Orthotopic Prostate Cancer Model Research Articles

Zinc ions activate AKT and promote prostate cancer cell proliferation via disrupting AKT intramolecular interaction.

Prostate is a zinc rich organ and the physiological function of the abundant zinc ions is relatively less understood. AKT kinase is a pivotal regulator downstream of cytokines, growth factors and other extracellular stimuli, and the attachment of its PH domain to PtdIns-3,4,5-P3 (PIP3) and the subsequent phosphorylation of its kinase domain by PDPK1 are considered important for its activation. Herein, we report a regulatory mechanism of AKT kinase by zinc ions. Mechanistically, zinc ions directly bind to AKT and facilitate AKT activation through disrupting the interaction between PH and kinase domains within AKT molecule. Consistently, AKT1-H89A/E91A mutant (zinc-binding-deficient) fails to respond to zinc ions and exhibits strong interaction between PH and kinase domains, and it is less oncogenic in orthotopic xenograft model of prostate cancer. On the other hand, the AKT1-W80L mutant with minimum intra-molecular interaction between PH and kinase domains shows strong tumor promoting capacity although it could not be further stimulated by zinc ions. Overall, this study reveals a distinctive regulatory mechanism of AKT activation and implies a tumor promoting role of the zinc ions in prostate cancer.

Silencing SPP1 in M2 macrophages inhibits the progression of castration-resistant prostate cancer via the MMP9/TGFβ1 axis.

M2 macrophages can promote the progression of castration-resistant prostate cancer (CRPC), but the specific mechanism is still unclear. Therefore, we are preliminarily exploring the molecular mechanism by which M2 macrophages regulate the progression of CRPC. The genes positively correlated with CRPC and with the most significant differences in the GEO32269 dataset were obtained. Database and immunofluorescence experiments were used to validate the localization of secreted phosphoprotein 1 (SPP1) in localized prostate cancer (PCa), hormone-sensitive prostate cancer (HSPC), and CRPC tumor tissues. The function of SPP1 in M2 macrophages was verified through cell scratch, Transwell, and an orthotopic PCa model. PCa database and Western blot were used to verify the relationship between SPP1 and matrix metallopeptidase 9 (MMP9), as well as the ability of MMP9 in M2 macrophages to promote epithelial-mesenchymal transition (EMT) in PCa cells. The primary localization of SPP1 in prostate and CRPC tissues is in macrophages. Silencing SPP1 expression in M2 macrophages promotes their polarization towards the M1 phenotype and significantly inhibits the malignant progression of PCa in vitro and in vivo. SPP1 promotes the expression of MMP9 through the PI3K/AKT signaling pathway in M2 macrophages. Furthermore, MMP9 enhances the EMT and migratory capabilities of PC3 cells by activating the TGFβ signaling pathway. We have found that the high expression of SPP1 in M2 macrophages promotes the progression of CRPC through cell-cell interactions. These findings can contribute to the development of novel therapeutic approaches for combating this deadly disease.

An orthotopic prostate cancer model for new treatment development using syngeneic or patient-derived tumors.

There are limited preclinical orthotopic prostate cancer models due to the technical complexity of surgical engraftment and tracking the tumor growth in the mouse prostate gland. Orthotopic xenografts recapitulate the tumor microenvironment, tumor stromal interactions, and clinical behavior to a greater extent than xenografts grown at subcutaneous or intramuscular sites. This study describes a novel micro-surgical technique for orthotopically implanting intact tumors pieces from cell line derived (transgenic adenocarcinoma mouse prostate [TRAMP]-C2)or patient derived (neuroendocrine prostate cancer [NEPC])tumors in the mouse prostate gland and monitoring tumor growth using magnetic resonance (MR) imaging. The TRAMP-C2 tumors grew rapidly to a predetermined endpoint size of 10 mm within 3 weeks, whereas the NEPC tumors grew at a slower rate over 7 weeks. The tumors were readily detected by MRand confidently identified when they were approximately 2-3 mm in size. The tumors were less well-defined on CT. The TRAMP-C2 tumors were characterized by amorphous sheets of poorly differentiated cells similar to a high-grade prostatic adenocarcinoma and frequent macroscopic peritoneal and lymph node metastases. In contrast, the NEPC's displayed a neuroendocrine morphology with polygonal cells arranged in nests and solid sheets and high count. There was a local invasion of the bladder and other adjacent tissues but no identifiable metastases. The TRAMP-C2 tumors were more hypoxic than the NEPC tumors. This novel preclinical orthotopic prostate cancer mouse model is suitable for either syngeneic or patient derived tumors and will be effective in developing and advancing the current selection of treatments for patients with prostate cancer.

