eoe sub articles

EoE Sub Articles

1. Implantation of Cells 
To implant Renca cells intrarenally, prepare Renca cells into a single-cell suspension at 2 x 106&#160;cells/mL in Hank&#39;s Balanced Salt Solution (HBSS).
<ol>
	<li>Remove adherent Renca cells from the tissue culture flask using 0.25% trypsin in HBSS. After ~5 min, add complete RPMI to the flask to neutralize the trypsin and transfer the cells to a centrifuge tube for washing. Centrifuge the cells at 300 relative centrifugal g force (rcf x g) for 5 min at 25 &#176;C.</li>
	<li>Decant the supernatant and resuspend the cells in HBSS. Count the cells using a hemocytometer and adjust the volume with HBSS to yield 2 x 106&#160;cells/mL.</li>
	<li>Draw the cells into a 1 mL syringe fitted with an 18-gauge needle to prevent shear stress on the cells. Replace the 18-gauge needle with a 28-gauge needle prior to the injection of the tumor cells. An injection of 0.1 mL delivers 2 x 105&#160;cells into the kidney.</li>
</ol>
2. Surgical Area Preparation
<ol>
	<li>Prepare a sterile surgical area in a biosafety cabinet by placing a sterile surgical drape over a heating pad. Gather all supplies, including forceps, scalpel, scissors, eye drops, 5% povidone-iodine antiseptic, and sterile gauze. Autoclave all instruments prior to use.</li>
	<li>Perform the surgery using an aseptic tip technique. Resterilize instruments between animals with a bead sterilizer. Wear sterile gloves, a lab coat, and a face mask.</li>
</ol>
3. Animal Preparation
<ol>
	<li>Anesthetize the mice with ketamine/xylazine (87.5 mg/kg and 12.5 mg/kg, respectively, i.p.) according to institutional guidelines. Determine anesthetic depth by performing a toe pinch.</li>
	<li>When the animal is considered non-responsive, prepare the injection site (e.g.,&#160;left flank) on Balb/c mice by plucking (with fingers) or shaving (using clippers) the fur.</li>
	<li>Move the mice from the surgery prep area (e.g.,&#160;the housing cage) to the surgical area and apply vet ointment to the eyes to prevent dryness. Liberally swab the prepared incision site with 5% povidone-iodine antiseptic.</li>
	<li>Inject bupivacaine hydrochloride (0.1 mL of a 2.5 mg/mL solution, s.c.) into the area where the incision will be made as a pre-operative analgesia.</li>
	<li>Make a single incision (~1.0 &#160;to 1.5 cm) on the left flank of the animal using a sterile scalpel or scissors carefully so as not to penetrate the peritoneum. Separate the dermis from the peritoneum with sterile scissors. Dab the incision site with sterile cotton gauze to remove any blood.</li>
	<li>Hold the mouse with both hands, using the left hand to hold the head and the right hand to support the flank.</li>
	<li>Identify the spleen through the peritoneum and, with one finger of the right hand, palpate the mouse from underneath; the kidney should now be visible through the peritoneum. Keep a firm hold of the animal to provide tension across the peritoneum and kidney.</li>
	<li>Have a second person inject the tumor cells (prepared in section 1.2) through the intact peritoneum into the center of the kidney using a 1-mL syringe fitted with a 28-gauge needle. Slowly deliver 0.1 mL (2 x 105) of cells. Once the cells are injected, hold the needle in place for 5&#8211;10 s to reduce the backflow of cells.&#160;It is important to note the need for extreme caution during this step, as the person injecting the cells will bring the needle into close proximity to the fingers of the person holding the mouse.</li>
	<li>Remove the needle and close the incision with a thin layer of cyanoacrylate tissue adhesive.</li>
	<li>Place the animal back into a clean cage with a sterile paper towel. Allow the animals to recover on a heating pad. Do not leave the animals unattended during the recovery period. Do not return the animals to standard housing conditions until they are upright and sternal. Follow institutional guidelines in all procedures using laboratory animals.</li>
</ol>

<table><tbody><tr><td>Artificial tears ophthalmic ointment</td><td>Akorn</td><td>NDC 17478-162-35</td><td></td></tr><tr><td>Vetbond Tissue Adhesive&amp;nbsp;&amp;nbsp;</td><td>3M</td><td>CBGBIW011019</td><td></td></tr><tr><td>Betadine Solution (Povidoneiodine, 5%)&amp;nbsp; NDC&amp;nbsp;</td><td>Purdue Products, L.P.</td><td>67618-155-32</td><td></td></tr><tr><td>0.25% Marcaine (Bupivavaine HCl injection)</td><td>Hospira</td><td>0409-1587-50</td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Sunbeam</td><td></td><td></td></tr><tr><td>Syringes BD</td><td></td><td>309659</td><td></td></tr><tr><td>Gauze&amp;nbsp;&amp;nbsp;</td><td>Venture</td><td>908291</td><td></td></tr><tr><td>Ketamine</td><td>Phoenix Pharmaceuticals NDC</td><td>57319-609-02</td><td></td></tr><tr><td>Xylazine&amp;nbsp;&amp;nbsp;</td><td>Akorn NDC</td><td>59399-111</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salts&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H2387</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Renca</td><td>ATCC</td><td>CRL-2947</td><td></td></tr></tbody></table>

generating renal cell carcinoma model: a protocol to develop orthotopic rcc murine model by intrarenal implantation of cancer cells

Source: Murphy, K. A. et al. <a href="https://www.jove.com/video/55080" target="_blank">A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies.</a> J. Vis. Exp. (2017)
In this video, we develop an orthotopic mouse model of renal cell carcinoma. This model results in reproducible primary tumors in the injected kidney and metastatic tumors in the lung, allowing the evaluation of disease progression and treatment.

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical ...

The implantation of renal cell carcinoma, or RCC, cells from a donor mouse to an immunocompromised recipient mouse&#39;s kidney leads to the development of an orthotopic RCC model.
To develop an orthotopic RCC model, begin by placing an anesthetized immunodeficient mouse in the prone position on a dissection board. Now, use a scalpel to make a surgical cut on the left flank of the mouse, keeping the underlying peritoneum intact.
Palpate the mouse by applying gentle pressure from the bottom. This step helps visualize the kidney through the translucent peritoneum. Next, take a syringe containing RCC cell suspension in an appropriate medium. Using a needle, inject the cell suspension into the kidney without damaging the peritoneum.
Retract the needle and seal the incision site using a tissue adhesive. Allow the mouse to recover and monitor the tumor growth at desired intervals. Initially, the injected renal cancer cells get implanted into the kidney. These cells then proliferate to form tumors.
Subsequently, a few cells may detach from the primary tumor and disseminate within the peritoneal cavity. These migrating tumor cells can invade the lung tissue and metastasize to form secondary tumors.

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Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Cancer Research

Urinary Tract Cancer

In vivo studies

1.4K Views. Source: Murphy, K. A. et al. A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies. J. Vis. Exp. (2017) In this video, we develop an orthotopic mouse model of renal cell carcinoma. This model results in reproducible primary tumors in the injected kidney and metastatic tumors in the lung, allowing the evaluation of disease progression and treatment. 

Video: Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells - Concept

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

JoVE Journal

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

Transcript

Generating Renal Cell Carcinoma Model: A Protocol to Develop Orthotopic RCC Murine Model by Intrarenal Implantation of Cancer Cells

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy