Orthotopic Renal Cell Carcinoma Research Articles

Preclinical assessment of the efficacy of B7-H3 CAR-T in renal cell carcinoma

B7-H3 is a type I transmembrane protein that belongs to the B7 immune checkpoint protein family, is aberrantly expressed in cancer cells, but rarely expressed in normal tissues, making it an attractive target for cancer therapy. Here, we found B7-H3 is highly expressed in the renal cell carcinoma (RCC) tumor tissues and RCC cell lines, but is undetectable in normal renal tissues. Therefore, we engineered second-generation CAR-T cells targeting B7-H3, incorporating either CD28 or 4–1BB co-stimulatory domains. Both CAR-T cell variants demonstrated potent antitumor activity against RCC tumors in vitro and in metastatic and orthotopic RCC mouse models. Furthermore, the B7-H3 CAR-T cells exhibited remarkable proliferation and robust cytokine release when co-cultured with RCC cancer cells. These findings demonstrated that targeting B7-H3 by CAR-T cells potentially offering a new treatment option for RCC patients.

A multifunctional PEGylated liposomal-encapsulated sunitinib enhancing autophagy, immunomodulation, and safety in renal cell carcinoma

BackgroundSunitinib is a multikinase inhibitor used to treat patients with advanced renal cell carcinoma (RCC). However, sunitinib toxicity makes it a double-edged sword. Potent immune modulation by sunitinib extends to nuclear interactions. To address these issues, there is an urgent need for delivery vectors suitable for sunitinib treatment.MethodsWe developed PEGylated liposomes as delivery vectors to precisely target sunitinib (lipo-sunitinib) to RCC tumors. Further investigations, including RNA sequencing (RNA-seq), were performed to evaluate transcriptomic changes in these pathways. DiI/DiR-labeled lipo-sunitinib was used for the biodistribution analysis. Flow cytometry and immunofluorescence (IF) were used to examine immune modulation in orthotopic RCC models.ResultsThe evaluation of results indicated that lipo-sunitinib precisely targeted the tumor site to induce autophagy and was readily taken up by RCC tumor cells. In addition, transcriptomic assays revealed that following lipo-sunitinib treatment, autophagy, antigen presentation, cytokine, and chemokine production pathways were upregulated, whereas the epithelial-mesenchymal transition (EMT) pathway was downregulated. In vivo data provided evidence supporting the inhibitory effect of lipo-sunitinib on RCC tumor progression and metastasis. Flow cytometry further demonstrated that liposunitinib increased the infiltration of effector T cells (Teffs) and conventional type 1 dendritic cells (cDC1s) into the tumor. Furthermore, systemic immune organs such as the tumor-draining lymph nodes, spleen, and bone marrow exhibited upregulated anticancer immunity following lipo-sunitinib treatment.ConclusionOur findings demonstrated that lipo-sunitinib is distributed at the RCC tumor site, concurrently inducing potent autophagy, elevating antigen presentation, activating cytokine and chemokine production pathways, and downregulating EMT in RCC cells. This comprehensive approach significantly enhanced tumor inhibition and promoted anticancer immune modulation.Graphical

Abstract 111: Establishing orthotopic renal cell carcinoma xenograft in PBMC-humanized immunodeficient NSG-MHC I/II double knock-out mice

