Murine Model Of Prostate Cancer Research Articles

MP70-17 DECIPHERING THE EFFECTS OF OMEGA-3 FATTY ACID SUPPLEMENTS ON PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2018MP70-17 DECIPHERING THE EFFECTS OF OMEGA-3 FATTY ACID SUPPLEMENTS ON PROSTATE CANCER Gabriel Lachance, Nikunj Geveriya, Karine Robitaille, Alain Bergeron, Valérie Picard, Mickael Leclerc, Charles Joly, Yves Fradet, Arnaud Droit, André Marette, and Vincent Fradet Gabriel LachanceGabriel Lachance More articles by this author , Nikunj GeveriyaNikunj Geveriya More articles by this author , Karine RobitailleKarine Robitaille More articles by this author , Alain BergeronAlain Bergeron More articles by this author , Valérie PicardValérie Picard More articles by this author , Mickael LeclercMickael Leclerc More articles by this author , Charles JolyCharles Joly More articles by this author , Yves FradetYves Fradet More articles by this author , Arnaud DroitArnaud Droit More articles by this author , André MaretteAndré Marette More articles by this author , and Vincent FradetVincent Fradet More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inflammation is one contributing factors to prostate cancer (PCa). We have shown that an omega-3 (?3)-rich diet decrease PCa tumor growth by favourably modulating the inflammatory response. In this study, we tested the specific effects of purified ?3 fatty acids (FA) sub-types on tumor growth, inflammatory response, gut microbiota and tumor gene expression compared to ?6 and ?9 in a murine PCa model. METHODS C57BL/6 mice were daily supplemented with purified FA (?3-eicosapentaenoic acid (EPA), ?3-docosahexaenoic acid (DHA), ?6-arachidonic acid (AA) or ?9-rich high-oleic sunflower oil (HOSO)), and injected s.c. with TRAMP-C2 mouse PCa cells. Feces were collected weekly while blood and tumors were collected at end point. FA incorporation was analyzed in blood and tumors. Bacterial content from fresh fecal DNA was profiled using 16S rRNA metagenomic sequencing. Cytokine and gene expression profiles were analyzed in tumors using BioPlex technology and high-throughput RNA sequencing, respectively. RESULTS We measured a specific FA incorporation of each group into red blood and tumor cells. Interestingly, only tumors of EPA-fed mice had a reduced tumor growth than HOSO-fed control mice. Many pro-inflammatory cytokines were expressed at a significantly lower level in tumors of ?3-EPA and –DHA fed mice compared to ?6-AA-fed mice. We found gene expression changes only in the EPA-fed mice group: differentially expressed genes were related to angiogenesis and inflammatory pathways. We confirmed reduced expression of VEGFR and VEGF as well as smaller CD31-stained blood vessels in EPA-fed tumors. Finally, gut microbiota metagenomic analysis revealed that most bacterial shifts were found in DHA-fed animals. Interestingly, while we observed little changes for the gut microbiota of AA-fed animals, we were able to predict metagenome functional content and found an increase in functions associated to butirosin and neomycin biosynthesis, renal cell carcinoma and glycerophospholipid metabolism compared to control animals. CONCLUSIONS Both ?3 subtypes modulated intra-tumoral inflammation. Interestingly, DHA was the FA driving the biggest changes in fecal microbiota. However, we found only EPA to significantly reduce PCa tumor growth, potentially achieved by gene expression modulation leading to a reduced angiogenesis and inflammation. Together, these results suggest that both ?3 subtypes modulate inflammation but possibly via separate mechanisms. Our work supports the use of EPA for targeting PCa progression. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e941 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Gabriel Lachance More articles by this author Nikunj Geveriya More articles by this author Karine Robitaille More articles by this author Alain Bergeron More articles by this author Valérie Picard More articles by this author Mickael Leclerc More articles by this author Charles Joly More articles by this author Yves Fradet More articles by this author Arnaud Droit More articles by this author André Marette More articles by this author Vincent Fradet More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Detection Threshold and Reproducibility of 68Ga-PSMA11 PET/CT in a Mouse Model of Prostate Cancer.

