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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 countries^2,3. 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 heterogeneity^8,9. 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 cancer^13,14. PDX models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment^15,16. 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.

Protokół

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. 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

1. Obtain gastric cancer tissues (P0 = passage 0) directly from the operation. The tumor specimen should be larger than 0.5 cm³.
2. 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.
3. Put the fresh tumor specimen in the stock-solution at 4 °C for no more than 4 hours.

2. Establishment of PDX model (Figure 1)

1. 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  30 min, before we clean the desk with dichloro isocyanuric acid sodium suppositoria disinfectant. Then povidone-iodine is used to disinfect the skin.
2. Using forceps and scissors, carefully dissect the tumor tissues and trim them into several small pieces (approximately 1 mm³ cubes) under sterile conditions.
3. Anesthetize female 5- to 7-week-old NOD-SCID-IL2rg (NSG) mice by exposure to 1-1.5% isoflurane with an anesthesia machine.
   1. 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.
4. Make a 1 cm incision on both dorsal flanks using sterile scissors, and implant one tumor piece into each flank of the mouse.
   1. 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.
5. Close the implant area by subcutaneous suture with surgical suture needles, and mark the mouse ears with labeled ear tags
6. Sterilize the wound with iodine.
7. 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.
8. Assess tumor size by palpation of the implantation site. Measure the tumors with a Vernier caliper twice a week.
9. 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.

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.

1. Euthanize the mice with an IRB approved method when the tumor is greater than 10 mm in diameter.
2. Using sterile forceps and scissors, slowly isolate the tumor from the mice.
3. 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.
4. Cut the tumor tissues to a maximum 1 mm thickness with a mold.
5. 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.
6. Transfer the slices into tube V1 with forceps, and incubate the tube at 4 °C for 4 min. Roll and invert the tube briefly, and place it at 4 °C for another 4 min.
7. 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 °C for 4 min. Roll and invert the tube briefly, and place it at 4 °C for another 4 min.
8. 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 °C for 5 min. Roll and invert the tube briefly, and place it at 4 °C for at least 5 min. Make sure the slices all sink to the bottom of the tube.
   1. If several slices remain floating, roll and invert the tube briefly, and place the tube at 4 °C again until all slices sink completely; if necessary, discard the floating slices.
9. Pour the V3 solution and slices into a 10 cm dish.
10. 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.
11. Label the cryogenic vials with the tissue information.
12. Transfer the holders with tissue slices into cryogenic vials, which are stored in liquid nitrogen.

4. Isolation of primary cells (Figure 2)

1. Sterilize forceps and scissors with high pressure steam at 121 °C for 30 min.
2. 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.
3. Dissect and remove necrotic areas, fatty tissue, blood clots and connective tissue with forceps and scissors.
4. Wash the tumor tissues once with DPBS containing 100 U/mL penicillin and 0.1 mg/mL streptomycin in a 10 cm dish.
5. Cut the tumor tissue into 1 mm³ pieces on the lid of the dish; the maximum thickness of each piece should be 1 mm.
6. 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.
7. Incubate the tube in a water bath at 37 °C for 30-40 min. Vortex the mixture every 5 min.
8. Add an equal volume of RPMI-1640 medium (1x) supplemented with 10% FBS to the tube. Vortex the mixture thoroughly.
9. Transfer the mixture into a new 50 mL centrifuge tube by slow filtration through a 40 μm filter.
   NOTE: Use 40 μm filters to ensure a higher ratio of cancer cells. If necessary, 100 μm filters can be used to preserve more types of cells, such as immunological cells.
10. Centrifuge the filtrates at 113-163 x g for 5-7 min at RT. Carefully remove the supernatant.
11. Wash the pellet with 5 mL of PBS, and carefully remove the supernatant.
12. If the pellet is red, it contains many erythrocytes. Gently resuspend the pellet with 500 μL of red blood cell lysis buffer. After a 5 min incubation, add 5 mL of PBS, and carefully remove the supernatant.
13. Resuspend the pellet with culture medium, and transfer the mixture to a sterile 10 cm dish.
14. Replace the medium with serum-containing medium every 2-3 days.
15. Passage the primary cells using trypsin/EDTA when they reach 50% confluence.

Wyniki

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...

Dyskusje

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 clinic^4,8,17. 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...

Materiały

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