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 c...

<ol>
	<li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> Ca-a Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Sugano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+gastric+cancer+in+Asia.">Screening of gastric cancer in Asia.</a> Best Practive &amp; Research in Clinical Gastroenterology. 29 (6), 895-905 (2015).</li><li>Nikfarjam, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+survey+of+four+thousand+patients+with+10+common+cancers+in+North+Eastern+Iran+over+the+past+three+decades.">Demographic survey of four thousand patients with 10 common cancers in North Eastern Iran over the past three decades.</a> Asian Pacific Journal of Cancer Prevention. 15 (23), 10193-10198 (2014).</li><li>Coccolini, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+gastric+cancer:+What+we+know+and+what+we+still+have+to+learn.">Advanced gastric cancer: What we know and what we still have to learn.</a> World Journal of Gastroenterology. 22 (3), 1139-1159 (2016).</li><li>Goetze, O. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+treatment+in+locally+advanced+gastric+cancer.">Multimodal treatment in locally advanced gastric cancer.</a> Updates in Surgery. 70 (2), 173-179 (2018).</li><li>Graziosi, L., Marino, E., Donini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+Treatment+of+Locally+Advanced+Gastric+Cancer:+Will+the+West+Meet+the+East?.">Multimodal Treatment of Locally Advanced Gastric Cancer: Will the West Meet the East?.</a> Annals of Surgical Oncology. 26 (3), 918 (2019).</li><li>Choi, Y. Y., Noh, S. H., Cheong, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Gastric+Cancer+Treatment:+From+the+Golden+Age+of+Surgery+to+an+Era+of+Precision+Medicine.">Evolution of Gastric Cancer Treatment: From the Golden Age of Surgery to an Era of Precision Medicine.</a> Yonsei Medical Journal. 56 (5), 1177-1185 (2015).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCGA+divides+gastric+cancer+into+four+molecular+subtypes:+implications+for+individualized+therapeutics.">TCGA divides gastric cancer into four molecular subtypes: implications for individualized therapeutics.</a> Chinese Journal of Cancer Research. 33 (10), 469-470 (2014).</li><li>Tirino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+New+in+Gastric+Cancer:+The+Therapeutic+Implications+of+Molecular+Classifications+and+Future+Perspectives.">What's New in Gastric Cancer: The Therapeutic Implications of Molecular Classifications and Future Perspectives.</a> International Journal of Molecular Sciences. 19 (9), (2018).</li><li>Roschke, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotypic+complexity+of+the+NCI-60+drug-screening+panel.">Karyotypic complexity of the NCI-60 drug-screening panel.</a> Cancer Research. 63 (24), 8634-8647 (2003).</li><li>Wilding, J. L., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+for+drug+discovery+and+development.">Cancer cell lines for drug discovery and development.</a> Cancer Research. 74 (9), 2377-2384 (2014).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Research. 73 (17), 5315-5319 (2013).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+mouse+models:+A+high+fidelity+tool+for+individualized+medicine.">Patient-derived xenograft mouse models: A high fidelity tool for individualized medicine.</a> Oncology Letters. 17 (1), 3-10 (2019).</li><li>Lai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+perspectives+of+patient-derived+xenograft+models+in+cancer+research.">Current status and perspectives of patient-derived xenograft models in cancer research.</a> Journal OF Hematology &amp; Oncology. 10 (1), 106 (2017).</li><li>Kawaguchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Update+of+Patient-Derived+Xenograft+Model+for+Translational+Breast+Cancer+Research.">Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research.</a> Journal of Mammary Gland Biology and Neoplasia. 22 (2), 131-139 (2017).</li><li>Cassidy, J. W., Caldas, C., Bruna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+Tumor+Heterogeneity+in+Patient-Derived+Tumor+Xenografts.">Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts.</a> Cancer Research. 75 (15), 2963-2968 (2015).</li><li>Liu, X., Meltzer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+Cancer+in+the+Era+of+Precision+Medicine.">Gastric Cancer in the Era of Precision Medicine.</a> Cellular and Molecular Gastroenterology and Hepatology. 3 (3), 348-358 (2017).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human cancer growth and therapy in immunodeficient mouse models. (7), 694-708 (2014).</li><li>McDermott, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+cord+blood+engraftment+between+immunocompromised+mouse+strains.">Comparison of human cord blood engraftment between immunocompromised mouse strains.</a> Blood. 116 (2), 193-200 (2010).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD/SCID/gamma+(c)+(null)+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/gamma (c) (null) mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Wege, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-transplantation+of+human+hematopoietic+stem+cells+and+human+breast+cancer+cells+in+NSG+mice:+a+novel+approach+to+generate+tumor+cell+specific+human+antibodies.">Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: a novel approach to generate tumor cell specific human antibodies.</a> MAbs. 6 (4), 968-977 (2014).</li></ol>

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 &#956;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&#38;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&#215;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. ...