To improve prostate-specific membrane antigen (PSMA)-targeted theranostic approaches, robust murine models of prostate cancer are needed. However, important characteristics of preclinical PSMA imaging-that is, the reproducibility of the imaging signal and the relationship between quantitative cell surface PSMA expression and lesion detectability with small-animal PET/CT-have not been defined yet. Methods: Murine prostate cancer RM1 sublines (ras myc transformed cells of C57BL/6 prostate origin) expressing varying levels of human PSMA were injected into the shoulder of C57BL/6 mice on day 0. 68Ga-PSMA11 PET/CT was performed on days 7 and 8 and interpreted by 2 masked readers to determine interday and interreader reproducibility. PSMA expression was quantified on days 7 and 8 by flow cytometry of fine-needle aspiration tumor biopsy samples. Cell surface PSMA expression was correlated with PET signal. The threshold for PET positivity was based on the clinical Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE) criteria. Results: The maximum and average percentages of injected 68Ga-PSMA11 activity per gram of tissue (%IA/g) correlated nearly perfectly as determined by 2 independent readers and on 2 separate days (intraclass correlation coefficient, 1.00/0.89 and 0.95/0.88, respectively). The number of PSMA molecules per cell increased from the RM1-yellow fluorescent protein subline (PSMA-; 2,000/cell) to the RM1-low subline (PSMA+; 17,000/cell), the RM1-medium subline (PSMA++; 22,000/cell), and the RM1-PGLS subline (PSMA-positive, green fluorescent protein-positive, and luciferase-positive; PSMA+++; 45,000/cell). Expression levels correlated with the visual positivity rate on 68Ga-PSMA11 PET and with the PSMA PET %IA/g. The PSMA threshold for PET positivity was approximately 20,000 per cell. Signal correlation was close at lower PSMA levels (RM1-low to RM1-medium; 10-23 %IA/g) but was lost at higher PSMA levels (RM1-medium to RM1-PGLS; 23-27 %IA/g). Conclusion: The in vivo relationship between 68Ga-PSMA11 PET/CT and PSMA expression level in a murine model of prostate cancer was robust for lower cell surface PSMA expression levels (≤22,000/cell). Thus, preclinical 68Ga-PSMA11 PET/CT can be used as an imaging biomarker to test PSMA-targeted interventions in murine models.

The dialyzable leukocyte extract TransferonTM inhibits tumor growth and brain metastasis in a murine model of prostate cancer

Prostate cancer (PCa) is the second most frequently diagnosed cancer in men worldwide. Dialyzed Leukocyte Extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that improve clinical responses in various diseases. Here, we analyzed the effects of TransferonTM, a commercial DLE with characterized active pharmaceutical ingredient and proven batch-to-batch reproducibility, in preclinical models of PCa. We employed v-Src-transformed murine prostate epithelial (PEC-Src) cells, which recapitulate the transcriptional profiles in human PCa, can be grown in immunocompetent mice, and consistently form bone and brain metastases. In vitro, TransferonTM did not induce cytotoxicity nor alterations in migration /invasion of PEC-Src cells. In vivo, TransferonTM reduced metastatic dissemination after intracardiac injection of PEC-Src and inhibited tumor growth of subcutaneous isotransplants. The antineoplastic effect of TransferonTM correlated with changes in tumor infiltration, increased serum concentrations of IL-12 and CXCL1, and reduced levels of VEGF. Our results suggest that the antineoplastic effect produced by TransferonTM is due to its immunomodulatory activity and not by a direct effect on cancer cells, and indicate that TransferonTM could be beneficial as adjuvant therapy in PCa patients.

Combining immune check-point blockade and cryoablation in an immunocompetent hormone sensitive murine model of prostate cancer.

