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 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.
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.
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 metastases of a spontaneously transformed BALB/C keratinocyte line (Pam 212)12,13. Both cell lines were kindly provided by Dr. Nadarajah Vigneswaran (UTHealth, Houston, TX, USA).
2. Cell Injection into Mice
3. Mouse Monitoring
4. Harvesting Tumors
5. Tumor Processing for Downstream Applications
6. Cell Staining and Data Acquisition
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 anticipated. LY2 tumors grew at a higher rate compared to B4B8 tumors. The average tumor volume on day 21 in LY2 tumor-bearing mice was 632 ± 10 mm3 compared to 162 ± 4 mm3 in B4B8 tumors (Figure 1B). Mice exhibiting jaw displacement rapidly developed weight loss due to dysphagia (Figure 1C). Mice exhibiting significant weight loss defined as >15% of their initial weight and/or having a tumor volume of >1 cm3 were euthanized within 1 day of the noted observation. The median survival in LY2 mice was 22.5 days, compared to 52.0 days in B4B8 tumor-bearing mice (Figure 1D).
Magnetic resonance imaging (MRI) of tumor-bearing mice showed well-demarcated tumors extending into the inner layer of the buccal mucosa (Figure 2A). Tumors did not invade into the tongue or other nearby organs (esophagus, bronchus, thymus) as shown with histological assessment. Signal heterogeneity on MR images was representative of the presence of vascularized hyperintense regions (denoted with V) and necrotic hypointense regions (denoted with N) (Figure 2A). A gross pathological examination showed enlarged DLNs (Figure 2B). We further assessed tumor volume with computed tomography (CT) to determine the reliability of the caliper measurements. Tumors were delineated using a digital imaging and communications in medicine (DICOM) image analysis software16. Assessment of LY2 tumor volume by calipers and CT imaging showed an excellent correlation between the two methods (R2 = 0.8493; Figure 2C). This indicates that tumor growth is primarily exophytic and caliper measurement is a reliable method for the assessment of tumor growth. Histologic examination showed that all LY2 tumor-bearing mice developed poorly differentiated squamous cell carcinoma (Figure 2D). Nine out of nine LY2 tumor-bearing mice developed metastases to the first and second echelon lymph nodes. Nodal metastases were primarily subcapsular (with seven out of nine mice) and within sinuses (with five out of nine mice), with two out of nine mice demonstrating intracapsular invasion. In contrast, mice bearing B4B8 tumors developed moderately differentiated squamous cell carcinoma (Figure 2E) and did not metastasize to regional or distant sites, including DLNs. Necrosis was assessed based on a previously established three-grade scale: 0 = no visible necrosis, 1 = scant, 2 = moderate, and 3 = severe17. All LY2 tumor-bearing mice had histologically confirmed necrosis with the majority (seven out of ten) demonstrating moderate to severe necrosis (Figure 2F). No evidence of necrosis was observed in B4B8 tumors.
We focused on the LY2 tumor model for further characterization of the tumor-immune microenvironment. Tumors were harvested at 3 weeks after the tumor inoculation and digested using collagenase III, followed by the staining of the cell surface and intracellular markers for flow/mass cytometry (Figure 3A). Samples were processed, using a mass cytometer, at the University of Colorado Denver Flow Cytometry Shared Resource. Gating and data analysis were performed using flow cytometry commercial software. The data obtained showed the presence of numerous immune cell populations to varying degrees. Total immune cells (CD45+) represented 7.3% of the total tumor (Figure 3B). In absolute numbers, we observed, on average, 26 CD45+ cells (SD = 0.81) per milligram of tumor tissue (Figure 3B). Myeloid cells (CD11b+) represented 37.8% of all CD45+ cells (Figure 3C). Myeloid cells were largely comprised of macrophages (F4/80+, 63.0%), followed by myeloid-derived suppressor cells (MDSCs) (Gr1+, 8.51%) and neutrophils (Ly6G+, 5.87%). Total T cells comprised 15.9% of CD45+ cells with CD4+ T cells comprising the majority of all T cells (79.4%), of which 53.4% were regulatory T cells (Tregs) (CD4+FoxP3+) (Figure 3D). Natural killer (NK) cells comprised 1.75% of all CD45+ cells. These data highlight the presence of various immune infiltrates in LY2 orthotopic HNSCC tumors which likely play a role in mediating tumor progression and immune evasion.
Figure 1: Monitoring mice bearing orthotopic HNSCC tumors. (A) Representative images of tumor-bearing mice. The inoculated buccal develops a tumor within 1 week of inoculation. At 3 weeks, displacement of the jaw is observed. (B) Tumor growth rate in B4B8 and LY2 tumor-bearing mice as determined by caliper measurements. (C) Changes in the body weight of mice bearing B4B8 or LY2 tumors. (D) Survival of B4B8 and LY2 tumor-bearing mice. The bars represent the standard error of the mean (SEM) of 7–10 mice per group. Please click here to view a larger version of this figure.
Figure 2: Imaging and histologic features of murine HNSCC tumors. (A) Representative coronal MR image of an LY2 tumor-bearing mouse. Anatomic sites are labeled. Areas of signal hyperintensity represent vascularized regions (denoted V). Regions of signal hypointensity represent necrotic areas (denote N). (B) Representative gross image of a dissected tumor-containing region. White arrows point to enlarged draining lymph nodes (DLNs). (C) Correlation of tumor volume as assessed by caliper measurement versus computed tomography (CT)-based assessment. Slope, correlation coefficient (R2), and line of best fit are shown. (D) Representative hematoxylin and eosin (H&E) image of LY2 tumor. (E) Representative H&E image of B4B8 tumor. (F) Quantification of necrosis in LY2 tumors, based on an H&E four-grade scoring system. Please click here to view a larger version of this figure.
Figure 3: Analysis of baseline intratumoral immune populations using cytometry by time-of-flight (CyTOF). Tumors were processed into a single-cell suspension using collagenase-based enzymatic digestion and, then, stained with cell surface and intracellular markers. (A) Mass cytometry was performed using the CyTOF platform. (B) Absolute and relative quantitative assessment of total immune cells (CD45+). (C) Quantification of myeloid-immune subpopulations. (D) Quantification of lymphoid immune subpopulations. Please click here to view a larger version of this figure.
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 human HNSCCs. Furthermore, numerous murine models employ a subcutaneous implantation of tumors which lack important physiological features of the head and neck region, including a high vascular density, an extensive lymphatic vasculature, and resident mucosal flora.
In this study, we characterized an orthotopic model of murine HNSCC. We chose the buccal mucosa as the site of tumor inoculation for a number of reasons, including easy access to the region without the need for image guidance, the ability to assess tumor growth using calipers, the presence of the tumor within the mucosal flora, the presence of surrounding lymphatics, and the ease of reproducibility. Although the injection of cells into the buccal mucosa is straightforward, the positioning of the needle and the depth of penetration are critical to prevent puncture through the skin and ensure cells are not injected subcutaneously. We recommend inserting the needle intra-orally while it is perfectly parallel to the buccal region and tilting at an angle no greater than 10° when ready to inject.
The cell lines we used were murine cell lines allowing their implantation in immunocompetent mice of the strain they were derived from. There are over 39 HNSCC human cell lines that have been studied in the literature but cannot be used in the presence of a native microenvironment18. Recent developments allowed for the design of humanized mouse models which integrate human bone-marrow-derived chimeras in NOD scid gamma mice (also known as NSG mice), allowing the development of a human immune system that can interact with human tumors in an immunodeficient mouse model19. However, such models are costly, require laborious breeding methods, and do not recapitulate all the components of the tumor microenvironment, including endothelial cells, fibroblasts, and lymphatics. The murine mouse models we employed in this study recapitulate critical components of human HNSCCs, including the presence of various immune infiltrates. To ensure maximum retrieval of viable single cells from tumors, the use of digestion enzymes is necessary. Collagenase-based digestion enzymes can be harsh on cells and the optimization of the concentration and type of collagenase may be necessary for different tumor types. We compared five digestion enzymes before determining that collagenase III, which was used in this study, is the optimal method for digestion of squamous cell tumors.
The tumor models used in this study are particularly useful for studying mechanisms of HNSCC immune evasion and preclinical assessment of various therapies. We previously demonstrated that both B4B8 and LY2 tumors are radioresistant and refractory to the immune checkpoint inhibitor anti-PD-L120. This is consistent with the majority of human HNSCCs. Recent clinical trials showed that less than 15% of HNSCC patients are responsive to anti-PD-1/PD-L1 therapy21,22,23,24,25. In addition, it is well established that HNSCCs are radioresistant26,27. Targeting pathways responsible for radioresistance in HNSCC is an important step for enhancing treatment response. The landmark Bonner et al. study showed a significant benefit in overall survival with the addition of cetuximab to RT compared to RT alone in locally advanced HNSCC patients28. Recent data demonstrate that the inhibition of EphB4-Ephrin-B2 signaling in orthotopic HNSCCs can further improve the response to RT or cetuximab-RT by promoting tumor apoptosis and inhibiting tumor proliferation29,30. In addition, combining RT with rational immune therapies can augment antitumor immune responses and enhance both local tumor control and control of distant metastases31. We have recently demonstrated that targeting Tregs in combination with immune checkpoint blockade and RT results in tumor eradication and immunologic memory32. Future studies employing orthotopic models of HNSCC will better inform the design of clinical trials and improve the understanding of mechanisms of tumor immune evasion and treatment resistance.
The authors have nothing to disclose.
None
Collagenase III | Worthington Biochemical Corp. | LS004183 | |
DNase I | Worthington Biochemical Corp. | LS006328 | |
Fc Block (CD16/32) | BD Biosciences | 553141 | Clone 2.4G2 |
Flow Cytometry Staining Buffer | eBioscience | 00-4222-26 | |
HBSS | ThermoFisher Scientific | 14175079 | no calcium, no magnesium, no pheno red |
Helois mass cytometer | Fluidigm | NA | |
Matrigel membrane matrix | Corning | CB-40234B | |
MRI Scanner | Bruker | NA | 7.4 Tesla |
RBC lysis buffer | BioLegend | 420301 | |
Trypsin inhibitor | Worthington Biochemical Corp. | LS002830 |