Murine Renal Cell Carcinoma Model Research Articles

Abstract B026: Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma

Abstract Purpose: Although renal cell carcinoma (RCC) is relatively more resistant to radiation therapy (RT) compared to other cancer indications, recent advances in single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) made it possible to achieve better outcome and reduce treatment related toxicities. In addition, with studies demonstrating that certain therapeutic agents including chemotherapies can act as radiosensitizers, the idea of combining radiotherapy with those agents are being actively pursued. Among them, mammalian target of rapamycin (mTOR) inhibitor everolimus (E) and survivin inhibitor YM155 (Y) are of special interest since they target multiple RT-resistance mechanisms simultaneously. Additionally, YM155 has been shown to help in overcoming resistance to mTOR inhibitors in various cancer including RCC. Hence, the present study aims to evaluate the efficacy of a tumor-targeted liposomal formulation combining everolimus and YM155 (EY-L) in sensitizing RCC tumors towards radiation. Experimental Design: Previously we developed a tumor-targeted liposomal formulation and used it successfully to deliver multiple drugs for the treatment of RCC in both immune-deficient and immune-competent mouse models. Here we developed a similar formulation combining everolimus and YM155 (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further evaluated the efficacy of EY-L in sensitizing RCC cell lines and tumors towards radiation in vitro using clonogenic assay and in vivo in RCC xenografts in immunodeficient mice and murine RCC models in immunocompetent mice. Results: EY-L (2 mg/kg E, 1.4 mg/kg Y, twice a week intravenous administration) was better than E-L or Y-L, the corresponding single drug loaded formulations in containing the primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. Furthermore, EY-L leads to significantly higher sensitization of RCC cells towards radiation in vitro than E-L or Y-L. Finally, EY-L was able to sensitize RCC tumors towards radiation therapy in both RCC xenograft and murine RCC model. Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy using in vitro clonogenic assay and in vivo radiosensitization studies in multiple RCC tumor models. Citation Format: Krishnendu Pal, Vijay S. Madamsetty, Hari K. Rachamala, Shamit K. Dutta, Enfeng Wang, Debabrata Mukhopadhyay. Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B026.

L-Serine-Modified Poly-L-Lysine as a Biodegradable Kidney-Targeted Drug Carrier for the Efficient Radionuclide Therapy of Renal Cell Carcinoma.

In the present study, L-serine (Ser)-modified poly-L-lysine (PLL) was synthesized to develop a biodegradable, kidney-targeted drug carrier for efficient radionuclide therapy in renal cell carcinoma (RCC). Ser-PLL was labeled with 111In/90Y via diethylenetriaminepentaacetic acid (DTPA) chelation for biodistribution analysis/radionuclide therapy. In mice, approximately 91% of the total dose accumulated in the kidney 3 h after intravenous injection of 111In-labeled Ser-PLL. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed that 111In-labeled Ser-PLL accumulated in the renal cortex following intravenous injection. An intrarenal distribution study showed that fluorescein isothiocyanate (FITC)-labeled Ser-PLL accumulated mainly in the renal proximal tubules. This pattern was associated with RCC pathogenesis. Moreover, 111In-labeled Ser-PLL rapidly degraded and was eluted along with the low-molecular-weight fractions of the renal homogenate in gel filtration chromatography. Continuous Ser-PLL administration over five days had no significant effect on plasma creatinine, blood urea nitrogen (BUN), or renal histology. In a murine RCC model, kidney tumor growth was significantly inhibited by the administration of the beta-emitter 90Y combined with Ser-PLL. The foregoing results indicate that Ser-PLL is promising as a biodegradable drug carrier for kidney-targeted drug delivery and efficient radionuclide therapy in RCC.

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma.

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c+CD8+ T cells. The CD11c+CD8+ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c+CD8+ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.