Abstract 4182: Development and evaluation of orthotopically and subcutaneously implanted luciferase-labeled prostate cancer xenograft models for therapeutic assessment

Abstract Introduction: Prostate cancer is the most common male-specific cancer in many western countries including the US and the second most prevalent cause of cancer deaths in American males after lung cancer. The establishment of clinically relevant prostate cancer models is crucial for advancing our understanding of the disease and evaluating potential therapeutic interventions. In this in vivo project we characterize the tumor growth and response to therapy of two luciferase (luc) tagged human prostate cancer xenograft models of metastatic PC3M and non-metastatic DU145 cell lines. These models aim to replicate the variable clinical behavior of prostate cancer to provide valuable pre-clinical platforms to test existing and novel therapies. As a first step, these studies evaluate the effect of standard of care cytotoxic agents paclitaxel and carboplatin and the newer AXL kinase inhibitor bemcentinib alone and in combination on tumor growth kinetics and agent tolerability. Methods: PC3M-luc and DU145-luc cells were implanted subcutaneously on right flank or orthotopically into the prostate of NCG triple immunodeficient mice (Charles River). Subcutaneous tumor progression and treatment responses were monitored using calipers and bioluminescent imaging (BLI), with the capacity to detect developing metastases prior to necropsy. Orthotopic implants were monitored exclusively using BLI. Results: Orthotopic and subcutaneous implants of PC3M-luc demonstrated spontaneous metastasis to lungs and bones, replicating the established behavior of this cell line and effectively modeling metastases observed in prostate cancer patients. In contrast, orthotopic and subcutaneous implants of DU145-luc model did not metastasize. Both PC3M-luc and DU145-luc models independent of the tumor implant placement responded to paclitaxel treatment, with enhanced effects observed when combined with carboplatin. Notably, bemcentinib exhibited no activity as a single agent but showed promise when used in combination with cytotoxic agents. Conclusion: The development of orthotopic and subcutaneous PC3M-luc and DU145-luc prostate cancer models provides a valuable platform for assessing the effectiveness of established and novel therapies. These models mimic various clinical presentations of prostate cancer and promise to offer insights into disease progression and therapeutic strategies. Future studies will focus on durable responses and the assessment of other combination therapies for improved treatment outcomes. Citation Format: Kyle Draheim, Elizabeth Rainbolt, Bincy John, Cara Clouse, Andrew Wong, Brooke Wolff, Justin Avery, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Development and evaluation of orthotopically and subcutaneously implanted luciferase-labeled prostate cancer xenograft models for therapeutic assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4182.

Abstract 1453: Development and evaluation of subcutaneous and orthotopic PC3M metastatic prostate cancer xenograft model in SRG rats for therapeutic assessment

Abstract Introduction: Prostate cancer is the most prevalent cause of cancer deaths in American males after lung cancer. The establishment of clinically relevant prostate cancer models is crucial for advancing our understanding of the disease and evaluating potential therapeutics. Oncology studies in rats provide a secondary species to mice with the advantage of extensive longitudinal blood sampling and tumor biopsies in pharmacodynamics analysis. This in vivo study evaluates the behavior of subcutaneous and orthotopic luciferase (luc) tagged metastatic PC3M-luc cells in immunodeficient SRG rats (Hera Biolabs) for tumor growth kinetics, metastasis, and response to paclitaxel and carboplatin alone and in combination. Methods: PC3M-luc cells were implanted subcutaneously into the right flank or orthotopically into the prostate of SRG rats. Subcutaneous tumor progression and treatment responses were monitored using calipers and bioluminescent imaging (BLI), with the capacity to detect developing metastases prior to necropsy. Orthotopic implants were monitored exclusively using BLI. Tolerability was evaluated based on biweekly changes in body weight, daily observations for signs of tumor- or treatment-related clinical signs, necropsy results, and complete blood count (CBC) and clinical chemistry marker comparisons with immunocompetent CD rats. Results: PC3M-luc prostate carcinoma was successfully established orthotopically and subcutaneously in SRG rats, with similar growth rates and the development of spontaneous metastases to distant organs, modeling metastases observed in prostate cancer patients. Paclitaxel treatment was less tolerated in SRG rats in comparison to nude rats, enhanced toxicity was observed when combined with carboplatin. Comparison of CBC and clinical chemistry blood markers in naïve immunocompetent CD and immunodeficient SRG rats differed mainly in White Blood Cell (WBC) counts that were significantly lower in SRG rats, as expected due to the lack of mature B, T, and NK cells in these animals. Conclusion: The development of orthotopic and subcutaneous PC3M-luc prostate cancer models in rats provides a valuable alternative platform for assessing the effectiveness of established and novel therapies. This rat-based models offers a secondary species for comparison of prostate cancer behavior in mice, successfully mimics the clinical development of metastases, and provides additional insights into disease progression and therapeutic strategies. Future studies will focus on the assessment of tolerability and combination therapy strategies for improved treatment outcomes. Citation Format: Kyle Draheim, Elizabeth Rainbolt, Bincy John, Cara Clouse, Karyn Shinn, Justin Avery, Koh Meng Aw Yong, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Development and evaluation of subcutaneous and orthotopic PC3M metastatic prostate cancer xenograft model in SRG rats for therapeutic assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1453.

