model from two weeks to four weeks. We discovered the peptide to be a multi-modal anti-angiogenic drug that also inhibits IGF1R and MET signaling pathways in HCC cell lines. We also found that SP2043 treatment reduced the microvascular density in both subcutaneous and autochthonous liver tumors with reduced tumor cell proliferation and increased apoptosis. This study shows the therapeutic potential of SP2043 in the treatment of hepatocellular carcinoma. Note: This abstract was not presented at the meeting. Citation Format: Mustafa A. Barbhuiya, Adam C. Mirando, Brian W. Simons, Ghali Lemtiri-Chlieh, Jordan J. Green, Aleksander S. Popel, Niranjan B. Pandey, Phuoc T. Tran. Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3201. doi:10.1158/1538-7445.AM2017-3201

The majority of prostate cancer (PCa) deaths are attributed to bone metastases, which may not arise until years after the primary tumor has been removed. Disseminated tumor cells (DTCs) have been shown to already be present in the bone marrow of patients at the time of prostatectomy, and it is thought that they undergo a period of dormancy, allowing them to remain undetected for years before manifesting into clinically detectable metastases. It is not known what allows PCa DTCs to undergo and persist in this dormant state, but it is likely that factors in the bone marrow niche play an essential role. Previously our group has shown that one of these niche factors, Gas6, can regulate growth of PCa cells. The growth restraining effects of Gas6 on PCa cells is likely mediated through the tyrosine kinase receptor Axl, whose expression was found to be increased on dormant DTCs compared to proliferating metastases in a xenograft mouse model. Here, we utilized the TRAMP model, a genetically engineered mouse model of spontaneous PCa progression, to compare the frequency of Axl-hi and Axl-low bone marrow DTCs that are proliferating versus non-proliferating. Further, we also compared Axl expression and proliferation in matched primary tumors. To investigate the functional significance of Gas6/Axl signaling in mediating PCa dormancy, we have generated Axl knockout PCa cells to test the requirement of Axl in maintaining dormancy, and Axl overexpressing PCa cells to test the sufficiency of Axl in mediating dormancy. Determining the role of Axl and any other regulatory mechanisms of PCa DTC dormancy will be crucial in efforts to prevent lethal and incurable metastasis. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Brian W. Simons, Kenneth J. Pienta. Axl-hi DTCs in the bone marrow of a genetically engineered mouse model of prostate cancer exhibit decreased proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2017-4945

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.

Background: Though about half of newly diagnosed prostate cancer may not require immediate treatment, it remains the second most common cause of cancer death in the United States indicating a need for novel preventive and therapeutic treatments. Here we use expression based analysis to identify “druggable” targets which were then used to tailor screens for commercially available candidate inhibitors of prostate cancer. Methods: A list of 537 differentially expressed genes was developed by comparing expression profiles of prostate cancer from men with high risk localized disease that either metastasized or was cured following radical prostatectomy. Differentially expressed genes were compared to PharmGKB and Drugbank to link them to commercially available drugs. Identified drugs were assessed for their effects on cell viability, migration and invasion in androgen responsive and independent prostate cancer cell lines and in an ex-vivo embryonic development assay of prostate epithelial invasion. Results: Putative prostate cancer inhibitors roughly grouped into anti-neoplastic agents, tyrosine kinase inhibitors, statins, beta- blockers/beta-agonists, aldosterone/ATII inhibitors, catecholamine pathway related drugs, anti-platelets, immunosuppresants, leukotriene antagonists and anti-diabetic drugs. Multiple classes of drugs inhibited cell viability in a dose-dependent manner in prostate cancer cell lines with individual inhibitors in each group having disparate effects. A subset of drugs showed significant inhibition of cell migration, including Celecoxib, Metformin, Simvastatin, Iressa and Sunitinib. In addition, inhibition of prostate cell invasion into the surrounding mesenchyme was seen with several agents including Sorafenib and Metformin. Conclusions: Use of gene expression data and bioinformatics allowed for smaller scale screening of inhibitors enriched for putative effects on prostate cancer. Through these methods we identified inhibitors already shown to effect prostate cancer and in addition currently available drugs with previously unknown effects on prostate cancer.

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. Cancer Res; 73(9); 2718–36. ©2013 AACR.

Twelve Australian water dragons (Intellagama lesueurii) presented with acute neurologic dysfunction over a 2-month period. Clinical signs included obtundation, head tilt, loss of proprioceptive and righting reflexes, ataxia, and hyperesthesia. Various treatment protocols had no apparent effect on clinical signs or disease progression, and all 12 animals died or were euthanized. A retrospective review was carried out on tissue sets that were available for nine individuals. Four (44%) of the nine water dragons were classified as cases with long (≥8 days) duration of neurologic signs, and five (56%) of nine were classified as short (≤7 days) duration. On histopathology, all cases with a long duration of neurologic signs had brain lesions characterized by encephalomalacia (i.e., softening of the brain due to necrosis) and gitter cell infiltrates. In the three (33%) water dragons with the longest duration of clinical signs (>13 days), the necrosis was more severe and numerous tortuous vascular formati...

INTRODUCTION AND OBJECTIVES B7-H3 (CD276), part of the B7 superfamily, has been shown to play an immunomodulatory role, however its regulation, receptor and mechanism of action remain unclear. Protein levels of B7-H3 have been previously shown to relate to prostate cancer outcomes and currently, humanized monoclonal antibodies are being developed for clinical use (MGA271, Macrogenics). Here we use genomic expression data to examine the relationship of B7-H3 to prostate cancer outcomes, and molecular subtypes and explore its relationship to other gene sets and pathways. METHODS Prostatectomy tissue from 905 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Kruskal-Wallis tests were used to identify significant associations of B7-H3 expression with clinico-pathologic variables, and survival analysis was used to evaluate the prognostic value of B7-H3. Pearson’s correlation analyses were also performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy of promoter regions was derived in silico from chromosomal immune- precipitation (ChIP) data. RESULTS B7-H3 expression was positively associated with Gleason score (p < 0.01) and tumor stage (p < 0.01). High B7-H3 expression also correlated with the development of metastasis and prostate cancer specific mortality (HR of 3.4 and 2.4 respectively, p < 0.05 for both), but this was not significant on multi-variable analysis. B7-H3 was positively associated with ERG+ disease (n = 670, r = 0.85, p < 0.05) and AR expression (n = 670, r = 0.46, p < 0.001). B7-H3 was found to be one of the most correlated genes with AR (95th percentile) and ChIP analysis revealed androgen dependent AR binding upstream of B7-H3. Gene set enrichment analysis demonstrated a positive correlation with androgen signaling and a negative correlation with the defensins reactome (FDR

// Filipe LF. Carvalho 1,3 , Brian W. Simons 1,2 , Emmanuel S. Antonarakis 3 , Zeshaan Rasheed 3 , Nora Douglas 1 , Daniela Villegas 1 , William Matsui 3 , David M. Berman 1,3,4 1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 4 Departments of Pathology and Molecular Medicine and Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, Ontario, Canada Correspondence: David M. Berman, email: // Keywords : Circulating tumor cells; EpCAM; prostate cancer; prostate-specific antigen; TRAMP mouse Received : February 21, 2013 Accepted : March 04, 2013 Published : March 05, 2013 Abstract 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.