Renal Cell Carcinoma Model Research Articles

Abstract 5323: Targeting PIM1 and CDK4/6 kinases in renal cell carcinoma

Abstract In this study we examine the hypothesis that combination therapy targeting the VEGFR, PIM1, and CDK4/6 kinases improves the efficacy of VEGFR-directed monotherapy in renal cell carcinoma (RCC). Two RCC cell lines, 786-O and Caki-1 (chosen for differences in VHL), were evaluated and the ED50s of SGI-1776 (PIM1 inhibitor), PD0332991 (CDK4/6 inhibitor), and LY2835219 (dual PIM1 and CDK4/6 inhibitor) assessed with an MTT assay. Cell Titer-Glo® was used to determine the effects of each of the inhibitors on cellular viability alone and in combination with sunitinib (VEFGR tyrosine kinase inhibitor). The effect of LY2835219 on phosphorylation of BAD, a PIM1 substrate, was assessed by Western blot. PIM1 and dual PIM1-CDK4/6 inhibition showed more potent effects on cellular viability than CDK4/6 inhibition. ED50 values of each inhibitor for RCC cells are reported in Table 1. Further assays showed greater decreased cellular viability in cells treated with the combination of a PIM1 inhibitor and sunitinib than in cells treated with either a PIM1 inhibitor or sunitinib alone. When combined with sunitinib, dual PIM1-CDK4/6 inhibition was more effective than PIM1 inhibition in 786-O cells. Sunitinib in combination with a CDK4/6 inhibitor was not as effective as in combination with either a PIM1 inhibitor or a dual PIM1-CDK4/6 inhibitor. Western blot analysis showed a decrease in phospho-BAD in cells treated with a dual PIM1-CDK4/6 inhibitor. Table 1.Potency of Kinase Inhibitors Against RCC Cell LinesInhibitorED50 (786-O; uM)ED50 (Caki-1; uM)SGI-17768.67 (8.13 - 9.25)6.94 (5.05 - 9.53)PD0332991Indeterminate*Indeterminate*LY28352197.46 (6.32 - 8.81)1.18 (1.02 - 1.36)95% confidence intervals are presented in parenthesis. *Highest dose level did not result in a sufficient response to allow for reliable curve fitting to determine the ED50 Our data reveal that targeting PIM1 in combination with sunitinib is superior to sunitinib alone in RCC cell lines. Additionally, in 786-O cells, added CDK4/6 inhibition further improves the efficacy of the combination therapy. Dual PIM1-CDK4/6 kinase targeted therapy in combination with sunitinib is an attractive therapeutic regimen with good potential for translation to the clinic. Ongoing studies include further characterization of the molecular events involved including apoptosis, cell cycle regulation, PIM1 kinase and CDK4/6 kinase activity, and evaluation of the combination therapy in a mouse xenograft model of RCC. Citation Format: Jeffery S. Small, Sheldon L. Holder. Targeting PIM1 and CDK4/6 kinases in renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5323. doi:10.1158/1538-7445.AM2015-5323

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

BackgroundCarbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated.MethodsThe role of human anti-CAIX mAbs on CAIX+ RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX+ RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining.ResultsOur studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ−/− mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX+ tumor growth.ConclusionsOur findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX+RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users.

Targeting Survivin Inhibits Renal Cell Carcinoma Progression and Enhances the Activity of Temsirolimus.