TLR9 Monotherapy in Immune-Competent Mice Suppresses Orthotopic Prostate Tumor Development.

Prostate cancer is ranked second in the world for cancer-related deaths in men, highlighting the lack of effective therapies for advanced-stage disease. Toll-like receptors (TLRs) and immunity have a direct role in prostate cancer pathogenesis, but TLR9 has been reported to contribute to both the progression and inhibition of prostate tumorigenesis. To further understand this apparent disparity, we have investigated the effect of TLR9 stimulation on prostate cancer progression in an immune-competent, syngeneic orthotopic mouse model of prostate cancer. Here, we utilized the class B synthetic agonist CPG-1668 to provoke a TLR9-mediated systemic immune response and demonstrate a significant impairment of prostate tumorigenesis. Untreated tumors contained a high abundance of immune-cell infiltrates. However, pharmacological activation of TLR9 resulted in smaller tumors containing significantly fewer M1 macrophages and T cells. TLR9 stimulation of tumor cells in vitro had no effect on cell viability or its downstream transcriptional targets, whereas stimulation in macrophages suppressed cancer cell growth via type I IFN. This suggests that the antitumorigenic effects of CPG-1668 were predominantly mediated by an antitumor immune response. This study demonstrated that systemic TLR9 stimulation negatively regulates prostate cancer tumorigenesis and highlights TLR9 agonists as a useful therapeutic for the treatment of prostate cancer.

UP14 - Exploring the efficacy of oncolytic reovirus jin-3 in a preclinical orthotopic prostate cancer model

UP14 - Exploring the efficacy of oncolytic reovirus jin-3 in a preclinical orthotopic prostate cancer model

Synergistic action of lactoferrin in enhancing the safety and effectiveness of docetaxel treatment against prostate cancer.

Tumor metastasis is promoted by an immunosuppressive environment. Lactoferrin (Lf) is known to regulate immunological activity in tumor cells and inhibit processes associated with tumor metastasis. A delivery of lactoferrin with docetaxel (DTX) in prostate cancer cells in the form of DTX-loaded lactoferrin nanoparticles (DTX-LfNPs) would provide a dual activity wherein the lactoferrin affects metastasis and DTX chemotherapeutically inhibits mitosis and cell division. DTX-LfNPs were prepared using sol-oil chemistry, and particles were characterized using transmission electron microscopy. Antiproliferation activity was analyzed in prostate cancer Mat Ly Lu cells. The target localization and efficacy of DTX-LfNPs were studied in an orthotopic prostate cancer induced by Mat Ly Lu cells in a rat model. Biomarkers were estimated using ELISA and biochemical reactions. DTX was loaded in pure Lf nanoparticles without involving any chemical modification and conjugation, thus when these nanoparticles are delivered in cancer cells both DTX and Lf will be present in biologically active forms. DTX-LfNps exhibit a spherical morphology of dimension of 60 ± 10nm with DTX Encapsulation Efficiency of 62.06 ± 4.07%. Competition experiments using soluble Lf confirm that DTX-LfNPs enter prostate cancer cells through the Lf receptor. DTX-LfNPs exhibit an improved anti-proliferative activity by 2.5 times compared to DTX. Further, analysis of the bioavailability of the drug in the prostate showed that DTX-LfNPs increased drug bioavailability in the prostate by two times more than the DTX. The analysis of efficacy in the Mat Ly Lu cells-induced orthotopic prostate cancer model showed that DTX-LfNPs significantly enhanced the anti-cancer activity compared to DTX in terms of regression of weight and volume of prostate tissue, the efficacy was confirmed by histochemical analysis. Lf provides synergistic activity along with DTX in inhibiting metastasis as assessed by the reduction of lactate dehydrogenase, alkaline phosphatase, TNF alpha, and IFNγ. LfNPs facilitate higher DTX localization along with Lf-mediated protection from DTX-associated toxicity to neutrophils and kidneys as assessed by C-reactive protein, creatinine, and uric acid. Thus, DTX LfNPs show a dual action by enhancing DTX bioavailability in prostate along with Lf-mediated suppression of metastasis as well as DTX-associated toxicity. In conclusion, DTX-LfNPs enhance the bioavailability of DTX in the prostate along with Lf-assisted improvement in inhibition of tumor metastasis and drug-associated toxicity.

The ADAM9/WISP-1 axis cooperates with osteoblasts to stimulate primary prostate tumor growth and metastasis.

Background: Metastatic prostate cancer (PCa) predicts a poor prognosis and lower likelihood of survival. Osteoblasts (OBs) are known to be responsible for the synthesis and mineralization of bone, although it is unclear as to whether PCa in the prostate gland cooperates with OBs in bone to promote PCa malignant transformation. We aimed to elucidate how primary PCa cells cooperate with distal OBs and contribute to the vicious cycle that leads to metastatic PCa. Methods: N-cadherin, E-cadherin, and Twist protein expression were measured by Western blot. Twist translocation into the nucleus was detected by the immunofluorescence (IF) assay. Enzyme-linked immunosorbent assay (ELISA) detected protein levels in human serum samples. Levels of candidate protein expression were examined by the human cytokine array. Prostate tumor growth and metastasis were analyzed by orthotopic and metastatic prostate cancer models, respectively. Immunohistochemistry (IHC) staining was used to observe ADAM metallopeptidase domain 9 (ADAM9) and WNT1 inducible signaling pathway protein 1 (WISP-1) expression in tissue. Results: Our in vitro and in vivo analyses have now discovered that primary PCa expressing ADAM9 protein enables the transformation of OBs into PCa-associated osteoblasts (PCa-OBs), inducing WISP-1 secretion from PCa-OBs in the bone microenvironment. The upregulation of WISP-1 in bone provided feedback to primary PCa and promoted PCa cell aggressiveness via epithelial-mesenchymal transition (EMT) activity. Elevated levels of WISP-1 expression were detected in the serum of patients with PCa. ADAM9 levels were overexpressed in tumor tissue from PCa patients; ADAM9 blockade interrupted OB-induced release of WISP-1 and also suppressed primary tumor growth and distal metastasis in orthotopic PCa mouse models. Conclusion: Our study suggests that the ADAM9/WISP-1 axis assists with metastatic PCa progression. Thus, targeting the ADAM9/WISP-1 axis may help to prevent the malignant phenotypes of PCa cells.

Head-up tilt does not enhance prostate tumor perfusion or oxygenation in young rats.

Solid tumors contain hypoxic regions that contribute to anticancer therapy resistance. Thus, mitigating tumor hypoxia may enhance the efficacy of radiation therapy which is commonly utilized for patients with prostate cancer. Increasing perfusion pressure in the prostate with head-up tilt (HUT) may augment prostate tumor perfusion and decrease hypoxia. The purpose of this study was to determine if an increase in the vascular hydrostatic gradient via 70° HUT increases tumor perfusion and decreases tumor hypoxia in a preclinical orthotopic model of prostate cancer. Male Copenhagen rats (n=17) were orthotopically injected with Dunning R-3327 (AT-1) prostate adenocarcinoma cells to induce prostate tumors. After tumors were established, prostate tumor perfusion and hypoxia were measured in rats during level (0°) and 70° HUT positions. To compare the magnitude of the hydrostatic column to that present in humans, ultrasound was used to measure the heart to prostate distance in male human subjects to estimate the prostate vascular hydrostatic pressure with the upright posture. In young rats, no differences were detected in prostate tumor perfusion or prostate tumor hypoxia with 70° HUT versus the level position. However, from the retrospective study, young rats increased prostate vascular resistance to HUT, whereas aged rats lacked this response. Tumor vessels co-opted from existing functional vasculature in young rats may be sufficient to negate increases in perfusion pressure with HUT seen in aged rats. Additionally, in humans, the estimated hydrostatic column at the level of the prostate is five times greater than that of the rat. Therefore, 70° HUT may elicit increases in prostate/prostate tumor blood flow in humans that is not seen in rats.

Developing a Translational Platform for Therapeutic Delivery of Image-Guided Ultra-Hypofractionated Prostate-Directed Radiotherapy in Orthotopic Prostate Cancer Models

<h3>Purpose/Objective(s)</h3> Prostate stereotactic body radiation therapy (SBRT) is rapidly evolving as a standard-of-care option for definitive management of localized prostate cancer. To our knowledge, there are currently no preclinical models that deliver SBRT-like treatment to orthotopic prostate tumors that reflect the precision and accuracy of clinical treatment. The objective of this study is to develop and credential a small animal radiation research platform (SAARP)-based therapeutic model of image-guided ultra-hypofractionated prostate-directed radiotherapy (RT) that is dosimetrically accurate with clinically relevant radiation doses. <h3>Materials/Methods</h3> Orthotopic prostate tumors are established following intraprostatic injection of 2 × 10⁵ PC3 tumor cells (stably expressing luciferase following lentiviral transduction) into the dorsal aspect of the prostate gland of nude mice. Serial orthotopic tumor growth is monitored with weekly bioluminescence (BLI) and Q2week MRI. Reverse-contrast cone-beam CT imaging (CBCT) is performed with intraperitoneal injection of 2mL of 60% omnipaque immediately prior to CBCT. Pre-treatment MRI is fused to CBCT to facilitate delineation of organs-at-risk (rectum, bladder, bowel bag) and target (prostate) for contouring. Individual plans (generally 4 or 5-field coplanar beam arrangement) were generated to target the prostate (SV not intentionally targeted) and avoid OAR. The SARRP was used to deliver 36.25Gy in 5 fractions of prostate-directed RT [day 30] and weekly serial BLI + MRI used to assess treatment response. <h3>Results</h3> Reverse contrast CBCT imaging +/- MR fusion was sufficient for OAR and target delineation and facilitated daily image-guidance whereas CBCT alone was insufficient. Radiation dosimetry noted a positive association between pH2AX mean fluorescence intensity (MFI) with escalating radiation doses of 0Gy, 2Gy, 8Gy and 20Gy. Preliminary DVH analysis of therapeutically irradiated orthotopic prostate tumors and adjacent OARs noted CTV prostate coverage>90%, rectumD50%<4Gy and bladderD50<4Gy in 3 of 5 serial fractions delivered. Surgically implanted intraprostatic TLD were harvested post-RT to confirm and quantify intraprostatic radiation dose received. Therapeutic response was observed with significantly lower luminescence counts in irradiated (n=13) vs non-irradiated controls (n=11) [p=0.049] and no treatment-related deaths were observed in irradiated mice. <h3>Conclusion</h3> We have developed a preclinical workflow for image-guided delivery of ultra-hypofractioned radiotherapy of orthotopic prostate tumors with ongoing optimization goals to achieve clinically relevant dose constraints and target coverage of NRG GU-005. Ongoing translational work involves DNA damage response assessment in irradiated prostate and adjacent organs-at-risks. This platform is now being explored in immunocompetent settings to better understand intraprostatic immune responses induced by prostate-directed RT.

Establishment and evaluation of ectopic and orthotopic prostate cancer models using cell sheet technology

BackgroundThe traditional prostate cancer (PCa) model is established by injecting cell suspension and is associated with a low tumor formation rate. Cell sheet technology is one of the advancements in tissue engineering for 3D cell-based therapy. In this study, we established ectopic and orthotopic PCa models by cell sheet technology, and then compared the efficiency of tumor formation with cell suspension injection.MethodsDU145 cells were seeded on 35 mm temperature-sensitive dishes to form PCa cell sheets, while the cell suspension with the same cell density was prepared. After transplanting into the nude mice, the tumor volumes were measured every 3 days and the tumor growth curves were conducted. At the time points of 2 weeks and 4 weeks after the transplantation, magnetic resonance imaging (MRI) was used to evaluate the transplanting site and distant metastasis. Finally, the mice were sacrificed, and the related tissues were harvested for the further histological evaluation.ResultsThe orthotopic tumor formation rate of the cell sheet injection group was obviously better than that in cell suspension injection group (100% vs 67%). Compared with cell suspension injection, the tumors of DU145 cell sheet fragments injection had the higher density of micro-vessels, more collagen deposition, and lower apoptosis rate. There was no evidence of metastasis in forelimb, lung and liver was found by MRI and histological tests.ConclusionWe successfully cultured the DU145 cell sheet and can be used to establish ectopic and orthotopic PCa tumor-bearing models, which provide an application potential for preclinical drug development, drug-resistance mechanisms and patient individualized therapy.