Patient-derived Xenograft Models and Primary Cell Lines are becoming an integral part of drug development and the initiation and progression of tumor biology research. Patient-derived Xenograft Models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. His models are for more accurate representations of human cancer then traditional cancer cell lines and have the potential to improve the preclinical evaluation of novel anticancer therapies.In addition, these models may be useful in comparing molecular characteristics or tumor signatures between different subgroups of cancer patients. NSG mice are recommended as immunodeficient mouse model. Due to the severe immunodeficiency of the mice, sterility must be maintained in all experiments.Under sterile conditions, use forceps and scissors to carefully dissect the tumor tissues and trim them into several small pieces. After anesthetizing the mouse, use sterile scissors to make a one centimeter incision on both dorsal flanks. Use sterile forceps to disrupt the subcutaneous tissue.Then, clip a piece of the tumor tissue, and place it into the deep site. Close the implant area by subcutaneous suture with surgical suture needles. Sterilize the wound with iodine.After this, gently place the mice in an empty cage while maintaining sternal recumbency. Pay close attention to the mice as they should wake up and begin walking after approximately three to four minutes. Place mice that underwent surgery in a new cage separated from those not subjected to surgery.Assess the tumor size by palpation of the implantation site and measure them with a Vernier caliper twice a week. After euthanizing the mice, use sterile forceps and scissors to slowly isolate the tumor from the mice. Wash the tumor tissues in DPBS in a 10 centimeter dish.You forceps and scissors to dissect and remove necrotic areas, fatty tissue, blood clots and connective tissue. Next, use a mold to cut the tumor tissues to a maximum thickness of one millimeter. Wash the tumor slices with DPBS in a 10 centimeter dish.To begin the vitrification process, use forceps to transfer the slices into tube V1 and incubate at four degrees Celsius for four minutes. Roll and invert the tube briefly and incubate at four degrees Celsius for another four minutes. Next, pour the V1 solution and slices into a 10 centimeter dish and use forceps to transfer the slices into tube V2.Incubate at four degrees Celsius for four minutes.Roll and invert the tube briefly and incubate it at four degrees Celsius for another four minutes. After this, pour the V2 solution and the slices into a 10 centimeter dish. Transfer the slices into tube V3 and incubate at four degrees Celsius for five minutes.Roll and invert the tube briefly and incubate at four degrees Celsius for another five minutes. Make sure that all of the slices sink to the bottom of the tube. If several slices remain floating, roll and invert the tube briefly and incubate at four degrees Celsius until all of the slices sink completely.Then, pour the V3 solution and slices into a 10 centimeter dish. Cut the tissue holders to the proper length and place them on sterile gauze. Transfer the slices onto the holders.Wrap the holders with gauze. Using forceps, place the holders in liquid nitrogen and let them incubate for five minutes. After this, label the cryogenic vials with the tissue information.Transfer the holders with tissue slices into the vials, which will be stored in liquid nitrogen. First, resect gastric cancer samples from resected specimens or harvested PDX tissues. Place the tissues on ice and transfer them to a 10 centimeter sterile culture dish.Use forceps and scissors to remove necrotic areas, fatty tissue, blood clots and connective tissue. Wash the tumor tissues once with DPBS containing penicillin and streptomycin in a 10 centimeter dish. Next, cut the tumor into pieces on the lid of the dish.The maximum thickness of each piece should be one millimeter. Transfer the pieces into a 50 milliliter centrifuge tube containing seven milliliters of a Type 1 collagenase and trypsin solution. Vortex the mixture briefly.Incubate in a water bath at 37 degrees Celsius for 30 to 40 minutes, making sure to vortex the mixture every five minutes. After this, add an equal volume of RPMI-1640 medium supplemented with 10%FBS and vortex the mixture thoroughly. Transfer this mixture into a new 50 milliliter centrifuge tube by slow filtration through a 40 microliter filter.Centrifuge the filtrates at a speed between 113 and 163 times g for five to seven minutes at room temperature. Carefully remove this supernatant. Wash the pellet with five milliliters of PBS and carefully remove the supernatant.If the pellet is red, it contains erythrocytes. Gently resuspend the pellet with 500 microliters of red blood cell lysis buffer and incubate at room temperature for five minutes. Then, add five milliliters of PBS.Centrifuge at a speed between 113 and 163 times g for five to seven minutes at room temperature. Carefully remove the supernatant and resuspend the pellet with culture medium and transfer the mixture into a sterile 10 centimeter dish. Incubate the culture at 37 degrees Celsius with 5%carbon dioxide, making sure to replace the medium with serum-containing medium every two to three days.In this study, gastric cancer PDX models and primary cell lines are established. First-generation tumors are seen to grow more slowly than those in later generations, taking three weeks or longer to reach the appropriate size. The success rate of first-generation subcutaneous tumor formation is over 80%The identity of the cancer cells from PDX models is confirmed from PDX models by H&E staining.The success rate of tumor formation from cryopreserved tumor tissue is seen to be approximately 95%Tumor cells can be easily recognized by differences in morphology. The primary cells are authenticated independently by two pathologists under a microscope. For further confirmation, H&E staining is used to observe cancer cell morphology after fixation.The rate of successful isolation of primary cell lines is approximately 40%These models have been shown to be predictive of clinical outcomes and are being used for preclinical drug evaluation by a marker identification, biological studies, and personalized medicine strategies.

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9.6K ビュー.  Sun Yat-sen University Cancer Center. 患者由来の異種移植片モデルと原細胞株は、医薬品開発の不可欠な部分となり、腫瘍生物学研究の開始と進行を行っています。患者由来の異種移植片モデルは、癌細胞の組織学的外観を維持し、腫瘍内不均一性を保持し、腫瘍微小環境の関連するヒト成分をよりよく反映する。彼のモデルは、ヒト癌の、そして伝統的な癌細胞株のより正確な表現のためのものであり、?...