Human Prostate Cancer Xenograft Model Research Articles

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.

Ultrasound-stimulated microbubbles enhancement of fractionated radiation for tumor treatment.

Radiation therapy (XRT) causes numerous biological changes in tumor microenvironment. Radiation vascular response, due to endothelial disruption, can influence treatment outcomes in a dose-dependent manner. Ultrasound-stimulated microbubbles (USMB) have also been demonstrated to create a vascular response in the tumor microenvironment and enhance tumor response when used in combination with XRT. Single doses of 8-10Gy are known to induce activation of acid sphingomyelinase (ASMase)-induced ceramide production, causing vascular damage. Destruction of vasculature results in endothelial apoptosis followed by tumor cell death. The effect of tumor response is known to be synergistic by 10-fold higher cell kill observed when USMB is combined with radiation. In this study, we used an USMB approach in combination with conventional low dose fractionated radiation to enhance endothelial cell responses to XRT in human PC3 prostate cancer xenograft model. Mice were divided into untreated, USMB therapy, fractionated XRT, and combined USMB therapy followed by XRT (USMB + XRT) groups. USMB therapy was delivered twice per week in the USMB-alone and combined USMB + XRT treatment groups over four weeks. Radiation treatments were delivered in fractions of 2Gy/day (total 40Gy in 20 fractions, BED10 = 48Gy) in the XRT-alone and combined USMB + XRT groups. The treatment outcome was evaluated using histopathology, power Doppler, and immunohistochemistry assays. Tumor growth assessment showed that sizes of tumors increased in the control and the single treatment groups over a treatment period of four weeks, but significantly decreased with the combined treatments of USMB + XRT. Immunohistochemical analysis indicated a statistically significant vascular disruption in mice that received treatment involving a full 4-week schedule of combined (USMB + XRT) treatments. A statistically significant increase in vascular disruption was demonstrated through CD68 and trichrome fibrosis staining. Changes in local perfusion assessed using high-frequency power Doppler imaging demonstrated attenuated blood flow in the combined group. This work demonstrates the efficacy of using USMB as a radiation sensitizer in a mouse model of human PC3 tumor xenograft. This radiation treatment enhancement modality has the advantage of targeting tumor vasculature with ultrasound stimulation that can be implemented prior to radiation treatment.

Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold

Overexpression of both human murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) is detected in tumor cells from several cancer types, including childhood acute leukemia lymphoma (ALL), neuroblastoma (NB), and prostate cancer, and is associated with disease progression and treatment resistance. In this report, we described the design and syntheses of a series of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold from our previously reported lead compound JW-2-107 and tested their cytotoxicity in a panel of human cancer cell lines. The best compound identified in this study is compound 3e. Western blot analyses demonstrated that treatments with 3e decreased MDM2 and XIAP protein levels and increased expression of p53, resulting in cancer cell growth inhibition and cell death. Furthermore, compound 3e effectively inhibited tumor growth in vivo when tested using a human 22Rv1 prostate cancer xenograft model. Collectively, results in this study strongly suggest that the tetrahydroquinoline scaffold, represented by 3e and our earlier lead compound JW-2-107, has abilities to dual target MDM2 and XIAP and is promising for further preclinical development.

Abstract 222: Exposure to dim light at night disrupts the nocturnal melatonin signal in male nude rats bearing tissue-isolated castration-sensitive VCaP human prostate cancer: Impact on tumor circadian dynamics of the Warburg effect, lipid signaling and proliferation

