This work was supported by the Sichuan Province Science and Technology Support Program (Grant Nos. 2019JDRC0019 and 2021ZYD0097), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (Grant No. ZYJC18026), the 1.3.5 project for disciplines of excellence-Clinical Research Incubation Project, West China Hospital, Sichuan University (Grant No. 2020HXFH023), the Fundamental Research Funds for the Central Universities (SCU2022D025), the International Cooperation Project of Chengdu Science and Technology Bureau (Grant No. 2022-GH02-00023-HZ), the Innovation Spark Project of Sichuan University (Grant No. 2019SCUH0015), and the Talent Training Fund for Medical-engineering Integration of West China Hospital - University of Electronic Science and Technology (Grant No. HXDZ22012).

All the animal experiments were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and protocols approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University. NOD-SCID immunodeficient mice aged 4-6 weeks old (of both sexes) and female Balb/c nude mice aged 4-6 weeks old were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The ethics committee of West China Hospital authorized the study with human subjects (protocol number 2020353). Each patient provided written informed consent.
1. Experimental preparation
<ol>
	<li>Arrange disposable blades, sterilized scissors and tweezers, and other instruments required for tumor transplantation, place them on the ultra-clean workbench, and irradiate them with ultraviolet light in advance.</li>
	<li>Prepare sterile saline and Petri dishes for use during the test.</li>
</ol>
2. Acquisition and transport of fresh tumor tissue
<ol>
	<li>Obtain fresh tumor samples (usually larger than 5 mm x 5 mm in size) from the operating room, and place them in a 15 mL or 50 mL centrifuge tube containing sterile HTK solution (see Table of Materials) or saline. Label the centrifuge tubes. 
	NOTE: Fresh tumor samples were obtained by surgical removal or puncture from patients with ATC or HNSCC.</li>
	<li>Put the centrifuge tubes in an ice box prepared in advance. 
	NOTE: During this time, the transplant operator must prepare the necessary items for transplantation (see Table of Materials).</li>
	<li>Ensure that the time between sample collection and transportation to the laboratory for PDX construction does not exceed 2 h. During transport, surround the tubes containing the tissues with an ice-water mixture or ice packs to preserve the tissue activity.</li>
</ol>
3. Tumor transplantation
<ol>
	<li>Once the tumor tissues arrive at the laboratory, record and renumber them. 
	NOTE: For the present study, the patient information was kept strictly confidential. The remaining steps of the procedure were performed in a biosafety level 2 (BSL-2) laboratory. When entering the laboratory, wearing a smock over the work clothes or protective clothing, a hat, and a mask is recommended. The treatment of tumor tissue is conducted in a biosafety cabinet.</li>
	<li>Disinfect the centrifuge tubes containing the tumor tissues with 75% alcohol, and place them on the operating table. Transfer the tumor tissues to 6 cm Petri dishes filled with saline using sterilized ophthalmic forceps. Next, cut them into small pieces of about 2 mm x 2 mm and 3 mm x 3 mm using a blade.</li>
	<li>Transfer the pieces of tumor tissues into a 6 cm Petri dish containing the appropriate amount of saline, wrap the dish with the sealing film, place it in an ice box, and carry it into the specific pathogen-free (SPF) animal room along with the necessary instruments (a pair of scissors, forceps, and inoculation needles).</li>
	<li>Prepare the animal following the steps below.
	<ol>
		<li>Remove the hair on the right lateral thorax of 4-6 week-old female or male NOD-SCID immunodeficient mice, and disinfect the skin with 75% alcohol. Anesthetize the mice by an intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine (see Table of Materials), and smear their eyes with vet ointment to prevent dryness. Confirm anesthesia depth via loss of pedal reflex.</li>
		<li>Make a 2 mm incision with scissors through the skin at the middle of the right lateral thorax of mice.</li>
	</ol>
	</li>
	<li>Take a tumor piece from the Petri dish, and place it into the 2.4 mm x 2.0 mm trocar needle (see Table of Materials) with forceps.</li>