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. 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. 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. 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.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

Novel PAP-derived vaccine for the treatment of advanced prostate cancer

Background: Treatment options for patients with advanced prostate cancer (PC) still remain limited and rarely curative. The prostatic acid phosphatase (PAP) is prostate specific protein over-expressed in more than 90% of prostate tumours. Although an FDA-approved vaccine for the treatment of advanced prostate disease, PROVENGE® (sipuleucel-T), has been shown to prolong survival, the precise sequence of the PAP protein responsible for the outcome remains unknown. The aim of the study is to develop a cheaper and more effective, Prostatic Acid Phosphatase (PAP)-based, vaccine for the treatment of advanced prostate cancer. Our laboratory has previously shown that a 15mer PAP-derived vaccine, when injected as a DNA vaccine, could induce PAP-specific T-cell responses and reduce tumor growth in a syngeneic heterotopic murine prostate cancer model. We have subsequently developed an elongated (42mer) PAP-derived peptide containing Leucine instead of an Alanine at position 14.

Prostate Cancer Cells Express More Androgen Receptor (AR) Following Androgen Deprivation, Improving Recognition by AR-Specific T Cells.

Androgen deprivation is the primary therapy for recurrent prostate cancer, and agents targeting the androgen receptor (AR) pathway continue to be developed. Because androgen-deprivation therapy (ADT) has immmunostimulatory effects as well as direct antitumor effects, AR-targeted therapies have been combined with other anticancer therapies, including immunotherapies. Here, we sought to study whether an antigen-specific mechanism of resistance to ADT (overexpression of the AR) may result in enhanced AR-specific T-cell immune recognition, and whether this might be strategically combined with an antitumor vaccine targeting the AR. Androgen deprivation increased AR expression in human and murine prostate tumor cells in vitro and in vivo The increased expression persisted over time. Increased AR expression was associated with recognition and cytolytic activity by AR-specific T cells. Furthermore, ADT combined with vaccination, specifically a DNA vaccine encoding the ligand-binding domain of the AR, led to improved antitumor responses as measured by tumor volumes and delays in the emergence of castrate-resistant prostate tumors in two murine prostate cancer models (Myc-CaP and prostate-specific PTEN-deficient mice). Together, these data suggest that ADT combined with AR-directed immunotherapy targets a major mechanism of resistance, overexpression of the AR. This combination may be more effective than ADT combined with other immunotherapeutic approaches. Cancer Immunol Res; 5(12); 1074-85. ©2017 AACR.

Inhibition of prostate cancer growth by immunization with a GM-CSF-modified mouse prostate cancer RM-1 cell vaccine in a novel murine model.

Advanced prostate cancer is difficult to treat owing to a lack of effective approaches for disrupting immune tolerance. C57BL/6 male and female mice implanted with viable RM-1 cells represent a novel murine model of advanced prostate cancer for studying antitumor effects following immunization with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified RM-1 cell vaccine, which has been described previously. In vitro cytotoxic activity and cytokine secretion experiments were conducted to investigate the antitumor response. The cytotoxicity profile of splenocytes from female mice immunized against RM-1 cells primarily involved cytotoxic T lymphocyte (CTL) lysis and, to a lesser extent, natural killer (NK) cell lysis. NK cell lysis was also observed in males, which exhibited no evidence of CTL lysis. The secretion of interferon-γ in the GM-CSF-modified cell vaccine group was significantly increased compared with the other groups. The level of interleukin-4 was low. To investigate the antitumor immune response further, cluster of differentiation 4 (CD4) T cells and CD8 T cells were analyzed in the spleens and tumors of female mice receiving the GM-CSF-modified RM-1 cell vaccine. Unlike female mice, males exhibited the highest proportion of NK cells in the spleen. NK cells were not detected in the tumor tissue in any of the groups. The difference between the sexes may explain the specificity of the immune response, as females are intolerant to prostate antigens whereas males are. This model is clinically relevant as it translates to human immunology and offers an effective and convenient method for studying immunotherapy for prostate cancer.