Abstract Over 248,000 men in the U.S. alone this year will be diagnosed with prostate cancer, and over 33,300 will die from the disease. The World Health Organization, in the case of breast cancer, has classified night-shift work involving light at night (LAN)-induced circadian disruption to be a probable carcinogen (Class 2A). Exposure to LAN suppresses nighttime pineal melatonin (MLT) production that influences normal and neoplastic tissue metabolism, physiology, and proliferation. Previously, we showed in rodent and human tumors in vivo that MLT inhibits linoleic acid (LA)-uptake and conversion to 13-hydroxyoctadecadienoic acid (13-HODE), a lipoxygenase product that enhances epidermal growth factor and insulin-like growth factor-I-induced mitogenesis. Recently, we developed a tissue-isolated, castration-sensitive VCaP human prostate cancer xenograft model to test the hypothesis that suppression of the nocturnal MLT signal due to exposure to dim LAN (dLAN) accelerates tumor LA-uptake and metabolism, aerobic glycolysis (Warburg Effect), and proliferative activity. Male nude rats (Crl:NIHFoxn1rnu; n=6/group) bearing castration-sensitive VCaP human prostate cancer xenografts were maintained on either a control L(300 lux)D(0 lux),12:12 or experimental LD,12:12dLAN (300 lux light phase followed by dLAN 0.2 lux dark phase intensity) light/dark cycle. Results revealed (Mean ± S.D.) plasma MLT levels in controls peaked in the mid-dark phase (183.4 ± 12.8 pg/mL) and were lowest (2.2 ± 0.4 pg/mL) in the mid-light phase, and low (< 10 pg/mL) throughout the 24-hr period in dLAN rats. Tumors in rats exposed to dLAN exhibited a significantly shorter latency to onset and a two-fold faster growth rate than controls. Harvested control group tumors revealed elevated cAMP levels, LA uptake, 13-HODE production, the Warburg effect, and [3H]thymidine incorporation into DNA, as well as elevated patterns of expression of signaling pathways phospho-ERK1/2, -AKT, -STAT3, -glycogen synthase kinase-3β (GSK3β), and -NF-κβ, and full-length androgen receptor and aldo-keto reductase (AKR1C3), during the light phase and markedly suppressed during the dark phase. In the dLAN group, these measures were markedly elevated throughout the 24-hr period. This is the first evidence in vivo, that dLAN-induced disruption of integrated circadian rhythms of signaling, metabolism, and proliferation results in accelerated growth of castration-sensitive human prostate cancer xenografts. Thus, lighting design strategies to minimize human exposure to LAN that preserve the integrity of the circadian MLT signal may offer a novel approach to suppress the growth progression of human castration-sensitive prostate cancer in our increasingly 24-hour society. Citation Format: Erin M. Dauchy, Robert T. Dauchy, Steven M. Hill, Yan Dong, Victoria P. Belancio, Shulin Xiang, George C. Brainard, John P. Hanifin, David E. Blask. Exposure to dim light at night disrupts the nocturnal melatonin signal in male nude rats bearing tissue-isolated castration-sensitive VCaP human prostate cancer: Impact on tumor circadian dynamics of the Warburg effect, lipid signaling and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 222.

Effects of green tea on prostate carcinogenesis in rat models and a human prostate cancer xenograft model.

There is evidence to suggest that green tea soy may have protective effects against prostate cancer, but there are several epidemiologic and clinical studies that did not identify such an effect. We tested the notion of protective effects in a rat model of prostate carcinogenesis that has been predictive of the effects of selenium and vitamin E in randomized clinical trials and a human prostate cancer xenograft model in nude mice and rat prostate tumor cells transplanted in immune-competent syngeneic animals. Prostate cancer was induced in rats with methylnitrosourea and testosterone and tumor incidence was determined. Subcutaneous tumor growth was measured resulting from injected cells isolated from rat prostate cancers grafted in syngeneic animals and from the prostate-specific antigen (PSA)-producing human prostate cancer PC346 xenografted in nude mice. Brewed decaffeinated green tea infusion or caffeinated green tea extract and the same 300 mg/ml concentration of caffeine were provided in drinking water of the rats and nude mice. Caffeinated green tea extract and caffeine provided in drinking water did not modify the induction of prostate cancer in the rat model compared with control rats. The same drinking water treatments also did not affect the growth and PSA production of PC346 human prostate cancer xenografts in nude mice and the growth of two transplantable rat prostate cancer tumor lines in Wistar Firth rats. Brewed green tea infusion as drinking water did also not affect tumor growth in these xeno- and allograft models. These animal studies with drinking water exposure to green tea and caffeine do not support the idea that green tea is protective against prostate cancer.