Tyrosine Kinase Inhibitor Cabozantinib Inhibits Murine Renal Cancer by Activating Innate and Adaptive Immunity

BackgroundAdvanced renal cell carcinoma (RCC) has a very dismal prognosis. Cabozantinib, a tyrosine kinase inhibitor, has been approved for the treatment of advanced RCC. However, the impact of cabozantinib on the immune microenvironment of RCC remains poorly understood.MethodsKaplan-Meier survival curves were constructed to examine the correlation between intratumor infiltration of neutrophils and patient prognosis in RCC. Infiltration and effector function of neutrophils and T cells in response to cabozantinib treatment were investigated in a murine RCC model.ResultsA retrospective study of 307 RCC patients indicated that neutrophils were recruited into tumor tissues, and increased neutrophil infiltration was associated with improved clinical outcomes. In a murine model of RCC, cabozantinib treatment significantly increased both intratumor infiltration and anti-tumor function of neutrophils and T cells. Mechanistically, we found that cabozantinib treatment induced expression of neutrophil-related chemokines (CCL11 and CXCL12) and T cell-related chemokines (CCL8 and CX3CL1) in the tumor microenvironment. Furthermore, depletion of neutrophils and CD8+ T cells compromised the therapeutic efficacy of cabozantinib. Importantly, cabozantinib treatment induced long-term anti-tumor T cell response.ConclusionsOur study revealed novel mechanisms of the therapeutic effects of cabozantinib on RCC by activating both neutrophil-mediated innate immunity and T cell-mediated adaptive immunity. These findings are of great significance for guiding the clinical use of cabozantinib and provide a good candidate for future combination therapy with T-cell therapies or other immunotherapies.

Development of a highly pulmonary metastatic orthotopic renal cell carcinoma murine model.

ABSTRACTThe incidence of renal cell carcinoma (RCC) is high, and its outcomes remain poor. Mortality is attributable largely to metastatic disease and a dearth of effective therapeutic interventions. The lungs are the most common metastatic site. To elucidate the biological mechanisms underlying pulmonary metastasis and identify superior therapeutic strategies, we developed a novel and clinically relevant murine RCC model exhibiting enhanced pulmonary metastasis. Mice underwent intrarenal implantation using luciferase-expressing Renca, a murine renal adenocarcinoma cell line. Primary renal tumor progression and development of metastatic lung lesions were monitored in live mice using bioluminescent imaging, followed by post-mortem organ assessment. Cells were isolated from pulmonary metastases for reimplantation, followed by repeat monitoring and assessment. This process was repeated once more for a total of two in vivo passages to select for pulmonary metastatic Renca cell subpopulations. However, a single round of in vivo selection was sufficient to produce a near-maximally metastatic subpopulation. Relative to Renca cell-implanted mice, subpopulation-implanted mice exhibited shorter implantation-metastasis intervals (5 days), shorter implantation-moribundity intervals (sacrificed at 18.6±2.9 versus 22.3±1.1 days), a higher number of metastatic lung lesions at 23 days (183.9±39.0 versus 172.6±38.2) and poorer survival. Implantation of cells derived from the second round of in vivo selection produced no further significant differences in the above metrics. This model consistently and efficiently recapitulates RCC pulmonary metastasis while allowing in vivo monitoring of tumor progression, thereby facilitating elucidation of the molecular mechanisms underlying pulmonary metastasis and evaluation of therapeutic modalities.