Elevated expression of the antiapoptotic factor survivin has been implicated in cancer cell survival and disease progression. However, its specific contribution to renal cell carcinoma (RCC) pathogenesis is not well defined. We investigated the roles of survivin in RCC tumor progression, resistance to mTOR inhibitors, and evaluated the therapeutic activity of the survivin suppressant YM155 in RCC models. Here, we report that survivin expression levels were significantly higher in RCC cell lines compared with normal renal cells. Stable targeted knockdown of survivin completely abrogated the ability of 786-O RCC tumors to grow in mice, thus demonstrating its importance as a regulator of RCC tumorigenesis. We next explored multiple strategies to therapeutically inhibit survivin function in RCC. Treatment with the mTOR inhibitor temsirolimus partially diminished survivin levels and this effect was augmented by the addition of YM155. Further analyses revealed that, in accordance with their combined anti-survivin effects, YM155 significantly improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in xenograft models in vivo. Similar to pharmacologic inhibition of survivin, shRNA-mediated silencing of survivin expression not only inhibited RCC tumor growth, but also significantly sensitized RCC cells to temsirolimus therapy. Subsequent experiments demonstrated that the effectiveness of this dual survivin/mTOR inhibition strategy was mediated by a potent decrease in survivin levels and corresponding induction of apoptosis. Our findings establish survivin inhibition as a novel approach to improve RCC therapy that warrants further investigation.

Anti-S1P Antibody as a Novel Therapeutic Strategy for VEGFR TKI-Resistant Renal Cancer.

VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC. In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC. Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI. Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

PD33-04 INVERSE RELATIONSHIP BETWEEN INSULIN RECEPTOR EXPRESSION AND CANCER PROGRESSION IN RENAL CELL CARCINOMA: CLINICAL AND EXPERIMENTAL EVALUATION

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2015PD33-04 INVERSE RELATIONSHIP BETWEEN INSULIN RECEPTOR EXPRESSION AND CANCER PROGRESSION IN RENAL CELL CARCINOMA: CLINICAL AND EXPERIMENTAL EVALUATION Makoto Takahashi, Takamitsu Inoue, Mingguo Huang, Hiroshi Tsuruta, Mitsuru Saito, Shintaro Narita, Norihiko Tsuchiya, and Tomonori Habuchi Makoto TakahashiMakoto Takahashi More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Mingguo HuangMingguo Huang More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2102AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin is not only a key regulator in the glucose metabolism pathway but also a strong growth factor in cell proliferation pathways. It has been reported that obesity increases the risk of renal cell carcinoma (RCC). However, epidemiological studies have suggested that obese patients with RCC have better prognosis. We investigated the relationship between insulin receptor (IR) expression and outcomes in patients who underwent radical nephrectomy for RCC and explored the role of insulin signaling in the progression of a murine RCC model using metformin to treat hyperinsulinemia induced by a high-carbohydrate diet. METHODS pT1-T4 tumor specimens from 99 patients with RCC were evaluated for IR expression by immunohistochemistry. Serum insulin and C-peptide levels were measured. The association between biomarker expression and serum concentration, prognostic factors, and clinical outcomes were assessed. C57BL/6 mice were randomized into four groups: low-carbohydrate diet, high-carbohydrate diet, low-carbohydrate diet + metformin, and high-carbohydrate diet + metformin groups. RENCA cells were injected subcutaneously. Body weight, glucose tolerance test, serum insulin level, and tumor volume were evaluated. Immunoblotting was performed to assess the insulin signaling pathway in the murine RCC model. RESULTS The IR expression level was significantly lower in patients with tumor stage pT2-4 and with a preoperative serum C-peptide level more than 5.14 ng/mL. High IR expression was an independent predictor of favorable progression-free survival after radical nephrectomy. In vivo progression of RENCA in the murine model was not significantly stimulated by hyperinsulinemia induced by a high-carbohydrate diet. However, in vivo progression of RENCA was significantly inhibited by metformin in both the low- and high-carbohydrate diet groups. IR expression was significantly higher in the tumors from the low-carbohydrate diet group and high-carbohydrate diet plus metformin group than that from the high-carbohydrate diet group. CONCLUSIONS IR expression in RCC was inversely associated with cancer progression and serum insulin levels in both clinical and murine models. The anticancer effect of metformin in the murine RCC model might be derived from the direct effect of the agent on cancer cells rather than the effect of the lower insulin level. The lower expression of IR in the tumor from patients with a poor outcome might be the result of a higher serum insulin level, which is not strong cause of cancer progression. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e710-e711 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Makoto Takahashi More articles by this author Takamitsu Inoue More articles by this author Mingguo Huang More articles by this author Hiroshi Tsuruta More articles by this author Mitsuru Saito More articles by this author Shintaro Narita More articles by this author Norihiko Tsuchiya More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MAPKs' status at early stages of renal carcinogenesis and tumors induced by ferric nitrilotriacetate.