Abstract PO030: A novel integrin-targeted therapeutic agent for prostate cancer

Abstract Disintegrins are disulfide-rich peptides, many containing an Arg-Gly-Asp (RGD) motif. They hold significant translational potential as anti-cancer agents based on their high-affinity/high-specificity interaction with tumor integrins and desirable pharmacological attributes. The integrin-binding activity of disintegrins depends on a mobile 11-amino acid loop displaying the RGD tripeptide motif or some other tripeptide motif. They bind only to the activated conformation of integrins on motile cancer cells and angiogenic endothelial cells. We designed a sequence-engineered RGD-disintegrin, vicrostatin (VCN), that can be produced at ~200mg/L from cell lysates of a bacterial recombinant system. VCN, a monomer (MW=7146), inhibits tumor cell adhesion and dissemination, and angiogenic endothelial cell invasion and tube formation. Integrins are cell surface adhesion receptors (containing a and b chains) operating at the interface between the extracellular matrix and cytoskeletal apparatus. A sub-group of integrins recognize the RGD sequence present in key extracellular matrix proteins involved in prostate cancer (PC) growth and dissemination. Integrins are involved in bi-directional signaling interactions that alter cellular functions. Among these interactions are those involved in PC growth, metastasis and angiogenesis. Integrin avb3 plays an important role in the development of bone metastases, supporting our belief that it, as well as other RGD-integrins, are important therapeutic targets in advanced PC. We examined the effect of treatment of a xenograft model of human PC using a liposomal formulation of VCN (LVCN). PC3 cells were implanted subcutaneously in 5-week old male nude mice. LVCN and VCN (100µg VCN equivalent per dose) administration was initiated IV when tumors became palpable (14 days). Tumor size was measured weekly by caliper in a blinded study. There was a 75% reduction in tumor growth in LVCN-treated animals compared to controls. VCN displayed little anti-tumor activity most likely due to its lack of availability to the PC cells. We also examined the anti-angiogenic activity of LVCN in this model using CD31/PECAM immunohistochemistry and observed a significant decrease in tumor-associated microvessel density. We then evaluated therapeutic efficacy of LVCN in treating bone metastases. CWR22rV1 PC cells were suspended in a solution of matrigel and injected into a drilled hole in the proximal left tibia of immunodeficient mice (100,000 cells/10µL). Following injection, the diameters of both tibias were measured by caliper twice weekly. Since CWR22rV1 cells are androgen dependent, animals received daily injections of testosterone. Tumors grew slowly initially, but at 20 days tumors were evident and treatment began. LVCN and VCN were administered twice weekly via IV injection (100µg per dose) for 5 weeks. LVCN displayed ~81% inhibition of tumor growth compared to VCN, PBS or empty liposomes. Our findings provide strong support for the effectiveness of LVCN as an inhibitor of PC growth and metastases. Citation Format: Francis S. Markland, Stephen Swenson, Radu Minea, Jacek Pinski. A novel integrin-targeted therapeutic agent for prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO030.

Low-frequency ultrasound combined with microbubbles improves gene transfection in prostate cancer cells in vitro and in vivo.

The aim of this study is to explore whether low-frequency ultrasound combined with microbubbles improves pEGFP genes transfection into human prostate cancer cells. Ultrasound with frequency of 80kHz and duty cycle of 50% was adopted in the study; in in vitro experiments, cell lysis, and membrane damage were evaluated after ultrasound exposure; and the membrane continuity and transfection efficiency were observed by transmission electron microscope and laser scanner, respectively. Human prostate cancer xenograft models were exposed to ultrasound and transfection efficiency and histological examination were analyzed. Compared with the control group, ultrasound combined with microbubbles significantly improves gene transfection efficiency (P<.05). In in vitro study, ultrasound combined with microbubbles resulted in cell lysis and the interruption of cell membrane continuity, and its average transfection efficiency was 9.9%; the green fluorescence intensity was 15.2% in the ultrasound combined with microbubbles group in vivo; both values were higher than that in the control group (P<.05). Low-frequency ultrasound combined with microbubbles could be used as a method to promote gene transfection in prostate cancer cells.

Abstract 5347: Preliminary evaluation of BB2r TAT using 212Pb-RM2 in a PC3 human prostate cancer xenograft model

