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.

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Research

JoVE Journal

Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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, ...