Male SCID Mice Research Articles

Evaluation of Lu-177 RM2 as a targeted radiopharmaceutical in a PC-3 xenograft model of androgen-independent prostate cancer.

e13522 Background: Prostate cancer is the most common cancer in American men (after nonmelanoma skin cancers). In 2010 more than 200,000 cases were diagnosed and more than 30,000 men died of prostate cancer. One in six men will be diagnosed with prostate cancer during his lifetime and one in thirty six men will die of prostate cancer. Human prostate cancer has been shown to widely express the bombesin receptor (BB2R). Lutetium-177 (177 Lu) radiolabeled BB2r agonist analogs are currently being investigated preclinically for their utility as targeted radiation therapy agents. (Maddalena, J Nucl Med, 50:2017, 2009 and Johnson, Can Biother & Radiopharm, 21(2):155, 2006) Recently, Maecke and co-workers identified a high affinity BB2r antagonist (Ga-68 and In-111-RM2) for potential use as a PET/SPECT diagnostic imaging agent. (Mansi, Clin Can Res, 15(16):5240, 2009 and Mansi, Eur J Nucl Med Mol Imaging, May 2010) The purpose of this work was to investigate the biodistribution of Lu-177-RM2 for potential as a radiotherapeutic agent using a PC-3 xenografted SCID mouse model. Methods: Male SCID mice were inoculated subcutaneously with 5 million PC-3 cells/site in 100uL. At 4 weeks post-inoculation, pharmacokinetic studies of 177Lu-VA RM2 were conducted at 1, 4, 24, 48, 72, and 96 hours post-injection. Micro-SPECT/CT imaging studies were done at 24, 48, 72, and 96 hrs. Results: See table; shown in %ID/g. Conclusions: Our study demonstrates that Lu-177-RM2 shows promising pharmacokinetic properties with high affinity for the BB2 receptor and excellent tumor to blood and tumor to organ ratios. The prostate tumor uptake of Lu-177-RM2 is equal or greater than any previously identified BB2r targeting radiotherapeutic agent. Lu-177-RM2 exhibits rapid washout from the pancreas and other non-target organs making it an ideal candidate for tumor targeted therapy research. 1 HR 4 HRS 24 HRS 48 HRS 72 HRS 96 HRS Blood 0.36 +/- 0.15 0.02 +/- 0.01 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 0.00 +/- 0.00 Pancreas 17.53 +/- 1.97 1.59 +/- 0.7 0.26 +/- 0.03 0.18 +/- 0.04 0.19 +/- 0.03 0.03 +/- 0.00 Tumor 9.25 +/- 3.35 11.46 +/- 5.38 5.56 +/- 1.59 5.28 +/- 1.42 4.03 +/- 0.92 3.14 +/- 0.33

Abstract 4767: C6 ceramide sensitizes multiple aggressive pancreatic cancer cell lines to gemcitabine, paclitaxel and cetuximab mediated anti tumor effects in vivo and in vitro

Abstract Introduction: Pancreatic adenocarcinoma continues to be a highly lethal malignancy (5 yr survival 5%) poorly responsive to all chemo radiation therapies, with only modest improvement with Gemcitabine the current “main line” drug. We have previously demonstrated the C6 Ceramide chemo enhancement of Gemcitabine and other pancreatic chemo agents. The current study explores the mechanism of C6 Ceramide effects on pancreatic chemo resistance pathways – pro-survival and ras-signaling in pancreatic cancer cells. Methods: In vivo: 2-3 month SCID male mice with pancreatic (pan) tumor implants (2 × 106 L3.6 cells) received intraperitoneal (I.P.) injections (3x/wk) of Paclitaxel (Pax) (3.0mg/gm), Gemcitabine (Gem, 10mg/gm) with/without C6 ceramide (10mg/ml) and were observed 6 weeks and tumor response/survival recorded. In vitro: Drug dose Pax 3 ug/ml, Gem 3 ug/ml, C6 Ceramide 10 ug/ml. The MTT anti tumor response was measured in 3 pancreatic lines L3.6, PANC-1, and MIA (Kras mutated). Signaling activation by Western blot utilized antibodies: LY294002, and Wortmannin to block PI3K/AKT pathway, U0126 and PD 98059 to block ERK pathway and Rapamycin to block mTORC1 pathway. Results: Combined therapy with C6 Ceramide induced significant inhibition of exponential tumor growth and prolonged survival by Gem and Pax. Administration of Gem or Pax with C6 Ceramide induced a significant increase in cell death and apoptosis in pancreatic cancer cells (L3.6, PANC-1 and MIA PaCa-2) which were associated with inhibiting perturbations of cell signaling pathways including pro-survival PI3K/Akt/mTOR, and ERK/MAPK, Ras signaling pathways. Pharmacological inhibition with specific signaling pathway inhibitors enhanced cytotoxicity by Gem or Pax, suggesting that survival pathway inhibition might be the key mechanism contributing to C6 Ceramide synergism of Pax or Gem anti-tumor effects. We also showed C6 ceramide potentiation of Cetuximab anti tumor effects on Kras mutant MIA cells thus overcoming (EGFR mAb) resistance by inhibiting pro-survival PI3K/AKT/mTOR pathways and bypassing K-RAS mutant Ras-ERK pathways. Conclusion: C6 Ceramide an apoptosis signal potentiates the anti tumor effects of chemo agents Gemcitabine and Paclitaxel and the biologic EGFR Inhibitor Cetuximab against 3 aggressive pancreatic cancer cell lines by apparent inhibition of the pro-survival PI3K/AKT/mTOR pathways and the Kras mutated ERK/MAPK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4767. doi:10.1158/1538-7445.AM2011-4767