Renal cell carcinoma (RCC) is asymptomatic at early stages, and thus, initial diagnosis frequently occurs at advanced or even metastatic stages, leading to a high rate of mortality. Ferric nitrilotriacetate (FeNTA)-induced RCC model is a useful tool to analyze molecular events at different stages of the carcinogenesis process in vivo. MAPKs' alterations seem to play an important role in the development and maintenance of human RCC tumors. Based on the above, p38α/β/γ, JNK1/2, and ERK1/2 statuses were studied at early stages of FeNTA-induced renal carcinogenesis (1 and 2 months of carcinogen treatment) as well as in tumor tissue. MAPKs showed distinct response along carcinogenesis process, either as total proteins and/or as their phosphorylated forms. While the increase in total and phospho-p38α/β levels became lower as carcinogenesis progressed, p38γ overexpression grew. Instead, total JNK2 diminished, but JNK1 was elevated at all studied times, and p-JNK1 levels increased at early stages, but not in tumors. In contrast, p-JNK2 rose at 2 months of treatment and in tumor tissue. Increased levels of p-ERK1/2 were observed at all stages analyzed. Very interestingly, at 1 and 2 months of FeNTA treatment, no alterations in MAPKs were found in liver or lung, where no primary tumors are induced with the scheme of FeNTA administration followed here. In conclusion, MAPKs' behavior evolved differentially as renal carcinogenesis advanced, even among isoforms of the same family, but it did not change in other tissues. All this strongly suggests a role of these kinases in FeNTA-induced RCC tumor development and maintenance.

Abstract 5016: Antitumor activity of anti-PD-1 in combination with tyrosine kinase inhibitors in a preclinical renal cell carcinoma model

Abstract Introduction: Nivolumab (BMS-936558; MDX-1106; ONO-4538) is a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), inhibiting the downregulation of antitumor T-cell functions. Nivolumab has shown activity in advanced solid tumors, including renal cell carcinoma (RCC), melanoma, and non-small cell lung cancer (Topalian SL, et al. NEJM 2012;366:2443-54). Sunitinib and sorafenib are anti-angiogenic tyrosine kinase inhibitors (TKIs) used for the treatment of RCC. We investigated the activity of nivolumab in combination with TKIs in a preclinical RCC murine model. Methods: The murine RCC (Renca) tumor cell line was maintained in vitro and implanted subcutaneously into 8-12 week old female Balb/c mice. When mean tumor volume reached approximately 90-100 mm3, mice were randomized into groups of eight. Vehicle control, sunitinib 120 mg/kg, or sorafenib 200 mg/kg were administered orally once daily for 14 days. The nivolumab surrogate antibody, an IgG1 anti-mouse PD-1 monoclonal antibody (clone 4H2), was administered at 10 mg/kg by intraperitoneal injection every four days for four cycles. Immunohistochemistry and flow cytometry analyses were used to assess immune cell infiltration of tumors. Results: Sunitinib monotherapy showed activity in the Renca murine RCC model producing tumor growth inhibition of 84% by the end of treatment; however, tumors grew progressively after cessation of therapy. Conversely, while the anti-PD-1 monoclonal antibody was inactive in this model, addition of anti-PD-1 to sunitinib produced significant antitumor activity, resulting in complete tumor regressions or marked delay in tumor growth. Combination anti-PD-1 plus sunitinib therapy also had no effect on the body weight of the mice. Immunohistochemical analysis demonstrated that sunitinib monotherapy led to an influx of immune cells predominantly into the tumor periphery, whereas greater infiltration of immune cells throughout the tumor was observed with combination anti-PD-1 and sunitinib therapy. In contrast, combination treatment with anti-PD-1 antibody and sorafenib did not enhance antitumor activity in this murine RCC model. Further exploration of the mechanisms contributing to this synergy will be presented. Conclusions: Combination therapy with sunitinib and anti-PD-1 antibody demonstrated an enhanced effect against murine RCC. Safety and response to nivolumab plus sunitinib, pazopanib, or ipilimumab in patients with metastatic RCC are being assessed in an ongoing phase 1 study (NCT01472081). Citation Format: Gregg Masters, Gennaro Dito, Becky Penhallow, Anne Lewin, Henry Kao, Maria N. Jure-Kunkel. Antitumor activity of anti-PD-1 in combination with tyrosine kinase inhibitors in a preclinical renal cell carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2014-5016

