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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.

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.

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.

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.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of about 65-70%. Thus, additional molecular insights and representative models are critical for further identifying and evaluating new treatment modalities. Using MyoD-Cre mediated introduction of mutant K-RasG12D and perturbations in p53 we have developed a novel genetically engineered mouse model (GEMM) for RMS. Specifically, we directly crossed mice expressing MyoD promoter-regulated Cre-recombinase with germline p53Flox or Lox-Stop-Lox (LSL) knock-in alleles expressing oncogenic p53R172H and/or K-RasG12D mutants. The anatomic sites of primary RMS development observed in these mice recapitulated human disease, with the most frequent sites of tumor growth seen in the head, neck, extremities, and abdomen.We have confirmed RMS histology and diagnosis through hematoxylin and eosin (H&E) staining, as well as positive immunohistochemistry (IHC) staining for desmin, myogenin, and phosphotungstic acid hematoxylin (PTAH). We established cell lines from several of the GEMM tumors with the ability to engraft and develop tumors in immunocompetent mice with similar histological and staining features as the primary tumors. Furthermore, injection of syngeneic RMS lines via tail vein 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. Specifically, we noted alterations in gene ontologies including immune response, metabolism and mRNA processing. Finally, pre-clinical use of these murine RMS lines demonstrated similar therapeutic responsiveness to relevant chemotherapy and targeted therapies as human cell line models.Summary StatementWe have developed a new conditional genetically engineered mouse model of rhabdomyosarcoma (RMS) with homologous molecular signature to human RMS that provides valuable pre-clinical models for evaluating novel therapies.

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.

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.

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.

// 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.

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.

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 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.

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.

BackgroundC-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.MethodsFirst, 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 pResultsIn vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (pConclusionCombinatorial 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.

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) post-translational 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 for the first time that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs. Citation Format: Takumi Shiraishi, Phuoc T. Tran, Reem Malek, Audrey Lafargue, Mustafa Barbhuiya, Xing Wang, Brian Simons, Matthew Ballew, Katriana Nugent, Jennifer Groves, Russell Williams, Hailun Wang, James Verdone, Gokben Yildirir, Roger Henry, Bin Zhang, John Wong, Ken Wang, Barry Nelkin, Kenneth Pienta, Dean Felsher, Natasha Zachara, Kekoa Taparra. O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B11.

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.

Objective: Dose optimization and pharmacokinetic evaluation of alpha emitting Radium-223 dichloride (223RaCl2) by planar gamma camera or single photon emission computed tomographic (SPECT) imaging are hampered by the low photon abundance and injection activities. Here, we demonstrate SPECT of 223Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223Ra were imaged using a next generation dedicated small animal SPECT by detecting the low energy photon emissions from 223Ra. Localization of the therapeutic agent was verified by whole body and whole limb autoradiography and its effect determined 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. Line sources of 223Ra in both air and in a water scattering phantom gave linear response functions with provide 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 were correlated with DNA damage from the alpha particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223Ra, a recently approved alpha emitting radionuclide. This approach allows quantitative assessment of 223Ra distribution and may provide radiation dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning.

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.

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...

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.

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.

Debunking the mucoadhesion myth: Nonsticky particles for enhanced pulmonary drug delivery., 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

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 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.

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.

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.

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

Here we show that activation of the canonical WNT/β-catenin pathway increases the expression of stem cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signaling alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this process in the regulation of glioma motility. Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of cells with intranuclear signal for β-catenin in the infiltrating edge of tumor compared to central tumor parenchyma. These findings suggest that canonical WNT/β-catenin pathway is a critical regulator of GBM invasion and may represent a potential therapeutic target.

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.

Zhenhua Huang 1,2,3 , Paula J Hurley 1 , Brian W Simons 1,2,3 , Luigi Marchionni 2,3 , David M Berman 1,2,3 , Ashley E Ross 1 and Edward M Schaeffer 1,2,3 1 Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. 2 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. 3 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. Received: June 26, 2012; Accepted: June 30, 2012; Published: July 2, 2012; Keywords: Sox9, prostate, development, cancer, initiation Correspondence: Edward M Schaeffer, email: // // Abstract Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. The molecular mechanisms driving prostate carcinogenesis are complex; with several lines of evidence suggesting that the re-expression of conserved developmental programs plays a key role. In this study, we used conditional gene targeting and organ grafting, to describe conserved roles for the transcription factor Sox9 in the initiation of both prostate organogenesis and prostate carcinogenesis in murine models. Abrogation of Sox9 expression prior to the initiation of androgen signaling blocks the initiation of prostate development. Similarly, Sox9 deletion in two genetic models of prostate cancer (TRAMP and Hi-Myc) prevented cancer initiation. Expression profiling of Sox9-null prostate epithelial cells revealed that the role of Sox9 in the initiation of prostate development may relate to its regulation of multiple cytokeratins and cell adherence/polarity. Due to its essential role in cancer initiation, manipulation of Sox9 targets in at-risk men may prove useful in the chemoprevention of prostate cancer.

The therapeutic goal in peripheral arterial disease (PAD) patients is to restore blood flow to ischemic tissue. Stem cell transplantation offers a new avenue to enhance arteriogenesis and angiogenesis. Two major problems with cell therapies are poor cell survival and the lack of visualization of cell delivery and distribution. To address these therapeutic barriers, allogeneic bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in alginate impregnated with a radiopaque contrast agent (MSC-Xcaps.) In vitro MSC-Xcap viability by a fluorometric assay was high (96.9% ± 2.7% at 30 days postencapsulation) and as few as 10 Xcaps were visible on clinical x-ray fluoroscopic systems. Using an endovascular PAD model, rabbits (n = 21) were randomized to receive MSC-Xcaps (n = 6), empty Xcaps (n = 5), unencapsulated MSCs (n = 5), or sham intramuscular injections (n = 5) in the ischemic thigh 24 hours postocclusion. Immediately after MSC transplantation and 14 days later, digital radiographs acquired on a clinical angiographic system demonstrated persistent visualization of the Xcap injection sites with retained contrast-to-noise. Using a modified TIMI frame count, quantitative angiography demonstrated a 65% improvement in hind limb perfusion or arteriogenesis in MSC-Xcap-treated animals versus empty Xcaps. Post-mortem immunohistopathology of vessel density by anti-CD31 staining demonstrated an 87% enhancement in angiogenesis in Xcap-MSC-treated animals versus empty Xcaps. MSC-Xcaps represent the first x-ray-visible cellular therapeutic with enhanced efficacy for PAD treatment. Disclosure of potential conflicts of interest is found at the end of this article.

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.

The ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic-modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific gene promoter DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming, we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis, and add to means of assessing iPS for safety. Cancer Res; 70(19); 7662–73. ©2010 AACR.

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.

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.

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.

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., Invasion of malignant cells involves changes in cytoskeleton dynamics. Here the authors identify absent in melanoma 1 as an actin binding protein and show that it regulates cytoskeletal remodeling and cell migration in prostate epithelial cells, acting as a metastatic suppressor in cancer cells.

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.

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. Cancer Prev Res; 8(8); 683–92. ©2015 AACR.

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. Cancer Res; 75(20); 4322–34. ©2015 AACR.