PD07-08 FIBROBLAST ACTIVATION PROTEIN-SPECIFIC MRI PROVIDES IMPROVED TUMOUR MAPPING IN AN ORTHOTOPIC MODEL OF PROSTATE CANCER COMPARED TO PROSTATE SPECIFIC MEMBRANE ANTIGEN

You have accessJournal of UrologyCME1 May 2022PD07-08 FIBROBLAST ACTIVATION PROTEIN-SPECIFIC MRI PROVIDES IMPROVED TUMOUR MAPPING IN AN ORTHOTOPIC MODEL OF PROSTATE CANCER COMPARED TO PROSTATE SPECIFIC MEMBRANE ANTIGEN Nicole Dmochowska, Valentina Milanova, Anil Shetty, Ramesh Mukkamala, Madduri Srinivasarao, Eric Walser, Phil Low, and Benjamin Thierry Nicole DmochowskaNicole Dmochowska More articles by this author , Valentina MilanovaValentina Milanova More articles by this author , Anil ShettyAnil Shetty More articles by this author , Ramesh MukkamalaRamesh Mukkamala More articles by this author , Madduri SrinivasaraoMadduri Srinivasarao More articles by this author , Eric WalserEric Walser More articles by this author , Phil LowPhil Low More articles by this author , and Benjamin ThierryBenjamin Thierry More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: MRI guided focal therapies of prostate cancer have the potential to reduce treatment related side effects without sacrificing oncological outcomes. Molecular MRI with agents targeted to tumour tissues enhances tumour margin detection and consequently treatment guidance/planning. Prostate specific membrane antigen (PSMA) expression is currently the gold standard for molecular imaging of prostate cancer. Fibroblast activation protein (FAP) is an extracellular transmembrane protein expressed on cells of the tumour microenvironment, particularly in stromal and vascular components. FAP has been gaining momentum as a pan-cancer marker(DOI: 10.2967/jnumed.119.227967) as its overexpression often correlates with cancer severity, including in prostate cancer (DOI: 10.1371/journal.pone.0116683) and has been found to be negligibly expressed in healthy tissue. Our objective was to compare MRI contrast of PSMA-targeting and FAP-targeting magnetic nanoparticles (MNPs) in an orthotopic murine model of prostate cancer. METHODS: Control (no ligand), FAP and PSMA-targeted MNPs were prepared with modification of an MRI agent (FerroTrace, Ferronova) currently undergoing a phase 1 clinical trial. In vitro binding was evaluated in PSMA and FAP positive cell lines. To form orthotopic tumours, LNCaP cells (PSMA+ human prostate cancer) were injected into the prostate. After 4-6 weeks of tumour growth, mice underwent 16.4T T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. Enhancement of MRI contrast in tumours was quantified by determining the proportion of pixels with low signal intensity throughout regions of interest. Accumulation of MNPs in prostate tumours at 24 hours post injection was confirmed ex vivo by Prussian blue staining. RESULTS: PSMA and FAP-MNPs demonstrated specific binding in vitro. MRI contrast of tumours was increased in FAP (p<0.001) and PSMA-MNPs (p<0.05) but not control MNPs (p>0.05), relative to orthotopic tumours with no injected MNPs. FAP-MNPs provided increased contrast relative to PSMA-MNPs (∼10% enhancement, p<0.05). Increased accumulation of FAP-MNPs in prostate tumours was observed on tissue sections compared to PSMA-MNPs. Control-MNPs showed minimal tumour accumulation. CONCLUSIONS: These results suggest that FAP-targeting MNPs could enhance the MRI of prostate tumours and assist in the delivery of precise focal treatment. Source of Funding: NHMRC Development Grant 1158755 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e101 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nicole Dmochowska More articles by this author Valentina Milanova More articles by this author Anil Shetty More articles by this author Ramesh Mukkamala More articles by this author Madduri Srinivasarao More articles by this author Eric Walser More articles by this author Phil Low More articles by this author Benjamin Thierry More articles by this author Expand All Advertisement PDF DownloadLoading ...