CpG-mediated modulation of MDSC contributes to the efficacy of Ad5-TRAIL therapy against renal cell carcinoma.

Tumor progression occurs through the modulation of a number of physiological parameters, including the development of immunosuppressive mechanisms to prevent immune detection and response. Among these immune evasion mechanisms, the mobilization of myeloid-derived suppressor cells (MDSC) is a major contributor to the suppression of antitumor T-cell immunity. Patients with renal cell carcinoma (RCC) show increased MDSC, and methods are being explored clinically to reduce the prevalence of MDSC and/or inhibit their function. In the present study, we investigated the relationship between MDSC and the therapeutic potential of a TRAIL-encoding recombinant adenovirus (Ad5-TRAIL) in combination with CpG-containing oligodeoxynucleotides (Ad5-TRAIL/CpG) in an orthotopic mouse model of RCC. This immunotherapy effectively clears renal (Renca) tumors and enhances survival, despite the presence of a high frequency of MDSC in the spleens and primary tumor-bearing kidneys at the time of treatment. Subsequent analyses revealed that the CpG component of the immunotherapy was responsible for decreasing the frequency of MDSC in Renca-bearing mice; further, treatment with CpG modulated the phenotype and function of MDSC that remained after immunotherapy and correlated with an increased T-cell response. Interestingly, the CpG-dependent alterations in MDSC frequency and function did not occur in tumor-bearing mice complicated with diet-induced obesity. Collectively, these data suggest that in addition to its adjuvant properties, CpG also enhances antitumor responses by altering the number and function of MDSC.

Inhibition of Hedgehog signalling by NVP-LDE225 (Erismodegib) interferes with growth and invasion of human renal cell carcinoma cells.

Background:Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth.Methods:We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib – a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC.Results:NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays.Conclusions:Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Identification and significance of mobilized endothelial progenitor cells in tumor neovascularization of renal cell carcinoma.

Neovascularization is a key role of renal cell carcinoma (RCC) and the status of neovascularization in RCC is closely correlated with the tumor development and patient prognosis. Endothelial progenitor cells (EPCs) are considered as important building blocks for neovascularization. However, the role of mobilized EPCs in RCC remains unknown. In this study, the orthotopic RCC model was established to investigate the distribution, frequency, and significance of mobilized EPCs. We found that circulating endothelial progenitor cell (CEPC) levels and plasma angiogenic factors (vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were higher in peripheral blood (PB) of the RCC than those in the normal group and positively correlated with each other. EPC levels in adjacent nonmalignant kidney tissue (AT) were significantly higher than those in tumor tissue (TT) and normal kidney tissue (NT), which were positively correlated with CEPC levels. VEGF, VEGF receptor-2 (Flk), and SDF-1 and its SDF-1 receptor (CXCR4) expression in AT was significantly higher than that in TT and NT. Levels of these angiogenic factors in AT were positively correlated with those in PB. Mean microvessel density (MVD) was higher in AT than in TT, and that in TT was slightly lower than that in NT. Our findings propose that mobilized EPCs play an important role in RCC neovascularization. EPCs in PB and AT can be used as a biomarker for predicting RCC progression.