Abstract 3504: Targeting the Akt pathway and HDAC signaling in prostate cancer

Abstract Loss of the tumor suppressor phosphatase and tensin homolog protein (PTEN), a negative regulator of the survival Akt pathway, and elevated levels of histone deacetylase (HDAC) enzymes which regulate DNA transcription, correlated positively with the prostate cancer progression, recurrence and metastasis. MK-2206 is an allosteric Akt inhibitor with a broad antitumor activity; clinical studies have recently been completed in patients with solid tumors, showing MK-2206 good tolerability and evidences of clinical activity. vorinostat, an HDAC inhibitor, is involved in more than 100 clinical trials in solid tumors and it showed antitumor activity in different prostate cell lines. In this study we hypothesized that combination of MK-2206 and vorinostat may provide a greater antitumor efficacy, targeting different survival pathways activated in prostate cancer. Different prostate cell lines were tested in both in vitro and in vivo xenograft models. LuCaP23.1. hormone-sensitive tumors were placed subcutaneously into male SCID mice. Tumor growth was assessed twice weekly by using serial caliper measurements. Luciferase-transfected PC3 hormone-independent tumor cells were injected in the right tibiae of male SCID mice and tumor progression was monitored weekly by bioluminescence assay using the Xenogen IVIS Living Imaging System. At the end of the experiment, Faxitron radiographs of the tibiae were taken. Mice were treated with MK-2206 at 120 mg/kg/d by oral gavage and i.p. injection of vorinostat at 100 mg/kg/d, 5 days per week. In vitro treatment of PC3 cells showed that both MK-2206 and vorinostat affected cell proliferation in a dose and time dependent manner, with a greater effect in combination. Western blot experiments validated p-Akt protein level inhibition due to MK-2206 treatment. Preliminary PC3 and LuCaP23.1. xenograft in vivo experiments confirmed the therapeutic activity of MK-2206 and vorinostat as single agents. The combination therapy showed a modest but greater inhibition of tumor growth in the PC3 xenograft bone metastatic model though not statistically significant. The antiproliferative effect of this combination was more evident in the subcutaneously LuCaP 23.1. xenograft model. This study showed for the first time the therapeutic association of MK-2206 and vorinostat in prostate cancer. We expect to further investigate the MK-2206 +/- vorinostat therapy and to provide new insight into the role of HDACs signaling and Akt pathway in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2011-3504

Abstract 4890: Diffusion-weighted MRI provides an early assessment of treatment response in a murine model of metastatic prostate cancer to the bone

Abstract Ninety percent of patients with advanced prostate cancer develop metastases to the bone1. At present, there is no definitive means for assessing response to therapy, with RECIST identifying tumors in the bone as “unmeasureable”. Diffusion-weighted MRI (DW-MRI) is highly sensitive to microscopic changes in tumor tissue. Our goal is to use DW-MRI to detect and quantify changes in tumor tissue throughout therapy where the magnitude of the change could be related to the effectiveness of the treatment. Human prostate cancer (PC3) cells were implanted by direct intra-tibial injection into male SCID mice, which served as a model for prostate bone metastases. MRI experiments were performed on a 9.4T MRI. Upon identifying a tumor volume of ∼10mm3, animals were randomly distributed into 4 treatment groups: docetaxel 20 mg/kg i.p. dose once a week for 3 doses (qd7×3; N=5), fractionated radiation IR at 2 Gy/day 5x per week for 2 weeks (N=7), combined treatment (N=5) and control (N=6). Animals were imaged by DW-MRI in which the apparent diffusion coefficient was calculated from the DW images. Tumor volume and ADC measurements were acquired pre-treatment and on days 0, 1, 4, 7, and once per week after post-treatment initiation. Animals were removed from the study when the tumor reached a percent increase of 400% (5x the initial tumor volume). Group comparisons in MRI metrics were obtained using an unpaired Student t-test. Mice treated by Dox or IR had significant smaller increase in tumor volume compared to control mice. Combined treatment resulted in improved efficacy over lone therapies. This resulted in a significant decrease in tumor size. The Dox group showed an increase in ADC following the first administration and a slow decline in ADC values following day 4 post-treatment initiations regardless of additional treatments. ADC value for IR treated animals did not elevate above control until day 7. Combined treatment generated increased ADC values that were maintained throughout the treatment cycle. IR and Dox alone and in combination were effective as assessed by anatomical and DW-MRI. Those results demonstrate the potential of quantitative ADC map for assessing early response of metastatic cancer to the bone.

Abstract 1543: Potential role of Schwann cells and laminin adhesion in prostate cancer perineural invasion

Abstract Prostate tumor cell migration and invasion of prostatic nerves, a process known as perineural invasion (PNI), is a major route for extracapsular extension during prostate cancer metastasis. The clinical significance of perineural invasion as a predictor for poor patient outcome has been extensively documented. The laminin family of extracellular matrix glycoproteins lines the neural route and plays a prominent role in the development and maintenance of the peripheral nervous system. Evidence has shown that the laminin receptor integrin α6β1 promotes prostate tumor cell migration and invasion in aggressive disease. We hypothesize that α6β1 regulates prostate cancer perineural invasion by regulating laminin dependent adhesion and migration on nerves. In this study we demonstrate remarkable integrin α6 expression in prostate tumors undergoing active perineural invasion using immunohistochemical analysis of human prostate tumors. The expression of integrin α6 correlates with expression of the laminin subunits α5 and γ1 which comprise laminin-511, the major ligand for integrin α6β1. The laminin extracellular matrix on the peripheral nerves of the prostate is derived from Schwann cells during the myelination process. We demonstrate here that Schwann cells are highly expressed in prostatic nerves using immunohistochemical analysis of the Schwann cell specific marker S-100, suggesting that tumor cells invade myelinated nerves. We also demonstrate two specific patterns of prostate tumor nerve invasion along prostatic nerves. Invasion along the external surface of the nerve sheath or in the perineural space maintains nerve structure while invasion of the endoneural space disrupts nerve structure and displaces Schwann cells. This suggests that tumor interactions with nerves and Schwann cells may illicit nerve damage during the PNI process. Using a DU-145 prostate tumor and neuron/glial cell in vitro co-culture model we demonstrate that DU-145 cells (cytokeratin positive) migrate towards glial cells (GFAP positive) and neurons (Beta-III tubulin positive) isolated from dorsal root ganglia of male SCID mice. Mature cultures of the prostate tumor cells and neuron/glial cells demonstrate distinct islands of growth. The patterns of colony formation were non-random and interspersed colonies of tumor and nerve cells were not observed. Taken together, our immunohistochemical results suggest that perineural invasion of laminin coated nerves is regulated by the laminin adhesion receptor α6β1. Our in vitro model system demonstrates that tumor cells migrate to neuron/glial cells and dominate the co-culture, correlating with our immunohistochemical data. We expect that blocking α6β1 function in this system will impede prostate tumor cell migration and invasion in the nerve environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2011-1543

Abstract 599: TMPRSS2-ERG gene fusion sensitizes VCaP human prostate tumors to the fully human anti-IGF-IR monoclonal antibody SCH 717454

Abstract A gene fusion of the androgen-regulated gene transmembrane protease, serine 2, (TMPRSS2) to the v-ets erythroblastosis virus E26 oncogene homolog (avian), ERG, of the erythroblast transformation-specific (ETS) family of transcription factors is a recently discovered genetic alteration in prostate cancer. Expression of the TMPRSS2-ERG fusion in prostate cancer patients is associated with an aggressive clinical phenotype and an early recurrence of the disease. Interestingly, in TMPRSS2-ERG positive prostate cancer cells, expression of this gene fusion is regulated by both estrogens and androgens. However, while the hormonal regulation of this fusion has been established the downstream effects of TPRSS2-ERG fusion gene have not been determined. Quantitative PCR analyses of 134 cancer cell lines, including 10 prostate cancer models, for expression of the TMPRSS2-ERG gene fusion showed that it was only present in VCaP, DuCaP, and NCI-H660 prostate cancer cells. We report here that VCaP human prostate cancer cells and tumor xenografts are sensitive to treatment with the fully human anti insulin-like growth factor-I receptor (IGF-IR) monoclonal antibody SCH 717454. After 28 days of treatment, single-agent SCH 717454 (0.5 mg, ip, 2 × week) caused complete inhibition of VCaP tumor growth. MDA PCa-2b, 22Rv1, DU-145, and LNCaP human prostate cancer tumor xenografts (negative for expression of the TMPRSS2-ERG gene fusion) did not respond to treatment with SCH 717454. Further, PTEN null NCI-H660 prostate tumors that also express the TMPRSS2-ERG gene fusion also did not respond to treatment with SCH 717454. When male SCID mice bearing established VCaP prostate cancer tumor xenografts were treated with the combination of surgical castration and SCH 717454 we observed 100% tumor regressions in all of the animals so that after 28 days of treatment there were no detectable tumors. When SCH 717454 was combined with the antiandrogen bicalutamide (50 mpk, po, bid), VCaP tumors regressed by 42%. That the combination of SCH 717454 and castration produced superior anti-tumor activity than the combination of SCH 717454 and bicalutamide provides further evidence that expression of the TMPRSS2-ERG gene fusion in VCaP prostate cancer tumors is regulated by both estrogens and androgens. TMPRSS2-ERG gene fusion expressing prostate cancer represents a new sub-classification of human prostate cancer. The precise role that the TMPRSS2-ERG fusion plays in the biology of human prostate cancer remains to be fully determined. These data indicate that PTEN wild-type VCaP prostate tumors that express the TMPRSS2-ERG gene fusion are sensitive to anti-IGF-IR treatment with SCH 717454 and that combining SCH 717454 with an androgen ablation therapy results in impressive anti-tumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 599. doi:10.1158/1538-7445.AM2011-599

Cell Origin of Human Mesenchymal Stem Cells Determines a Different Healing Performance in Cardiac Regeneration

The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105+-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105+ hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function.

Inhibitory effect of dietary atorvastatin and celecoxib together with voluntary running wheel exercise on the progression of androgen-dependent LNCaP prostate tumors to androgen independence

We determined the inhibitory effect of dietary atorvastatin, dietary celecoxib and voluntary running wheel exercise (RW) alone or in combination on the formation and growth of androgen-independent LNCaP tumors in castrated SCID mice. Male SCID mice were injected subcutaneously with androgen-dependent prostate cancer LNCaP cells. When the tumors reached a moderate size, the mice were surgically castrated and treated with atorvastatin (0.02% in the diet), celecoxib (0.05% in the diet) or RW alone or in combination for 42 days. RW or celecoxib alone had a moderate inhibitory effect on the androgen-independent growth of LNCaP tumors, but atorvastatin alone had little or no effect on tumor growth. Combinations of atorvastatin and celecoxib had a stronger inhibitory effect on the formation and growth of androgen-independent LNCaP tumors than either drug alone. A combination of RW together with atorvastatin and celecoxib had the most potent inhibitory effect on the progression of LNCaP tumors to androgen independent growth. The serum concentration of atorvastatin after two weeks of oral administration of atorvastatin was 6.1 ng/ml. The serum concentration of celecoxib after treatment with dietary celecoxib for two weeks was 1090 ng/ml. The serum concentration of atorvastatin but not that of celecoxib was substantially reduced when the two drugs were given in combination. The drug concentrations observed in our animal studies are comparable or less than those commonly found in humans treated with atorvastatin or celecoxib. Our results indicate that administration of atorvastatin and celecoxib together with voluntary exercise may be an effective strategy for the prevention of prostate cancer progression from androgen dependence to androgen independence.