Abstract 2242: Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway

Abstract Lenvatinib mesilate (LEN) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. LEN plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase1b/2 clinical trial of LEN in combination with pembrolizumab with selected solid tumors including RCC and Phase3 study of LEN in combination with everolimus or pembrolizumab for metastatic RCC patients are in progress. In this study, we investigated the antitumor and immunomodulatory activities of LEN alone and in combination with anti-PD1 Ab and explored the mechanism of action underlying the activities of LEN and PD1 blockade in preclinical syngeneic RCC models. We examined antitumor activity of combination treatment of LEN at 10 mg/kg (po, qd) and anti-PD1 Ab at 10 mg/kg (ip, twice weekly) in the murine subcutaneous RCC model using RAG cell line. Immune cell population analyses in tumor tissues were performed by flow cytometer. Immune-related gene expression profile in tumor tissues or cultured cells was analyzed by qPCR and RNA seq. Effects on FGFR and IFN gamma (IFNγ) signaling pathways and their downstream molecules in cultured cells were analyzed by western blot. In the murine syngeneic RAG tumor model, the combination treatment of LEN and anti-PD1 Ab showed tumor shrinkage, and some of tumors were regressed to nonpalpable sizes with long duration of response. LEN alone and in combination with anti-PD1 Ab significantly decreased the population of tumor associated macrophage in tumor tissues by FCM analysis. Gene expression analysis for immune cell responses in tumor tissues revealed the upregulation of IFNγ and its downstream genes including PD-L1 with combination of LEN and anti-PD1 Ab. Western blot analysis elucidated that FGFR signaling pathway inhibited the activation of IFNγ signaling pathway and the induction of PD-L1 expression in murine RCC cells, and LEN-treatment reactivated the IFNγ signaling pathway by inhibition of FGFR signaling. Combination of LEN and anti-PD1 Ab showed greater antitumor activity including tumor shrinkage with long duration of response in the murine RCC model. Modulation of immune microenvironment by LEN and combination of LEN with PD1 Ab may underlie the strong antitumor activity in preclinical RCC model. Inhibitory activity of LEN against FGFR reactivated the IFNγ pathway and PD-L1 expression suppressed by activation of FGFR signaling pathway, and this inhibitory activity of LEN may potentiate the antitumor activity of combination treatments with anti-PD1 Ab. These preclinical results provide one of the unique mechanisms of combinational effect of LEN with PD1 blockade in the preclinical RCC model. Citation Format: Yusuke Adachi, Kenji Ichikawa, Yu Kato, Yasuhiro Funahashi. Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2242.

Abstract 12: Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models

Abstract Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. Lenvatinib plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase3 clinical study of lenvatinib in combination with everolimus or pembrolizumab for metastatic RCC patients is in progress. However, the detailed mechanisms underlying the efficacy of these combination treatments remain to be elucidated. In this study, we investigated the antitumor activities of lenvatinib in combination with everolimus or anti-PD1 mAb in preclinical RCC models. We examined antitumor activities in four human RCC (A-498, VMRC-RCW, Caki-1, and ACHN) xenograft models orally treated with lenvatinib, everolimus, and their combination. Expression of Ki67 and phosphorylation of S6 in RCC xenograft models were analyzed by immunofluorescence (IF) staining. Inhibitory effects of lenvatinib and everolimus on the phosphorylation of S6K (T389 and T421/S424), and S6 (S235/S236) were analyzed by western blot in vitro. We also examined antitumor activity of combination treatment of lenvatinib and anti-PD-1 mAb in RAG murine RCC model. The combination of lenvatinib with everolimus showed greater antitumor activity than that of either mono-treatment in all of 4 RCC xenograft models we examined and led to tumor regression in three out of 4 models (A-498, Caki-1, and ACHN). IF imaging showed expression of Ki67 was further suppressed with the combination treatment compared to lenvatinib and everolimus mono-treatments. Phospho-S6 (p-S6) signal were localized in perivascular regions of control group. Lenvatinib mono-treatment decreased p-S6 in the region apart from vessels, but some staining of p-S6 in perivascular regions were remained. The combination treatment further suppressed p-S6 staining regardless of the regions. In RAG syngeneic model, the combination treatment of lenvatinib at 10 mg/kg and anti-PD-1 mAb showed tumor shrinkage and tumors were regressed to nonpalpable sizes in 3 of 6 mice, although lenvatinib and anti-PD-1 mAb mono-treatments only slowed growths of RAG tumor. Our results indicate that combination of lenvatinib and everolimus enhances the inhibitory effects of mTOR signaling and tumor proliferation and showed greater antitumor activities. Combination of lenvatinib and anti-PD-1 mAb showed greater antitumor activity including tumor shrinkage, which was not demonstrated by each monotherapy in the murine RCC syngeneic model. These preclinical results provide one of the mechanisms of combinational effect of lenvatinib and everolimus, or PD-1 blockade in RCC. Citation Format: Yusuke Adachi, Takayuki Kimura, Masahiro Matsuki, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 12.

