Renca Cells Research Articles

CD8+ T�cells stimulated by exosomes derived from RenCa cells mediate specific immune responses through the FasL/Fas signaling pathway and, combined with GM‑CSF and IL‑12, enhance the anti‑renal cortical adenocarcinoma effect

A satisfactory cure rate for renal cell carcinoma (RCC) is difficult to achieve through traditional immunotherapy. RCC has a relatively high spontaneous regression rate due to tumor immune escape. However, tumor‑derived exosomes (TEXs), which effectively carry tumor‑associated antigens (TAAs) and trigger stronger antigen‑specific tumor immunity against autologous tumors than against other tumors, have been widely viewed as attractive potential vaccines for tumor treatment, although improvements are needed. Therefore, in our study, we determined whether RenCa cell‑derived exosome (RDE)‑stimulated CD8+ T cells exert a stronger specific cytotoxic effect on autologous tumor cells than on other types of tumor cells through the Fas ligand (FasL)/Fas signaling pathway, and whether the combination of RDE‑stimulated CD8+ T cells with GM‑CSF and IL‑12 enhances the anticancer effect. The results showed that RDEs were isolated, as expected, and promoted an increased percentage of CD8+/CD4+ T cells. RDE‑stimulated CD8+ T cells also more effectively facilitated cytotoxicity against RenCa cells when combined with GM‑CSF and IL‑12 in vitro. Furthermore, immunization with RDEs restrained the growth of RenCa tumors in mouse models, and facilitated the stimulation of a stronger specific cytotoxic CD8+ T cell response via the FasL/Fas signaling pathway in vitro. However, these results were observed less frequently for other types of tumor cells after treatment with RDEs, suggesting that RDEs depend on their antigen specificity to trigger antitumor immune responses. These findings revealed that RDE‑stimulated CD8+ T cells combined with GM‑CSF and IL‑12 can more effectively exert a stronger cytotoxic effect than RDEs alone and that RDEs can induce immunization more effectively against renal cortical adenocarcinoma than against other types of cancer. Therefore, according to our study, exosomes are promising potential vaccines, and the combination of exosome‑stimulated CD8+ T cells with GM‑CSF and IL‑12 may be a novel strategy for the treatment of RCC.

Preclinical development of combination therapy targeting the dominant cytokine interleukin-1β for renal cell carcinoma.

e14237 Background: Targeting myeloid-derived suppressor cells (MDSCs), key mediators of intrinsic and adaptive immune resistance, remains a significant challenge in kidney cancer immunotherapy. One potential driver of MDSC mediated tumor immunosuppression is the cytokine interleukin-1 beta (IL-1β). Although large cancer prevention studies show that targeting IL1 decreases cancer risk, the exact mechanism of anti-IL-1 mediated anti-tumor efficacy is not yet defined. To understand the immunogenic effects of IL-1β blockade and effects on MDSCs, we used the RENCA model of RCC. Methods: We inoculated BALB/c mice with 1x106 RENCA cells. On Day +10 we treated mice (n = 8/group) with vehicle, αPD1, αIL1β, cabozantinib, combination αPD1/αIL1β, or cabozantinib/αIL1β. We harvested day +18 tumors to quantify tumor size and immune cells by flow cytometry. We then isolated immune cells for differential gene expression profiling by RNAseq. Results: Treatment with αIL1β significantly decreased tumor size compared to vehicle or αPD1 monotherapy. Combining αPD1/αIL1β or cabozantinib/αIL1β potentiated the anti-tumor effects of αPD1 or cabozantinib monotherapy respectively. Anti-IL1β therapy decreased polymorphonuclear (PMN)-MDSC infiltration (αIL1β: 0.76% +/- 0.21 vs. vehicle: 1.89% +/- 0.37, P = 0.014). Relative to PD1 monotherapy, combining αPD1 and αIL1β decreased PMN-MDSCs (αPD1: 2.14% +/- 0.56 vs αPD1/αIL1β: 0.91% +/- 0.15, P = 0.033) and increased M1-like tumor associated macrophages (TAM) (αPD1: 0.798% +/- 0.21 vs. αPD1/αIL1β: 12.04% +/- 2.9, P = 0.0008). Gene expression profiling from sorted immune cells from αIL1β treated tumors revealed multiple differentially regulated cytokines including interleukin-6, IFN-γ, and IL-10. Conclusions: Blockade of IL-1β depletes intratumoral PMN-MDSCs and increases M1-like TAM with minimal effects observed on the T cell compartment. Treatment with cabozantinib or αPD1 in combination with αIL1β led to sustained control of tumor growth. IL-1β represents a promising target for kidney cancer immunotherapy. Based on the preclinical data, a clinical trial of neoadjuvant αPD1 and αIL1β therapy is planned for patients with high risk RCC.

