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...

<ol>
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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>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 ...

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