Synergistic effects of the immune checkpoint inhibitor CTLA-4 combined with the growth inhibitor lycorine in a mouse model of renal cell carcinoma.

Renal cell carcinoma (RCC) management has undergone a major transformation over the past decade; immune checkpoint inhibitors are currently undergoing clinical trials and show promising results. However, the effectiveness of immune checkpoint inhibitors in patients with metastatic RCC (mRCC) is still limited. Lycorine, an alkaloid extracted from plants of the Amaryllidaceae family, is touted as a potential anti-cancer drug because of its demonstrative growth inhibition capacity (induction of cell cycle arrest and inhibition of vasculogenic mimicry formation). Moreover, T cell checkpoint blockade therapy with antibodies targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) has improved outcomes in cancer patients. However, the anti-tumor efficacy of combined lycorine and anti-CTLA-4 therapy remains unknown. Thus, we investigated a combination therapy of lycorine hydrochloride and anti-CTLA-4 using a murine RCC model. As a means of in vitro confirmation, we found that lycorine hydrochloride inhibited the viability of various RCC cell lines. Furthermore, luciferase-expressing Renca cells were implanted in the left kidney and the lung of BALB/c mice to develop a RCC metastatic mouse model. Lycorine hydrochloride and anti-CTLA-4 synergistically decreased tumor weight, lung metastasis, and luciferin-staining in tumor images. Importantly, the observed anti-tumor effects of this combination were dependent on significantly suppressing regulatory T cells while upregulating effector T cells; a decrease in regulatory T cells by 31.43% but an increase in effector T cells by 31.59% were observed in the combination group compared with those in the control group). We suggest that a combination of lycorine hydrochloride and anti-CTLA-4 is a viable therapeutic option for RCC patients.

Repurposing Sunitinib with Oncolytic Reovirus as a Novel Immunotherapeutic Strategy for Renal Cell Carcinoma.

In addition to their direct cytopathic effects, oncolytic viruses are capable of priming antitumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the antitumor immune response generated by oncolytic reovirus. In vitro, oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines after exposure to reovirus, sunitinib, or their combination. In vivo, the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses after treatment with reovirus (intratumoral), sunitinib, or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation. Reovirus-mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive antitumor immune response evidenced by increased numbers of tumor-specific CD8+ IFNγ-producing cells. Coadministration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells, and the establishment of protective immunity upon tumor rechallenge. Similar results were observed for KLN205 tumor-bearing mice, highlighting the potential broad applicability of this approach. The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839-50. ©2016 AACR.

Promising pharmacological profile of a Kunitz-type inhibitor in murine renal cell carcinoma model.

Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate.

MCAM and LAMA4 Are Highly Enriched in Tumor Blood Vessels of Renal Cell Carcinoma and Predict Patient Outcome.

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma, or colorectal liver metastasis. We identified a panel of genes consistently upregulated by tumor blood vessels, of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes. This result was subsequently confirmed by immunohistochemical analysis of MCAM and LAMA4 expression in RCC and colorectal carcinoma blood vessels. Strong MCAM and LAMA4 expression was also shown to predict poor survival in RCC, but not in colorectal carcinoma. Notably, MCAM and LAMA4 were enhanced in locally advanced tumors as well as both the primary tumor and secondary metastases. Expression analysis in 18 different cancers and matched healthy tissues revealed vascular MCAM as highly specific in RCC, where it was induced strongly by VEGF, which is highly abundant in this disease. Lastly, MCAM monoclonal antibodies specifically localized to vessels in a murine model of RCC, offering an opportunity for endothelial-specific targeting of anticancer agents. Overall, our findings highlight MCAM and LAMA4 as prime candidates for RCC prognosis and therapeutic targeting. Cancer Res; 76(8); 2314-26. ©2016 AACR.

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 ...

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

Tumor-specific immunity induced by cryoablation in a murine renal cell carcinoma model.

PurposeTo evaluate tumor-specific immunity and define the mechanisms involved in the cryoimmunologic response, we compared the tumor control efficacy and immunologic responses of cryoablation with those of surgical excision in a tumor rechallenge model.Materials and MethodsSixty BALB/c mice with RENCA tumors that were generated in the left flank area underwent cryoablation or radical excision. The mice successfully treated were rechallenged with RENCA or an undifferentiated colon carcinoma cell line, CT26, in the contralateral right flank area. The recurrence rate after tumor rechallenge in each group was then observed. To assess the immunologic response of each treatment modality, fluorescent-activated cell sorting (FACS) analysis and a cytotoxicity assay using 51Cr release were performed.ResultsAfter reinoculation of the RENCA cells, the rate of tumor growth was significantly higher in the surgical excision group than in the cryoablation group (94.4% vs. 11.1%, p=0.001). In the cryoablation group, the tumor growth rate was significantly increased after rechallenge of CT26 cells compared with RENCA (94.1% vs. 11.1%, p=0.001). The cryoablation group showed an elevated CD3, CD4, CD8 T, and natural killer cell count in the FACS analysis and also showed significantly increased cytotoxicity in the 51Cr release assay compared with the excision group.ConclusionsThese results showed that cryoablation, compared to surgical resection, was more effective in preventing tumor growth after rechallenge with RENCA cells and that this response was tumor-specific, because the CT26 cells did not have the same effect.

169 SUNITINIB AUGMENTS ONCOLYTIC VIRUS MEDIATED ANTI-TUMOR IMMUNITY AGAINST RENAL CELL CARCINOMA - A NOVEL THERAPEUTIC PARADIGM?

You have accessJournal of UrologyKidney Cancer: Basic Research (I)1 Apr 2013169 SUNITINIB AUGMENTS ONCOLYTIC VIRUS MEDIATED ANTI-TUMOR IMMUNITY AGAINST RENAL CELL CARCINOMA - A NOVEL THERAPEUTIC PARADIGM? Keith Lawson, Zhong-Qiao Shi, Jason Spurrell, Wenqian Chen, Jun Kawakami, and Don Morris Keith LawsonKeith Lawson Calgary, Canada More articles by this author , Zhong-Qiao ShiZhong-Qiao Shi Calgary, Canada More articles by this author , Jason SpurrellJason Spurrell Calgary, Canada More articles by this author , Wenqian ChenWenqian Chen Calgary, Canada More articles by this author , Jun KawakamiJun Kawakami Calgary, Canada More articles by this author , and Don MorrisDon Morris Calgary, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1549AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Here, we investigated the ability of the oncolytic virus, reovirus (RV), to prime both innate and adaptive immune responses against renal cell carcinoma (RCC). Moreover, based on clinical trials demonstrating sunitinib?s ability to reverse RCC induced immunosuppression, we sought to determine the utility of combining this agent with reovirus for superior anti-tumor immunity. METHODS Following RV infection of RCC cell lines (ACHN, 786-O, A498, RENCA) cytotoxicity, chemokine production and viral progeny were quantified by WST-1, luminex and plaque titration assays, respectively. In vivo, 8-9 week old Balb/c mice were inoculated with 2.5 x 106 RENCA cells subcutaneously to establish an immunocompetent murine model of RCC and treated with intraperitoneal (i.p) phosphate buffered saline (PBS), UV-irradiated RV (DV) intratumorally (i.t), RV (i.t), sunitinib (i.p) or a combination of these agents. Mice were followed for tumor size and overall survival. To determine the anti-tumor immune response, CD8+ splenocytes were isolated magnetically and stimulated with RV infected RENCA cells; IFN-ψ production was subsequently determined by ELISA assay. Additionally, Gr-1+/CD11b+ splenic myeloid derived suppressor cells (MDSC) were quantified from all mice by flow cytometry. Adoptive transfer of splenocytes from mice treated with RV, sunitinib or a combination these agents into treatment naive mice followed by subcutaneous RENCA tumor challenge was utilized to assess protective immunity. RESULTS In vitro, RV induced an oncolytic response associated with chemokine (RANTES, MIP-1α, MCP-1, KC, IP-10, MIG) production in all cell lines tested. In vivo, combination RV/sunitinib therapy decreased tumor burden and improved overall survival more significantly than either agent used as a monotherapy [132mm2 (PBS) vs. 61mm2 (RV) vs. 82mm2 (S) vs. 21mm2 (RV+S); p < 0.05]. This was associated with a downregulation of splenic MDSC and enhanced anti-tumor CD8+ splenocyte IFNκ response [p < 0.02]. Decreased tumor burden was observed in treatment naive mice receiving splenocytes from RV/sunitinib treated mice versus RV or sunitinib alone following RENCA tumor challenge [80mm2 (RV) vs. 74mm2 (S) vs. 35 mm2 (RV+S); p < 0.05]. CONCLUSIONS Our results demonstrate that reovirus has both direct oncolytic as well as immunotherapeutic activity against RCC. Moreover, we highlight that combination of this agent with sunitinib leads to decreased tumor burden, increased overall survival and improved protective immunity. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e69-e70 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Keith Lawson Calgary, Canada More articles by this author Zhong-Qiao Shi Calgary, Canada More articles by this author Jason Spurrell Calgary, Canada More articles by this author Wenqian Chen Calgary, Canada More articles by this author Jun Kawakami Calgary, Canada More articles by this author Don Morris Calgary, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Combination therapy with the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin achieves complete remissions and improves tolerability in an ovarian A2780 xenograft model

