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).
<ol>
	<li>Use Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) F/12 supplemented with 10% fetal bovine serum (FBS) and 1% antimicrobial reagent for the cell line maintenance. Maintain these cell lines in a sterile incubator at 37 &#176;C and 5% CO2. Cells should be used for inoculation before exceeding 15 passages.</li>
	<li>Plate 2 x 106 to 4 x 106 cells in 175 cm2 cell culture flasks. 
	NOTE: Ensure the cells are less than 90% confluent to avoid inducing a stress response.</li>
	<li>When the cells are 70% confluent (~48 h), remove the flask from the incubator and wash the cells 3x with cold phosphate-buffered saline (PBS).</li>
	<li>Detach the cells from the flask using 0.25% trypsin, enough to cover the surface of the plate.
	<ol>
		<li>To do this, dispense 4 mL of trypsin in a 175 cm2 flask containing 70% confluent cells. Incubate the cells with trypsin for 3&#8211;4 min in the cell culture incubator at 37 &#176;C and 5% CO2.</li>
		<li>Visualize the cells under a microscope to ensure cell detachment.</li>
		<li>Add 12 mL of DMEM F/12 media containing FBS to neutralize trypsin activity.</li>
	</ol>
	</li>
	<li>Aspirate the cell suspension and place it into a 50 mL conical tube.</li>
	<li>Shake the tube containing the cells by inverting it 3x&#8211;4x.</li>
	<li>Optionally, mix a 10 &#181;L aliquot of cell suspension with 10 &#181;L of Trypan blue in a microcentrifuge tube and count the cells using a hemocytometer. Determine cell viability by subtracting the number of Trypan-blue-positive cells from the total number of cells and divide by the total number of cells.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cells in serum-free and antibiotic-free DMEM at an appropriate volume so that 1 x 106 cells are present in 50 &#181;L of media. 
	NOTE: Based on the in vivo cell line aggressiveness (determined empirically), 1 x 106 cells per injection site per mouse was deemed appropriate for B4B8 and LY2 cells.</li>
	<li>Place the vial containing the cell suspension on ice.</li>
	<li>Place the pre-thawed basement membrane matrix on ice. 
	NOTE: The basement membrane matrix was thawed overnight at 4 &#176;C.</li>
</ol>
2. Cell Injection into Mice
<ol>
	<li>Prepare a 1:1 mixture of cells:basement membrane matrix (50 &#181;L each).</li>
	<li>Add the cells first; then, gradually pipette the basement membrane matrix. Avoid introducing air bubbles. Ensure that the mixture is made immediately before the animal injection. Adding cells to basement membrane matrix for an extended period of time can result in cell settling in the matrix mixture, which makes the mixture difficult to shake rigorously. This will cause a considerable variability in tumor size between mice.</li>
	<li>Mix gently. Ensure all steps involving matrix are performed on ice. Basement membrane matrix will polymerize at room temperature.</li>
	<li>Prepare syringes for inoculation.</li>
	<li>Load 0.5 mL insulin syringes (23 G) with 100 &#181;L of the cell/basement membrane matrix solution.</li>
	<li>Keep the syringes on ice to avoid basement membrane matrix polymerization.</li>
	<li>Anesthetize mice by placing them in a chamber with isoflurane and oxygen (2.5%).</li>
	<li>Ensure the mice are deeply anesthetized before performing the injection (by ensuring a lack of response to a toe pinch).</li>
	<li>Insert the needle into the right or left buccal region. This is performed through the available open space on either side of the mouth.</li>
	<li>Ensure that the mouse&#8217;s tongue is not in the way. 
	NOTE: It is easy to poke the tongue, which will result in tongue tumors. Move the tongue to the opposite side if necessary.</li>
	<li>Keep the syringe parallel to the buccal region while inside the oral cavity.</li>
	<li>When ready to inject, pull the syringe back and slowly insert the syringe at a 10&#176; angle.</li>
	<li>Inject 100 &#181;L of the cell/basement membrane matrix suspension over a period of 5 s.</li>
	<li>Hold the syringe in place for an additional 5 s to ensure all material is injected. 
	NOTE: For control nontumor-bearing mice, inject a mixture of serum-free media and matrix (as described above) without the tumor cells.</li>
	<li>Withdraw the syringe gently.</li>
	<li>Continue the above procedure with the remaining mice.</li>
	<li>Allow for 1 week until tumors begin to appear grossly (50&#8211;200 mm3 for B4B8 and LY2 cells).</li>
</ol>
3. Mouse Monitoring
<ol>
	<li>Perform the first measurement using calipers at 1 week after the tumor cell injection. In order to calculate the tumor volume using external calipers, determine the greatest longitudinal diameter (length) and the greatest transverse diameter (width) and use the modified ellipsoidal formula14,15. 
	<img alt="Equation" src="/files/ftp_upload/59195/59195eq1.jpg" /></li>
	<li>Continue to perform regular caliper measurements for tumor volume (1x&#8211;2x per week at regular intervals).</li>
	<li>Measure animal weight to assess the effects of tumor growth on feeding.</li>