Modeling spontaneous metastatic renal cell carcinoma (mRCC) in mice following nephrectomy.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.

Development of a realistic in vivo bone metastasis model of human renal cell carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastases. The incidence of RCC is increasing and bone metastatic RCC merits greater focus. Realistic preclinical bone metastasis models of RCC are lacking, hampering the development of effective therapies. We developed a realistic in vivo bone metastasis model of human RCC by implanting precision-cut tissue slices under the renal capsule of immunodeficient mice. The presence of disseminated cells in bone marrow of tissue slice graft (TSG)-bearing mice was screened by human-specific polymerase chain reaction and confirmed by immunohistology using human-specific antibody. Disseminated tumor cells in bone marrow of TSG-bearing mice derived from three of seven RCC patients were detected as early as 1 month after tissue implantation at a high frequency with close resemblance to parent tumors (e.g., CAIX expression and high vascularity). The metastatic patterns of TSGs correlated with disease progression in patients. In addition, TSGs retained capacity to metastasize to bone at high frequency after serial passaging and cryopreservation. Moreover, bone metastases in mice responded to Temsirolimus treatment. Intratibial injections of single cells generated from TSGs showed 100 % engraftment and produced X-ray-visible tumors as early as 3 weeks after cancer cell inoculation. Micro-computed tomography (μCT) and histological analysis revealed osteolytic characteristics of these lesions. Our results demonstrated that orthotopic RCC TSGs have potential to develop bone metastases that respond to standard therapy. This first reported primary RCC bone metastasis model provides a realistic setting to test therapeutics to prevent or treat bone metastases in RCC.

Targeted Contrast-Enhanced Ultrasound Imaging of Angiogenesis in an Orthotopic Mouse Tumor Model of Renal Carcinoma

Previous studies have reported that microbubbles bearing targeting ligands to molecular markers of angiogenesis can be successfully detected by ultrasound imaging in various animal models of solid cancer. In the present study, we sought to investigate the activity of microbubbles targeted to vascular endothelial growth factor receptor 2 (VEGFR2) in an orthotopic model of renal cell carcinoma (RCC). Microbubbles conjugated to an anti-VEGFR2 antibody (MBV) were compared with microbubbles conjugated to an isotype control antibody (MBC) or naked microbubbles (MBN). An orthotopic mouse model of human RCC was established by surgically implanting an established tumor within the renal capsule in mice. Tumor growth and blood flow were verified by B-mode and color Doppler ultrasound imaging. VEGFR2 expression within the tumor and renal parenchyma was detected by immunohistochemistry. The duration of contrast enhancement of MBV was much longer than those of MBN and MBC when assessed over 10 min. The baseline-subtracted contrast intensity within the tumor was higher for MBV than for MBC and MBN (p < 0.01). Additionally, the contrast intensity for MBV was significantly higher in the tumor region than in normal parenchyma (p < 0.01). Microbubbles targeting VEGFR2 exhibit suitable properties for imaging angiogenesis in orthotopic models of renal cell carcinoma, with potential applications in life science research and clinical medicine.

Effect of carbon dioxide pneumoperitoneum on human renal cell carcinoma proliferation and metastasis in an orthotropic xenograft nude mouse model.