We recently reported on the in superior in vivo antitumor efficacy of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206, formerly DOXO-EMCH), an albumin-binding prodrug of doxorubicin with acid-sensitive properties (see Fig. 1), in three tumor models in this journal, i.e. in three xenograft models (breast carcinoma M3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreatic carcinoma model (AsPC-1) [1] Previously, INNO-206 has shown significantly improved activity over doxorubicin in two xenograft breast cancer models and an orthotopic murine renal cell carcinoma model [2] and recently against resistant and non resistant myeloma (see http//:www.cytrx.com). INNO-206 binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site due to incorporation of an acid-sensitive hydrazone bond. The accumulation of INNO-206 in tumor tissue combined with a favorable biodistribution [3] is due to the pathophysiology of tumor tissue, characterized by a high metabolic turnover, angiogenesis, hypervasculature, a defective vascular architecture and an impaired lymphatic drainage, the so-called EPR (enhanced and retention) effect [4]. As a carrier for INNO-206, we selected albumin, which is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptideor protein-based drugs [5]. Albumin is a potential carrier for tumor targeting because it accumulates in solid tumors. In addition, we discovered that the HS-group of cysteine-34 of albumin is a unique and accessible functional group of a plasma protein for in situ prodrug conjugation considering that free thiol groups are absent from most circulating serum proteins except for albumin, which makes this group by far the most abundant thiol group in human plasma After injection directly into the blood stream, INNO-206 binds in situ to the cysteine-34 position of circulating albumin and is then transported in its albumin-bound form to the tumor site. INNO-206 is a prodrug of the anticancer agent doxorubicin in which doxorubicin is derivatized at its C-13 keto-position with a thiol-binding spacer molecule, i.e. 6-maleimidocaproic acid hydrazide. It contains an acidsensitive hydrazone linker that allows doxorubicin to be released either extracellularly in the slightly acidic environment often present in tumor tissue (pH range 6.0-6.8) [6] or intracellularly in acidic endosomal (pH 5.0-6.5) or lysosomal (pH 4.0-5.0) compartments after cellular uptake of the albumin conjugate by the tumor cell. INNO-206 was selected as an investigational product for clinical evaluation after toxicology studies in mice, rats, and dogs had shown a 2.5to 5-fold increase in the maximum tolerated dose (MTD) when compared to conventional doxoElectronic supplementary material The online version of this article (doi:10.1007/s10637-011-9686-5) contains supplementary material, which is available to authorized users.