Celecoxib steigert die Strahlenempfindlichkeit von sekundären Knochentumoren eines nicht-kleinzelligen humanen Lungenkarzinoms durch Anti-Angiogenese in vivo

Cyclooxygenase-2 (COX-2) inhibitors mediate a systemic antitumor activity via antiangiogenesis and seem to enhance the response of primary tumors to radiation. Radiosensitizing effects of COX-2 inhibition have not been reported for bone metastases. Therefore, the aim of this study was the investigation of the radiosensitizing effects of the selective COX-2 inhibitor celecoxib in secondary bone tumors of a non-small cell lung carcinoma in vivo. Human A549 lung carcinomas were implanted into a cranial window preparation in male SCID mice (n = 24). Animals were treated with either celecoxib or radiation (7 Gy single photon dose) alone or a combination of celecoxib and radiation, respectively. Untreated animals served as controls. The impact of radiation and COX-2 inhibition on angiogenesis, microcirculation, and tumor growth was analyzed over 28 days by means of intravital microscopy and histological methods. Monotherapies with radiation as well as celecoxib had significant antitumor effects compared to untreated controls. Both therapies reduced tumor growth and vascularization to a similar extent. The simultaneous administration of celecoxib and radiation further enhanced the antitumor and antiangiogenic effects of single-beam radiation. With the combined treatment approach, tumor vascularization and tumor size were decreased by 57% and 51%, respectively, as compared to monotherapy with radiation. The combined application of radiation therapy and COX-2 inhibition showed synergistic effects concerning the inhibition of tumor growth and tumor angiogenesis. Therefore, the combination of radiation with COX-2 inhibitor therapy represents a promising approach to improve the therapeutic efficacy of radiotherapy of bone metastases.

Neuroendocrine prostate cancer xenografts with large‐cell and small‐cell features derived from a single patient's tumor: Morphological, immunohistochemical, and gene expression profiles

Small-cell carcinoma (SCC) of the prostate is an AR-negative variant of prostate cancer found at progression in 10-20% of castrate-resistant disease. Its finding predicts a distinct clinical course and a poor prognosis. Large-cell neuroendocrine carcinoma (LCNEC) is a much rarer variant that behaves similarly to SCC. The biological mechanisms that drive these disease variants are poorly understood. Eight tumor fragments from the salvage pelvic exenteration specimen of a patient with castrate-resistant prostate carcinoma were subcutaneously implanted into 6- to 8-week-old male CB17 SCID mice. Serial tissue sections and tissue microarrays of the resulting MDA PCa 144 xenograft lines were used for histopathologic and immunohistochemical characterization of the xenografts and their tissue of origin. RNA from two representative xenograft sublines was used for gene-expression profiling. All eight fragments formed tumors: four of the MDA PCa 144 xenograft sublines had morphologic characteristics of SCC and four, of LCNEC. All retained high fidelity to their parent tumor tissue, which remained stable through serial passages. Morphological transitions in the specimen of origin suggested LCNEC represents an intermediate step between adenocarcinoma and SCC. Over 2,500 genes were differentially expressed between the SCC (MDA PCa 144-13) and the LCNEC (MDA PCa 144-4) sublines and enriched in "Nervous System Development" Gene Ontology subtree. The eight xenograft models described represent the spectrum of neuroendocrine carcinomas in prostate cancer and will be valuable preclinical tools to study the pathogenesis of and therapy targets for this increasingly recognized subset of lethal prostate cancer.

Properties and effects of a novel liquid crystal nanoparticle formulation of docetaxel in a prostate cancer mouse model

Treatment with docetaxel is the standard of care as first line chemotherapy in castration resistant prostate cancer. Due to serious side effects from the commercially available Taxotere formulation, we aimed to develop a safe and effective nanoparticle formulation of docetaxel. Liquid crystal nanoparticles (LCNPs), based on phosphatidyl choline, glycerol dioleate and polysorbate 80 dispersed in excess aqueous solution, were produced by simple procedures as carriers of docetaxel. Their effect on tumor growth in male SCID mice inoculated with PC-3 cells was compared to the effect of Taxotere and empty LCNP vehicle. Immunohistochemistry was performed to evaluate cell proliferation, angiogenesis and apoptosis in tumor tissue. Docetaxel and lipid excipients were dispersed into well-defined LCNP, stable during long-term storage. Mice subjected to LCNP/docetaxel formulation showed a better tumor regression than mice treated with Taxotere, with an indication of better tolerability. Immunohistochemical staining showed a decreased expression of Ki-67 in tumors from LCNP/docetaxel treated animals, especially in the cores of the tumors, suggesting better penetration/absorption compared to Taxotere. A new lipid-based nanoparticle formulation has been developed as carrier for docetaxel. Treatment effects in SCID mice indicate that this may be an interesting alternative to the current marketed formulation product.