	<li>Hold the mouse, tighten the skin at the puncture site, use the trocar containing the tumor pieces to insert the tumor through the initial 2 mm skin incision, move to the back of the shoulder, and push the trocar core.</li>
	<li>Ensure that the tumor piece is pushed out and is left in the transitional sinus formed by the trocar puncture, and then pull out the trocar.</li>
	<li>If the tumor moves with the needle when it is withdrawn, use the trocar to reset it and suture the incision. 
	NOTE: In this study, each mouse was inoculated at the dorsal fore and hind limbs. One to three mice were inoculated per tumor sample from each patient based on the tumor size.</li>
</ol>
4. Tumor tissue preservation, fixation, and protein freezing
NOTE: The remaining tumor tissues were used for seed preservation, fixation, and DNA/RNA/protein freezing, respectively.
<ol>
	<li>Remove the saline from the tumor surface with a sterile gauze before placing it in the cryopreservation tube to ensure that the tumor surface is not excessively wet.</li>
	<li>Put four to six pieces of 2 mm x 2 mm tumor tissue in a 2 mL cell cryopreservation tube, add 1 mL of cryopreservation solution composed of 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) into the tube, put the tube in a gradient cooling box, freeze it at &#8722;80 &#176;C overnight, and finally, transfer it to liquid nitrogen.</li>
	<li>Place the 3 mm x 3 mm tumor tissue blocks in 10% buffered formalin for tissue fixation for pathological examination.</li>
	<li>Put the 3 mm x 3 mm tissue block into a 2 mL cell cryopreservation tube, quickly freeze it in liquid nitrogen, and then transfer to a &#8722;80 &#176;C refrigerator for DNA/RNA and protein extraction.</li>
	<li>Collect the clinical information of the patients, such as the smoking history, tumor size, differentiation, pathological subtype, cancer grade, cancer stage, distant metastasis, origin, medical history, immunohistochemistry, human papillomavirus (HPV) infection in HNSCC patients, and treatment medication.</li>
</ol>
5. Passaging, cryopreservation, and resuscitation of PDX model tumors
<ol>
	<li>Measure the length and width of the subcutaneous tumors in mice by using vernier calipers once a week, and calculate the tumor volume according to the formula: tumor volume = 0.5 &#215; length &#215; width2. Draw the tumor growth curve.</li>
	<li>When the PDX tumor reaches 2,000 mm3, passage it to the next generation of mice, and perform tumor re-transplantation. Perform the preparation of the instruments following step 4.</li>
	<li>Euthanize the mice by cervical dislocation after anesthetizing with 80 mg/kg ketamine.</li>
	<li>Disinfect the skin with 75% alcohol. Then, cut the skin surrounding the tumor using scissors, then remove the tumor with forceps, and place it in a Petri dish.&#160;</li>
	<li>Perform the tumor transplantation procedure following step 3.</li>
	<li>Perform the preservation and cryopreservation of the PDX model tumors following step 4.</li>
	<li>For the resuscitation of the tumor tissue, follow the principle of slow freezing and quick dissolving. After taking out the cryovials from liquid nitrogen, quickly place them in a water bath at 37 &#176;C for rapid dissolving.</li>
	<li>Gently shake the cryovials in the water bath to accelerate the thawing process.</li>
	<li>Thaw, transfer the tumor pieces to the prepared normal saline for washing, and then inoculate the next generation of mice. For the specific operation, please see the tissue transplantation procedure in step 3.</li>
</ol>
6. Determining the therapeutic efficacy of lenvatinib and cisplatin in the ATC PDX model
NOTE: The ATC PDX model was used to test the therapeutic effect of the tyrosine kinase inhibitor lenvatinib and the chemotherapeutic drug cisplatin25,26,27.
<ol>
	<li>Select the P5 generation tumor tissue of an ATC PDX model (THY-017), cut into 2-4 mm3 tissue pieces, and inoculate subcutaneously (step 3) to the right back of ten 4-6 week female Balb/c nude mice.</li>
	<li>Select 15 mice with tumor volumes between 50-150 mm3, and divide them into three groups.</li>
	<li>Administer lenvatinib (10 mg/kg) intragastrically to one group once daily for 15 days, administer cisplatin (3 mg/kg) intraperitoneally to one group every 3 days for a total of six doses, and administer the control group with the same volume of normal saline.</li>
	<li>Measure the body weight and tumor volume of the mice twice per week.</li>
	<li>At the end of the test, euthanize the mice (step 5.3), and weigh the tumors.</li>
</ol>

