Protocol for Using Cultrex™ Basement Membrane Extract to Improve the Take and Growth of Xenografts in Mice

Introduction

One of the most challenging aspects of modeling human cancers in animals is the achievement of tumor growth of cancer cell lines, cancer stem cells, or tumor biopsies implanted in animal hosts. Most frequently, mice are used as recipients of human cancer cells or biopsy specimens, and the model is collectively known as a xenograft or tumorgraft. When a specific patient tumor sample is used in the xenograft, it is referred to as a patient-derived xenograft (PDX). PDXs involve the extraction of primary cells derived from tumors or small pieces of tumor tissue from human patients followed by implantation of these cells into the recipient animal. Although tumorgafts/PDXs were developed almost fifty years ago, positive results from grafting experiments remain uncertain. One promising advance was the finding that the co-injection of Basement Membrane Matrix, or Cultrex Basement Membrane Extract (BME), with tumor cells improved tumor take and growth. Currently, a number of cell lines have shown increased take and growth when mixed and co-injected with Cultrex BME.

Cultrex BME, Type 3 (R&D Systems, Catalog # 3632-010-02) is a version of BME that has been developed, produced, and qualified specifically for use in in vivo studies. Cultrex BME, Type 3 mimics the in vivo microenvironment, including increased stiffness, low glucose, and low pH, to improve take rate and growth of implanted cells for xenograft and tumorgraft models. The use of Cultrex BME, Type 3 may not entirely solve problematic or slow growing tumors, as other possible causes may contribute to difficulties in culture, such as the mouse strain and the tumor or cell processing technique chosen to perform the experiment. The following protocol and protocol tips have been designed to solve frequently asked questions from researchers working with stalled or slow growing xenografts.

Protocol Tips for Improved Initiation and Growth of Tumor Cells in Mice

1. Processing the starting material and improving reproducibility

 a. Tissue from tumor biopsies:

• It is extremely important that all surgically removed tissue or biopsy material is used as soon as possible after harvest, within 24 hours. For improved take and growth, keep transport material at 2–8 °C in tissue culture medium, without serum, containing 1–5% Cultrex BME, Type 3.

• Using a scalpel or razor, finely mince the biopsy to about 1 mm cubes.

• Per injection site, implant 1 to 10 cubes of tissue suspended in BME using a trocar or an 18 gauge needle. Take into consideration that only tumor material will grow, implanted healthy normal cells do not proliferate. Also note that the growth takes place along the cut edges, so smaller tumor pieces are preferred to larger ones.

b. Cancer stem cells and primary or established tumor cell lines:

• If a single cell suspension is injected, perform a quick trypan blue test to check the viability of the cells. This will ensure that the culture is exponentially growing and viable on the day of the experiment.

• Do not use the cells if they appear more than 80% confluent.

• Always passage the cells at least twice after thawing from liquid nitrogen before implantation.

• A considerable amount of material is lost between injections and during loading of each needle. Plan to grow twice as many cells as you calculate will be needed for implantation.

• Be sure the cells are passaged or fed the day before the injection.

• Do not over trypsinize the cells.

• Be prompt and inject the cells right after they are ready (i.e. have everything set up and ready to start once the suspension of cells is made). Cells lose viability if maintained as a suspension at low temperatures for long periods of time.

• Settling of cells occurs in the bottom of the tube, so mix the cells gently but thoroughly before loading the syringe each time.

2. Determining how many cells and how much volume to inject

The optimal amount of material or the number of cells injected will vary in every case, as the reported number of cells used ranges from 1 × 10⁶ – 1 × 10⁷ million cells. In Table 1, some examples from the literature are shown. It is also important to note that some specific cell types may be injected as a complex mix, for example, involving both tumor epithelial and healthy stromal cells. Always try to use published papers as a guide to determine the number of cells, location, and type of recipient mouse based on your needs.