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors.

Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu+ RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo, in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo. Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts.

Abstract A184: T-cell rejuvenation is associated with vorinostat-induced immune response in combination with immune checkpoint blockade

Abstract Background: Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown clinical benefit in solid tumor patients, including renal cell carcinoma (RCC). However, the rate of clinical response remains modest. Growing evidence suggests that epigenetic modifying agents may have an immunomodulatory role. Our group has previously demonstrated that the selective class I histone deacetylase (HDAC) inhibitor entinostat decreases the function of regulatory T-cells (Treg) and myeloid-derived suppressor cells (MDSC), and synergizes with PD-1 blockade. In this study, we assessed the immunomodulatory activity and efficacy of combining PD-1 blockade with the pan-HDAC inhibitor vorinostat in a RCC model. Methods: To test the efficacy of a combination therapy with a PD-1 inhibitor, mDX-400 (10 and 20 mg/kg I.P) (Merck & Co., Inc.) and a pan-HDAC inhibitor, vorinostat (100 and 150 mg/kg I.P) (Merck & Co., Inc.), we utilized a syngeneic mouse model of metastatic RCC following orthotopic implantation of luciferase expressing RENCA cells in immunocompetent BALB/c mice. Antitumor activity was assessed by bioluminescence technique as well as end point measurements of tumor weights. Immune landscape profiling of tumor infiltrating lymphocytes (TILs) was performed by flow cytometry, immunohistochemistry, and immunofluorescence. Survival analysis was performed by Kaplan–Meier estimates and log-rank statistic. Differences in chromatin accessibility in peripheral blood mononuclear cells (PBMC) were assessed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Results: Statistically significant reductions in end point tumor weights, as well as lung metastases nodules, were observed in mice treated with the combination of vorinostat (100 mg/kg P=0.0391; 150 mg/kg P=0.0165) and mDX-400 (20 mg/kg) compared to vehicle, while no statistical significant reduction was observed in those treated with single-agent mDX-400. Combination therapy also significantly lengthened the survival of the mice (median survival = 60 days; P=0.009) compared to treatment with the single agent mDX-400 (median survival=42 days). Immune landscape profiling did not demonstrate a significant increase in CD8+ tumor infiltration (P=0.479), but a statistically significant increase in natural killer cell infiltration (P=0.048) was observed. Though the CD8+ tumor infiltration was unchanged, a significant reduction (P=0.049) of exhausted CD8+ T-cells (CD8+PD1+) was observed in the combination treatment compared to mDX400 alone. Furthermore, a decrease was observed in the immunosuppressive Tregs (CD4+FOXP4+) and MDSC (CD11b+Gr1+) in the combination group compared to mDX400 alone. Bioinformatic analyses of ATAC-seq data from the PBMC cells of mice in the combination treatment and mDX400 alone showed increased chromatin accessibility between the two conditions. Pathway analysis of genes associated with more accessible chromatin in the combination treatment than mDX400 treatment identified enrichment of cell cycle control and immune cell activation pathways. Conclusions: Our results demonstrate that the pan-HDAC inhibitor vorinostat augments the antitumor effect of immune checkpoint inhibitor mDX-400 and prolongs survival in the RENCA model. This combination advantage was achieved by changing the immune landscape in TILs, especially by decreasing the exhausted subset of T-cells. The combination of these drugs is associated with higher chromatin accessibility near genes involved in cell cycle progression and immune cell activation. Taken together, our results support the clinical testing of pan-HDAC inhibitors in combination of PD-1 inhibitors and provide a novel potential immunomodulatory effect of epigenetic drugs. Citation Format: Nur P. Damayanti, Justin A. Budka, Josue D. Ordaz, Ashley Orillion, Khunsha Ahmed, Xioana Chu, Yue Wang, Yunlong Liu, Roberto Pili. T-cell rejuvenation is associated with vorinostat-induced immune response in combination with immune checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A184.

