Name: establishment of gastric cancer patient-derived xenograft models and primary cell lines
Uploaded: 2019-07-19T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines at JoVE.com

This work was supported by the National Natural Science Foundation of China (81572392); the National Key Research and Development Program of China (2016YFC1201704); Tip-top Scientific and Technical Innovative Youth Talents of Guangdong Special Support Program (2016TQ03R614).
We specifically thank Guangzhou Sagene Biotech Co., Ltd. for aid in the preparation of the figures.

This human study was approved by the Institutional Ethics Review Board of Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China). The animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.&#160;Note: all experiments were performed in compliance with the relevant laws and institutional guidelines, including the Guideline for occupational exposure protection against blood-borne pathogens.
1. Sample preparation
<ol>
	<li>Obtain gastric cancer tissues (P0 = passage 0) directly from the operation. The tumor specimen should be larger than 0.5 cm3.</li>
	<li>Prepare 3-4 mL of stock solution: for example, RPMI-1640 medium (1x) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin.</li>
	<li>Put the fresh tumor specimen in the stock-solution at 4 &#176;C for no more than 4 hours.</li>
</ol>
2. Establishment of PDX model (Figure 1)
<ol>
	<li>To ensure a sterile surgical area, disinfect all materials with ultraviolet light for more than 30 min before transfer into the animal laboratory. 
	Note: the operation room need to be disinfected with ultraviolet light for&#160; 30 min, before we clean the desk with dichloro isocyanuric acid sodium suppositoria disinfectant. Then povidone-iodine is used to disinfect the skin.</li>
	<li>Using forceps and scissors, carefully dissect the tumor tissues and trim them into several small pieces (approximately 1 mm3 cubes) under sterile conditions.</li>
	<li>Anesthetize female 5- to 7-week-old NOD-SCID-IL2rg (NSG) mice by exposure to 1-1.5% isoflurane with an anesthesia machine.
	<ol>
		<li>Anesthetize the mouse until it stops struggling but maintains even breathing. 
		NOTE: The 50 mL tube is a simple equipment that functions similar to an anesthesia machine. The use of veterinary eye ointment to prevent dryness is unnecessary due to the short operation time.</li>
	</ol>
	</li>
	<li>Make a 1 cm incision on both dorsal flanks using sterile scissors, and implant one tumor piece into each flank of the mouse.
	<ol>
		<li>To ensure that the tumor piece does not slip out, use sterile forceps to disrupt the subcutaneous tissue. Then, clip a piece of the tumor tissue, and place it into the deep site.</li>
	</ol>
	</li>
	<li>Close the implant area by subcutaneous suture with surgical suture needles, and mark the mouse ears with labeled ear tags</li>
	<li>Sterilize the wound with iodine.</li>
	<li>Gently place the mice in an empty cage after surgery while maintaining sternal recumbency. Pay close attention to the condition of the mice; they wake up and begin to walk approximately 3-4 min later. Place mice that underwent surgery in another new cage separated from those not subjected to surgery.</li>
	<li>Assess tumor size by palpation of the implantation site. Measure the tumors with a Vernier caliper twice a week.</li>
	<li>Once the tumor reaches 10 mm in diameter, the animal condition worsens, or the tumor ulcerates, euthanize the mouse with an IRB approved method. Reimplant harvested fresh tumor fragments into 2 new mice for passaging, or temporarily store the specimens in PBS on ice for the isolation of primary cell lines.</li>
</ol>
3. Tissue cryopreservation
NOTE: This part primarily references the methods for the Live Tissue Kit Cryo Kit. The main kits and equipment are listed in the Table of Materials.
<ol>
	<li>Euthanize the mice with an IRB approved method when the tumor is greater than 10 mm in diameter.</li>
	<li>Using sterile forceps and scissors, slowly isolate the tumor from the mice.</li>
	<li>Wash the tumor tissues with DPBS in a 10 cm dish. Dissect and remove necrotic areas, fatty tissue, blood clots and connective tissue with forceps and scissors.</li>
	<li>Cut the tumor tissues to a maximum 1 mm thickness with a mold.</li>
	<li>Wash the slices with DPBS in a 10 cm dish. 
	NOTE: The vitrification process involves the use of tubes labeled V1/V2/V3 in steps 3.6-3.8. The main ingredients are DMSO and sucrose.</li>
	<li>Transfer the slices into tube V1 with forceps, and incubate the tube at 4 &#176;C for 4 min. Roll and invert the tube briefly, and place it at 4 &#176;C for another 4 min.</li>
	<li>Pour the V1 solution and slices into a 10 cm dish. Transfer the slices into tube V2 with forceps, and incubate tube V2 at 4 &#176;C for 4 min. Roll and invert the tube briefly, and place it at 4 &#176;C for another 4 min.</li>
	<li>Pour the V2 solution and slices into a 10 cm dish. Transfer the slices into tube V3 with forceps, and incubate the tube at 4 &#176;C for 5 min. Roll and invert the tube briefly, and place it at 4 &#176;C for at least 5 min. Make sure the slices all sink to the bottom of the tube.
	<ol>
		<li>If several slices remain floating, roll and invert the tube briefly, and place the tube at 4 &#176;C again until all slices sink completely; if necessary, discard the floating slices.</li>
	</ol>
	</li>
	<li>Pour the V3 solution and slices into a 10 cm dish.</li>
	<li>Cut the tissue holders to the proper length, and place them on sterile gauze. Transfer the slices onto the holders. Wrap the holders with the gauze, and place them in liquid nitrogen using forceps, followed by incubation for 5 min.</li>
	<li>Label the cryogenic vials with the tissue information.</li>
	<li>Transfer the holders with tissue slices into cryogenic vials, which are stored in liquid nitrogen.</li>
</ol>
4. Isolation of primary cells (Figure 2)
<ol>
	<li>Sterilize forceps and scissors with high pressure steam at 121 &#176;C for 30 min.</li>
	<li>Resect gastric cancer samples from resected specimens or harvested PDX tissues. Place the tissues on ice, and then, transfer the tissues to a 10 cm sterile culture dish.</li>
	<li>Dissect and remove necrotic areas, fatty tissue, blood clots and connective tissue with forceps and scissors.</li>
	<li>Wash the tumor tissues once with DPBS containing 100 U/mL penicillin and 0.1 mg/mL streptomycin in a 10 cm dish.</li>
	<li>Cut the tumor tissue into 1 mm3 pieces on the lid of the dish; the maximum thickness of each piece should be 1 mm.</li>
	<li>Transfer the tissues into a 50 mL centrifuge tube with approximately 7 mL of type 1 collagenase and trypsin (1:14) solution. Vortex the mixture briefly.</li>
	<li>Incubate the tube in a water bath at 37 &#176;C for 30-40 min. Vortex the mixture every 5 min.</li>
	<li>Add an equal volume of RPMI-1640 medium (1x) supplemented with 10% FBS to the tube. Vortex the mixture thoroughly.</li>
	<li>Transfer the mixture into a new 50 mL centrifuge tube by slow filtration through a 40 &#956;m filter. 
	NOTE: Use 40 &#956;m filters to ensure a higher ratio of cancer cells. If necessary, 100 &#956;m filters can be used to preserve more types of cells, such as immunological cells.</li>
	<li>Centrifuge the filtrates at 113-163 x g for 5-7 min at RT. Carefully remove the supernatant.</li>
	<li>Wash the pellet with 5 mL of PBS, and carefully remove the supernatant.</li>
	<li>If the pellet is red, it contains many erythrocytes. Gently resuspend the pellet with 500 &#956;L of red blood cell lysis buffer. After a 5 min incubation, add 5 mL of PBS, and carefully remove the supernatant.</li>
	<li>Resuspend the pellet with culture medium, and transfer the mixture to a sterile 10 cm dish.</li>
	<li>Replace the medium with serum-containing medium every 2-3 days.</li>
	<li>Passage the primary cells using trypsin/EDTA when they reach 50% confluence.</li>
</ol>

