Compound C Inhibits Renca Renal Epithelial Carcinoma Growth in Syngeneic Mouse Models by Blocking Cell Cycle Progression, Adhesion and Invasion.

Compound C (CompC), an inhibitor of AMP-activated protein kinase, reduces the viability of various renal carcinoma cells. The molecular mechanism underlying anti-proliferative effect was investigated by flow cytometry and western blot analysis in Renca cells. Its effect on the growth of Renca xenografts was also examined in a syngeneic BALB/c mouse model. Subsequent results demonstrated that CompC reduced platelet-derived growth factor receptor signaling pathways and increased ERK1/2 activation as well as reactive oxygen species (ROS) production. CompC also increased the level of active Wee1 tyrosine kinase (P-Ser642-Wee1) and the inactive form of Cdk1 (P-Tyr15-Cdk1) while reducing the level of active histone H3 (P-Ser10-H3). ROS-dependent ERK1/2 activation and sequential alterations in Wee1, Cdk1, and histone H3 might be responsible for the CompC-induced G2/M cell cycle arrest and cell viability reduction. In addition, CompC reduced the adhesion, migration, and invasion of Renca cells in the in vitro cell systems, and growth of Renca xenografts in the BALB/c mouse model. Taken together, the inhibition of in vivo tumor growth by CompC may be attributed to the blockage of cell cycle progression, adhesion, migration, and invasion of tumor cells. These findings suggest the therapeutic potential of CompC against tumor development and progression.

Investigating Changes Driving Cumulative Impacts on Native Vegetation in Mining Regions in the Northeastern Brazilian Amazon.

Developing conservation strategies to mitigate cumulative impacts requires the understanding of historic land use and land cover changes at the regional scale. By using a multisensory and multitemporal approach, we identified the major changes driving cumulative impacts on native vegetation in northeastern Amazon. Comparing two regions, one with mining as the key driver and another where mining is associated with other industrial activities (cellulose), we explore the land use and land cover historic dynamics and derive implications for the assessment of cumulative impacts. Transitions of forest cover to pastureland, silviculture, and urban expansion were mapped in detail over a 20-year period, revealing that silviculture growth cleared more forests than pastureland expansion when associated with pulp mill activities and kaolin mining. In contrast, in a region with gold and iron mining, pastureland expansion was more relevant, clearing mainly areas surrounding new roads. This research shows that the interplay of major mining and industrial investments can produce cumulative losses of native vegetation, depending on the associated industries and infrastructure required for the project development. Our findings emphasize that the definition of spatial and temporal boundaries for the assessment of cumulative impacts must consider different trends in impact accumulation and changes in their spatial distribution over time.

Intratumoral submicron particle docetaxel inhibits syngeneic Renca renal cancer growth and increases CD4+, CD8+, and Treg levels in peripheral blood.

Administration of chemotherapeutics as direct injections into tumors offers increased anti-tumor activity and reduced systemic toxicity. In this study, the Renca syngeneic murine xenograft model of renal cancer was used to evaluate the effects of intratumoral (IT) submicron particle docetaxel (NanoDoce®) on tumor growth and immunomodulation. Tumor volume (TV) was compared to controls, including intravenous (IV) chemotherapy. Flow cytometry of peripheral bloods and tumors was used to evaluate immune cell populations. Groups of animals were inoculated with a second Renca tumor at a site distant from the primary tumor. IT NanoDoce significantly reduced primary TV and reduced the growth rates of untreated secondary tumors. CD4+, CD8+ and Treg populations were increased in peripheral bloods from animals administered IT NanoDoce. Additional evaluation of the effect of IT NanoDoce on peripheral and local immune cell populations as well as the impact on sites of distant tumor growth are warranted.

Clinical laboratory and imaging evidence for effectiveness of agarose-agarose macrobeads containing stem-like cells derived from a mouse renal adenocarcinoma cell population (RMBs) in treatment-resistant, advanced metastatic colorectal cancer: Evaluation of a biological-systems approach to cancer therapy (U.S. FDA IND-BB 10091; NCT 02046174, NCT 01053013).

OBJECTIVE: The complexity, heterogeneity and capacity of malignant neoplastic cells and tumors for rapid change and evolution suggest that living-cell-based biological-systems approaches to cancer treatment are merited. Testing this hypothesis, the tumor marker, metabolic activity, and overall survival (OS) responses, to the use of one such system, implantable macrobeads [RENCA macrobeads (RMBs)], in phase I and IIa clinical trials in advanced, treatment-resistant metastatic colorectal cancer (mCRC) are described here. METHODS: Forty-eight mCRC patients (30 females; 18 males), who had failed all available, approved treatments, underwent RMB implantation (8 RMB/kg body weight) up to 4 times in phase I and phase IIa open-label trials. Physicals, labs [tumor and inflammation markers, lactate dehydrogenase (LDH)] and positron emission tomography-computed tomography (PET-CT) imaging to measure number/volume and metabolic activity of the tumors were performed pre- and 3-month-post-implantation to evaluate safety and initial efficacy (as defined by biological responses). PET-CT maximum standard uptake value (SUVmax) (baseline and d 90; SUVmax ≥2.5), LDH, and carcinoembryonic antigen (CEA) and/or cancer antigen 19-9 (CA 19-9) response (baseline, d 30 and/or d 60) were assessed and compared to OS. RESULTS: Responses after implantation were characterized by an at least 20% decrease in CEA and/or CA 19-9 in 75% of patients. Fluorodeoxyglucose (FDG)-positive lesions (phase I, 39; 2a, 82) were detected in 37/48 evaluable patients, with 35% stable volume and stable or decreased SUV (10) plus four with necrosis; 10, increased tumor volume, SUV. LDH levels remained stable and low in Responders (R) (d 0-60, 290.4-333.9), but increased steadily in Non-responders (NR) (d 0-60, 382.8-1,278.5) (d 60, P=0.050). Responders to RMBs, indicated by the changes in the above markers, correlated with OS (R mean OS=10.76 months; NR mean OS=4.9 months; P=0.0006). CONCLUSIONS: The correlations of the tumor marker, tumor volume and SUV changes on PET-CT, and LDH levels themselves, and with OS, support the concept of a biological response to RMB implantation and the validity of the biological-systems approach to mCRC. A phase III clinical trial is planned.

Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

Although aldosterone is a known regulator of renal and cardiovascular function, its role as a regulator of cancer growth and spread has not been widely considered. This study tested the hypothesis that aldosterone regulates cancer cell growth/spread via G protein-coupled estrogen receptor (GPER) activation. In vitro in murine renal cortical adenocarcinoma (RENCA) cells, a widely used murine in vitro model for the study of renal cell adenocarcinoma, aldosterone increased RENCA cell proliferation to a maximum of 125 ± 3% of control at a concentration of 10 nM, an effect blocked by the GPER antagonist G15 or by GPER knockdown using short interfering (sh) RNA techniques. Further, aldosterone increased RENCA cell migration to a maximum of 170 ± 20% of control at a concentration of 100 nM, an effect also blocked by G15 or by GPER down-regulation. In vivo, after orthotopic RENCA cell renal transplantation, pulmonary tumor spread was inhibited by pharmacologic blockade of aldosterone effects with spironolactone (percentage of lung occupied by metastasis: control = 68 ± 13, spironolactone = 26 ± 8, P < 0.05) or inhibition of aldosterone synthesis with a high dietary salt diet (percentage of lung: control = 44 ± 6, high salt = 12 ± 3, P < 0.05), without reducing primary tumor size. Additionally, adrenalectomy significantly reduced the extent of pulmonary tumor spread, whereas aldosterone infusion recovered pulmonary metastatic spread toward baseline levels. Finally, inhibition of GPER either with the GPER antagonist G15 or by GPER knockdown comparably inhibited RENCA cell pulmonary metastatic cancer spread. Taken together, these findings provide strong evidence for aldosterone serving a causal role in renal cell cancer regulation via its GPER receptor; thus, antagonism of GPER represents a potential new target for treatment to reduce metastatic spread.-Feldman, R. D., Ding, Q., Hussain, Y., Limbird, L. E., Pickering, J. G., Gros, R. Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

Therapeutic effects and underlying mechanism of poly (L-glutamic acid)-g-methoxy poly (ethylene glycol)/combretastatin A4/BLZ945 nanoparticles on Renca renal carcinoma.

Introduction: The prognosis of advanced renal carcinoma is not ideal, necessitating the exploration of novel treatment strategies. Poly(L-glutamic acid)-g-methoxy poly(ethylene glycol)/Combretastatin A4 (CA4)/BLZ945 nanoparticles (CB-NPs) possess the dual capability of CA4 (targeting blood vessels to induce tumor necrosis) and BLZ945 (inducing M2 macrophage apoptosis), thereby inhibiting tumor growth. Methods: Here, the therapeutic effects and underlying mechanism was explored by CCK-8 cytotoxicity experiment, transwell cell invasion and migration experiment, H&E, western blot analysis, immunohistochemistry, flow cytometry, and other techniques. Results: These results demonstrated that CB-NPs could inhibit the growth of Renca cells and subcutaneous tumors in mice, with an impressive tumor inhibition rate of 88.0%. Results suggested that CB-NPs can induce necrosis in renal carcinoma cells and tissues, downregulate VEGFA expression, promote renal carcinoma cell apoptosis, and reduce the polarization of M2 macrophages. Discussion: These findings offer innovative perspectives for the treatment of advanced renal carcinoma.

Development of a New Focal Mouse Model of Bone Metastasis in Renal Cell Carcinoma.

BACKGROUND/AIM: Developing animal models of bone metastasis in renal cell carcinoma (RCC) is challenging as immunodeficient mice are required. The aim of this study was to develop a simple immune model of RCC bone metastasis. MATERIALS AND METHODS: RENCA tumor cells were injected into the right femurs of BALB/c mice. Sixty mice were grouped into each twenty-mouse group according to the tumor cell concentration, and the presence or absence and extent of bone metastasis in the total length of the femur were compared using hematoxylin and eosin staining of the excised tissues. RESULTS: Bone metastasis was significantly higher in the high concentration group than in the other groups (p<0.05), with 10 mice developing bone metastasis at two weeks and nine mice developing bone metastasis at three weeks. The extent of bone metastasis was significantly greater in the high concentration group than in the other groups (p<0.05). Multiple logistic regression analysis was performed to examine the factors influencing bone metastasis, and only the high concentration was a significant factor (p<0.05). CONCLUSION: We developed a normal immunity mouse model of local bone metastasis from RCC. This model could prove valuable for research into the treatment of bone metastases in RCC.

Localized PD-1 Blockade in a Mouse Model of Renal Cell Carcinoma.

Herein we report the impact of localized delivery of an anti-mouse PD-1-specific monoclonal antibody (aPD1) on Renca tumors in the resulting T cell responses and changes in broader immune gene expression profiles. Renca is a BALB/c mice syngeneic tumor that has been used to model human renal cell carcinoma In this study, T cell subsets were examined in tumors and draining lymph nodes of mice treated with localized PD-1 with and without the addition of adenosine deaminase (ADA), an enzyme that catabolizes adenosine (ADO), identified as an immune checkpoint in several types of human cancers. The biologics, aPD1, or aPD1 with adenosine deaminase (aPD1/ADA), were formulated with the self-assembling peptides Z15_EAK to enhance retention near the tumor inoculation site. We found that both aPD1 and aPD1/ADA skewed the local immune milieu towards an immune stimulatory phenotype by reducing Tregs, increasing CD8 T cell infiltration, and upregulating IFNÉ£. Analysis of tumor specimens using bulk RNA-Seq confirmed the impact of the localized aPD1 treatment and revealed differential gene expressions elicited by the loco-regional treatment. The effects of ADA and Z15_EAK were limited to tumor growth delay and lymph node enlargement. These results support the notion of expanding the use of locoregional PD-1 blockade in solid tumors.

Animal models of spontaneous renal carcinoma: A possible rebirth?

Introduction and Objectives The animal models for renal carcinogenesis have allowed researchers to better understand the physiopathological, genetic, molecular, and immunological mechanisms underlying the development of human renal cancers from the induction of precancerous lesions to the metastatic process. Our study aimed to review and discuss the characteristics of all the spontaneous animal models of renal carcinogenesis described in scientific literature to date and see how they could address future challenges in the field of personalized medicine. Methods and Materials We reviewed all the papers on PubMed which focus on spontaneous animal models of renal carcinogenesis. To do so, we used the keywords "spontaneous" AND "animal model" AND "renal cancer" in the PubMed search engine. We also conducted a search using the keywords "spontaneous" AND "animal model" AND "kidney cancer." PRISMA recommendations were used for the literature review. Results A total of 213 publications were found on PubMed with the keywords "spontaneous" AND "animal model" AND "kidney cancer." 219 publications were found on PubMed with the keywords "spontaneous" AND "animal model" AND "renal cancer." After pooling these 2 searches and removing the duplicate publications, 233 publications remained. Among these, 220 full-text articles were assessed for eligibility; 160 were removed because they were irrelevant to our topic, and the remaining 60 studies were included in our qualitative synthesis. Conclusions Many spontaneous animal models have been developed to study renal cancer. So far, these models have enabled the understanding of pathophysiological mechanisms underlying renal cancer. Though less appropriate than patient xenografts from the perspective of personalized medicine, we believe animal models can help medical professionals better understand the hows and whys of the genetic events underlying the intratumoral heterogeneity and spatial distribution of metastatic subclones.

Inhibition of Tumor Growth and Sensitization to Sunitinib by RNA Interference Targeting Programmed Death-ligand 1 in Mouse Renal Cell Carcinoma RenCa Model.

BACKGROUND/AIM: There are several unresolved issues regarding the combined treatment with an immune checkpoint inhibitor and anti-angiogenic agent for renal cell carcinoma (RCC) patients. The purpose of this study was to address the inhibitory effects of programmed death-ligand 1 (PD-L1) expression on growth and sensitivity to sunitinib in the mouse RCC RenCa model. MATERIALS AND METHODS: We established RenCa/sh-PD-L1 by transfecting RenCa cells with a plasmid carrying a short hairpin RNA targeted against PD-L1. The growth pattern of RenCa/sh-PD-L1 with or without sunitinib was compared to that of RenCa cells transfected with control plasmid alone (RenCa/Co). RESULTS: No significant difference in growth or sensitivity to sinitinib was noted between RenCa/sh-PD-L1 and RenCa/Co cells in vitro. The tumor volume in mice subcutaneously injected with RenCa/sh-PD-L1 was significantly smaller than that with RenCa/Co. Treatment of mice bearing each tumor with sunitinib resulted in a significant reduction of the RenCa/sh-PD-L1 tumor compared to the RenCa/Co tumor. Moreover, infiltration by CD8+ T cells of RenCa/sh-PD-L1 tumors was significantly higher than that of RenCa/Co tumors, irrespective of treatment with sunitinib. CONCLUSION: Suppressed expression of PD-L1 could increase tumor-infiltrating CD8+ T cells and result in growth inhibition as well as enhanced sensitivity to sunitinib in the RenCa model.

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

PURPOSE: 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. METHODS: 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). RESULTS: Intensity of luciferase signals from Renca-luc was positively correlated with cell number. Bioluminescence signal was detected 1 day after the establishment of the renal carcinoma model using Renca-luc and was significantly increased after 7 days. Tumour size at 7 days 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. CONCLUSION: Luciferase labelling could provide a sensitive and non-invasive evaluation method for immunological and tumour therapy of renal carcinomas.

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).

Neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis.

Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to progressive recurrences.

Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors.

In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease. To that purpose we inoculated renal tumors orthotopically, intra-kidney (IK), in mice. Treg depletions were performed using anti-CD4 antibody in wild type mice or diphtheria toxin (DT) in Foxp3DTR transgenic mice. Our main observation was that Treg were not the key immunosuppressive component of the IK tumoral microenvironment, compared to the same tumors located SC. We demonstrated that the CD8+ effector immune response was still suppressed in IK tumors when compared to SC tumors, following Treg depletion. Furthermore, the level of program cell death protein (PD)-1 was increased on the surface of CD4+ T cells infiltrating IK tumors compared to SC tumors. Finally, the Treg-independent immunosuppression, occurring in IK tumors, was potent enough to inhibit regression of concomitant SC tumors, normally responsive to Treg depletion. Our findings provide further insight into the immunosuppressive nature of the immune response generated in the kidney microenvironment, suggesting that it can have additional mechanisms in addition to Treg. These observations might help to identify better targets from the kidney tumor microenvironment for future cancer therapies.

Treatment of agarose-agarose RENCA macrobeads with docetaxel selects for OCT4(+) cells with tumor-initiating capability.

The cancer stem cell (CSC) theory depicts such cells as having the capacity to produce both identical CSCs (symmetrical division) and tumor-amplifying daughter cells (asymmetric division). CSCs are thought to reside in niches similar to those of normal stem cells as described for neural, intestinal, and epidermal tissue, are resistant to chemotherapy, and are responsible for tumor recurrence. We recently described the niche-like nature of mouse renal adenocarcinoma (RENCA) cells following encapsulation in agarose macrobeads. In this paper we tested the hypothesis that encapsulated RENCA colonies function as an in vitro model of a CSC niche and that the majority of cells would undergo chemotherapy-induced death, followed by tumor recurrence. After exposure to docetaxel (5 µg/ml), 50% of cells were lost one week post-treatment while only one or two cells remained in each colony by 6 weeks. Surviving cells expressed OCT4 and reformed tumors at 16 weeks post-treatment. Docetaxel-resistant cells also grew as monolayers in cell culture (16-17 weeks post-exposure) or as primary tumors following transplantation to Balb/c mice (6 of 10 mice) or NOD.CB17-Prkdc(scid)/J mice (9 of 9 mice; 10 weeks post-transplantation or 28 weeks post-exposure). These data support the hypothesis that a rare subpopulation of OCT4(+) cells are resistant to docetaxel and these cells are sufficient for tumor recurrence. The reported methodology can be used to obtain purified populations of tumor-initiating cells, to screen for anti-tumor-initiating cell agents, and to investigate the in vitro correlate of a CSC niche, especially as it relates to chemo-resistance and tumor recurrence.