Progrès dans l’étude du cancer de la prostate : la culture cellulaire en trois dimensions reproduit ex vivo les caractéristiques des tumeurs prostatiques

Despite new therapeutics options, Prostate Cancer (PCa) remains a public health challenge because of its high incidence and mortality. Limits in PCa research come from the lack of in vitro and in vivo models that mimic the human disease. Currently, 2D in vitro tissue culture models of PCa are widely used but they present numerous limits. They do not reproduce cellular morphology, tissue architecture, inter-patients and intratumor heterogeneity. Furthermore, they lack two key components of PCa tumors, the tumoral microenvironment and the cancer stem cells. In vivo murine models of PCa cannot be representative of all the genetic alterations known in prostate tumors and they hardly reproduce the pathophysiology of human metastatic progression. Consequently, the physiology of these in vitro and in vivo models do not well represent patients tumors. 3D cell cultures overcome many of these limits by sharing morphologic characteristics with in vivo tumors as well as reproducibility of in vitro models. 3D models of PCa include spheroids derived from tumor cell lines, and organoids, derived from patient. In 3D cell cultures, cell fitness is maintained, the physiological cells-cells and cell-matrix interactions are restored and an extracellular matrix surrounds the cells. Organoids, generated from PCa primary tumors or metastases, allow studies on cancer stem cells and their microenvironment. Moreover, organoids retain genetic integrity of PCa tumors. PCa organoid model is an innovative tool that offers great perspectives of therapeutic screening. In the future, organoids generated from patients' biopsies may also lead to personalized medicine.

Synthesis, Positron Emission Tomography Imaging, and Therapy of Diabody Targeted Drug Lipid Nanoparticles in a Prostate Cancer Murine Model.

The blood clearance of chemotherapeutic drugs such as doxorubicin (Dox) can be extended by incorporation into lipid nanoparticles (LNPs) and further improved by tumor targeting with antibody fragments. We used positron emission tomography (PET) imaging in a murine prostate cancer model to evaluate tumor targeting of LNPs incorporating Dox and antiprostate-specific membrane antigen (PSMA) diabodies. Dox-LNPs were generated by mixing or covalent attachment to water soluble distearoylphosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG)2000. Cu-64 PET imaging was performed with DOTA-conjugated Dox, PEG-LNP, or an anti-PSMA site-specific cysteine-diabody. Since the mixture Dox+PEG-LNP was unstable in serum, further studies utilized Dox covalently bound to LNP ± covalently bound DOTA-cys-diabody (cys-DB)-LNP. Blood clearance of covalent Dox-PEG-LNP was slower than Dox alone or Dox+PEG-LNP. PET imaging of 64Cu-DOTA-Dox-PEG-LNP reached a maximum of 10% ID/g in tumors compared with 3% ID/g of 64Cu-DOTA-Dox, due to the prolonged blood clearance. Mixing 64Cu-DOTA-cys-DB-PEG-LNP with covalent Dox-PEG-LNP gave LNPs containing both drug and tumor targeting cys-DB. The mixed LNPs exhibited increased tumor uptake (15% ID/g) versus untargeted 64Cu-DOTA-Dox-PEG-LNPs (10% ID/g) demonstrating feasibility of the approach. Based on these results, a therapy study with mixed LNPs containing cys-DB-LNP and either Dox-LNP or the antitubulin drug auristatin-LNP showed significant reduction of tumor growth with the auristatin-diabody-LNP mixture, but not the Dox-diabody-LNP mixture.

Fibroblast growth factor modulates mast cell recruitment in a murine model of prostate cancer.

Mast cells are important modifiers of prostate tumor microenvironment. The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) system plays a non-redundant autocrine/paracrine role in the growth, vascularization and progression of prostate tumors. Accordingly, the FGF antagonist long pentraxin-3 (PTX3) and the PTX3-derived small molecule FGF-trap NSC12 have been shown to inhibit the growth and vascularization of different FGF-dependent tumor types, including prostate cancer. In this study, we show that recombinant FGF2 is able to cause mast cell recruitment in vivo in the Matrigel plug assay. Conversely, PTX3 overexpression in transgenic mice or treatment with the FGF inhibitor NSC12 result in a significant inhibition of the growth and vascularization of TRAMP-C2 tumor grafts, a murine model of prostate cancer, that were paralleled by a decrease of mast cell infiltrate into the lesion. These data confirm and extend previous observations about the capacity of mast cells to respond chemotactically to FGF2 stimulation and provide evidence about a relationship among mast cell recruitment, angiogenesis, and tumor growth in human prostate adenocarcinoma.

