Name: a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies
Uploaded: 2017-04-12T14:00:00
Description: Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

This work was supported by grants from the National Cancer Institute (R15CA173657 to A.W. and R01CA109446 to T.S.G.), the Simmons Cancer Institute (to A.W.), and the University of Minnestoa Climb 4 Kidney Cancer Foundation (to T.S.G.). We thank Dr. Kristin Anderson for assistance with the immunofluorescence.

The following protocol describes an experimental procedure for inducing and monitoring the growth of experimental orthotopic renal tumors and any potential spontaneous metastases. All of the following procedures are done in accordance with institutional policies and approved procedures regarding the humane use of experimental animals.
1. Maintenance of Cell Lines
<ol>
	<li>Maintain murine renal adenocarcinoma cell line, Renca, in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf...

<ol>
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We present a protocol for an orthotopic RCC mouse model. Implantation of mouse renal adenocarcinoma tumor cells into the mouse kidney provides a clinically-relevant model of mRCC. This model results in a primary tumor within the kidney and distant metastasis in the lungs, both of which are hallmarks of advanced RCC. The description presented herein is specific for the Renca cell line in immunocompetent Balb/c mice. Using this orthotopic model, we have shown that the Renca tumors are responsive to immunotherapy, demonstra...

Renal cell carcinoma (RCC) accounts for the majority of malignant kidney neoplasms and roughly 3% of all adult malignancies worldwide1,2. Due to lack of symptoms, misdiagnosis, and insufficient screening tools for RCC, almost 30% of patients will present with metastatic RCC (mRCC) at the time of diagnosis, with an additional 20-30% of patients progressing to a metastatic stage2. These cases result in ~13,500 deaths annually1,3. Though earlier detection and treatment of primary RCC has improved, the rate of RCC-related death continues to increase, suggesting that metastatic disease is largely responsible for mortality3. Therapy development for RCC has progressed over the last decade with the discovery and implementation of targeted therapies and immunotherapies. Unfortunately, progression-free survival and overall survival of advanced cases are still well under 2 years for the majority of patients4,5,6. These statistics warrant the need for further research into the identification and development of efficacious treatments for RCC. To adequately advance therapeutic interventions for mRCC, translationally-relevant pre-clinical models of the disease are first needed.
Development of a relevant and consistent model to recapitulate the human condition of advanced RCC should address several key questions: 1) Is the model anatomically relevant; 2) Does the tumor progress similarly to the human pathology; 3) Do metastases arise from the primary tumor; and 4) Can primary and metastatic tumor progression be monitored over time? Depending on the type of treatment being investigated, mouse RCC tumor cell lines, human RCC tumor cell lines, and human RCC patient-derived xenografts may be used in immunocompetent or immunodeficient mice. A host with an intact and functional immune system is required for those studies evaluating some aspect of immunotherapy, necessitating the use of the well-described Renca cell line derived from a spontaneous renal adenocarcinoma of Balb/c mice7. Most studies inject Renca cells subcutaneously (s.c.), which forms an easy-to-measure local tumor, or intravenously (i.v.) into Balb/c mice to produce experimental lung &#34;metastases&#34;8,9,10,11,12. Use of an s.c.-implanted Renca tumor to model human RCC has a number of limitations, including inaccurate innervation of vasculature13, differences in microenvironment14,15, and a lack of organ/tumor cellular communication13,16. Additionally, many s.c. tumors (especially Renca) do not metastasize to distal organs, inhibiting the study of an event that is a common clinical characteristic17. To study mRCC, Renca cells can be injected i.v. to establish tumor burden in the lungs-the primary location of metastases in RCC patients. The i.v. injection method of initiating metastatic tumors, however, does not allow the investigation of how, when, or why the cells have migrated from the primary organ (i.e., the kidney) to the distant site. A model with both primary and metastatic disease evident in the same animal is crucial to study the progression and treatment of advanced disease, especially considering that metastases are typically the cause of mortality in these patients.
Our lab has developed a murine model of mRCC that incorporates all of the features outlined above. To establish primary, orthotopic tumors, Renca cells are implanted directly into the kidney of the animal through the translucent peritoneum. A small incision in the left flank allows for visualization of the spleen (as a landmark) and the left kidney. Using a small-gauge needle, Renca tumor cells are injected directly into the kidney through the peritoneum for orthotopic implantation. Compared to other methods of implanting Renca cells below the kidney capsule18, this method of implantation allows for a higher throughput, as it is fairly non-invasive, does not require suturing, is of a low pain class, and is time-efficient when practiced.
This well-characterized model results in reproducible primary tumor burden (~ 99% take rate) in the injected kidney as well as metastatic tumor load in the lungs. The significant advantages of this model include its syngeneic nature, allowing for immunotherapy investigations; its spontaneous metastases, to study advanced disease; and its orthotopic implantation, to model the anatomical impact on disease progression and treatment. Therapies aimed at targeting RCC would greatly benefit from the utilization of this model during preclinical development.

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a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

The successful implantation of Renca cells will result in tumor development in the kidney and metastasis to the lungs of the mice. After the excision of the tumor-bearing and the contralateral kidneys, wet tissue weights (g) were measured to demonstrate the tumor burden based on increased weight (Figure 1). Renca tumors can be identified by immunohistochemistry through cytokeratin 8 and 18 staining (Figure 2). For longitudinal studies, a luciferase-expres...

Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

The overall goal of this procedure is to establish mouse orthotopic renal tumors through the intrarenal implantation of tumor cell for the study of tumor growth and metastasis and the efficacy of anti-tumor therapies. This method can be used to answer key questions in the urologic oncology field such as how effective is a novel therapy in a pre-clinical model of kidney cancer. The main advantage of this technique is that intrarenal tumor implantation establishes a primary lung metastasizing renal tumor mimicking clinical renal cell carcinoma pathogenesis.To prepare the Renca tumor cells first detach the cell culture with 0.25%Trypsin and HBSS after rinsing with PBS. Neutralize the reaction after five minutes with Complete RPMI Medium. Transfer the cells to a conical tube for centrifugation and re-suspend the pellet in fresh HBSS.After counting, adjust the volume to a two times ten to the six cells per milliliter concentration in HBSS, and draw the cells into a one milliliter syringe equipped with an 18 gauge needle. Next, place a sterile surgical drape over a heating pad and confirm the appropriate level of sedation by toe pinch. Remove the hair from the left flank of the mouse and apply vet ointment to the eyes.Then swab the implantation site with 5%povidone iodine antiseptic and use a sterile scalpel to make a single one to one and a half centimeter incision on the left flank, taking care not to penetrate the peritoneum. Using scissors, separate the dermis from the peritoneum, dabbing the incision site with sterile cotton gauze to remove any blood. Then grasp the head with the left hand and support the flank with the right hand to locate the spleen through the peritoneum.Using one finger from the right hand, palpate the mouse from underneath. The kidney should become visible. Keeping a firm hold on the animal to provide tension across the peritoneum and kidney, have an assistant slowly deliver 0.1 milliliters of the Renca cells through the intact peritoneum into the center of the kidney.Hold the needle in place for five to 10 seconds to reduce the backflow of the cells. Then, close the incision with a thin layer of cyanoacrylate tissue adhesive. At the experimental end point, use scissors to make a midline incision, starting at the mid-abdomen through the ribcage, neck, and salivary glands.Using scissors and forceps, carefully remove the ribs without compromising the lung tissue, and remove the connective tissue to expose the trachea. Fill a three milliliter syringe equipped with an 18 gauge needle with 10%India Ink and PBS. Then use forceps to carefully thread a silk suture under the trachea and then tie a loose knot.Next, carefully insert the needle into the trachea and slowly inflate the lungs with one to one and a half milliliters of the India Ink solution. Pull the knot tight around the trachea to inhibit the backflow of the ink, then remove the needle and carefully cut away the connective tissue to remove the inflated lungs. Fix the lungs in a 15 milliliter conical tube containing five milliliter of Fekete's solution in a chemical fume hood at room temperature.After 24 to 48 hours, remove the intact de-stained lungs and carefully pull apart the lobes in a 35 by 10 millimeter petri dish. Then count the number of white tumor nodules under a dissecting microscope. A successful mouse intrarenal Renca cell implantation results in tumor development in the kidney and metastasis to the lungs with tumor-bearing animals demonstrating a robust tumor burden as measured by the increased weight of the tumor-implanted kidneys.As observed, Renca tumors can be identified by immunohistochemical staining for Cytokeratin eight and 18. The implantation of a luciferase expressing Renca cell line allows tracking of the tumor burden in the kidney and metastasis to the lung by bioluminescent imaging as further confirmed by india link lung inflation. Once mastered, this technique can be completed in less than five minutes per mouse if performed properly.While attempting this procedure it's important to remember to clearly identify the kidney through the peritoneum to ensure an accurate intrarenal tumor implantation. Following this procedure, other methods, like preclinical drug testing can be performed to answer additional questions, such as to what extent does novel drug effect the growth or metastasis of intrarenal tumors? After watching this video, you should have a good understanding of how to perform intrarenal tumor cell implantation for establishing orthotopic kidney tumors in the mouse.Don't forget that working with live animals and surgical instruments can be hazardous and that precautions, such as following institutional animal handling guidelines and maintaining a sterile surgical area and instruments should always be taken while performing this procedure.

Research

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