Abstract Introduction: Targeted alpha therapy (TAT) employing radiolabeled receptor targeted peptides holds the potential to be clinically effective for the treatment of advanced prostate cancer. We are evaluating the use of the TAT agent, Pb-212-RM2, for targeting the BB2r receptor shown to be expressed in prostate cancer. The current study is the initial therapeutic efficacy evaluation of Pb-212-RM2 TAT administered as a single bolus injection using the androgen independent/insensitive PC3 human prostate cancer xenograft model. Methods: SCID mice (4-5 weeks of age) were injected subcutaneously with 5 million cultured PC3 cells in both rear flanks as a suspension in RPMI 1640 media. The RM2 peptide was obtained commercially. Pb-212 was eluted from a Ra-224/Pb-212 generator and synthesis of Pb-212-RM2 performed using an Eckert &amp; Ziegler PharmTracer. QC was conducted using HPLC. PC3 xenografted SCID mice were administered a single intravenous bolus of saline (NTC), 0.3ug Pb-RM2 (Cold RM2), 50uCi or 100uCi of 212Pb-RM2 in approximately 100uL via the tail vein. Weekly caliper tumor measurements, complete blood counts (CBC), body weight (BW), and body condition score (BCS) were taken predose and up to 17 weeks post injection (pi). Results: At 18 days pi, tumor regression was observed in the 100uCi group with a maximum percent change of -49.3%, while the 50uCi dose group revealed slight tumor growth (+17.2%). As expected, the NTC and Cold RM2 groups demonstrated rapid tumor growth (+325.3% and +304.5% change, respectively). By day 40 pi, tumor regrowth was observed in the 100uCi group (+91.6% change from predose with a +221.1% change by day 47). Body weights of the NTC, Cold RM2, 50uCi and 100uCi groups at 7 days pi were 101.5%, 101.5%, 98.5%, and 95.4%, respectively. Platelet count comparisons between the NTC group and the 50uCi (P=0.0544) and 100uCi (P=0.0624) groups were comparable. Conclusions: Both Pb-212-RM2 TAT treatment groups (50uCi and 100uCi) demonstrated initial tumor control for 4-5 weeks post treatment. While tumor growth suppression was evident in only the 100uCi dose group, this suppression was transient with tumor volume increasing steadily beginning at 32 days pi. As expected, the non-radioactive Pb-RM2 had no effect on tumor growth. Body weights for all dose groups remained &amp;gt;95% of predose weights until disease progression required subject removal from the study. These results warrant further evaluation of multi-dose administration of Pb-212-RM2 with and without standard of care chemotherapy to evaluate optimal tumor control properties of BB2r TAT. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Elisabeth A. Devanny, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Preliminary evaluation of BB2r TAT using 212Pb-RM2 in a PC3 human prostate cancer xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5347.

Abstract 5346: Preclinical evaluation of maximum tolerated dose (MTD) following single dose administration of 212Pb-RM2

Abstract Introduction: Lead-212 (Pb-212)-RM2 is under evaluation as a potential BB2r targeted alpha therapy (TAT) agent for the treatment of BB2r positive prostate cancers. The purpose of this study was to define maximum tolerated dose (MTD) levels for conducting initial therapeutic efficacy studies using human prostate cancer xenograft models and to identify targeted organs and tissues for assessing potential radiotoxicity effects. Methods: The RM2 peptide (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was commercially synthesized and radiolabeled with Pb-212 eluted from a Ra-224/Pb-212 generator. Radiosynthesis was accomplished using an Eckert &amp; Ziegler PharmTracer. HPLC QC confirming &amp;gt;93% product purity was performed prior to and post administration of Pb-212 RM2. CF1 mice were administered a single intravenous bolus of either saline (non-treated controls / NTC), 50uCi, 100uCi, or 200uCi of 212Pb-RM2. Weekly complete blood counts (CBC), body weight (BW), and body condition score (BCS) were obtained up to 30 weeks post administration. Subsets of 3-5 mice/group were harvested at 30, 50, 70, 90 days, and 30 weeks for histopathology analysis and pooled serum analysis. An additional set of mice (n=3/group/time point) were administered saline (NTC), 50uCi or 200uCi of Pb-212 RM2 and tissues/serum were obtained at 2, 5, and 7 days to evaluate ALT, AST, BUN, lipase, amylase, and creatinine levels at early timepoints. Results: We observed BW loss in all treatment groups compared to the NTC. We further observed that only the high dose (200uCi) treatment group experienced weight loss that was significantly (P=0.0168) below that of the NTC at 30 weeks. No animals were removed from the study due to BW loss. WBC counts were suppressed at Week1(100uCi and 200uCi groups) and fully recovered by Week 2. WBC counts for all treatment groups were not significantly different at 30 weeks. Platelet counts were slightly suppressed at Week1 (all groups) and fully recovered by Week 2. Week 30 platelet counts were consistent between all treatment groups. Significantly elevated amylase/lipase levels were observed (Day 2) in the 200uCi treatment group with recovery to normal by Day 5. Liver (ALT and AST) and kidney (BUN and creatinine) markers showed nominal changes between groups. Marked changes in pancreas and kidney histopathology were observed only at 30 weeks (100uCi and 200uCi group). Conclusions: Based on BW, CBC, histopathology, and serum analysis, the 50uCi and 100uCi dose levels of Pb-212 RM2 appear to be well tolerated. The high dose Pb-212 RM2 group (200uCi) presented with initially elevated pancreatic enzymes (Day 2) and altered histopathology of the pancreas and kidney appearing only at 30 weeks post treatment. These data suggest that preclinical single dose administration of Pb-212 RM2 TAT at 100uCi or lower should be tolerable. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Elisabeth A. Devanny, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Preclinical evaluation of maximum tolerated dose (MTD) following single dose administration of 212Pb-RM2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5346.