Dual-Inhibition of mTOR and Bcl-2 Enhances the Anti-tumor Effect of Everolimus against Renal Cell Carcinoma In Vitro and In Vivo.

Renal cell carcinoma (RCC) is the predominant type of kidney cancer. Mammalian target of rapamycin (mTOR) inhibitor everolimus is currently used as a second-line therapy for sorafenib or sunitinib-refractory metastatic RCC patients. The clinical limitation confronted during everolimus therapy is the onset of drug resistance that decreases the efficacy of the drug. Elevated level of anti-apoptotic Bcl-2 protein is proposed to be an emerging feedback loop for the acquired drug-resistance in various cancer types. In this study, the Bcl-2 inhibitor ABT-737 was used in combination with everolimus to enhance its anti-tumor effectiveness in everolimus-resistant RCC cell lines. Everolimus and ABT-737 combination synergistically led to a decrease in the proliferation of primary site A-498 and metastatic site Caki-1 RCC cell lines, which was accompanied by a reduction in protein levels of cell cycle and mTOR pathway proteins. In both RCC cell lines, everolimus-ABT-737 combination not only induced apoptosis, caspase and PARP-1 cleavage but also a decrease in Bcl-2 protein levels in parallel with a concomitant increase in Bim and Noxa levels.In order to confirm our in vitro findings, we have generated everolimus-resistant RenCa cell line (RenCares) to establish a RCC mouse xenograft model. Animals co-treated with everolimus and ABT-737 exhibited a complete suppression of tumor growth without any notable toxicity. This study thus proposes the everolimus-ABT-737 combination as a novel therapeutic strategy for the treatment of RCC to overcome the current clinical problem of everolimus resistance.

Combination immunotherapy with interleukin-2 surface-modified tumor cell vaccine and programmed death receptor-1 blockade against renal cell carcinoma.

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4+, CD8+ and CD8+interferon‐γ+ but not CD4+Foxp3+ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.

MEF2 plays a significant role in the tumor inhibitory mechanism of encapsulated RENCA cells via EGF receptor signaling in target tumor cells

BackgroundAgarose encapsulated murine renal adenocarcinoma cells (RENCA macrobeads) are currently being investigated in clinical trials as a treatment for therapy-resistant metastatic colorectal cancer. We have previously demonstrated the capacity of RENCA macrobeads to produce diffusible substances that markedly inhibit the proliferation of epithelial-derived tumor cells outside the macrobead environment. This study examined the molecular mechanisms underlying the observed inhibition in targeted tumor cells exposed to RENCA macrobeads.MethodsWe evaluated changes in transcription factor responses, participating intracellular signaling pathways and the involvement of specific cellular receptors in targeted tumor cells exposed to RENCA macrobeads.ResultsFactors secreted by RENCA macrobeads significantly up-regulated the activity of the MEF2 transcription factor as well as altered the transcription of MEF2b and MEF2d isoforms in targeted tumor cells. Suppression of individual or multiple MEF2 isoforms in target tumor cells markedly reduced the growth inhibitory effects of RENCA macrobeads. Furthermore, these effects were linked to the activation of the EGF receptor as attenuation of EGFR resulted in a substantial reduction of the cancer cell growth-inhibitory effect.ConclusionsSince interruption of the EGFR signaling cascade did not eliminate RENCA macrobead-induced growth control, our data suggests that RENCA macrobeads exert their full growth inhibitory effects through the simultaneous activation of multiple signaling pathways. In contrast to a precision medicine approach targeting single molecular abnormalities, the RENCA macrobead functions as a biological-systems therapy to re-establish regulation in a highly dysfunctional and dysregulated cancer system.

Abstract 719: A tumor mitochondria vaccine protects against experimental renal cell carcinoma

Abstract Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer. Citation Format: Stefano Pierini, Andrea Facciabene, Mireia Uribe-Herranz, Stavros Rafail, Chongyun Fang, Janos Tanyi. A tumor mitochondria vaccine protects against experimental renal cell carcinoma [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 719.

Knockdown of NF-κB1 by shRNA Inhibits the Growth of Renal Cell Carcinoma In Vitro and In Vivo.

Renal cell carcinoma (RCC) accounts for approximately 2%–3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance, and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-κB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-κB in RCC, and many have implicated NF-κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse RCC (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-κB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNA-NF-κB1 tumors. Thus, this study indicates that downregulation of NF-κB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-κB1 may be a potential therapeutic target for RCC.

A Non-integrating Lentiviral Approach Overcomes Cas9-Induced Immune Rejection to Establish an Immunocompetent Metastatic Renal Cancer Model

The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (VHL) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).

Function of miR-146a-5p in Tumor Cells As a Regulatory Switch between Cell Death and Angiogenesis: Macrophage Therapy Revisited.

Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME) that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS), miR-146a-5p inhibits nitric oxide (NO) production in a mouse renal carcinoma cell line (RENCA), thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p’s activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001). Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001), leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05), and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001) and angiogenesis (twofolds, p < 0.01), and increased apoptosis (twofolds, p < 0.01). This combination therapy increased nitrites and reduced TGFβ concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage therapy approach to induce the immune system to successfully attack the tumor, and should be further explored as a new therapy for the treatment of cancer.

Abstract 123: Enhanced anti-tumor activity of the combination of entinostat and NKTR214 in renal and colon cancer tumor models

Abstract Background: Combination strategies are required to improve outcomes for immune mediated anti-tumor therapies. Entinostat is a class 1 selective histone deacetylase inhibitor shown in preclinical studies to target immune suppressive mechanisms in the tumor microenvironment to improve the efficacy of immune checkpoint blockade, vaccines and cytokines. NKTR-214 is a novel CD122-biased agonist with promising safety, tolerability and anti-tumor efficacy. A previous phase 2 clinical trial of entinostat combined with high dose interleukin 2 demonstrated enhanced clinical efficacy and a well-tolerated safety profile in advanced renal cell cancer patients. Based on these data and potential for combinatorial anti-tumor activity, we investigated the antitumor efficacy and immune response of entinostat combined with NKTR-214 in CT26 colon cancer and RENCA renal cell cancer syngeneic tumor models. Methods: CT26 and RENCA cells were subcutaneously inoculated into 6-8-week-old BALB/c mice (n=12 per arm) and when tumors reached ~140mm3 were randomized and treated with entinostat (p.o., 1 mg/kg or 5 mg/kg, QD x 28), NKTR-214 (i.v., 0.8mg/kg, Q9D x 4), the combination, or vehicle control. Phenotypic and functional analysis of tumor infiltrating cells in each group was conducted on tumors taken from 3 mice per arm, at day 7 post-treatment. Remaining mice (n=9 per arm) continued treatment until tumors reached 2,000mm3. Results: In the CT26 model, the combination of entinostat (1mg/kg and 5mg/kg) and NKTR-214 was tolerated and demonstrated prolonged survival with a medium survival time not reached for the combination groups (7 of 9 animals still alive) as compared to NKTR-214 (44 days; 3 of 9 alive), entinostat or controls (16 days; none living). In RENCA, the treatments were less well tolerated and tumor growth inhibition is reported only for the entinostat 1mg/kg dose level with that combination demonstrating 60% tumor growth inhibition (n=8), compared to 34% in NKTR-214 alone (n=8), or 27% in entinostat alone (n=8) arms. Analysis of tumor infiltrating immune and myeloid cells after treatment indicated no significant phenotypic changes in any of the cell populations studied. However, measurement of IFNγ, TNFα and Granzyme B expression in T cell and NK cells revealed enhanced anti-tumor cytotoxicity. IFNγ was increased &amp;gt;15-fold in CT26 and &amp;gt;9-fold in RENCA treated with the entinostat (5mg/kg) and NKTR-214 combination along with increases in granzyme B (CT26 &amp;gt;17-fold; RENCA &amp;gt; 166-fold) and TNFα (CT26&amp;gt;3-fold; RENCA &amp;gt;2-fold). Conclusions: These data demonstrate that entinostat combined with NKTR-214 results in enhanced cytotoxic Teff function which translates to significant anti-tumor effects. These results provide support for clinical testing of the combination with or without additional immune checkpoint blockade. Citation Format: Lei Wang, Fei Chen, Tingting Li, Peter Ordentlich, Jonathan Zalevsky. Enhanced anti-tumor activity of the combination of entinostat and NKTR214 in renal and colon cancer tumor 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 123.