Abstract Clear cell renal cell carcinoma (ccRCC), the predominant histological subtype of RCC, is highly correlated with immune cells, an association that is linked to a worse prognosis. Our study aimed to establish an orthotopic ccRCC mouse model reconstituted with human peripheral blood mononuclear cells (PBMCs) and implanted with human ccRCC SKRC-59 cells under the kidney capsule in immunodeficient NSG-MHC I/II double knock-out (DKO) mice. The study was conducted using two experimental designs to optimize humanization and tumor formation. In the first experimental design, the mice were engrafted with SKRC-59 cells under the left kidney capsule 24 hours post-irradiation on study day 0. The PBMCs were then injected intravenously on study day 9. The second experimental design involved irradiating mice on study day -1, followed by engraftment with PBMCs either perfectly (6/6) or partially (3/6) matched human leukocyte antigens (HLA) with the tumors. Subsequently, SKRC-59 cells were placed under the kidney capsule on study day 0. Daily post-operative observations were conducted for six days following surgery to ensure the well-being of the mice. Tumor burden was assessed twice weekly using an in vivo imaging system, along with monitoring body weights and clinical signs. The humanization rates were evaluated 7, 14, and 21 days post-PBMC engraftment, as well as at the end of the study, using peripheral blood. Approximately 5 to 6 weeks post-tumor implantation, the mice were euthanized, and both left and right kidneys were collected and weighed for further analysis. The humanization rates were significantly higher when the PBMCs were engrafted on the same day as irradiation compared to 10 days post-irradiation. The PBMC humanization, and HLA-type PBMC donors did not affect tumor burden. Additionally, we found that distant metastasis in the lungs developed in several mice. Using flow cytometry and immunohistochemistry (IHC) to analyze tumor infiltrating leukocytes (TILs), a predominant number of CD3+ T cells were found in human CD45+ TILs, indicating that this model could be useful for evaluating T cell-mediated immunotherapies. Moreover, lympho-myeloid aggression (LMA) was observed in this peripheral blood leukocytes (PBL) mouse model, showing early stage tertiary lymphoid structures (TLS), which potentially provides a powerful tool to recapitulate TLS formation and study associated mechanisms and therapies. Our study demonstrated the growth of ccRCC SKRC-59 tumors under the renal capsule and observed TILs and metastatic patterns in PBMC humanized DKO mice. This PBMC-humanized orthotopic ccRCC xenograft model can recapitulate tumor microenvironment (TME), and be potentially used to assess the safety and efficacy of various immunotherapeutics. Citation Format: Wonyoung Kang, Yufei Wang, Yasmin N. Laimon, Oanh Pham, Ben Matran, Lauren Bottoms, Sheng Khang, Hsien-Chi Yuan, Nithyassree Murugan, Aseman B. Sheshdeh, Li-chin Yao, Jiwon Yang, Mingshan Cheng, Sabina Signoretti, Wayne A. Marasco, James G. Keck. Establishing orthotopic renal cell carcinoma xenograft in PBMC-humanized immunodeficient NSG-MHC I/II double knock-out mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 111.

PD17-07 COMBINATION THERAPY FOR TKI RESISTANT KIDNEY CANCER VIA BLOCKING ERΒ/CIRCRNA-DGKD/MIR-125-5P/VE-CADHERIN SIGNAL INDUCED VASCULOGENIC MIMICRY

You have accessJournal of UrologyCME1 Apr 2023PD17-07 COMBINATION THERAPY FOR TKI RESISTANT KIDNEY CANCER VIA BLOCKING ERΒ/CIRCRNA-DGKD/MIR-125-5P/VE-CADHERIN SIGNAL INDUCED VASCULOGENIC MIMICRY Jie Ding, Yixi Hu, Chawnshang Chang, Edward M. Messing, Jean Joseph, and Shuyuan Yeh Jie DingJie Ding More articles by this author , Yixi HuYixi Hu More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean JosephJean Joseph More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Tyrosine kinase inhibitors (TKIs), pazopanib and sunitinib, are widely used in renal cell carcinoma (RCC) therapy by inhibiting tumor angiogenesis. However, most patients become insensitive or develop resistance toward TKI therapy eventually, and the underlying mechanisms have not yet been fully elucidated. Our goal is to reveal the molecular mechanisms of TKI resistant RCC to find a potential combination therapy for TKI-resistant RCC. METHODS: The human RCC cell lines: 786-O, A498, Caki-1, and SW-839 were used. Lentiviral plasmids were used to generate gene engineering tools. The qPCR and Western blot were used to evaluate the gene expressions at RNA and protein levels, respectively. Vasculogenic mimicry (VM) assays were performed to examine vascular-like structure formation ability of cancer cells. Protein-DNA binding complexes were detected by Chromatin immunoprecipitation (ChIP) assays. The human DGKD promoter region was cloned into pGL3 vector for Luciferase Assays. Preclinical mouse RCC model was used to evaluate the anti-cancer effect of pazopanib and circRNA-DGKD blockage in TKI resistant RCC. RESULTS: TKI resistant RCC cells showed increased ability of forming VM to counteract the TKI-suppression function both in vivo and in vitro. Mechanism dissection revealed that pazopanib (or sunitinib)-resistant RCC cells had an increased ERβ, consequently enhancing the circRNA-DGKD expression. In turn, circRNA-DGKD could increase VE-cadherin expression via sponging the miR-125-5p. Supportively, targeting circRNA-DGKD intercepted the RCC VM formation, reduced metastases and improved survival in an orthotopic RCC mouse model. Collectively, the findings may aid in the development of novel therapies for metastatic or TKI-resistant RCC. CONCLUSIONS: Our study reveals that the resistance to TKIs (pazopanib or sunitinib) occurs by increasing vasculogenic mimicry (VM) ability to counteract the TKI angiogenesis suppression function. Furthermore, we find a potential therapy to more effectively treat TKI resistant RCC by blocking ERβ/circRNA-DGKD/miR-125-5p/VE-cadherin pathway together with pazopanib administration. Source of Funding: This work was partially supported by URMC Urology Department Research fund and George H. Whipple Professorship Endowment © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e498 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Ding More articles by this author Yixi Hu More articles by this author Chawnshang Chang More articles by this author Edward M. Messing More articles by this author Jean Joseph More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement PDF downloadLoading ...

A Listeria-based vaccine targeting ISG15 exerts anti-tumor efficacy in renal cell carcinoma.

Renal cell carcinoma (RCC) is the deadliest form of urological cancer and is projected to be the fourth most common neoplasm in the USA in males by 2040. In addition to the current poor prognosis with 5-year survival rates hardly reaching 15%, the prevalence of resistance to currently available systemic therapies has also established an urgent need to develop new treatment regimen(s) for advanced RCC. Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been intensively studied for its central role in innate immunity against intracellular pathogens. However, in this study, we identified ISG15 as a novel tumor-associated antigen and prognostic marker in RCC. Further, we therapeutically targeted elevated ISG15 expression by means of a Listeria monocytogenes (Lm)-based vaccine, designated Lm-LLO-ISG15, in both subcutaneous and orthotopic RCC mouse models. Treatment with Lm-LLO-ISG15 resulted in an influx of tumor-infiltrating effector T cells and significant anti-tumor efficacy in both subcutaneous and orthotopic RCC tumor models. Treatment with Lm-LLO-ISG15 also generated a robust interferon-gamma response and attracted a larger pool of polyfunctional T cells into the tumor microenvironment. Importantly, the therapeutic efficacy of Lm-LLO-ISG15 in RCC is comparable to that of anti-PD-1 and sunitinib, the current frontline therapies for RCC patients. Collectively, our work illustrates that targeting ISG15 in RCC with a CTL-based immunotherapy such as Lm-LLO-ISG15 is a promising and potentially translatable therapeutic strategy to enhance survival in RCC patients.

Near-Infrared Fluorescence Imaging of Renal Cell Carcinoma with ASP5354 in a Mouse Model for Intraoperative Guidance.

Renal cell carcinoma is a prevalent disease associated with high morbidity and mortality rates. Partial nephrectomy is a first-line surgical option because it allows the preservation of renal function. Clear differentiation between normal and cancerous tissues is critical for increasing the negative margin rates. This study investigated the capability of the near-infrared (NIR) fluorescent imaging agent ASP5354 for in vivo fluorescence imaging of renal cell carcinoma. ASP5354 at a single dose of 12 nmol (0.037 mg)/kg body weight was intravenously administered to healthy and orthotopic renal cell carcinoma mice under anesthesia. NIR images of the abdominal cavity were obtained using a near-infrared fluorescence (NIRF) camera system. In addition, the cancerous kidneys were harvested, and the NIRF in their sections was measured using an NIRF microscope. Normal renal tissue emitted strong NIRF but the cancer tissue did not. The difference in NIRF intensity between the normal and cancer tissues clearly presented the boundary between the normal and cancer tissues in macro and micro NIRF imaging. ASP5354 can distinguish cancer tissue from normal tissue using NIRF. Thus, ASP5354 is a promising agent for renal cell carcinoma tissue imaging in partial nephrectomy for renal cell carcinoma patients.

An activated excretion-retarded tumor imaging strategy towards metabolic organs

Intraoperative fluorescence-based tumor imaging plays a crucial role in performing the oncological safe tumor resection with the advantage of differentiating tumor from normal tissues. However, the application of these fluorescence contrast agents in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) was dramatically hammered as a result of lacking active targeting and poor retention time in tumor, which limited the Signal to Noise Ratio (SNR) and narrowed the imaging window for complicated surgery. Herein, we reported an activated excretion-retarded tumor imaging (AERTI) strategy, which could be in situ activated with MMP-2 and self-assembled on the surface of tumor cells, thereby resulting in a promoted excretion-retarded effect with an extended tumor retention time and enhanced SNR. Briefly, the AERTI strategy could selectively recognize the Integrin αvβ3. Afterwards, the AERTI strategy would be activated and in situ assembled into nanofibrillar structure after specifically cleaved by MMP-2 upregulated in a variety of human tumors. We demonstrated that the AERTI strategy was successfully accumulated at the tumor sites in the 786-O and HepG2 xenograft models. More importantly, the modified modular design strategy obviously enhanced the SNR of AERTI strategy in the imaging of orthotopic RCC and HCC. Taken together, the results presented here undoubtedly confirmed the design and advantage of this AERTI strategy for the imaging of tumors in metabolic organs.

P0555 - Successful therapy of experimental, orthotopic renal cell carcinoma with two different types of Axl inhibiting agents

P0555 - Successful therapy of experimental, orthotopic renal cell carcinoma with two different types of Axl inhibiting agents

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.

SNU-333 Cells as an Appropriate Cell Line for the Orthotopic Renal Cell Carcinoma Model.

Objective: To investigate a feasible candidate for an appropriate cell line for the orthotopic renal cell carcinoma (RCC) model. Methods: Normal human proximal tubule cells (HK-2) and RCC cells were used for MTT assay, Western blotting, sphere-forming assay, and orthotopic injection of BALB/c-nude mice. Immunohistochemistry was adopted in tissue arrays and orthotopic tumors. Results: Primary RCC cells showed resistance to a GPX4 inhibitor compared to HK-2 and to metastatic RCC cells, Caki-1. Caki-2 and SNU-333 cells showed resistance to ferroptosis via increased GPX4 and FTH1, respectively. RCC cells showed increased αSMA, in which Caki-2 and SNU-333 cells exhibited different epithelial–mesenchymal transition and cancer stem cell markers. Caki-1 and SNU-333 cells formed spheres in vitro and orthotopic tumor masses in vivo. The injected SNU-333 tumor only showed high intensities of CD10 and PAX8, markers of renal origin. Conclusion: SNU-333 cell line exhibited resistance via iron metabolism and stemness, and had tumor-initiating capacities in vitro and in vivo. These results suggest that among the cells tested, SNU-333 cells were the most promising for the establishment of an orthotopic RCC model for further researches.

A Near-Infrared Peptide Probe with Tumor-Specific Excretion-Retarded Effect for Image-Guided Surgery of Renal Cell Carcinoma.

Image-guided surgery plays a crucial role in realizing complete tumor removal, reducing postoperative recurrence and increasing patient survival. However, imaging of tumor lesion in the typical metabolic organs, e.g., kidney and liver, still has great challenges due to the intrinsic nonspecific accumulation of imaging probes in those organs. Herein, we report an in situ self-assembled near-infrared (NIR) peptide probe with tumor-specific excretion-retarded (TER) effect in tumor lesions, enabling high-performance imaging of human renal cell carcinoma (RCC) and achieving complete tumor removal, ultimately reducing postoperative recurrence. The NIR peptide probe first specifically recognizes αvβ3 integrin overexpressed in renal cancer cells, then is cleaved by MMP-2/9, which is up-regulated in the tumor microenvironment. The probe residue spontaneously self-assembles into nanofibers that exhibit an excretion-retarded effect in the kidney, which contributes to a high signal-to-noise (S/N) ratio in orthotopic RCC mice. Intriguingly, the TER effect also enables precisely identifying eye-invisible tiny lesions (<1 mm), which contributes to complete tumor removal and significantly reduces the postoperative recurrence compared with traditional surgery. Finally, the TER strategy is successfully employed in high-performance identification of human RCC in an ex vivo kidney perfusion model. Taken together, this NIR peptide probe based on the TER strategy is a promising method for detecting tumors in metabolic organs in diverse biomedical applications.

Gankyrin is a novel biomarker for disease progression and prognosis of patients with renal cell carcinoma.

BackgroundIn the clinic, how to stratify renal cell carcinoma (RCC) patients with different risks and to accurately predict their prognostic outcome remains a crucial issue. In this study, we assessed the expression and prognostic value of gankyrin in RCC patients.MethodsThe expression of gankyrin was examined in public databases and validated in specimens from two independent centers. The clinical practice and disease correlation of gankyrin in RCC were evaluated in RCC patients, various cell lines and an orthotopic RCC model.FindingsUpregulation of gankyrin expression in RCC was corroborated in two independent cohorts. High gankyrin expression positively associated with disease progression and metastasis of RCC patients. A positive correlation between gankyrin and sunitinib-resistance was also observed in RCC cell lines and in an orthotopic RCC model. Kaplan-Meier analysis revealed that patients with higher gankyrin expression presented worse prognosis of RCC patients in the two cohorts. Gankyrin served as an independent prognostic factor for RCC patients even after multivariable adjustment by clinical variables. Time-dependent AUC and Harrell's c-index analysis presented that the incorporation of the gankyrin classifier into the current clinical prognostic parameters such as TNM stage, Fuhrman nuclear grade or SSIGN score achieved a greater accuracy than without it in predicting prognosis of RCC patients. All results were confirmed in randomized training and validation sets from the two patient cohorts.InterpretationGankyrin can serve as a reliable biomarker for disease progression and for prognosis of RCC patients. Combining gankyrin with the current clinical parameters may help patient management.FundNational Natural Science Foundation of China (No. 81773154, 81772747 and 81301861), Medical Discipline Construction Project of Pudong New Area Commission of Health and Family Planning (PWYgf2018-03), the Shanghai Medical Guidance (Chinese and Western Medicine) Science and Technology Support Project (No. 17411960200), Outstanding Leaders Training Program of Pudong Health Bureau of Shanghai (No. PWR12016-05)