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The authors have nothing to disclose.

Gastric cancer is an aggressive disease with limited therapeutic options; thus, models of gastric cancer have become a critical resource to enable functional research studies with direct translation to the clinic4,8,17. Here, we have described the methods and protocol of establishing gastric cancer PDX models and primary cell lines. Importantly, both morphological and biological characteristics of gastric cancer specimens were m...

Gastric cancer is the fifth-most common cancer worldwide and the third leading cause of cancer death. In 2018, over 1,000,000 new cases of gastric cancer were diagnosed globally, and an estimated 783,000 people were killed by this disease1. The incidence and mortality of gastric cancer remain very high in northeastern Asian countries2,3. Despite significant progress in the field of cancer therapeutics, the prognosis of patients with advanced gastric cancer remains poor, with a five-year survival rate of approximately 25%4,5,6,7,. Thus, there is an urgent need for the development of new therapeutic strategies for gastric cancer
The treatment of gastric cancer is challenging because of its high heterogeneity8,9.&#160;Thus, the question of how to address the challenges of tumor heterogeneity to realize precision medicine is central to cancer research. In vitro and in vivo models play crucial roles in elucidating the heterogeneous mechanisms and biology of gastric cancer. However, although there are numerous gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these models limit their applications10. 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 humans11. These issues severely limit the possibility of identifying subgroups of cancer patients that will respond to targeted drugs. The short-term culture of primary tumors provides a relatively rapid and personalized way to investigate anticancer pharmacological properties, which will likely be the hallmark of personalized cancer treatment.
Patient-derived xenografts (PDXs) are preferred as an alternative preclinical model for drug response profiling12. In addition, PDX models offer a powerful tool for studying the initiation and progression of cancer13,14. PDX models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment15,16. However, the limitation of the widely used PDX models is the low success rate for establishing and serially propagating human solid tumors. In this study, decently successful methods for establishing PDX models and primary cell lines are described.

<table><tbody><tr><td>40 &amp;mu;m Cell Strainer</td><td>Biologix, Shandong, China</td><td>15-1040</td><td></td></tr><tr><td>Biological Microscope</td><td>OLYMPUS, Tokyo, Japan</td><td>OLYMPUS CKX41</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Mittelsachsen, Germany.</td><td>5427R</td><td></td></tr><tr><td>CO2 Incubator</td><td>Thermo Fisher Scientific, Carlsbad, California, USA</td><td>HERACELL 150i</td><td></td></tr><tr><td>DPBS</td><td>Basalmedia Technology, Shanghai, China</td><td>L40601</td><td></td></tr><tr><td>Electro-Thermostatic Water Cabinet</td><td>Yiheng, Shanghai, China</td><td>DK-8AXX</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Wisent Biotechnology, Vancouver, Canada</td><td>86150040</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter, China</td><td>CN2L9100</td><td></td></tr><tr><td>Live Tissue Kit Cryo Kit</td><td>Celliver Biotechnology, Shanghai, China</td><td>LT2601</td><td></td></tr><tr><td>Live Tissue Thaw Kit</td><td>Celliver Biotechnology, Shanghai, China</td><td>LT2602</td><td></td></tr><tr><td>NSG</td><td>Biocytogen, Beijing, China</td><td>B-CM-002-4-5W</td><td></td></tr><tr><td>Penicilin&amp;amp;streptomycin</td><td>Thermo Fisher Scientific, Carlsbad, California, USA</td><td>15140122</td><td></td></tr><tr><td>Red blood cell lysis buffer</td><td>Solarbio, Beijing, China</td><td>R1010</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Thermo Fisher Scientific, Carlsbad, California, USA</td><td>8118367</td><td></td></tr><tr><td>Surgical Suture Needles with Thread</td><td>LingQiao, Ningbo, China</td><td>3/8 arc 4&amp;times;10</td><td></td></tr><tr><td>Tissue-processed molds and auxiliary blades</td><td>Celliver Biotechnology, Shanghai, China</td><td>LT2603</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific, Carlsbad, California, USA</td><td>2003779</td><td></td></tr><tr><td>Type 1 collagenase</td><td>Thermo Fisher Scientific, Carlsbad, California, USA</td><td>17100017</td><td></td></tr></tbody></table>

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.

Here, tumor tissues from an operation were preserved in stock solution until the next step. Within 4 hours, tumor tissues were cut into small pieces and implanted into the dorsal flanks of NSG mice that had been anesthetized using isoflurane-soaked cotton. Tumors larger than 1 cm3 could be resected for implantation into new mice (Figure 1) or sliced carefully and preserved in liquid nitrogen following the protocol. In this study, the first-generation tumors grew more slowly than t...

Watch this Scientific Journal Video about Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines at JoVE.com

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

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. ...

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9.8K Views.  Sun Yat-sen University Cancer Center. 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.

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

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.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

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 ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Immunology and Infection

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

Medicine

Generating Human Gastric Organoids from Upper Endoscopy Biopsies

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Author Spotlight: Generation of and Comparison Between Patient-Derived Gastric Organoids from Different Regions of the Stomach 

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a ...