Anticancer activity of Schiff base-Poloxamer P85 combination against kidney cancer.

Renal cell carcinoma (RCC) accounts for approximately 80% of the primary renal cancers, and current treatment strategies are not sufficient to provide a certain solution. Since there are not many treatment options, interest in discovery of alternative drugs has increased. In the current study, anticancer activity of a novel heterodinuclear Cu(II)-Mn(II) complex (Schiff base-SB) in combination with poly(ethylene oxide) and poly(propylene oxide) block copolymer (pluronic) P85 was tested against RCC. Cell viability, apoptosis and gene expression analysis were conducted in vitro by using Renca cells. The results revealed that the SB-P85 combination decreased cell proliferation by increasing the apoptotic gene expressions and apoptosis. Renca-injected BALB/c mice were used to mimic early stage of RCC model. Treatment with SB-P85 combination suppressed tumor formation and growth compared to baseline. Overall, SB-P85 showed promising anticancer activity against RCC in vitro and in vivo.

Self-Assembled Polypeptide Nanogels with Enzymatically Transformable Surface as a Small Interfering RNA Delivery Platform.

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.

Establishment of a bioluminescent Renca cell line for renal carcinoma research.

Luciferase modification of tumour cells enables early and non-invasive imaging to detect tumour growth in situ and could provide sensitive and effective detection of carcinoma during research and therapy. Renca cells, a murine renal carcinoma cell line, were infected with lentivirus expressing luciferase to obtain Renca-luc. The proliferation, invasion, and migration of Renca and Renca-luc cell lines were compared using colorimetric, Boyden chamber, and wound-healing assays. Orthotopic tumour models were established in BALB/c mice using Renca and Renca-luc cells, and tumour growth in vivo was detected using bioluminescence imaging and magnetic resonance imaging (MRI). Intensity of luciferase signals from Renca-luc was positively correlated with cell number. Bioluminescence signal was detected 1day after the establishment of the renal carcinoma model using Renca-luc and was significantly increased after 7days. Tumour size at 7days following the establishment of renal carcinoma models using Renca and Renca-luc was determined using MRI. The presence of renal model tumours was confirmed by histological staining. The expression of luciferase did not affect Renca cell characteristics in vitro or tumour growth in vivo. Luciferase labelling could provide a sensitive and non-invasive evaluation method for immunological and tumour therapy of renal carcinomas.