Degalactotigonin Exerts Effective Inhibition on the Growth, Metastasis and Sunitinib-Resistance of Renal Cell Carcinoma Through Inactivating YAP Signaling

Background: The pervasive progression and drug-resistance of renal cell carcinoma (RCC) after treatments demands for more effective drugs with little side effects. Recently, extracts from natural resources have been reported to effectively suppress the progression of certain tumors. In this study, we examined the effects of degalactotigonin extracted from Solanum nigrum L. on the progression and sunitinib-resistance of RCC, and the underlying mechanisms. Methods: The in vitro effects of degalactotigonin on the proliferation, apoptosis, invasion, migration and sunitinib-resistance were assessed by CCK-8, flow cytometry, Western blot, Matrigel invasion and transwell migration assays. The in vivo effects were evaluated in murine models of subcutaneous and in situ tumor and lung metastasis. The related molecular characteristics were delineated by RNA-sequencing. Findings: Degalactotigonin induced apoptosis and suppressed proliferation, invasion, migration, and sunitinib-resistance of RCC cells in vitro in a dose-dependent manner. Furthermore, degalactotigonin effectively inhibited the tumorigenicity and lung metastasis of RCC cells in vivo. Mechanistically, RNA-sequencing revealed that inactivation of YAP signaling occurred in degalactotigonin-treated RCC cells with reduced expression of YAP and its target genes. We also observed that degalactotigonin activated p-LATS1/2 to phosphorylate YAP, which induced more YAP retention in the cytoplasm but less YAP entering the nucleus of RCC cells. Moreover, re-expressed YAP in degalactotigonin-treated RCC cells mediated the anti-tumor activities of degalactotigonin on RCC. Furthermore, degalactotigonin ameliorated sunitinib-resistance in an orthotopic RCC model. Interpretation: Degalactotigonin is an effective therapeutic agent in inhibiting the growth, progression and sunitinib-resistance of RCC. Funding Statement: National Natural Science Foundation of China (No. 81773154, 81772747 and 81301861), Shanghai Natural Science Foundation of China (No. 13ZR1450700), the Shanghai Medical Guidance (Chinese and Western Medicine) Science and Technology Support Project (No. 17411960200). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All experimental animal procedures were approved by the Animal Care and Use Committee of the Second Military Medical University (Shanghai, China).

Experimental imaging in orthotopic renal cell carcinoma xenograft models: comparative evaluation of high-resolution 3D ultrasonography, in-vivo micro-CT and 9.4T MRI

In this study, we aimed to comparatively evaluate high-resolution 3D ultrasonography (hrUS), in-vivo micro-CT (μCT) and 9.4T MRI for the monitoring of tumor growth in an orthotopic renal cell carcinoma (RCC) xenograft model since there is a lack of validated, non-invasive imaging tools for this purpose. 1 × 106 Caki-2 RCC cells were implanted under the renal capsule of 16 immunodeficient mice. Local and systemic tumor growth were monitored by regular hrUS, μCT and MRI examinations. Cells engrafted in all mice and gave rise to exponentially growing, solid tumors. All imaging techniques allowed to detect orthotopic tumors and to precisely calculate their volumes. While tumors appeared homogenously radiolucent in μCT, hrUS and MRI allowed for a better visualization of intratumoral structures and surrounding soft tissue. Examination time was the shortest for hrUS, followed by μCT and MRI. Tumor volumes determined by hrUS, μCT and MRI showed a very good correlation with each other and with caliper measurements at autopsy. 10 animals developed pulmonary metastases being well detectable by μCT and MRI. In conclusion, each technique has specific strengths and weaknesses, so the one(s) best suitable for a specific experiment may be chosen individually.