241 TUMOR ANTIGENS CAN BE COVALENTLY CROSSLINKED TO ENHANCE ANTITUMOR IMMUNITY

INTRODUCTION AND OBJECTIVES: Cancer vaccines are promising therapies for immunoresponsive solid tumors such as renal cell carcinoma (RCC) and melanoma. A simple and novel strategy is described for enhancing the immunostimulatory properties of cancer vaccines. Covalently crosslinking a tumor antigen in vitro produces a tumor vaccine that can generate antitumor immunity even when an adjuvant is not used. METHODS: Antitumor immunity was assessed using mouse models for RCC (RENCA) and melanoma (B16). CA9 was the target antigen for RENCA tumors implanted in Balb/c mice. Gp100 was the target antigen for B16 tumors implanted in C57/Bl mice. Cancer vaccines consisted of recombinant CA9 or gp100 covalently crosslinked (CL) by incubating with 3,3’dithiobis(sulfosuccinimidylpropionate), which was then dialyzed off. Dendritic cells (DCs) were freshly generated from mouse bone marrow. The specificity of immune stimulation was studied using T-cell receptor transgenic mouse models (OT-1 and Pmel-1). In these transgenic mice, nearly all the CD8 T cells recognize a single, well-characterized epitope. RESULTS: In a murine RCC model, vaccination with CA9 alone produced little immune-mediated antitumor response; however, CLCA9 was highly effective in inhibiting tumor growth. Crosslinking enhanced antitumor immunity even when CA9 was substituted by an immunodominant peptide of CA9. Similarly, in a murine melanoma model, CL-gp100 inhibited tumor growth, demonstrating that crosslinking enhances vaccines in multiple animal models. Crosslinking of tumor antigen enhanced the cellular immune response as measured using an ELISPOT assay or a Cr release cytotoxicity assay. Potential mechanisms were explored. CL-antigens had enhanced binding to fresh DCs when FITC-labeled antigens were monitored by fluorescence microscopy, flow cytometry or immunoblotting. Crosslinking did not enhance markers of DC activation such as MHC I, MHC II, CD80 and CD86; however, crosslinking preferentially enhanced antigen processing through the proteosomal pathway in DCs, which is expected to result in crosspresentation of antigens. Finally, DCs stimulated with CL-gp100 or CL-ovalbumin were capable of stimulating antigen-specific lymphocytes derived from Pmel-1 or OT-1 mice, respectively. CONCLUSIONS: Crosslinking of tumor antigens is a simple and effective strategy for enhancing tumor vaccines, which results in greater capacity to bind DCs and stimulate antigen processing. DCs stimulated with crosslinked antigens are highly effective in activating antigen-specific lymphocytes.

Antitumor effect of the vascular-disrupting agent ZD6126 in a murine renal cell carcinoma model

ZD6126 is a vascular-disrupting agent that affects the endothelial tubulin cytoskeleton causing selective occlusion of tumor vasculature and extensive tumor cell necrosis. The present study evaluated the antitumor and antivascular activities of ZD6126 in the clinically relevant murine renal cell carcinoma (RENCA) model and also evaluated biological response to therapy using color Doppler imaging as biomarker. Mice were implanted with RENCA tumor cells (day 0) and established tumors were treated with ZD6126 (100 mg/kg i.p.) or vehicle with repeated intermittent doses on day 10, 14 and 18. ZD6126 treatment led to a significant reduction in tumor size and was associated with extensive tumor necrosis and a reduction in tumor blood flow versus controls. MVD increased with intermittent treatment (day 10, 14 and 18). In an additional study, animals were treated at day 19 and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Corrosion casting showed that tumor vessel architecture is affected by treatment, whereas pre-existing vessels in control tissues are practically not affected. Inter-vessel and inter-branch distances as well as vessel diameters are influenced by treatment. In conclusion, ZD6126 showed potent antitumor efficacy in the RENCA model and our data suggest that decrease in tumor blood flow may be a useful surrogate marker of treatment effect.