No potential conflicts of interest are disclosed.

This study has successfully established the subcutaneous PDX models of ATC and HNSCC. There are many aspects to pay attention to during the process of PDX model construction. When the tumor tissue is separated from the patient, it should be put into the ice box and sent to the laboratory for inoculation as soon as possible. After the tumor arrives at the laboratory, the operator must pay attention to maintaining a sterile field and practice aseptic procedures. For needle biopsy samples, because the tumor tissue is partic...

The PDX model is an animal model in which human tumor tissue is transplanted into immunodeficient mice and grows in the environment provided by the mice1. Traditional tumor cell line models suffer from several disadvantages, such as the lack of heterogeneity, the inability to retain the tumor microenvironment, the vulnerability to genetic variations during repeated in vitro passages, and the poor clinical application2,3. The main drawbacks of genetically engineered animal models are the potential loss of the genomic features of human tumors, the introduction of new unknown mutations, and the difficulty in identifying the degree of homology between mouse tumors and human tumors4. In addition, the preparation of genetically engineered animal models is expensive, time-consuming, and relatively inefficient4.
The PDX model has many advantages over other tumor models in terms of reflecting tumor heterogeneity. From the perspective of histopathology, although the mouse counterpart replaces the human stroma over time, the PDX model preserves the morphological structure of the primary tumor well. In addition, the PDX model conserves the metabolomic identity of the primary tumor for at least four generations and better reflects the complex inter-relationships between tumor cells and their microenvironment, making it unique in simulating the growth, metastasis, angiogenesis, and immunosuppression of human tumor tissue5,6,7. At the cellular and molecular levels, the PDX model accurately reflects the inter- and intra-tumor heterogeneity of human tumors, as well as the phenotypic and molecular characteristics of original cancer, including gene expression patterns, mutation status, copy number, and DNA methylation and proteomics8,9. PDX models with different passages have the same sensitivity to drug therapy, indicating that the gene expression of PDX models is highly stable10,11. Studies have shown an excellent correlation between the response of the PDX model to a drug and the clinical responses of patients to that drug12,13. Therefore, the PDX model has emerged as a powerful preclinical and translational research model, particularly for drug screening and clinical prognosis prediction.
Thyroid cancer is a common malignant tumor of the endocrine system and is a human malignancy that has shown a rapid increase in incidence in recent years14. Anaplastic thyroid carcinoma (ATC) is the most malignant thyroid cancer, with a median patient survival of only 4.8 months15. Although only a minority of thyroid cancer patients are diagnosed with ATC each year in China, the mortality rate is close to 100%16,17,18. ATC usually grows rapidly and invades the adjacent tissues of the neck as well as the cervical lymph nodes, and about half of the patients have distant metastases19,20. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and one of the leading causes of cancer deaths, with an estimated 600,000 people suffering from HNSCC each year21,22,23. HNSCC includes a large number of tumors, including those in the nose, sinuses, mouth, tonsils, pharynx, and larynx24. ATC and HNSCC are two of the main head and neck malignancies. In order to facilitate the development of novel therapeutic agents and personalized treatments, it is necessary to develop robust and advanced preclinical animal models such as PDX models of ATC and HNSCC.
This article introduces detailed methods for establishing the subcutaneous PDX model of ATC and HNSCC, analyzes the key factors affecting the tumor take rate in model construction, and compares the histopathological characteristics between the PDX model and the primary tumor. Meanwhile, in this work, in vivo pharmacodynamic tests were performed using the successfully constructed PDX models in order to validate their clinical relevance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.4 mm x 2.0 mm trocar</td>
			<td>Shenzhen Huayang Biotechnology Co., Ltd</td>
			<td>18-9065</td>
			<td></td>
		</tr>
		<tr>
			<td>Balb/c nude mice</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td>
			<td>401</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Suzhou Antai</td>
			<td>BSC-1300IIA2</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Shenzhen Huayang Biotechnology Co., Ltd</td>
			<td>18-0823</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube&#160;</td>
			<td>Corning</td>
			<td>430791/430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryopreservation tube</td>
			<td>Chengdu Dianrui Experimental Instrument Co., Ltd</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol HTK-Solution</td>
			<td>Custodiol</td>
			<td>2103417</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>SIGMA-ALORICH</td>
			<td>D5879-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>METTLER</td>
			<td>ME104</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic digital caliper</td>
			<td>Chengdu Chengliang Tool Group Co., Ltd</td>
			<td>0-220</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum(FBS)</td>
			<td>VivaCell</td>
			<td>C04001-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IBM SPSS Statistics 26</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd&#160;</td>
			<td>100761663</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenvatinib</td>
			<td>ApexBio</td>
			<td>A2174</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD SCID immunodeficient mice</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td>
			<td>406</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep Solution</td>
			<td>Biological Industries</td>
			<td>03-03101BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>WHB</td>
			<td>WHB-60/WHB-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline&#160;</td>
			<td>Sichuan Kelun</td>
			<td>W220051705</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Shenzhen Huayang Biotechnology Co., Ltd</td>
			<td>18-0110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Shenzhen Huayang Biotechnology Co., Ltd</td>
			<td>18-1241</td>
			<td></td>
		</tr>
		<tr>
			<td>Vet ointment</td>
			<td>Pfizer Inc.</td>
			<td>P10015353</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Dunhua Shengda Animal Medicine Co., Ltd</td>
			<td>070031777</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A total of 18 thyroid cancer specimens were transplanted, and five PDX models of thyroid cancer were successfully constructed (27.8% tumor take rate), including four cases of undifferentiated thyroid cancers and one case of anaplastic thyroid cancer. The correlation between the success rate of model construction and the age, gender, tumor diameter, tumor grade, and differentiation were analyzed. Although the model success rate of grade 4 tumor samples was higher than for samples with lower grades, and the success rate of...

Watch this Scientific Journal Video about Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma at JoVE.com

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

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 ...

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1.5K Views.  Sichuan University. 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.

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

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 ...

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.

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 ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.