An antisense oligonucleotide targeting TGF-β2 inhibits lung metastasis and induces CD86 expression in tumor-associated macrophages

The transforming growth factor (TGF)-β pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-β pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-β isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-β2. We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis. We observed that ISTH0047 is able to significantly reduce TGF-β2 mRNA and protein levels without altering the levels of TGF-β1 and TGF-β3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-β2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86. Overall, our data point to TGF-β2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Abstract 4216: Experimental imaging in orthotopic xenograft models of renal cell carcinoma: comparative evaluation of high-resolution ultrasonography, in vivo micro-CT, and 9.4T MRI

Abstract Introductory Sentence Orthotopic xenografts are increasingly used as preclinical in-vivo models for the study of renal cell carcinoma (RCC). However, monitoring of these tumors requires sophisticated imaging techniques. In this study, we comparatively evaluated modern small animal imaging tools like high-resolution 3D-ultrasonography (3D-US), contrast-enhanced in-vivo micro-CT (CE-CT) and 9,4T MRI (MRI) to non-invasively monitor tumor growth and progression in an orthotopic RCC xenograft model. Material and Methods 106 CAKI-1 cells were injected under the renal capsule of 18 Balb/c-nude mice. Every 14 days from week 4 imaging was performed by CE-CT and 3D-US. 10 weeks after tumor cell inoculation, all animals additionally underwent multiparametric MRI (T1, T2, T2*, DWI). At autopsy, tumor volumes were determined using a caliper and compared to in-vivo imaging results. CE-CT, MRI and 3D-US were evaluated regarding tumor detection, analysis of tumor volume, radiographic imaging properties and examination time. Finally, similar experiments were performed with the RCC cell lines KTCTL30 (n = 2) and 786-0 (n = 1) to test their applicability for the orthotopic xenograft model. Results Tumors were detected in 16/18 animals (89%) 4 weeks after orthotopic inoculation. All methods enabled to adequately visualize orthotopic tumors and to display their growth over time. While the tumors had a homogenously radiolucent signal in CT scans, sonography and MRI were better able to visualized intratumoral structures (e.g. solid areas or intratumoral bleedings) and surrounding soft tissue. CT had the best spatial resolution, followed by 3D-US and MRI. The median examination time was 4.8min per 3D-US, 12.5min per CT and 37.9min per MRI, respectively. Of interest, tumor volumes determined by KM-CT and ultrasonography showed strong correlation with each other (n = 85, R = 0.985) as well as with caliper measurements at autopsy (CT: n = 16. R = 0.922; sonography: n = 16, R = 0.934). Similarly, tumor volumes measured with T2-weighted MRI correlated well with those determined by CT, sonography and caliper. No side effects due to radiation exposure were observed. Mice inoculated with KTCTL30 cells developed partly cystic, partly solid tumors. 786-O cells yielded purely solid, exponentially growing tumors. Conclusion All three imaging modalities are feasible tools for the non-invasive monitoring of orthotopic tumor growth and show excellent correlation with each other. While sonography allows for a fast analysis of tumor volume with excellent resolution and no radiation exposure, MRI provides the best visualization of soft tissue and can give information about tumor biology when acquiring additional sequences. CT has the best spatial resolution of all methods and enables simultaneous screening for bone and lung metastases but relies on the use of contrast agent and ionizing radiation. Citation Format: Johannes Linxweiler, Christina Körbel, Andreas Müller, Volker Jung, Eva Jüngel, Stefan Siemer, Michael Stöckle, Kerstin Junker, Michael D. Menger, Matthias Saar. Experimental imaging in orthotopic xenograft models of renal cell carcinoma: comparative evaluation of high-resolution ultrasonography, in vivo micro-CT, and 9.4T MRI. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4216.

MP92-19 EXPERIMENTAL IMAGING IN ORTHOTOPIC XENOGRAFT MODELS OF RENAL CELL CARCINOMA: COMPARATIVE EVALUATION OF HIGH-RESOLUTION ULTRASONOGRAPHY, IN-VIVO MICRO-CT AND 9.4T MRI

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-19 EXPERIMENTAL IMAGING IN ORTHOTOPIC XENOGRAFT MODELS OF RENAL CELL CARCINOMA: COMPARATIVE EVALUATION OF HIGH-RESOLUTION ULTRASONOGRAPHY, IN-VIVO MICRO-CT AND 9.4T MRI Johannes Linxweiler, Christina Körbel, Andreas Müller, Volker Jung, Eva Jüngel, Stefan Siemer, Michael Stöckle, Kerstin Junker, Michael D. Menger, and Matthias Saar Johannes LinxweilerJohannes Linxweiler More articles by this author , Christina KörbelChristina Körbel More articles by this author , Andreas MüllerAndreas Müller More articles by this author , Volker JungVolker Jung More articles by this author , Eva JüngelEva Jüngel More articles by this author , Stefan SiemerStefan Siemer More articles by this author , Michael StöckleMichael Stöckle More articles by this author , Kerstin JunkerKerstin Junker More articles by this author , Michael D. MengerMichael D. Menger More articles by this author , and Matthias SaarMatthias Saar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2652AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Orthotopic xenografts are increasingly used preclinical in-vivo models for the study of renal cell carcinoma (RCC). However, monitoring of these tumors requires sophisticated imaging techniques. Herein we comparatively evaluated modern small animal imaging tools like high-resolution 3D-ultrasonography (3D-US), contrast-enhanced in-vivo micro-CT (CE-CT) and 9,4T MRI (MRI) to monitor tumor growth in an orthotopic RCC xenograft model. METHODS 106 CAKI-1 cells were injected under the renal capsule of 18 Balb/c-nude mice. Every 14 days from week 4 imaging was performed by CE-CT and 3D-US. 10 weeks after cell inoculation, all animals additionally underwent MRI. At autopsy, tumor volumes were determined with a caliper and compared to in-vivo imaging results. CE-CT, MRI and 3D-US were evaluated regarding tumor detection, tumor volume analysis, radiographic imaging properties and examination time. Finally, RCC cell lines KTCTL30 (n=2) and 786-0 (n=1) were used to test their applicability for the orthotopic xenograft model. RESULTS In 16/18 animals (take rate 89%) all methods enabled to adequately visualize orthotopic tumors and to display their growth over time. While the tumors had a homogenously radiolucent signal in CE-CT, 3D-US and MRI could better visualize intratumoral structures and surrounding soft tissue. CT had the best spatial resolution, followed by 3D-US and MRI. Median examination time was 4.8min per 3D-US, 12.5min per CT and 37.9min per MRI, respectively. Of interest, tumor volumes determined by CE-CT and 3D-US showed strong correlation with each other (n=85, R=0.985) as well as with caliper measurements at autopsy (CT: n=16. R=0.922; 3D-US: n=16, R=0.934). Similarly, tumor volumes measured with T2w MRI correlated well with those determined by CT, 3D-US and caliper. No side effects due to radiation exposure were seen. Mice inoculated with KTCTL30 cells developed partly cystic, partly solid tumors. 786-O cells yielded purely solid, fast growing tumors. CONCLUSIONS All three imaging modalities are feasible tools for a non-invasive monitoring of orthotopic tumor growth and show excellent correlation with each other. While 3D-US allows for a fast analysis of tumor volume with excellent resolution, MRI provides the best soft tissue contrast and can give additional information about tumor biology (e.g. diffusion, perfusion). CT has the best spatial resolution and enables simultaneous screening for bone and lung metastases but relies on the use of contrast agent and ionizing radiation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1169 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Johannes Linxweiler More articles by this author Christina Körbel More articles by this author Andreas Müller More articles by this author Volker Jung More articles by this author Eva Jüngel More articles by this author Stefan Siemer More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Michael D. Menger More articles by this author Matthias Saar More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP92-20 DIFFERENTIATION BETWEEN NORMAL AND CANCEROUS RENAL TISSUES USING ELECTRICAL IMPEDANCE SPECTROSCOPY NEEDLE.

INTRODUCTION AND OBJECTIVES: Orthotopic xenografts are increasingly used preclinical in-vivo models for the study of renal cell carcinoma (RCC). However, monitoring of these tumors requires sophisticated imaging techniques. Herein we comparatively evaluated modern small animal imaging tools like high-resolution 3D-ultrasonography (3D-US), contrast-enhanced in-vivo micro-CT (CE-CT) and 9,4T MRI (MRI) to monitor tumor growth in an orthotopic RCC xenograft model. METHODS: 10 CAKI-1 cells were injected under the renal capsule of 18 Balb/c-nude mice. Every 14 days from week 4 imaging was performed by CE-CT and 3D-US. 10 weeks after cell inoculation, all animals additionally underwent MRI. At autopsy, tumor volumes were determined with a caliper and compared to in-vivo imaging results. CE-CT, MRI and 3D-US were evaluated regarding tumor detection, tumor volume analysis, radiographic imaging properties and examination time. Finally, RCC cell lines KTCTL30 (n1⁄42) and 786-0 (n1⁄41) were used to test their applicability for the orthotopic xenograft model. RESULTS: In 16/18 animals (take rate 89%) all methods enabled to adequately visualize orthotopic tumors and to display their growth over time. While the tumors had a homogenously radiolucent signal in CE-CT, 3D-US and MRI could better visualize intratumoral structures and surrounding soft tissue. CT had the best spatial resolution, followed by 3D-US and MRI. Median examination time was 4.8min per 3D-US, 12.5min per CT and 37.9min per MRI, respectively. Of interest, tumor volumes determined by CE-CT and 3D-US showed strong correlation with each other (n1⁄485, R1⁄40.985) as well as with caliper measurements at autopsy (CT: n1⁄416. R1⁄40.922; 3D-US: n1⁄416, R1⁄40.934). Similarly, tumor volumes measured with T2w MRI correlated well with those determined by CT, 3D-US and caliper. No side effects due to radiation exposure were seen. Mice inoculated with KTCTL30 cells developed partly cystic, partly solid tumors. 786-O cells yielded purely solid, fast growing tumors. CONCLUSIONS: All three imaging modalities are feasible tools for a non-invasive monitoring of orthotopic tumor growth and show excellent correlation with each other. While 3D-US allows for a fast analysis of tumor volume with excellent resolution, MRI provides the best soft tissue contrast and can give additional information about tumor biology (e.g. diffusion, perfusion). CT has the best spatial resolution and enables simultaneous screening for bone and lung metastases but relies on the use of contrast agent and ionizing radiation.