Dynamic Contrast-Enhanced Magnet Resonance Imaging of Sunitinib-Induced Vascular Changes to Schedule Chemotherapy in Renal Cell Carcinoma Xenograft Tumors

In an attempt to develop better therapeutic approaches for metastatic renal cell carcinoma (RCC), the combination of the antiangiogenic drug sunitinib with gemcitabine was studied. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), we have previously determined that a sunitinib dosage of 20 mg/kg per day increased kidney tumor perfusion and decreased vascular permeability in a preclinical murine RCC model. This sunitinib dosage causing regularization of tumor vessels was selected to improve delivery of gemcitabine to the tumor. DCE-MRI was used to monitor regularization of vasculature with sunitinib in kidney tumors to schedule gemcitabine. We established an effective and nontoxic schedule of sunitinib combined with gemcitabine consisting of pretreatment with sunitinib for 3 days followed by four treatments of gemcitabine at 20 mg/kg given 3 days apart while continuing daily sunitinib treatment. This treatment caused significant tumor growth inhibition resulting in small residual tumor nodules exhibiting giant tumor cells with degenerative changes, which were observed both in kidney tumors and in spontaneous lung metastases, suggesting a systemic antitumor response. The combined therapy caused a significant increase in mouse survival. DCE-MRI monitoring of vascular changes induced by sunitinib, gemcitabine, and both combined showed increased tumor perfusion and decreased vascular permeability in kidney tumors. These findings, confirmed histologically by thinning of tumor blood vessels, suggest that both sunitinib and gemcitabine exert antiangiogenic effects in addition to cytotoxic antitumor activity. These studies show that DCE-MRI can be used to select the dose and schedule of antiangiogenic drugs to schedule chemotherapy and improve its efficacy.

Extracts from Citrus unshiu promote immune-mediated inhibition of tumor growth in a murine renal cell carcinoma model

Citrus unshiu (Satsuma mandarin, SM) is a citrus fruit the peel of which has been used as a traditional Chinese medicine to treat common cold, relieve exhaustion, and cancer. In this study, we examined how effectively the content and peel extracts of SM can suppress cancer growth. The mechanism underlying cancer-suppressing properties of SM was investigated in tumor-bearing mice with renal carcinoma cell, Renca. Effectiveness of SM in tumor suppression was evaluated by measuring size of tumor mass in tumor-bearing mice treated with various doses of SM content and peel extracts. Proliferation of tumor cells and splenocytes was determined by MTT assay and [³H]TdR uptake, respectively. Relevant immunological mechanisms were chased by assaying cytokines including TGF-β, IL-6, IFN-γ, and TNF-α by ELISA. The content and peel extracts of SM inhibited the growth of tumor cells in tumor-bearing mice. Especially, average tumor volume of two groups treated with 3 and 30 mg peel extracts per mouse weight (kg) were significantly decreased to 52.32% (p<0.05) and 68.72% (p<0.01), respectively. To identify tumor regression mechanism, anti-tumor cytokines measured in Con A-activated splenocytes from tumor-bearing mice. IFN-γ was increased in both of the peel extract-treated groups, while TNF-α, which had been decreased by tumor growth, was rescued to the normal level in SM content and peel extracts-treated groups. However, SM content and peel extracts did not inhibit proliferation and tumor-proliferative cytokines including TGF-β and IL-6 production of tumor cells. These results indicate that SM content and peel extracts have anti-tumor properties in the tumor-bearing murine model. The mechanism underlying the anti-tumor effects of SM extracts is strongly suggested to be via boosting cytokines such as IFN-γ and TNF-α, enhancing immune-mediated anti-tumor properties.