</ol>
4. Harvesting Tumors
<ol>
	<li>When the experimental endpoint is reached, euthanize the animals using appropriate measures (e.g., CO2 asphyxiation, decapitation, or cervical dislocation). 
	NOTE: In this study, the endpoint was reached if the mice became moribund (with a weight loss of &#62;15% of their initial weight, a lack of grooming, cachexia) and/or if the tumor size reached 1,000 mm3.</li>
	<li>Begin dissecting the animal by creating a long incision through the midline in the neck region.
	<ol>
		<li>Use blunt forceps to grab the skin and sharp scissors to cut the skin.</li>
	</ol>
	</li>
	<li>Insert scissors gently under the skin covering the tumor and create air pockets by pushing the scissors across and into the skin.</li>
	<li>Once the skin is sufficiently detached from the tumor, identify the draining lymph nodes (DLNs) and excise them to avoid having the tumor tissue confounded by the presence of lymph nodes. 
	NOTE: Large tumors may reach and/or cover the DLN. Depending on the type of assay being performed, the isolation of intact lymph nodes is essential to avoid spillage of immune cells into the tumor, which will result in skewing the assay results.</li>
	<li>Cut through the borders of the tumor until the entire volume is detached.</li>
</ol>
5. Tumor Processing for Downstream Applications
<ol>
	<li>For the downstream histologic examination, place tumors in 10% formalin at room temperature. To avoid overfixation, replace the formalin with 70% ethanol. For flow cytometry analysis, process the tumors as described below. 
	NOTE: The tissue can be kept in formalin for 72 h before overfixation can become problematic for certain types of staining.</li>
	<li>Cut the tumor into 1&#8211;2 mm-sized pieces using a sterile razor blade or sharp scissors.</li>
	<li>Place the cut tumor pieces in a 50 mL conical tube with collagenase III (4,250 units per sample), DNase I (0.1 mg per sample), and trypsin inhibitor (1 mg per sample).</li>
	<li>Incubate at 37 &#176;C for 30 min with shaking every 10 min. 
	NOTE: The samples can be placed in a resealable bag, sterilized with 90% ethanol, and placed in a cell culture incubator.</li>
	<li>After 30 min, add 20 mL of Hank&#8217;s balanced salt solution (HBSS) and spin at 300 x g for 5 min. 
	NOTE: HBSS is comprised of potassium chloride, sodium chloride, sodium bicarbonate, sodium phosphate dibasic, sodium phosphate monobasic and glucose.</li>
	<li>Discard the supernatant and resuspend the pellet in 2&#8211;3 mL of red blood cell (RBC) lysis buffer. Pipette rigorously. 
	NOTE: RBC lysis buffer is comprised of ammonium chloride, sodium bicarbonate, and disodium.</li>
	<li>Incubate for 2 min at room temperature. 
	NOTE: Longer incubation can be toxic to other cell populations.</li>
	<li>Add 20 mL of HBSS to neutralize the effect of lysis buffer.</li>
	<li>Centrifuge at 300 x g for 5 min and discard the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of HBSS.</li>
	<li>Pass the solution through a 70 &#181;m nylon restrainer and centrifuge at 300 x g for 5 min. Discard the supernatant.</li>
	<li>Repeat steps 5.8&#8211;5.10 and pass the cell suspension through a 40 &#181;m nylon restrainer to ensure any additional debris is removed from the suspension.</li>
</ol>
6. Cell Staining and Data Acquisition
<ol>
	<li>Resuspend the cell pellet in 1 mL of HBSS.</li>
	<li>Count the cells using a hemocytometer or an automated cell counter, as described in step 1.7</li>
	<li>Determine the total cell number and appropriate concentration for staining. 
	NOTE: The ideal cell concentration for flow cytometry staining is 1&#8211;2 million cells. For example, if staining is performed in a 96-well plate and there are 10 million cells, resuspend the sample in 1 mL and plate 100 &#181;L for 1 million cells per well.</li>
	<li>Add Fc block (CD16/CD32) at a concentration of 1:100 in order to prevent nonantigen-specific binding of immunoglobulins to the Fc&#947;III and Fc&#947;II receptors. Incubate for 5 min at room temperature.
	<ol>
		<li>Centrifuge and resuspend the cell pellet in 100 &#181;L of flow cytometry cell staining buffer (comprised of saline solution containing 5% FBS, 2% EDTA, and 1% HEPES).</li>
	</ol>
	</li>
	<li>Perform cell surface staining according to supplier-provided instructions (adding each antibody at the appropriate dilution). Incubate for 60 min at room temperature.
	<ol>
		<li>Centrifuge and resuspend the cell pellet in 100 &#181;L of HBSS. Repeat 2x to get rid of excess antibody.</li>
	</ol>
	</li>
	<li>Run samples on an appropriate flow cytometer.
	<ol>
		<li>If analyzing intracellular markers (e.g., foxp3), perform cell permeabilization and staining according to the supplier&#8217;s instructions.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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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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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JoVE Journal

Cancer Research

11.3K Views.  University of Colorado Denver - Anschutz Medical Campus. 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.

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

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 important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

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

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor 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, 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, ...

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 relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

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

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 ...

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 decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

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

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

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

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

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

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive 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 contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

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 human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

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

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

Prognostic Significance of Immune Cell Infiltration in Hepatocellular Carcinoma

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies investigating the expression and localization of immune cells within tumor tissues are associated with several challenges, including a lack of precise annotation for tumor regions and random selection of microscopic fields of view. QuPath is an open-source, user-friendly software that could meet the growing need for digital pathology in whole-slide image (WSI) analysis.
The infiltration of HCC and adjacent tissues by CD1a+ immature dendritic cells (iDCs), CD117+ mast cells, and NKp46+ natural killer cells (NKs) cells was assessed immunohistochemically in representative specimens of 67 patients with HCC who underwent curative resection. The area fraction (AF) of positively stained cells was assessed automatically in WSIs using QuPath in the tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS).
The AF of mast cells was significantly greater than the AF of NKs, and the AF of iDCs was significantly lower compared to NKs in each region of interest. High AFs of mast cells in the IM and PT areas were associated with longer DFS. In addition, high AF of mast cells in IM was associated with longer OS.
Computer-assisted analysis using this software is a suitable tool for obtaining prognostic information for tumor-infiltrating immune cells (iDCs, mast cells, and NKs) in different regions of HCC after resection. Mast cells displayed the greatest AF in all regions of interest (ROIs). Mast cells in the peritumor region and IM showed a positive prognostic significance.

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies ...

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