Estradiol suppresses tissue androgens and prostate cancer growth in castration resistant prostate cancer

BackgroundEstrogens suppress tumor growth in prostate cancer which progresses despite anorchid serum androgen levels, termed castration resistant prostate cancers (CRPC), although the mechanisms are unclear. We hypothesize that estrogen inhibits CRPC in anorchid animals by suppressing tumoral androgens, an effect independent of the estrogen receptor.MethodsThe human CRPC xenograft LuCaP 35V was implanted into orchiectomized male SCID mice and established tumors were treated with placebo, 17β-estradiol or 17β-estradiol and estrogen receptor antagonist ICI 182,780. Effects of 17β-estradiol on tumor growth were evaluated and tissue testosterone (T) and dihydrotestosterone (DHT) evaluated by mass spectrometry.ResultsTreatment of LuCaP 35V with 17β-estradiol slowed tumor growth compared to controls (tumor volume at day 21: 785 ± 81 mm3 vs. 1195 ± 84 mm3, p = 0.002). Survival was also significantly improved in animals treated with 17β-estradiol (p = 0.03). The addition of the estrogen receptor antagonist ICI 182,780 did not significantly change survival or growth. 17β-estradiol in the presence and absence of ICI 182,780 suppressed tumor testosterone (T) and dihydrotestosterone (DHT) as assayed by mass spectrometry. Tissue androgens in placebo treated LuCaP 35V xenografts were; T = 0.71 ± 0.28 pg/mg and DHT = 1.73 ± 0.36 pg/mg. In 17β-estradiol treated LuCaP35V xenografts the tissue androgens were, T = 0.20 ± 0.10 pg/mg and DHT = 0.15 ± 0.15 pg/mg, (p < 0.001 vs. controls). Levels of T and DHT in control liver tissue were < 0.2 pg/mg.ConclusionsCRPC in anorchid animals maintains tumoral androgen levels despite castration. 17β-estradiol significantly suppressed tumor T and DHT and inhibits growth of CRPC in an estrogen receptor independent manner. The ability to manipulate tumoral androgens will be critical in the development and testing of agents targeting CRPC through tissue steroidogenesis.

Abstract 4172: Multi-parametric characterization of an experimental model of cancer cachexia

Abstract Cachexia is a complex syndrome that is characterized by a massive loss of adipose tissue and skeletal mass. It is encountered in up to 50% of cancer patients and accounts for more than 20% of cancer related deaths. Currently, there is no known cure for cachexia, since mechanisms underlying its manifestation are not defined clearly enough to design effective therapeutic strategies. Here we have used magnetic resonance imaging (MRI) to characterize the vascular properties of cachectic vs. non-cachectic tumors. We have also analyzed the glucose metabolism of these tumors by using [18F]fluorodeoxyglucose (FDG) with positron emission tomography (PET). These studies will provide further insight into the cachectic phenotype that can be used to design treatments to arrest or reverse this condition. We used cachectic (MAC16) and non-cachectic (MAC13) murine colon adenocarcinoma tumors. The cell lines, originally from Dr. Tisdale's laboratory, were obtained from Dr. Sidransky with Dr. Tisdale's permission. Approximately 2 × 106 cells were inoculated in the flank of male SCID mice. MRI studies were performed on a 4.7T Bruker Avance spectrometer. Mice were anesthetized with an i.p. injection of ketamine and acepromazine. For vascular imaging, the tail vein was catheterized before placing the animal in the spectrometer. Multislice relaxation rate maps were obtained by a saturation recovery method combined with fast T1 SNAPSHOT-FLASH imaging. An M0 map with a recovery delay of 7 s was acquired from four 1 mm thick slices through the tumor followed by corresponding images obtained with three relaxation delays (100 ms, 500 ms, and 1 s). The T1 maps were obtained before i.v. injection of albumin-GdDTPA and repeated over a 21 minute period. For PET imaging, mice fasted overnight were injected with 0.2 mCi of FDG. At 60 min post injection, a 15 min static image was acquired over the tumors. Images were decay corrected and reconstructed using 2D OSEM. Cachectic MAC16 tumors induced extensive weight loss, unlike the non-cachectic MAC13 tumors. The difference in the weight of mice became significant within 2 weeks after inoculation. Mice with comparable tumor volumes were imaged 3 to 4 weeks after inoculation. While vascular volumes were not significantly different between the two groups, we did observe significantly lower permeability in the cachectic tumors. Consistent with the MRI data we found significantly lower levels of VEGF mRNA in quantitative-RT-PCR of tumor extracts of MAC16 tumors compared to MAC13 tumors but not in MAC16 cells compared to MAC13 cells. FDG PET imaging revealed increased glycolytic activity in a cachectic MAC16 tumor compared to a non-cachectic MAC13 tumor. These studies are part of our ongoing work to obtain a comprehensive characterization of the cachectic phenotype using noninvasive multi-modality imaging that will allow us to detect cancer-induced cachexia and identify new targets to prevent or reverse this condition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4172.

Abstract 344: Targeting fibroblast growth factor signaling in prostate cancer

Abstract Currently no therapy effectively controls the progression of advanced human prostate cancer. We previously reported that fibroblast growth factor 9 (FGF9) contributes to the osteoblastic progression of human prostate cancer in bone (J Clin Invest 2008;118:2697). We examined the effect of TKI258 (a receptor tyrosine kinase inhibitor [TKI] with strong activity against FGF receptor 1-3 [FGFR1-3; IC50 &amp;lt; 40 nM]; [Novartis Pharma Corp.]) on primary mouse osteoblasts treated with and without FGF9 and TKI258. TKI258 blocked FGF9's induction of p-FRS2α (a gatekeeper that mediates FGFR downstream signals) an indication that TKI258 specifically blocks FGF signaling in osteoblasts. These observations provide strong evidence implicating FGF signaling in the osteoblastic progression of prostate cancer in bone. We hypothesized that pharmacologic blockade of the FGFR signaling has an antitumor effect in prostate cancer. To test our hypothesis, we studied the antitumor efficacy of TKI258 in the osteoblastic growth of MDA PCa 118b, a prostate cancer tumor graft that depends on FGF9 for growth. We assessed the effect of TKI258 on the growth of MDA PCa 118b prostate cancer cells in the femurs of male SCID mice. Ten mice were treated with TKI258 (20 and 60 mg/kg body weight daily by oral gavage), and another 10 mice were treated with vehicle only. Treatment started the day after we identified tumor on MRI scanning at the site of tumor cell injection and continued for 3 weeks, when we used MRI to assess tumor volumes in the femurs and microCT to assess bone mass. Femurs bearing MDA PCa 118b in mice treated with TKI258 had significantly smaller tumors (P = 0.019) and less prostate cancer-induced cortical bone (P = 0.034) than did control mice. Histopathologic analysis indicated that tumors in the treated mice were smaller than in the controls. Initial analysis of tumor-associated osteoclasts (assessed by their expression of tartrate-resistant acidic phosphatase) shows no difference between the treated and control mice. The femurs bearing MDA PCa 118b tumors were then analyzed by Western blotting to measure signaling; we found that TKI258 specifically inhibited p-ERK1/2 (an FGF signaling target gene) but not p-AKT. Taken together, these results suggest that TKI258 inhibits the growth of prostate cancer cells in bone by targeting both those cells and osteoblasts (but not osteoclasts), possibly by blocking FGF signaling. Subsequent molecular analysis of FGF downstream target genes will help elucidate the mechanism underlying TKI258's inhibition of prostate cancer growth in bone. Results from these studies will also help identify markers of response to TKI258 that will be used to interpret an ongoing clinical study with TKI258 in selected men with castrate-resistant prostate cancer with bone marrow infiltration. The results will serve as the foundation for the development of candidate predictive markers and further therapies based on targeting the FGF pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 344.

Abstract 3243: Differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a mouse model

Abstract Background: Caloric restriction (CR) has been shown to be anti_cancer. However, whether these effects are the result of generalized reduction in overall caloric intake or reduced intake of macronutrients (i.e. carbohydrates) are unknown. We sought to investigate the differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a xenograft model. Materials and Methods: A total of 100 male SCID mice were injected subcutaneously with the LAPC-4 cell line. Mice were randomized to 1 of 4 diets: ad libitum Western diet (WD, 49% carbohydrates, 35% fat, and 16% protein), pair-fed No Carbohydrate Ketogenic Diet (NCKD; 1% carbohydrates, 83% fat, 16% protein), Western diet calorically restricted (WD-CR), and NCKD calorically restricted (NCKD-CR). Calorically restricted arms were fed 75% of the calories of their reference diet group. Tumor volumes and body weights were measured twice weekly. Mice were sacrificed when tumor volumes reached 1,000 mm3. Results: Relative to WD, there was a suggestion of smaller body weights in both NCKD-CR and WD-CR arms (rank-sum, p≤0.06). There was trend for heavier mice in the NCKD group (rank-sum, p=0.09). By day 50, median tumor volumes for NCKD, WD-CR, and NCKD-CR were 45, 51, and 62% smaller than WD (rank-sum, all p≤0.001). At any time point, there were no significant differences in tumor volume among the NCKD, NCKD-CR, and WD-CR arms. However, there was a suggestion of smaller tumors in the NCKD-CR arm by day 42 relative to NCKD and WD-CR (p&amp;lt;0.11). Diet was significantly associated with mouse survival (log_rank, p=0.004). In 2_way comparisons, NCKD, NCKD-CR, and WD-CR had significantly prolonged survival relative to WD (log_rank, all p≤0.02). However, no difference in survival was seen among any caloric or carbohydrate restricted arms (log rank, p≤0.16). A total of 34 mice died due to infection prior to their tumors reaching 1,000mm3 and therefore were not included in the survival analyse (WD-CR, n=14; NCKD, n=5; NCKD-CR, n =15). Conclusions: Both CR and carbohydrate restriction without weight loss are effective in delaying tumor growth and improving mouse survival. We found no differences between caloric and carbohydrate restricted arms, though this may relate to loss of study power due to mouse infections. CR may further weaken the immune system of immunocompromised mice subjecting them to increased risk of infections. A confirmatory study is underway in a high-level barrier facility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3243.

Abstract 2816: Low-carbohydrate diets and prostate cancer growth: How low is “low enough”

Abstract Introduction: Previous dietary studies indicate carbohydrate intake may influence prostate cancer biology, as LAPC-4 and LNCaP xenograft mice fed a no-carbohydrate ketogenic diet (NCKD; 84% fat-0% carbohydrate-16% protein kcal) had significantly smaller tumors and longer survival times compared to mice fed a Western diet (40% fat-44% carbohydrate-16% protein kcal). The NCKD mice were also found to have higher levels of circulating IGFBP-3 and the lowest levels of insulin, IGF-1, and IGF-1:IGFBP-3 ratio despite consuming more calories than the Western group. As it is nearly impossible for a human to consume and maintain a no-carbohydrate diet similar to that in the previous xenograft studies, we sought to determine whether diets containing 10% or 20% kcal from carbohydrates could slow tumor growth in a similar manner to the NCKD in a xenograft model. METHODS: A total of 150 male SCID mice were injected with LAPC-4 cells and placed on a Western diet (35% fat-49% carbohydrate-16% protein kcal) ad libitum. Two weeks post-injection, all mice were randomized to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate. Ten mice not injected with tumor were fed an ad libitum low-fat diet (12% fat-72% carbohydrate-16% protein kcal) and served as the reference group in a modified-paired feeding protocol for the other three groups. Calorie intake and body weights were measured thrice weekly and tumor volumes twice per week. Mice were sacrificed when tumors reached 1,000mm3. RESULTS: Despite consuming 5-10% extra calories on average, all mice receiving low-carbohydrate diets were significantly lighter than the mice consuming the low-fat diet (p&amp;lt;0.04). Overall, the mice fed a NCKD were significantly lighter than the other two arms at multiple time points (p&amp;lt;0.05). There were no significant differences in tumor volumes among groups at any time except at Day 52 and 59, where 10% carbohydrate mice had larger tumors (p&amp;lt;0.05). However, after adjusting for the fact that tumor volumes were compared at multiple time points (i.e. multiple comparisons), these were no longer significant. Dietary treatment did not impact overall survival (p=0.34). NCKD mice had significantly higher glucose levels at sacrifice compared to the mice fed 10% and 20% carbohydrates (p=0.001), but similar levels of urinary ketones (p=0.37). CONCLUSIONS: LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival to mice consuming a NCKD (0% carbohydrate kcal). Thus, the survival benefit of a NCKD may be achievable with less restrictive low-carbohydrate diets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2816.