Exposure to maternal obesogenic diet worsens some but not all pre-cancer phenotypes in a murine genetic model of prostate cancer.

Prostate cancer research has been predominantly focused on adult exposures and risk factors. However, because the prostate develops during gestation and early life, exposure to external factors, such as obesity, during development could affect the prostate cancer progression in adults. Our previous work demonstrated that exposure to a high fat/high sugar (HF/HS) diet during gestation and until weaning stimulated prostate hyperplasia and altered the Pten/Akt pathway in adult mice fed a normal diet after weaning. Here, we asked whether maternal exposure to HF/HS would worsen prostate phenotypes in mice lacking Pten, a widely accepted driver of prostate cancer. We found that, at six weeks of age, both Chow (control)—and HF/HS-exposed Pten knockout mice showed evidence of murine PIN that included ducts with central comedo necrosis but that the HF/HS exposure did not influence murine PIN progression. The Pten knockout mice exposed to HF/HS in utero had significantly more mitotic cells than Pten knockouts exposed to Chow diet. In the Pten null background, the maternal HF/HS diet enhanced proliferation but did not have an additive effect on Akt activation. We observed neuroendocrine differentiation in Pten knockout mice, a phenotype that had not been previously described in this model.

AKT-mediated stabilization of histone methyltransferase WHSC1 promotes prostate cancer metastasis.

Loss of phosphatase and tensin homolog (PTEN) and activation of the PI3K/AKT signaling pathway are hallmarks of prostate cancer (PCa). However, these alterations alone are insufficient for cells to acquire metastatic traits. Here, we have shown that the histone dimethyl transferase WHSC1 critically drives indolent PTEN-null tumors to become metastatic PCa. In a PTEN-null murine PCa model, WHSC1 overexpression in prostate epithelium cooperated with Pten deletion to produce a metastasis-prone tumor. Conversely, genetic ablation of Whsc1 prevented tumor progression in PTEN-null mice. Molecular characterization revealed that increased AKT activity due to PTEN loss directly phosphorylates WHSC1 at S172, preventing WHSC1 degradation by CRL4Cdt2 E3 ligase. Increased WHSC1 expression transcriptionally upregulates expression of RICTOR, a pivotal component of mTOR complex 2 (mTORC2), to further enhance AKT activity. Therefore, the AKT/WHSC1/mTORC2 signaling cascade represents a vicious feedback loop that elicits unrestrained AKT signaling. Furthermore, we determined that WHSC1 positively regulates Rac1 transcription to increase tumor cell motility. The biological importance of a WHSC1-mediated signaling cascade is substantiated by patient sample analysis in which WHSC1 signaling is tightly correlated with disease progression and recurrence. Taken together, our findings highlight a pivotal link between an epigenetic regulator, WHSC1, and key intracellular signaling molecules, AKT, RICTOR, and Rac1, to drive PCa metastasis.

Interleukin-6 and prostate cancer: Current developments and unsolved questions

Interleukin (IL)-6 is a pro-inflammatory cytokine that is expressed in prostate tumors and in the stromal tumor micro-enviroment. It is known to regulate proliferation, apoptosis, angiogenesis, and differentiation. The signaling pathway of Janus kinase and signal transducer and activator of transcription (STAT)3, which is activated by IL-6, is in the focus of scientific investigations for improved treatment approaches. Different effects of IL-6 and/or STAT3 on tumor cell growth have been observed in human and murine prostate cancer (PCa) models. Experimental therapies have been proposed in order to block the IL-6/STAT3 signaling pathway. In this context, the anti-IL-6 antibody siltuximab (CNTO 328) has been demonstrated to inhibit growth of prostate tumors in vitro and in vivo and delays progression towards castration resistance. However, clinically, the anti-IL-6 antibody was not successful as a monotherapy in phase II studies in patients with metastatic PCa. IL-6 is implicated in regulation of cellular stemness by increasing phosphorylation of STAT3. The cytokine has also a role in development of resistance to the non-steroidal anti-androgen enzalutamide. Endogenous inhibitors of IL-6 are suppressors of cytokine signaling and protein inhibitors of activated STAT. Although they inhibit signal transduction through STAT3, they may also exhibit anti-apoptotic effects. On the basis of complexity of IL-6 action in PCa, an individualized approach is needed to identify patients who will benefit from anti-IL-6 therapy in combination with standard treatments.