Abstract 167: Ibrutinib regulates tumor microenvironment and enhances response to everolimus in renal cell carcinoma mouse models

Abstract Introduction: Ibrutinib (ibr), a first-in-class, once-daily, oral inhibitor of Bruton’s tyrosine kinase (BTK), is indicated for the treatment of patients with CLL/SLL, MCL and WM. Ibr also inhibits EGFR/HER2 and has demonstrated efficacy against EGFR+ NSCLC and HER2+ breast cancer in vitro and in xenograft models (Gao 2014; Chen 2016). Further, ibr modulated host immunity and enhanced anti-PD-L1 activity in solid tumor models otherwise insensitive to BTK or HER kinase inhibition (Sagiv-Barfi 2015), suggesting that ibr may be active in renal cell carcinoma (RCC) via multiple mechanisms. Here we determined the impact of ibr alone and in combination with everolimus (eve) on tumor growth and the tumor microenvironment in syngeneic and xenograft RCC mouse models. Methods: Cell proliferation was determined with CellTiter-Glo using a 3-day treatment regimen. Subcutaneous implantation of 786-0 and Renca cells established xenograft and syngeneic models in nude and immune-competent mice. Treatment started when tumor volume reached 150 mm3 (786-0) or 55 mm3 (Renca) and was measured twice weekly. Immunophenotying of tumor infiltrates was determined by flow cytometry. For Treg differentiation, magnetically-sorted CD4+CD25- mouse splenic T cells were stimulated with anti-CD3/CD28 antibodies and TGF-β1 for 5 days. Results: Ibr significantly inhibited the growth of syngeneic Renca tumors (TGI%=32.4 at Day 19, p≤0.05) in Balb/c mice and enhanced the efficacy of eve, although Renca cells were not sensitive to ibr in vitro. The combination significantly reduced the numbers of splenic and tumor Tregs in addition to reducing PD-1 expression on tumor CD8+ T cells. Consistently, in vitro Treg differentiation assays revealed that, while the combination of ibr and eve significantly reduced Treg differentiation, either drug alone showed little effect. In 786-0 xenograft model, ibr alone slowed tumor growth in a subgroup of mice and significantly enhanced the effect of eve (p≤0.05 or 0.01). To identify whether BTK or EGFR/HER2 is the target, CGI-1746, a BTK inhibitor, and lapatinib, an EGFR/HER2 inhibitor, were tested. Surprisingly, both CGI-1746 and lapatinib were active in inhibiting tumor growth (TGI%=26.7 or 37.6 respectively, p≤0.05 or 0.01), and slightly potentiated the efficacy of eve. However, consistent with ibr results, neither CGI-1746 nor lapatinib showed any effect on the cell growth of 786-0 cells. Conclusion: This study suggests that ibr has antitumor activity against RCC alone and when combined with eve in animal models. This effect may be mediated by modulation of the tumor microenvironment, such as inhibiting Treg differentiation and suppressing PD-1 expression on CD8+ T cells, and both BTK and EGFR/HER2 are involved. Further investigation is needed to clarify the mechanism of action, but the results here provide a rationale for ibr as a novel agent for RCC in combination with mTOR inhibitors. Citation Format: Jun Chen, Chun-Te Chen, Jing Liu, Jeff Hsu, Taisei Kinoshita, Betty Y. Chang. Ibrutinib regulates tumor microenvironment and enhances response to everolimus in renal cell carcinoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2017-167

Abstract 811: Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma

Abstract Introduction: The receptor tyrosine kinase (RTK) inhibitor sunitinib is the first line treatment for advanced clear cell renal cell carcinoma (ccRCC). Sunitinib inhibits angiogenesis via blocking signaling through VEGFR. About 80% of patients develop resistance after a drug-sensitive period. Molecular changes early in treatment may impact drug resistance, but are poorly understood. Experimental Procedures: ACHN, 786-O and Renca cell lines were treated with 1 µM sunitinib. NSG mice were s.c. xenografted with the model cell lines and were treated with sunitinib at 40 mg/kg/day dose. mRNA expression was screened using Illumina HT-12 bead chip array and miRNA expression was assessed by Nanostring nCounter assay. R statistical packages were used for data processing. Reactome and miRPath softwares were used for downstream analysis. Results: Sunitinib treatment of ccRCC xenografts led to several early changes in tumor histology, such as the emergence of live tumor areas within the necrotic spaces. These areas showed membranous staining for E-cadherin, and β-catenin, while the rest of the tumor and vehicle-treated tumors were negative. In vitro model cell lines developed cancer spheroids when treated with sunitinib. Cancer spheroids were highly tumorigenic and metastatic, and expressed several established cancer stem cell markers. ccRCC cancer spheres, but not the 2D adherent cells, showed membranous staining for E-cadherin and β-catenin; similarly to the live tumor areas observed in in vivo sunitinib treatment. In vitro inhibition of E-cadherin by EGTA or by siRNA, interfered with viability of sunitinib treated ccRCC cell lines. Conclusions: Sunitinib treatment causes early phenotypic changes of the tumor in vivo and in vitro. The formation of highly metastatic and tumorigenic cancer spheres in model cell lines is the most prominent effect in vitro. We provide preliminary evidence that sunitinib induced in vitro cancer spheres and the live tumor areas that survive within necrotic patches of the sunitinib-treated xenografts, are related. Finally, membranous expression of E-cadherin enhances the survival of ccRCC cell lines under sunitinib treatment. Citation Format: Zsuzsanna Lichner, Rola Saleeb, Henriett Butz, Roy Nofech-Mozes, Sara Riad, Mina Farag, Andras Kapus, George Yousef. Histological heterogeneity contributes to sunitinib resistance in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2017-811

Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model.

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.

Intermittent hypoxia increases kidney tumor vascularization in a murine model of sleep apnea.

We investigate the effects of intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea (OSA), on renal cancer progression in an animal and cell model. An in vivo mouse model (Balb/c, n = 50) of kidney cancer was used to assess the effect of IH on tumor growth, metastatic capacity, angiogenesis and tumor immune response. An in vitro model tested the effect of IH on RENCA cells, macrophages and endothelial cells. Tumor growth, metastatic capacity, circulating vascular endothelial growth factor (VEGF) and content of endothelial cells, tumor associated macrophages and their phenotype were assessed in the tumor. In vitro, VEGF cell expression was quantified.Although IH did not boost tumor growth, it significantly increased endothelial cells (p = 0.001) and circulating VEGF (p<0.001) in the in vivo model. Macrophages exposed to IH in vitro increased VEGF expression, whereas RENCA cells and endothelial cells did not. These findings are in keeping with previous clinical data suggesting that OSA has no effect on kidney cancer size and that the association observed between OSA and higher Fuhrman grade of renal cell carcinoma may be mediated though a proangiogenic process, with a key role of macrophages.

Therapy with transcutaneous administration of imiquimod combined with oral administration of sorafenib suppresses renal cell carcinoma growing in an orthotopic mouse model.

Imiquimod is an imidazoquinoline immune response modifier that is used in antiviral and antiallergic creams. Combination therapy using transcutaneous imiquimod and oral sorafenib was previously demonstrated to reduce the tumor burden of renal cell carcinoma growing cutaneously in a mouse model. In the present study, an orthotopic mouse model was used to investigate whether combined treatment with oral sorafenib and transcutaneous imiquimod inhibited renal cell carcinoma growing in the kidney. Kidneys of female BALB/c mice were orthotopically implanted with RENCA mouse kidney cancer cells, and the mice were transcutaneously treated with cream containing imiquimod and/or with orally administered sorafenib 5 days following cell implantation. Tumor burden and incidence were determined 28 days following the start of therapy. Splenocyte activity was quantified using the 51Cr release assay and the fluorescence-activated cell sorting assay with cluster of differentiation (CD) 4 and CD8 antibodies. Imiquimod, sorafenib and combination therapy were tolerated well. A combination of transcutaneous imiquimod and oral sorafenib inhibited the growth of RENCA tumors in the kidney significantly compared with the control. The 51Cr release assay demonstrated that transcutaneous imiquimod therapy significantly induced the release of 51Cr from RENCA cells compared with the control. The fluorescence-activated cell sorting assay demonstrated that transcutaneous imiquimod therapy induced CD8+ and CD4- splenocytes compared with the control. In summary, the results of the present study demonstrated that combined treatment with transcutaneous imiquimod and oral sorafenib may be a promising strategy for the treatment of patients with renal cell carcinoma.