1439 DIFFERENTIAL EFFECTS OF CALORIC AND CARBOHYDRATE RESTRICTION UPON PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research VIII1 Apr 20101439 DIFFERENTIAL EFFECTS OF CALORIC AND CARBOHYDRATE RESTRICTION UPON PROSTATE CANCER Jean Thomas, Elizabeth Masko, Jodi Antonelli, Susan Poulton, Tameika Phillips, Tonisha Coburn, and Stephen J. Freedland Jean ThomasJean Thomas More articles by this author , Elizabeth MaskoElizabeth Masko More articles by this author , Jodi AntonelliJodi Antonelli More articles by this author , Susan PoultonSusan Poulton More articles by this author , Tameika PhillipsTameika Phillips More articles by this author , Tonisha CoburnTonisha Coburn More articles by this author , and Stephen J. FreedlandStephen J. Freedland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1132AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Caloric restriction has been shown to anti-cancer in its effects. However, whether these effects are the result of generalized reduction in overall caloric intake or reduced intake of macronutrients (i.e. carbohydrates) are unknown. We sought to investigate the differential effects of caloric and carbohydrate restriction upon prostate cancer (CaP) tumor growth. METHODS 100 7-8 week old male SCID mice were injected subcutaneously with 1x105 LAPC-4 prostate cancer cells. Mice were randomized to 1 of 4 diets: ad libitum Western diet (no intervention, 45% carbohydrates, 40% fat, and 15% protein), ad libitum No carbohydrate Ketogenic diet (NCKD; 1% carbohydrates, 83% fat, 16% protein), Western diet calorically restricted (WCR), and NCKD calorically restricted (NCKD CR). Calorically restricted arms were fed 75% of the calories of their reference diet group. Tumor volumes and mouse bodyweights were measured twice weekly. Mice were sacrificed when tumor volumes reached 1,000 mm3. RESULTS Relative to Western mice, there was a suggestion of smaller body weights in both NCKD CR and WCR arms (rank sum, p≤0.06). In contrast, there was trend for heavier mice in the NCKD fed group (rank sum, p=0.09). By day 50, median tumor volumes for NCKD, WCR, and NCKD CR were 45, 51, and 62% smaller compared to Western (rank sum, p≤0.001). At any time point, there were no significant differences in terms of tumor volume amongst the intervention arms. However, there was a suggestion of smaller tumors in the NCKD CR arm by day 42 (p=0.09). Dietary treatment was significantly associated with mouse survival (log-rank, p=0.004). In 2-way comparisons, NCKD, NCKD CR, and WCR had significantly prolonged survival relative to Western (log-rank, all p≤0.02). However, no difference in survival was seen between any calorically or carbohydrate restricted arm (log rank, p≥0.16). Over the course of the study, 34 mice were loss secondary to infection (WCR n=14, NCKD n=4, NCKD CR n =15). CONCLUSIONS Caloric and carbohydrate restriction are effective in delaying tumor growth and improving mouse survival. NCKD may provide anti-cancer benefits in the absence of weight loss. We were unable to detect a difference between caloric and carbohydrate restricted arms. This may be secondary to diminished power. Despite this, our findings support previous experimental findings and suggest a role for either caloric or carbohydrate restriction in the management of CaP. Durham, NC© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e554-e555 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jean Thomas More articles by this author Elizabeth Masko More articles by this author Jodi Antonelli More articles by this author Susan Poulton More articles by this author Tameika Phillips More articles by this author Tonisha Coburn More articles by this author Stephen J. Freedland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1437 LOW-CARBOHYDRATE DIETS AND PROSTATE CANCER GROWTH: HOW LOW IS “LOW ENOUGH”?