Raloxifene Suppresses Tumor Growth and Metastasis in an Orthotopic Model of Castration-Resistant Prostate Cancer.

Androgen receptor (AR)-castrate-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer that does not have clinically approved targeted treatment options. To this end, the cytotoxic potential of raloxifene and the synthetic curcumin derivative 2,6-bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) was examined in AR-(PC3 and DU145) cells and AR+ (LnCaP) CRPC cells. The results showed that both raloxifene and RL91 elicited significant cytotoxicity across three cell lines with the lowest EC50 values in PC3 cells. Additionally, the two drugs were synergistically cytotoxic toward the PC3, DU-145 and LNCaP cell lines. To determine the effect of the drug combination in vivo, an orthotopic model of CRPC was used. Male mice were injected with PC3 prostate cancer cells and then treated with vehicle (5 mL/kg), raloxifene (8.5 mg/kg, po), RL91 (8.5 mg/kg, po) or a combination of raloxifene and RL91 for six weeks. Sham animals were subjected to the surgical procedure but were not implanted with PC3 cells. The results showed that raloxifene decreased tumor size and weight as well as metastasis to renal lymph nodes. However, combination treatment reversed the efficacy of raloxifene as tumor volume and metastasis returned to control levels. The results suggest that raloxifene has tumor suppressive and anti-metastatic effects and has potential for further clinical use in AR-CRPC.

Development of a novel castration-resistant orthotopic prostate cancer model in New Zealand White rabbit.

Prostate cancer (PCa) models in mice and rats are limited by their size and lack of a clearly delineated or easily accessible prostate gland. The canine PCa model is currently the only large animal model which can be used to test new preclinical interventions but is costly and availability is sparse. As an alternative, we developed an orthotopic human prostate tumor model in an immunosuppressed New Zealand White rabbit. Rabbits are phylogenetically closer to humans, their prostate gland is anatomically similar, and its size allows for clinically-relevant testing of interventions. Rabbits were immunosuppressed via injection of cyclosporine. Human PC3pipGFP PCa cells were injected into the prostate via either (a) laparotomy or (b) transabdominal ultrasound (US) guided injection. Tumor growth was monitored using US and magnetic resonance imaging (MRI). Contrast-enhanced ultrasound (CEUS) imaging using nanobubbles and Lumason microbubbles was also performed to examine imaging features and determine the optimal contrast dose required for enhanced visualization of the tumor. Ex vivo fluorescence imaging, histopathology, and immunohistochemistry analyses of the collected tissues were performed to validate tumor morphology and prostate-specific membrane antigen (PSMA) expression. Immunosuppression and tumor growth were, in general, well-tolerated by the rabbits. Fourteen out of 20 rabbits, with an average age of 8 months, successfully grew detectable tumors from Day 14 onwards after cell injection. The tumor growth rate was 39 ± 25 mm2 per week. CEUS and MRI of tumors appear hypoechoic and T2 hypointense, respectively, relative to normal prostate tissue. Minimally invasive US-guided tumor cell injection proved to be a better method compared to laparotomy due to the shorter recovery time required for the rabbits following injection. Among the rabbits that grew tumors, seven had tumors both inside and outside the prostate, three had tumors only inside the prostate, and four had tumors exclusively outside of the prostate. All tumors expressed the PSMA receptor. We have established, for the first time, an orthotopic PCa rabbit model via percutaneous US-guided tumor cell inoculation. This animal model is an attractive, clinically relevant intermediate step to assess preclinical diagnostic and therapeutic compounds.

Primary Tumor Resection Decelerates Disease Progression in an Orthotopic Mouse Model of Metastatic Prostate Cancer

Simple SummaryIn this preclinical in-vivo study, we used an orthotopic prostate cancer mouse model to analyze the effects of primary tumor resection on further disease progression and survival in metastatic prostate cancer. Sixty-four mice with metastatic prostate cancer, induced by intraprostatic injection of three-dimensional prostate cancer spheroids, were randomized into two groups: one group received resection of their primary tumor while the other group received a sham operation. After this, the mice were followed-up for 10 weeks. In comparison with the sham operation group, mice with primary tumor resection showed significantly slower PSA progression, less lung metastases, and significantly longer survival. These results are a hint towards a beneficial oncological effect of primary tumor resection in metastatic prostate cancer. Furthermore, the established versatile in-vivo model can be used to study the molecular mechanisms of primary tumor/metastasis interaction in prostate cancer.Radical prostatectomy in oligometastatic prostate cancer is a matter of intense debate. Besides avoiding local complications, it is hypothesized that primary tumor resection may result in better oncological outcomes. The aim of our study was to analyze the effect of primary tumor resection on disease progression in an orthotopic prostate cancer mouse model. First, the optimal time point for primary tumor resection, when metastases have already occurred, but the primary tumor is still resectable, was determined as 8 weeks after inoculation of 5 × 105 LuCaP136 cells. In a second in vivo experiment, 64 mice with metastatic prostate cancer were randomized into two groups, primary tumor resection or sham operation, and disease progression was followed up for 10 weeks. The technique of orthotopic primary tumor resection was successfully established. Compared with the sham operation group, mice with primary tumor resection showed a significantly longer survival (p < 0.001), a significantly slower PSA increase (p < 0.01), and a lower number of lung metastases (p = 0.073). In conclusion, primary tumor resection resulted in slower disease progression and longer survival in an orthotopic mouse model of metastatic prostate cancer. In future studies, this model will be used to unravel the molecular mechanisms of primary tumor/metastasis interaction in prostate cancer.

Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis.

SUMMARYFBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and −2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.

P0424 - Effects of cytoreductive primary tumor resection in an orthotopic mouse model of metastatic prostate cancer

P0424 - Effects of cytoreductive primary tumor resection in an orthotopic mouse model of metastatic prostate cancer

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Effect of Moderate and High Intensity Exercise on Hypoxia and Radiosensitivity in Tumor‐Bearing Rats

Background Prostate tumors typically have hypoxic regions that are resistant to traditional radiation therapy. Prior evidence from our laboratory has demonstrated improved perfusion and reduced hypoxia of prostate tumors with moderate intensity exercise training; such changes have potential to enhance radiation therapy. However, there is a dearth of evidence for the magnitude of alterations of intra-tumor hypoxia markers with exercise, as well as the ideal intensity of exercise for combating hypoxia in solid tumors. Using a pre-clinical orthotopic model of prostate cancer, we tested the hypothesis that exercise training at both moderate and high intensities would result in a decrease in hypoxia assessed via immunohistochemical analysis of hypoxic markers. Further, that enhanced radiotherapy responses would occur in exercise trained tumor bearing animals compared to sedentary counterparts. Methods Immunocompetent male Copenhagen rats aged 6 months (n=48) were injected orthotopically (ventral lobe of prostate) with 1x105 Dunning R-3327 AT-1 prostate adenocarcinoma cells. All animals were first acclimated to treadmill exercise and randomized into three groups: tumor bearing sedentary (TBS, n=20), moderate intensity training, (TBMIT, n=12), or high intensity training (TBHIT, n=16). After 7 days of recovery from surgery, both exercise groups began progressive exercise training on a motorized treadmill at either 20/min with a 5% incline, or 30m/min 15% incline for 10 minutes a day progressing to 60 min/day for TBMIT and TBHIT, respectively, for approximately 5 weeks. Immunohistochemical analysis and whole-body ionizing radiation (2 Gy) were performed at the end of the 5-week period of training followed by clonogenic cell survival assay to assess survival fraction. Results There were no significant differences (p>0.05) in tumor mass between groups (TBS 7.8±1.1; TBMIT 7.2±1.3; TBHIT 6.4±0.9 g). Overall expression levels of hypoxia inducible factor 1-alpha (HIF-1a, n=40) were significantly greater in the TBS group vs both the TBMIT and TBHIT (TBS 37.9±7.4; TBMIT 25.5±6.1; TBHIT 14.4±5.7%, p<0.05), with a trend for increased expression in TBMIT vs TBHIT (p=0.059). The survival fraction (n=8) was also significantly higher in TBS when compared to both TBMIT and TBHIT (TBS 39.9±5.3; TBMIT 23.2±3.2; TBHIT 20.7±4.9%, p<0.05). Conclusion This study suggests that compared to sedentary counterparts, both chronic moderate and high intensity exercise training modify the tumor microenvironment (decreased levels of HIF-1a) favorably. Given exercise therapy is becoming increasingly recommended to mitigate fatigue and increase quality of life in patients, a potential therapeutic component could add to the efficacy of exercise prescription in a clinical setting.