IntroductionThis study aimed to explore the effect of carbon dioxide (CO2) pneumoperitoneum on tumor proliferation and metastasis in an orthotropic xenograft nude mice model of human renal cell carcinoma (RCC) and evaluate the safety of CO2 pneumoperitoneum laparoscopy for treating RCC.Material and methodsRCC 786-0 cells were injected to establish an orthotropic xenograft model. Fifty nude mice were given orthotropic inoculations and randomized to five groups: group A (control); group B (CO2 pneumoperitoneum for 2 h); group C (CO2 pneumoperitoneum for 4 h); group D (CO2 pneumoperitoneum for 4 h and 24 h after waking); group E (CO2 pneumoperitoneum for 4 h and 48 h after waking). The proliferation status was observed in RCC specimens by immunohistochemical staining for Ki67. The protein levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were examined by western blotting.ResultsAll groups showed similar Ki67-positive staining in RCC samples (p > 0.05). The relative expression of HIF-1α and VEGF gradually increased in both group B and group C, as compared with group A, but only the difference between group C and group A reached statistical significance (p < 0.05). The protein levels of HIF-1α and VEGF decreased in both group D and group E, as compared with group B and group C; however, the differences between group D, group E, and group A did not reach statistical significance (p > 0.05).ConclusionsIn an orthotropic xenograft nude mice model of RCC, CO2 pneumoperitoneum has no effect on expression of the cellular proliferation marker Ki67. However, CO2 pneumoperitoneum rapidly induces transient expression of HIF-1α and VEGF. Thus, CO2 pneumoperitoneum laparoscopy may be a safe method for treating RCC.

Underactivation of the adiponectin–adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression

Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.

Assessing the accuracy and generalizability of the preoperative and postoperative Karakiewicz nomograms for renal cell carcinoma: results from a multicentre European and US study

What's known on the subject? and What does the study add? The preoperative and postoperative Karakiewicz models for RCC are considered among the best prognostic tools available for clinical counseling. Nevertheless, their predictive acuracy was externally validated only in two papers: by the same author and in an independent sample of Asian patients. However, these models have not been externally validated in truly independent multicentre series of patients. Our study demonstrated that these models 1) provide robust prognostic information; 2) were robustly built; 3) are useful also in population far from the original series. The present results are the first to show the validity and generalizability of Karakiewicz nomograms, which are based on surgical series from European centres, for large-, mid- and small-volume European and American centres. Objective To assess the accuracy and generalizability of the pre- and postoperative Karakiewicz nomograms for predicting cancer-specific survival (CSS) in patients with renal cell carcinoma (RCC). Patients and Methods This retrospective study included 3231 patients from European and US centres, who were treated by radical or partial nephrectomy for RCC between 1992 and 2010. Prognostic scores for each patient were calculated and the primary endpoint was CSS. Discriminating ability was assessed by Harrell's c-index for censored data. The ‘validation by calibration’ method proposed by Van Houwelingen was used for checking the calibration of covariate effects. Calibration was graphically explored. Results Local and systemic symptoms were present in 23.2% and 9.1% of the patients, respectively. The median follow-up (FU) was 49 months. At the last FU, 408 cancer-related deaths were recorded, Kaplan–Meier estimates of CSS (with 95% confidence intervals [CIs]) at 5 and 10 years were 0.86 (0.84–0.87) and 0.77 (0.75–0.80), respectively. Both nomograms discriminated well. Stratified c-indices for CSS were 0.784 (95% CI 0.753–0.814) for the preoperative nomogram, and 0.842 (95% CI 0.816–0.867) for the postoperative one, with a significant difference between the two values (P < 0.001). The covariate-based predictions on our data for both nomograms were valid. The calibration plots showed no relevant departures from ideal predictions. Conclusions The results suggest that the postoperative Karakiewicz nomogram discriminates substantially better than the preoperative one. These nomogram-based predictions may be used as benchmark data for pretreatment and postoperative decision-making in patients at various stages of RCC.

Tissue slice grafts of human renal cell carcinoma: An authentic preclinical model with high engraftment rate and metastatic potential

ObjectiveDiscovery of curative therapies for renal cell carcinoma (RCC) is hampered by lack of authentic preclinical models. Tumorgrafts, generated by direct implantation of patient-derived tissues into mice, have demonstrated superior ability to predict therapeutic response. We evaluated “tissue slice grafts” (TSGs) as an improved tumorgraft model of RCC. Materials and methodsCores of fresh RCC were precision-cut at 300µm and implanted under the renal capsule of RAG2−/−γC−/− mice. Engraftment rate, histology, biomarker expression, genetic fidelity, and metastatic potential were evaluated. Magnetic resonance imaging (MRI) was tested as a noninvasive method to measure tumor volume, and response to a targeted therapy was determined. ResultsAll 13 cases of RCC engrafted and displayed characteristic histology and biomarkers. TSG volume quantified noninvasively by MRI highly correlated with graft weights, providing a unique tool for monitoring orthotopic growth. Moreover, in 2 cases, cancer cells from TSGs metastasized to clinically relevant sites, including bone. Microarray analysis and DNA sequencing demonstrated a high degree of correlation of global gene expression and von Hippel-Lindau (VHL) status between TSGs and parental tumors. Treatment of TSGs with sunitinib significantly decreased graft weight and mean vessel density compared with controls. ConclusionThe TSG model of RCC faithfully recapitulates tumor pathology, gene expression, genetic mutation, and drug response. The high engraftment rate and metastatic potential of this authentic model, in conjunction with the ability to generate large first-generation animal cohorts and to quantitate tumor volume at the orthotopic site by MRI, proffer significant advantages compared with other preclinical platforms.

Predictive Markers in Advanced Renal Cell Carcinoma

Predictive markers of response to therapy are increasingly important in advanced renal cell carcinoma (RCC) due to the proliferation of treatment options in recent years. Different types of potential predictive markers may include clinical, toxicity-based, serum, tissue, and radiologic biomarkers. Clinical factors are commonly used in overall prognostic models of RCC but have limited utility in predicting response to therapy. Correlation between development of particular toxicities and response to therapy has been noted, such as the correlation between hypertension and response to angiogenesis-targeted therapy. Serum and tissue biomarkers will be covered in detail elsewhere, but factors such as serum lactate dehydrogenase (LDH) and circulating cytokines show promise in this regard. Finally, baseline or early treatment radiology studies may have predictive ability for longer term efficacy, with most studies to date focusing on functional imaging modalities such as positron emission tomography (PET) scans, dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), and DCE ultrasound (US). The ultimate goal of developing predictive biomarkers is to enable rational and personalized treatment strategies for patients with advanced RCC.

Angiogenic and signalling proteins correlate with sensitivity to sequential treatment in renal cell cancer

Background:We aimed to study key signalling proteins involved in angiogenesis and proliferation on the response to inhibitors of tyrosine kinases and mammalian target of rapamycin in first- and in second-line treatment of renal cell carcinoma (RCC).Methods:In a panel of human RCC tumours, in vitro and in nude mice, we evaluated the effect of sunitinib, sorafenib and everolimus, alone and in sequence, on tumour growth and expression of signalling proteins involved in proliferation and resistance to treatment.Results:We demonstrated that, as single agents, sunitinib, sorafenib and everolimus share similar activity in inhibiting cell proliferation, signal transduction and vascular endothelial growth factor (VEGF) secretion in different RCC models, both in vitro and in tumour xenografts. Pre-treatment with sunitinib reduced the response to subsequent sunitinib and sorafenib but not to everolimus. Inability by sunitinib to persistently inhibit HIF-1, VEGF and pMAPK anticipated treatment resistance in xenografted tumours. After first-line sunitinib, second-line treatment with everolimus was more effective than either sorafenib or rechallenge with sunitinib in interfering with signalling proteins, VEGF and interleukin-8, translating into a significant advantage in tumour growth inhibition and mice survival.Conclusion:We demonstrated that a panel of angiogenic and signalling proteins can correlate with the onset of resistance to sunitinib and the activity of everolimus in second line.

A Potent Anti-CD70 Antibody–Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.