Evaluation of [131I]I- and [177Lu]Lu-DTPA-A11 Minibody for Radioimmunotherapy in a Preclinical Model of PSCA-Expressing Prostate Cancer.

Radioimmunotherapy uses tumor-specific antibodies to deliver therapeutic radionuclides, but hematological toxicity due to the long serum half-life of intact antibodies remains a challenge. We evaluated a smaller antibody fragment, the minibody, with faster kinetics and a potentially improved therapeutic index. The anti-prostate stem cell antigen (PSCA) minibody (A11 Mb) was radiolabeled with iodine-124 ([124I]I-A11 Mb) or conjugated with deferoxamine (DFO) and labeled with zirconium-89 ([89Zr]Zr-DFO-A11 Mb) for surrogate immunoPET to profile pharmacokinetics in a human prostate cancer xenograft model. Subsequently, minibodies labeled with two therapeutic beta emitters, directly iodinated [131I]I-A11 Mb (non-residualizing) and 177Lu chelated using DTPA ([177Lu]Lu-DTPA-A11 Mb) (residualizing), were compared for in vitro antigen-specific cytotoxicity. Full biodistribution studies (in 22Rv1-PSCA tumor bearing and hPSCA knock-in mice) were conducted for dosimetry calculations. Finally, the lead candidate [131I]I-A11 Mb was evaluated in a radioimmunotherapy experiment. Escalating single doses (3.7, 11, or 37MBq) and saline control were administered to 22Rv1-PSCA tumor bearing mice and anti-tumor effects (tumor volume) and toxicity (body weight) were monitored. Minibodies radiolabeled with therapeutic beta emitters [131I]I-A11 Mb and [177Lu]Lu-DTPA-A11 Mb exhibited comparable tumor cell growth inhibition in vitro. In vivo surrogate immunoPET imaging using [89Zr]Zr-DFO-A11 Mb showed activity retention in liver and kidney up to 72h, while [124I]I-A11 Mb cleared from liver, kidney, and blood by 48h. Based on full biodistribution and dosimetry calculations, administering 37MBq [131I]I-A11 Mb was predicted to deliver a favorable dose to the tumor (35Gy), with a therapeutic index of 22 (tumor:bone marrow). For [177Lu]Lu-DTPA-A11 Mb, the kidneys would be dose-limiting, and the maximum tolerated activity (7.4MBq) was not predicted to deliver an effective radiation dose to tumor. Radioimmunotherapy with a single dose of [131I]I-A11 Mb showed dose-dependent tumor inhibition with minimal off-target toxicity and improved median survival (19 and 24days, P < 0.001) compared with untreated mice (12days). These findings show the potential of the anti-PSCA minibody for targeted radioimmunotherapy with minimal toxicity, and the application of immunoPET and dosimetry for personalized treatment.

Androgens modify therapeutic response to cabazitaxel in models of advanced prostate cancer.

Disruption of the phenotypic landscape via epithelial-mesenchymal transition (EMT) enables prostate cancer cells to metastasize and acquire therapeutic resistance. Our previous studies demonstrated that cabazitaxel (CBZ) (second-generation Food and Drug Administration-approved taxane chemotherapy), used for the treatment of castration-resistant prostate cancer (CRPC), causes reversal of EMT to mesenchymal-epithelial transition (MET) and reduces expression of kinesin motor protein KIFC1 (HSET). The present study examined the effect of sequencing CBZ chemotherapy mediated MET on prostate tumor redifferentiation overcoming therapeutic resistance in models of advanced prostate cancer. To examine the impact of androgens on the antitumor effect of CBZ, we used human prostate cancer cell lines with different sensitivity to androgens and CBZ, in vitro, and two human prostate cancer xenograft models in vivo. Tumor-bearing male mice (with either the androgen-sensitive LNCaP or the CRPC 22Rv1 xenografts) were treated with CBZ (3 mg/kg) alone, or in combination with castration-induced androgen-deprivation therapy (ADT) for 14 days. Cell viability assays indicate that the presence of 5α-dihydrotestosterone (1 nM) confers resistance to CBZ in vitro. CBZ treatment in vivo induced MET in LNCaP-derived tumors as shown by increased E-cadherin and decreased N-cadherin levels. Sequencing CBZ after ADT improves tumor response in androgen-sensitive LNCaP, but not in CRPC 22Rv1 xenografts. Mechanistic dissection revealed a novel association between the androgen receptor and HSET in prostate cancer cells that is inhibited by CBZ in an androgen-dependent manner. Our findings provide new insights into the phenotypic reprogramming of prostate cancer cells to resensitize tumors to CBZ action. This evidence is of translational significance in treatment sequencing (CBZ and ADT) towards improved therapeutic benefit in patients with lethal CRPC.

Optimizing live-animal bioluminescence imaging prediction of tumor burden in human prostate cancer xenograft models in SCID-NSG mice.

Noninvasive live-animal longitudinal monitoring of xenograft tumor growth and metastasis by bioluminescent imaging (BLI) has been widely reported in cancer biology and preclinical therapy literature, mainly in athymic nude mice. Our own experience at calibrating BLI readout with tumor weight/volume in human prostate cancer xenograft models in haired, SCID-NSG mice through intraprostatic (orthotopic) and subcutaneous (SC) inoculations revealed either nonexistent or poor correlation (coefficient of determination, R 2 = ~0.01-0.3). The present work examined several technical and biological factors to improve BLI utility. After ruling out promoter-luciferase (luc) specificity and luc gene loss in the cell inoculum with LNCaP-AR-luc cells expressing an androgen receptor (AR) and taggedwith AR-responsive probasin promoter-luc gene, we evaluated different routes of d-luciferin administration, imaging time during the day, charge-coupled device camera image acquisition settings, and hair removal methods to improve the imaging protocol. For most imaging sessions, BLI was carried out within the same day of tumor volume measurement. After necropsy, histological and immunohistochemical (IHC) analyses were performed on the tumors to evaluate necrosis and expression of luciferase and AR, respectively. Injection of d-luciferin by SC route, robust image-capture setting (30 000 counts and autoexposure), imaging in the morning and thorough hair removal resulted in a substantial improvement of R2 to ~0.6. Histological analyses confirmed the lack of BLI signal in necrotic tumor masses consistent with luciferase-mediated light emission only in oxygenated adenosine triphosphate-producing viable cells. IHC staining detected heterogeneous expression of luciferase tracking generally with AR expression in nonnecrotic tumor tissues. Our body of work highlighted a framework to validate imaging protocols to ensure the acquisition of interpretable BLI data as an indicator of xenograft tumor burden. The vast tissue heterogeneity in prostate tumor xenografts and variable luciferase expression constrained this technology from achieving a high correlation.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

Hypoxia-Induced Reporter Genes with Different Half-Lives.

The utility of reporter genes has gained significant momentum over the last three decades. Reporter genes are used to understand the transcriptional activity of a gene both in vitro and in vivo, and in pathway analysis and drug screening for diseases involving protozoan parasites, and in anti-cancer drug developments. Here, using a human prostate cancer xenograft model (PC3), we describe a method to construct and validate hypoxia reporter genes with different half-lives. Using molecular biology and optical imaging techniques, we have validated the expression of long half-life enhanced green fluorescence protein (EGFP) expression and short half-life luciferase gene expression to report on the spatial and temporal evolution of hypoxia in vivo.

Pharmacokinetic/Pharmacodynamic Modeling of Schedule-Dependent Interaction between Docetaxel and Cabozantinib in Human Prostate Cancer Xenograft Models.

In this work, a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively describe the antitumor activity of docetaxel (Doc) and cabozantinib (Cab) under monotherapy, concurrent therapy, interval therapy, and different sequential therapy in mouse xenograft models of castration-resistant prostate cancer was developed and evaluated. The pharmacokinetics (PK) of Doc and Cab when administered separately and simultaneously were investigated in nude mice, and PD study was conducted in tumor-bearing mice treated with different dosing schedules. The PK interaction between Doc and Cab was expressed by adding the effect of Cab on the clearance of Doc in the PK model. And the PD interaction between the two drugs was demonstrated by the developed PK/PD model through the combination index "φ" Our results showed that the concurrent therapy and Doc followed by Cab (Doc ∼ Cab) sequential therapy exhibited better tumor inhibitory efficacy than monotherapy. The Cab followed by Doc (Cab ∼ Doc) sequential schedule was less effective than monotherapy, and the interval therapy did not enhance the antitumor efficacy compared with the concurrent therapy. The parameter φ estimated from the PK/PD model quantitatively characterized the action between Doc and Cab. There was no significant PD interaction between Doc and Cab in both the concurrent schedule and the interval schedule, whereas the effect of the two drugs in the "Doc ∼ Cab" and "Cab ∼ Doc" sequential schedule was synergistic and antagonistic, respectively. The proposed model properly described the antitumor effects of Doc and Cab under different treatment schedules and could be used for dose optimization through model-based simulation.

MP83-12 TISSUE-SPECIFIC RADIATION SENSITIZATION OF PROSTATE CANCER BY APTAMER TARGETED SIRNA KNOCK-DOWN OF DNA REPAIR PATHWAY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-12 TISSUE-SPECIFIC RADIATION SENSITIZATION OF PROSTATE CANCER BY APTAMER TARGETED SIRNA KNOCK-DOWN OF DNA REPAIR PATHWAY Kenji Zennami, Yonggang Zhang, Haoming Zhou, Daniel Thorek, Theodore DeWeese, and Shawn Lupold Kenji ZennamiKenji Zennami More articles by this author , Yonggang ZhangYonggang Zhang More articles by this author , Haoming ZhouHaoming Zhou More articles by this author , Daniel ThorekDaniel Thorek More articles by this author , Theodore DeWeeseTheodore DeWeese More articles by this author , and Shawn LupoldShawn Lupold More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2580AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radiosensitizing agents can improve ionizing radiation (IR) potency. We have previously reported that an A10-3-DNAPK chimera can deliver radiation-sensitizing short interfering RNA (anti-DNA-PK siRNA) to prostate cancer cells and subcutaneous tumors through the PSMA-targeting RNA aptamer, A10-3. Here we describe the DNA-PK knock-down and radiation-sensitizing effect of the A10-3-DNAPK chimera on a human prostate cancer xenograft model in the mouse tibia, and its evaluation by bioluminescent imaging, immunohistochemistry and micro-CT. METHODS Five to six-week-old male athymic nude mice were anesthetized by isoflurane, then luciferase expressing human prostate cancer cells (LNCaP-luc) were inoculated into tibias. Tumors were evaluated by IVIS bioluminescent imaging 6 weeks after implantation, then IVIS positive mice were separated into three groups and treated by PBS, A103-ctrl and A103-DNAPK via tail vein injection. For the confirmation of DNAPK knock-down, tibias were harvested 72hrs post injection and evaluated by HE staining and immunohistochemistry. In order to confirm radiation-sensitizing effect, half of the tumors received IR therapy (6 Gy) directly to the tumored leg. Four weeks after IR, the tibias were harvested and tumor burden assessed ex vivo by nanoScan PET/CT and VivoQuant software. The CT value (HU) from these images were applied to automatically assign regions of interest for bone (red, HU 5,000-10,000) and marrow (green, HU <5,000) (Fig 1B). The sum HU were then calculated for each treatment group to estimate tumor burden four weeks after treatment. RESULTS Immunohistochemistry demonstrated reduction of DNAPK protein after A103-DNAPK treatment (Fig 1A: 50%, 38% less than PBS, A103-ctrl, respectively). The results of CT indicate significantly reduced tumor volume in A103-DNAPK pre-treated animals, with IR, when compared to animals pre-treated with PBS or A103-Ctrl (Fig 1C: p =0.045 and p=0.026, respectively). CONCLUSIONS Systemically injected aptamer siDNAPK can induce PSMA specific radiosensitization through DNAPK knock-down in human prostate tumor within mouse tibia. This system could be used to enhance radiation therapy of locally advanced PCa. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1110 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kenji Zennami More articles by this author Yonggang Zhang More articles by this author Haoming Zhou More articles by this author Daniel Thorek More articles by this author Theodore DeWeese More articles by this author Shawn Lupold More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models.

Intratumoral inoculation of viruses with tumor-selective cytotoxicity may induce cancer cell death and, thereby, shrink neoplastic lesions. It is unlikely, however, that viral tumor cell killing alone could produce meaningful, durable clinical responses, as clinically suitable ‘oncolytic’ viruses are severely attenuated and their spread and propagation are opposed by host immunity. Thus, a more propitious event in this context is the innate antiviral response to intratumoral virus administration, in particular for recruiting durable adaptive immune effector responses. It may represent a double-edged sword, as innate immune activation may eliminate infected tumor cells early, intercept viral spread and block any meaningful therapeutic response. The innate response to viral infection of tumors may be very different from that in non-malignant target tissues, owing to the unusual composition/tissue properties of tumor stroma. In this work, we report investigations of the innate immune response to the oncolytic poliovirus recombinant, PVSRIPO, in two mouse xenotransplantation models for breast and prostate cancer. Our observations indicate short-term virus persistence in infected tumors and virus recovery indicative of modest intratumoral propagation and persistence. Yet, a powerful innate inflammatory response coincided with chemokine induction and myeloid cell infiltration into tumors that was, interestingly, dominated by neutrophils. The combined effect of PVSRIPO tumor infection and the innate response it elicits was significant tumor regression in both models.

