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 &#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. ...

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9.7K 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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

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 ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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 ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

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 ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...