Investigating RUNX1 and RUNX2 in prostate cancer.

232 Background: Prostate cancer is the most commonly diagnosed cancer in men and is increasing in incidence. Current treatment for metastatic disease is initially hormone treatment and chemotherapy. While this produces a response in the vast majority of patients, it is not curative. Novel therapeutic agents could provide a further treatment strategy and it is therefore important to understand the molecular pathways which contribute to this disease. The RUNX family of genes have been implicated as both oncogenes and tumor suppressors, with their role being context dependent. It has been reported that RUNX2 may have a tumor suppressor role in early stage prostate cancer but that this may switch to an oncogenic role in later stage disease. Methods: This project examines the expression of both RUNX1 and RUNX2 in a tissue microarray (TMA) of human prostate cancers. In parallel to the human studies both RUNX1 and RUNX2 were investigated in murine models of prostate cancer. Results: Analysis of the TMA showed that increasing levels of RUNX2 were associated with increasing Gleason grade, shorter time to relapse and poorer survival; the opposite effect was seen in RUNX1, where low levels of protein expression correlated with shorter survival. In concordance with the human results, reducing levels of RUNX2 in mouse models was associated with delayed tumor initiation, and smaller, less cystic tumors at endpoint. The opposite effect was seen with Runx1 where low levels resulted in more aggressive and invasive disease. Finally, when loss of both genes was combined in a model with loss of Pten and stimulation of WNT, survival was significantly reduced compared to controls. Conclusions: This study suggests that RUNX2 expression may correlate with a poorer prognosis in prostate cancer, while expression of RUNX1 is likely to be associated with an improved outcome.

Diffusion radiomics analysis of intratumoral heterogeneity in a murine prostate cancer model following radiotherapy: Pixelwise correlation with histology.

To investigate the biological meaning of apparent diffusion coefficient (ADC) values in tumors following radiotherapy. Five mice bearing TRAMP-C1 tumor were half-irradiated with a dose of 15 Gy. Diffusion-weighted images, using multiple b-values from 0 to 3000 s/mm2 , were acquired at 7T on day 6. ADC values calculated by a two-point estimate and monoexponential fitting of signal decay were compared between the irradiated and nonirradiated regions of the tumor. Pixelwise ADC maps were correlated with histological metrics including nuclear counts, nuclear sizes, nuclear spaces, cytoplasmic spaces, and extracellular spaces. As compared with the nonirradiated region, the irradiated region exhibited significant increases in ADC, extracellular space, and nuclear size, and a significant decrease in nuclear counts (P < 0.001 for all). Optimal ADC to differentiate the irradiated from nonirradiated regions was achieved at a b-value of 800 s/mm2 by the two-point method and monoexponential curve fitting. ADC positively correlated with extracellular spaces (r = 0.74) and nuclear sizes (r = 0.72), and negatively correlated with nuclear counts (r = -0.82, P < 0.001 for all). As a radiomic biomarker, ADC maps correlating with histological metrics pixelwise could be a means of evaluating tumor heterogeneity and responses to radiotherapy. 1 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:483-489.

Oxygen Enhanced Optoacoustic Tomography (OE-OT) Reveals Vascular Dynamics in Murine Models of Prostate Cancer.

Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes, hence non-invasive imaging of oxygen supply and demand in tumours could improve disease staging and therapeutic monitoring. Optoacoustic tomography (OT) is an emerging clinical imaging modality that provides static images of endogenous haemoglobin concentration and oxygenation. Here, we demonstrate oxygen enhanced (OE)-OT, exploiting an oxygen gas challenge to visualise the spatiotemporal heterogeneity of tumour vascular function. We show that tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. LNCaP tumours showed a spatially heterogeneous response within and between tumours, with a substantial but slow response to the gas challenge, aligned with ex vivo analysis, which revealed a generally perfused and viable tumour with marked areas of haemorrhage. PC3 tumours had a lower fraction of responding pixels compared to LNCaP with a high disparity between rim and core response. While the PC3 core showed little or no dynamic response, the rim showed a rapid change, consistent with our ex vivo findings of hypoxic and necrotic core tissue surrounded by a rim of mature and perfused vasculature. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vessel function assessed ex vivo. OE-OT provides a non-invasive approach to reveal the complex dynamics of tumour vessel perfusion, permeability and vasoactivity in real time. Our findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease as well as in patient stratification for chemo- and radio-therapy.

Role of Epigenetic Modification and Immunomodulation in a Murine Prostate Cancer Model.

Decreased expression of highly immunogenic cancer-testis antigens (CTA) might help tumor to achieve low immunogenicity, escape immune surveillance and grow unimpeded. Our aim was to evaluate CTA expression in tumor and normal tissues and to investigate possible means of improving the immune response in a murine prostate cancer (CaP) model by using the combination of epigenetic modifier 5-azacitidine (5-AzaC) and immunomodulator lenalidomide. No study to date has examined the effect of this combination on the prostate cancer or its impact on antigen-presenting cells (APC). Gene microarrays were performed to compare expression of several CTA in murine prostate cancer (RM-1 cells) and normal prostate. RM-1 cells were treated with 5-AzaC and real-time PCR was performed to investigate the expression of several CTA. Western blotting was used to determine whether expression of CTA-specific mRNA induced by 5-AzaC resulted in increase in the corresponding protein. Effect of the epigenetic agents and immunomodulators was assessed on dendritic cells (DC) using flow cytometry, ELISA and T-cell proliferation assay. Gene arrays demonstrated decreased expression of 35 CTA in CaP tissue compared to normal prostate. 5-AzaC treatment of RM-1 prostate cancer cells upregulated the expression of all 13 CTA tested in a dose-dependent fashion. DC were treated with 5-AzaC and lenalidomide and the expression of surface markers MHC Class I, MHC Class II, CD80, CD86, CD 205, and CD40 was increased. Combination of 5-AzaC and lenalidomide enhances the ability of DC to stimulate T-cell proliferation in mixed leukocyte reaction. Secretion of IL-12 and IL-15 by DC increased significantly with addition of 5-AzaC or 5-AzaC and lenalidomide. Decreased expression of CTA by prostate cancer may be a means of escaping immune monitoring. Combination of epigenetic modifications and immunomodulation by 5-AzaC and lenalidomide increased tumor immunogenicity and enhanced DC function and may be used in the treatment of advanced prostate cancer. Prostate 77: 361-373, 2017. © 2016 Wiley Periodicals, Inc.

TAT modified and lipid - PEI hybrid nanoparticles for co-delivery of docetaxel and pDNA.

Co-delivery of anticancer drugs and gene is promising to generate synergistic anticancer effects. Surface modification of nanocarriers with specific ligands could further assist in targeting and internalization of the nanocarriers into specific cell populations, such as cancers and disease organs. The aim of the study reported here is to develop Cell-penetrating peptides (CPPs) modified lipid - PEI hybrid nanoparticles (LPNs) for effective co-delivery of docetaxel (DTX) and plasmid DNA (pDNA) for combination chemotherapy. RKKRRQRRR peptide (TAT) modified, DTX and pDNA loaded LPNs (TAT-DTX/pDNA LPNs) were prepared and evaluated in PC3 cancer cells (in vitro) and in a murine prostate cancer model (in vivo). The results illustrated that the in vitro anticancer effect, in vitro transfection efficiency, in vivo antitumor and gene delivery efficacy of TAT-DTX/pDNA LPNs have advantages over other formulation tested. The results demonstrated that TAT-DTX/pDNA LPNs could be a promising co-delivery nano-system to achieve therapeutic efficacy for treatment of cancer.