MP92-07 NETRIN-1: A NOVEL PROTEIN IDENTIFIED IN DISEASE PROGRESSION OF SUNITINIB RESISTANT RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-07 NETRIN-1: A NOVEL PROTEIN IDENTIFIED IN DISEASE PROGRESSION OF SUNITINIB RESISTANT RENAL CELL CARCINOMA Sebastian Frees, Claudia Chavez-Munoz, Betty Zhou, Alexander Wong, Peter Raven, and Alan So Sebastian FreesSebastian Frees More articles by this author , Claudia Chavez-MunozClaudia Chavez-Munoz More articles by this author , Betty ZhouBetty Zhou More articles by this author , Alexander WongAlexander Wong More articles by this author , Peter RavenPeter Raven More articles by this author , and Alan SoAlan So More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2640AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America with an incidence that has been steadily increasing at a rate of 3% yearly over the last three decades. Patients with ccRCC can be treated with surgery; however, 30% of first-time diagnosed patients present with metastatic disease. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies target the formation of new vessels allowing nutrient depravation and cell death. A family of drugs that uses this mechanism is called Tyrosine kinase inhibitors (TKI), and one of the members of this family is Sunitinib. Sunitinib is the first-line of therapy currently used in patients with ccRCC. However, recent studies have shown that after approximately 1 year of treatment patients develop resistance, showing disease progression while on therapy. Therefore in this study we propose to identify the protein(s) responsible for increase migration and invasion with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease. METHODS Human renal cancer cell lines were used (CAKI-1, CAKI-2, ACHN) and treated with Sunitinib at increasing doses to develop a Sunitinib conditioned renal cell carcinoma cell line. A mRNA microarray was performed to compare the differences in gene expression between CAKI-1 Sunitinib conditioned cell line and CAKI-1 non-conditioned cell line. Moreover, we have successfully established an orthotopic renal cell carcinoma Sunitinib resistant animal model. Western blots and q-PCR were used to confirm microarray results and to evaluate the expression of Netrin-1 in-vitro. XCELLigence system was used to evaluate migration and invasion of CAKI-1 Sunitinib conditioned cell lines. Immunostaining of our in vivo Sunitinib resistant model confirms the in vitro findings. RESULTS Human renal cell carcinoma Sunitinib conditioned cell lines showed a highly upregulated expression of Netrin-1 in the microarray results as well as in Western blotting and q-PCR when compared to the un-treated renal cell carcinoma cell lines. XCELLigence system demonstrated an increased migration in CAKI-1 Sunitinib conditioned cell lines when compared to the non-treated ones as well as, increased endothelial cell migration when co-cultured with CAKI-1 Sunitinib conditioned cell line. Our results also demonstrated an increase expression of UNC5B and DCC Netrin-1 receptors in CAKI-1 Sunitinib conditioned cell line. Silencing of Netrin-1 in CAKI-1 Sunitinib conditioned cell line demonstrated a significant reduction in cell migration and invasion. Interestingly, our orthotopic renal cell carcinoma Sunitinib resistance animal model as well as human renal cell carcinoma Sunitinib resistant tumors confirmed a high expression of Netrin-1 when compared to those of control. CONCLUSIONS In conclusion we have found that Netrin-1 plays a key role in the migration and progression of renal cell carcinoma. Development of therapies targeting Netrin-1 suggests to be a promising approach to suppress renal cell carcinoma tumor progression and potentially prolong patients' survival. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1164 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Sebastian Frees More articles by this author Claudia Chavez-Munoz More articles by this author Betty Zhou More articles by this author Alexander Wong More articles by this author Peter Raven More articles by this author Alan So More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression.