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.

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Research

JoVE Journal

Cancer Research

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 &amp;#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 ...