Name: intramucosal inoculation of squamous cell carcinoma cells in mice for tumor immune profiling and treatment response assessment
Uploaded: 2019-04-22T14:00:00
Description: Watch this Scientific Journal Video about Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment at JoVE.com

All animal procedures were performed in accordance with an approved institutional animal care and use committee (IACUC) protocol of the University of Colorado Denver (protocol # 00250).
1. Tumor Cell Culture
NOTE: B4B8 and LY2 cell lines were used to generate orthotopic HNSCC tumors: B4B8 tumor cells were derived from carcinogen-transformed mucosal keratinocytes (from BALB/C mice)11. LY2 tumor cells were derived from lymph node...

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Rigorous analysis and characterization of the tumor microenvironment represent an important strategy for understanding mechanisms of tumor development, progression, and metastases and for the development of effective therapies. Head and neck cancer is a complex disease that can originate from multiple anatomical sites within the head and neck region. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine mouse cell lines that resemble the aggressive and metastatic nature of...

HNSCC is the fifth most common malignancy globally, with over 600,000 patients diagnosed annually1. Despite aggressive treatment involving chemotherapy and radiotherapy (RT), the overall survival (OS) rate for HNSCC patients without human papillomavirus (HPV) infection remains below 50% after 5 years2. This is largely attributed to a highly complex tumor microenvironment as tumors can originate from several distinct anatomical sites within the head and neck region, including the buccal mucosa, tongue, floor of the mouth, nasal cavity, oral cavity, pharynx, oropharynx, and hypopharynx. In addition, the head and neck region is highly vascularized and contains nearly half of all lymph nodes in the body3. A majority of studies investigating head and neck tumor biology rely on tumor models in the flank region. Such models can offer insight into tumor-intrinsic mechanisms, but the lack of the native head and neck microenvironment can significantly impact the translational potential of such findings. One method that has been used to induce oral tumors is through exposure to the carcinogen 9,10-dimethyl-1,2-benzanthracene (DMBA)4. However, this method is associated with a lengthy process, induces tumors in rats and hamsters but not in mice, and the resulting tumors do not possess many of the histological features of differentiated SCCs5,6. The introduction of the carcinogen4-nitroquinoline 1-oxide (4-NQO), a water-soluble quinolone derivative, resulted in mouse oral tumors when applied orally but also suffered from long exposure times (16 weeks) and a limited take rate within and between batches of mice7,8,9. In order to develop clinically relevant models, several groups utilized genetically engineered models involving the manipulation of driver oncogenes or tumor suppressor genes, including TP53, TGFB, KRAS, HRAS, and SMAD410. These models can offer insight into tumors with known driver genes but do not recapitulate the complex heterogeneity of human HNSCCs.
In this work, we demonstrate the feasibility of performing an intramucosal inoculation of squamous cell carcinoma cells in mice. Inoculated cells develop into aggressive tumors within 1 week of injection. Similar to human HNSCCs, the tumors metastasize to the regional lymph nodes. We characterize histological and clinical features of the disease and provide insight into the tumor immune microenvironment. We propose that this orthotopic model of HNSCC has applications in cancer biology, tumor immunology, and preclinical studies. Mechanisms of immune evasion, tumor progression, treatment resistance, and metastases represent areas of clinical significance that can be addressed using the proposed model.

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			<td style="width:217px;">Worthington Biochemical Corp.</td>
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			<td>Worthington Biochemical Corp.</td>
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			<td>BD Biosciences</td>
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			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer</td>
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			<td>ThermoFisher Scientific</td>
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			<td style="width:327px;">no calcium, no magnesium, no pheno red</td>
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			<td>Worthington Biochemical Corp.</td>
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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.

The in vitro assessment of LY2 and B4B8 cell proliferation showed that both cell lines have similar doubling times (21 h and 23 h, respectively). In vivo, both cell lines formed a single, visible, and palpable mass within 1 week of inoculation (Figure 1A). In mice bearing LY2 tumors, the jaw was displaced by 3 weeks due to tumor burden (Figure 1A). Control mice that did not receive tumor cells did not develop tumors as anticipate...

Watch this Scientific Journal Video about Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment at JoVE.com

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

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 ...

Cancers of the head and neck region are becoming increasingly prevalent. Yet, there's limited understanding of the tumor microenvironment and the mechanisms of treatment resistance in this region. This technique can be used to recapitulate the native microenvironment of head/neck tumors in an accessible manner and produces clinical manifestations similar to those seen in humans.When the tumor cell culture has reached 70%confluency, wash the cells three times with cold PBS per wash and attach the cells with enough 0.25%trypsin to cover the bottom surface of the culture flask. After three to four minutes in the cell culture incubator, confirm detachment under a light microscope and neutralize the trypsin with 12 milliliters of DMEM F12 medium, supplemented with fetal bovine serum. Transfer the cell suspension into a 50 milliliter conical tube and mix the cells three to four times by inversion.Then collect the cells by centrifugation and re-suspend the pellet in serum and antibiotic-free DMEM at a one times 10 to the sixth tumor cells per 50 microliters of medium concentration on ice. Immediately before the injection, mix the cells at a one to one tumor cells suspension to basement membrane matrix ratio on ice. Load one 0.5 milliliter syringe equipped with a 23-gauge needle with 100 microliters of cells per recipient animal.Place the syringes on ice and confirm a lack of response to toe pinch in an anesthetized mouse. Next, insert the needle into the right or left buccal region through the available open space on either side of the mouth keeping the syringe parallel to the buccal region while it is inside the oral cavity to facilitate injection of the full 100 microliter volume of the cell basement membrane matrix suspension over a period of five seconds. Keep the syringe in place for an additional five seconds to ensure that all of the material has been injected before withdrawing the syringe gently.The tumors will become grossly visible in about a week. One week after the injection, use calipers to measure the length and width of each tumor to determine the tumor volume one to two times a week. And measure the weight of each animal to assess the effects of the tumor growth on feeding.At the appropriate experimental endpoint, use sharp scissors and blunt forceps to make a skin incision through the midline in the neck. And insert the scissors gently under the skin covering the tumor to create air pockets by pushing the scissors across and into the skin. Once the skin is sufficiently detached from the tumor, excise the draining lymph nodes to avoid having the tumor tissue confounded by the presence of lymph tissue and cut through the borders of the tumor until the entire volume is detached.To process the tumor for downstream analysis, cut the tumor into one to two millimeter sized pieces and place the pieces into a 50 milliliter conical tube containing collagenase three, DNAs one, and trypsin inhibitor. After incubating the tumor pieces at 37 degrees Celsius for 30 minutes, with shaking every 10 minutes, add 20 milliliters of HBSS to the tube and pass the suspension containing the tumor pieces through a 70 micrometer pour nylon strainer. Use a five milliliter syringe plunger to mash the tumor pieces in the strainer and add an additional 10 milliliters of HBSS through the strainer.Spin down the cells by centrifugation. Re-suspend the pellet in two to three milliliters of red blood cell lysis buffer with rigorous pipetting, neutralizing the lysis with 20 milliliters of fresh HBSS after two minutes at room temperature. Then re-suspend the cells in an additional 20 milliliters of HBSS for another centrifugation and strain the cells through a 40 micrometer pour filter to remove any final debris from the tumor cell suspension.LY2 tumors grow at a higher rate, compared to B4B8 tumors. And mice that exhibit jaw displacement rapidly develop weight loss, due to dysphagia. The median survival in LY2 mice is also less than half that observed for B4B8 tumor-bearing mice.Magnetic resonance imaging of tumor-bearing mice shows well-demarcated tumors extending into the inner layer of the buccal mucosa, but not into the tongue or other nearby organs. Histologic examination indicates that all LY2 tumor-bearing mice develop poorly differentiated squamous cell carcinoma, while mice bearing B4B8 tumors develop moderately differentiated squamous cell carcinoma. All LY2 tumor bearing mice also have histologically confirmed necrosis, with the majority demonstrating moderate to severe necrosis.In this representative experiment, a LY2 tumor was harvested and processed three weeks after buccal region tumor cell injection, as demonstrated. CD45-positive immune cells represented 7.3%of the total tumor cell population. CD11b-positive myeloid cells represented 37.8%of all of the CD45-positive cells, the majority of which were determined to be F480-positive macrophages, with small populations of neutrophils and myeloid-derived suppressor cells also observed.T cells comprised 15.9%of the CD45-positive immune cell population, 53.4%of which were CD4-positive FoxP3-positive regulatory T cells. Natural killer cells comprised less than 2%of all all of the CD45-positive cells. It is important to practice injection in order to develop confidence and comfort in holding the needle and the recipient mouse and in exposing the mouth of the animal.Once the procedure has been successfully performed, experiments involving the assessment of tumor response to therapy or assessment of the tumor intrinsic and microenvironmental factors can be performed. This technique has paved the way in designing an investigator-initiated clinical trial, assessing the effects of combining radiotherapy with anti-PL1 therapy in head and neck cancer patients.

Research

JoVE Journal

Cancer Research

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

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, ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

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 ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...