Prostate-specific membrane antigen-targeted liposomes specifically deliver the Zn(2+) chelator TPEN inducing oxidative stress in prostate cancer cells.

To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

Discovery of BMS-641988, a Novel Androgen Receptor Antagonist for the Treatment of Prostate Cancer.

BMS-641988 (23) is a novel, nonsteroidal androgen receptor antagonist designed for the treatment of prostate cancer. The compound has high binding affinity for the AR and acts as a functional antagonist in vitro. BMS-641988 is efficacious in multiple human prostate cancer xenograft models, including CWR22-BMSLD1 where it displays superior efficacy relative to bicalutamide. Based on its promising preclinical profile, BMS-641988 was selected for clinical development.

MP37-14 RNA INTERFERENCE-MEDIATED KNOCKDOWN OF CHD1 IN HUMAN PROSTATE XENOGRAFT TUMORS ALTERS TUMOR GROWTH AND METASTATIC BEHAVIOR

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2015MP37-14 RNA INTERFERENCE-MEDIATED KNOCKDOWN OF CHD1 IN HUMAN PROSTATE XENOGRAFT TUMORS ALTERS TUMOR GROWTH AND METASTATIC BEHAVIOR Su Jung Oh, Derya Tilki, Hüseyin Sirma, Ronald Simon, and Tobias Lange Su Jung OhSu Jung Oh More articles by this author , Derya TilkiDerya Tilki More articles by this author , Hüseyin SirmaHüseyin Sirma More articles by this author , Ronald SimonRonald Simon More articles by this author , and Tobias LangeTobias Lange More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1277AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chromodomain helicase DNA-binding protein 1 (CHD1) is a tumor suppressor which is frequently homozygously deleted genes in prostate cancer (PCa). It still remains unclear whether this molecule is involved in metastatic behavior of PCa. To elucidate the role of CHD1 in tumor metastatic dissemination, we analyzed the tumorigenic and metastatic potential of PCa cells after RNAi-mediated knockdown (KD) of CHD1. METHODS CHD1 was stably depleted in PC-3 cells by lentiviral transduction of short hairpin RNA (shRNA) with puromycin resistance (PC-3-shCHD1). Control cells had an empty vector (PC-3-shNeg). The KD of CHD1 was confirmed by western blot. In vivo, PC-3-shCHD1 and -shNeg were subcutaneously xenografted into immunodeficient pfp−/−/rag2−/− mice. After 6 weeks, mice were euthanized. Both primary tumors and right lungs were processed for histopathological analysis. Quantification of spontaneous lung metastases was performed by direct microscopic visualization and counting. DNA from blood, left lungs and bone marrow were isolated for quantification of DTC and CTC by Alu-PCR. Effects of CHD1 depletion on cell adhesion were analyzed by static and dynamic flow adhesion assays. RESULTS KD of CHD1 by shRNA resulted in down-regulation of CHD1 protein levels by about 50%. In vivo, xenograft primary tumors were significantly larger in the CHD1-depleted group accompanied by a reduced number of DTC in the bone marrow compared to the control. Strikingly, the number of DTC in the lungs was increased. Spontaneous lung metastases consisted of several cells per metastasis after CHD1-depletion, whereas control cells predominantly formed single cell lung metastases as known from parental PC-3 in this model. Moreover, CHD1-depletion significantly increased the number of adhesive events of PC-3 under dynamic conditions. Static adhesive interactions and CTC-levels did not change upon CHD1-depletion. CONCLUSIONS KD of the tumor suppressor CHD1 increases tumorigenicity and drastically changes metastatic organotropism in a spontaneous metastasis xenograft model of human PCa. In particular, CHD1-depletion specifically promoted spontaneous dissemination into the lung, whereas dissemination into the bone marrow was abrogated. This might be reflected by the increased adhesive properties of CHD1-depleted PC-3 on pulmonary endothelium. We moreover hypothesize that CHD1 might suppress the switch of ‘dormant’ single cell DTC to proliferating metastatic colonies in the lung and indicate a possible novel role of altered CHD1 in metastatic behavior of PCa. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e443 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Su Jung Oh More articles by this author Derya Tilki More articles by this author Hüseyin Sirma More articles by this author Ronald Simon More articles by this author Tobias Lange More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...