You have accessJournal of UrologyProstate Cancer: Basic Research VIII1 Apr 20101437 LOW-CARBOHYDRATE DIETS AND PROSTATE CANCER GROWTH: HOW LOW IS “LOW ENOUGH”? Elizabeth Masko, Jean-Alfred Thomas, Jodi A. Antonelli, Jessica C. Lloyd, Tameika E. Phillips, Susan H.M. Poulton, Salvatore V. Pizzo, and Stephen J. Freedland Elizabeth MaskoElizabeth Masko More articles by this author , Jean-Alfred ThomasJean-Alfred Thomas More articles by this author , Jodi A. AntonelliJodi A. Antonelli More articles by this author , Jessica C. LloydJessica C. Lloyd More articles by this author , Tameika E. PhillipsTameika E. Phillips More articles by this author , Susan H.M. PoultonSusan H.M. Poulton More articles by this author , Salvatore V. PizzoSalvatore V. Pizzo More articles by this author , and Stephen J. FreedlandStephen J. Freedland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1130AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previous dietary studies indicate carbohydrate intake may influence prostate cancer biology, as LAPC-4 and LNCaP xenograft mice fed a no-carbohydrate ketogenic diet (NCKD; 84% fat-0% carbohydrate-16% protein kcal) had significantly smaller tumors and longer survival times compared to mice fed a Western diet (40% fat-44% carbohydrate-16% protein kcal). The NCKD mice were also found to have higher levels of circulating IGFBP-3 and the lowest levels of insulin, IGF-1, and IGF-1:IGFBP-3 ratio despite consuming more calories than the Western group. As it is nearly impossible for a human to consume and maintain a no-carbohydrate diet similar to that in the previous xenograft studies, we sought to determine whether diets containing 10% or 20% kcal from carbohydrates could slow tumor growth in a similar manner to the NCKD in a xenograft model. METHODS A total of 150 male SCID mice were injected with LAPC-4 cells and placed on a Western diet (35% fat-49% carbohydrate-16% protein kcal) ad libitum. Two weeks post-injection, all mice were randomized to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate. Ten mice not injected with tumor were fed an ad libitum low-fat diet (12% fat–72% carbohydrate–16% protein kcal) and served as the reference group in a modified-paired feeding protocol for the other three groups. Calorie intake and body weights were measured thrice weekly and tumor volumes twice per week. Mice were sacrificed when tumors reached 1,000mm3. RESULTS Despite consuming 5-10% extra calories on average, all mice receiving low-carbohydrate diets were significantly lighter than the mice consuming the low-fat diet (p<0.04). Overall, the mice fed a NCKD were significantly lighter than the other two arms at multiple time points (p<0.05). There were no significant differences in tumor volumes among groups at any time except at Day 52 and 59, where 10% carbohydrate mice had larger tumors (p<0.05). However, after adjusting for the fact that tumor volumes were compared at multiple time points (i.e. multiple comparisons), these were no longer significant. Dietary treatment did not impact overall survival (p=0.34). NCKD mice had significantly higher glucose levels at sacrifice compared to the mice fed 10% and 20% carbohydrates (p=0.001), but similar levels of urinary ketones (p=0.37). CONCLUSIONS LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival to mice consuming a NCKD (0% carbohydrate kcal). Thus, the survival benefit of a NCKD may be achievable with less restrictive low-carbohydrate diets. Durham, NC© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e554 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Elizabeth Masko More articles by this author Jean-Alfred Thomas More articles by this author Jodi A. Antonelli More articles by this author Jessica C. Lloyd More articles by this author Tameika E. Phillips More articles by this author Susan H.M. Poulton More articles by this author Salvatore V. Pizzo More articles by this author Stephen J. Freedland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of Isocaloric Low Fat Diet on Prostate Cancer Xenograft Progression in a Hormone Deprivation Model

Previous mouse studies suggesting that low fat diets slow prostate cancer growth often used corn oil (omega-6), which enhances prostate cancer growth, as the primary fat. Using a saturated fat based diet we previously found no significant difference in tumor growth between low and high fat fed SCID mice (Taconic Farms, Hudson, New York) xenografted with LAPC-4 cells. Whether similar results would hold in a castration model is unclear. A total of 80 male SCID mice were fed a Western diet (40% fat and 44% carbohydrate) and injected with LAPC-4 human prostate cancer cells. When tumors were 200 mm(3), the mice were castrated and randomized to an isocaloric Western or a low fat diet (12% fat and 72% carbohydrate). Animals were sacrificed when tumors were 1,000 mm(3). Serum was collected and assayed for prostate specific antigen, insulin, insulin-like growth factor 1 and insulin-like growth factor binding protein 3. Tumors were assayed for total and phosphorylated Akt. Mouse weight was equivalent in the 2 groups. Overall dietary group was not significantly associated with survival (log rank p = 0.32). There were no statistically significant differences in prostate specific antigen (p = 0.53), insulin-like growth factor axis parameters (each p >0.05) or p-Akt-to-t-Akt ratios (p = 0.22) between the groups at sacrifice. In this xenograft model we found no difference in tumor growth or survival between low fat vs Western fed mice when the fat source was saturated fat. These results conflict with those of other studies in which corn oil was used to show that low fat diets delay prostate cancer growth, suggesting that fat type may be as important as fat amount in the prostate cancer setting.

Effect of intermittent fasting with or without caloric restriction on prostate cancer growth and survival in SCID mice

Caloric restriction (CR) delays cancer growth in animals, though translation to humans is difficult. We hypothesized intermittent fasting (i.e., intermittent extreme CR), may be better tolerated and prolong survival of prostate cancer (CaP) bearing mice. We conducted a pilot study by injecting 105 male individually-housed SCID mice with LAPC-4 cells. When tumors reached 200 mm(3), 15 mice/group were randomized to one of seven diets and sacrificed when tumors reached 1,500 mm(3): Group 1: ad libitum 7 days/week; Group 2: fasted 1 day/week and ad libitum 6 days/week; Group 3: fasted 1 day/week and fed 6 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 4: 14% CR 7 days/week; Group 5: fasted 2 days/week and ad libitum 5 days/week; Group 6: fasted 2 day/week and fed 5 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 7: 28% CR 7 days/week. Sera from mice at sacrifice were analyzed for IGF-axis hormones. There were no significant differences in survival among any groups. However, relative to Group 1, there were non-significant trends for improved survival for Groups 3 (HR 0.65, P = 0.26), 5 (0.60, P = 0.18), 6 (HR 0.59, P = 0.16), and 7 (P = 0.59, P = 0.17). Relative to Group 1, body weights and IGF-1 levels were significantly lower in Groups 6 and 7. This exploratory study found non-significant trends toward improved survival with some intermittent fasting regimens, in the absence of weight loss. Larger appropriately powered studies to detect modest, but clinically important differences are necessary to confirm these findings.