The optimum number of cells to be injected is determined by three variables:

a. The type of tumor cell (slow growing vs. metastatic, with or without additional cells such as stromal cells)

b. Location of the injection (subcutaneous vs. orthotopic)

c. Type of mouse (low immunodeficiency-nude to high immunodeficiency-NSG™)

TABLE 1. Examples of Published Xenograft Models

3. Choosing the right implantation site

The site of injection is also important, but any location that suits your research is acceptable. Subcutaneous implantations on the flank of animals are the most common choice, as they are fast and easy. The back of the neck is highly vascular and is also a good location for injection. An orthotopic site might also be considered as more physiologically relevant.

a. Subcutaneous xenografts:

• A recommended site for cell injection is the upper region of the back near the neck as it contains a considerable amount of fat tissue. This is a highly vascularized area, good for tumor growth, and hard to reach for mice. We recommend injecting 100 to 300 μL of tumor cells or biopsy plus Cultrex BME, Type 3 with a final BME concentration of 7 to 10 mg/mL.

b. Orthotopic site (i.e. mammary fat pad, brain, pancreas, liver, etc.):

• Orthotopic sites may result in a more relevant model and may increase the chances of physiological metastasis. Inject a final volume of 20 to 100 μL of tumor cells or biopsy plus Cultrex BME, Type 3 with a final BME concentration of 7 to 10 mg/mL for optimal results.

4. Using Cultrex BME, Type 3 and co-injecting it with the cells

If this is the first time using Cultrex BME, Type 3 there are some important considerations to note before proceeding with the protocol.

• Cultrex BME is a soluble form of basement membrane purified from the Engelbreth-Holm-Swarm (EHS) tumor. This extract provides a natural extracellular matrix hydrogel that is liquid at 2–8 °C and polymerizes at 24–37 °C to form a reconstituted basement membrane.

• Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between epithelial, endothelial, muscle, or neuronal cells and their adjacent stroma and play an essential role in tissue organization by influencing cell adhesion, migration, proliferation, and differentiation.

• The major components of Cultrex BME include laminin, collagen IV, entactin, and heparan sulfate proteoglycans.

• Cultrex BME is sterile, mycoplasma-free and LDEV-free. Since Cultrex BME remains in a liquid form at 2–8 °C, but rapidly gels when the temperature rises, all steps of the protocol must be performed on ice, and all material should be pre-chilled and kept cold at all times. Partially gelled samples will be difficult to inject.

• Some cells grow better when co-injected with Cultrex BME because BME is prepared from a tumor and contains all the components found in authentic basement membrane matrix. Among these components are a series of growth factors and ligands that can stimulate growth and attachment of tumor cells.

5. Choosing the recipient mouse strain

If you are working with a cancer cell line that was never implanted in an animal before, it will be difficult to know which mouse strain will prove to be the best.

• A preliminary study including a few animals of different immunodeficient strains is always recommended.

• It is recommended to work with female mice aged 5–6 weeks, if grouped in cages, male mice may fight and compromise the implant. Female mice are easier to handle for the injection.

• Do not use older mice as tumors grow more slowly.

• If working with male tumors such as prostate, male mice must be used. Likewise, female cancers such as breast cancer require female mice. Some tumor cells such as MCF-7 require hormone supplementation.

6. Boosting growth with more Cultrex BME

About 15% of biopsy material and some slow growing tumors injected with Cultrex BME, Type 3 will “stall” or cease growth. If stalling of the xenograft is detected, one possible option is to re-inject BME to boost growth. The tumors can be rescued with a 50 to 100 μL injection of undiluted Cultrex BME, Type 3 into the edge of the tumor. This approach will successfully re-initiate tumor growth, but it may need to be repeated if a second stall occurs.

Protocol for Tumorgraft/Xenograft Injections Using Cultrex BME

1. Thaw a vial of Cultrex BME, Type 3 overnight at 2–8 °C (put the vial on ice in an ice bucket with a cover in the refrigerator), dispense in working aliquots for future use. Keep aliquots at ≤ –70 °C and avoid more than three thaw-freeze cycles.

2. The day before the injection, thaw an aliquot of Cultrex BME, Type 3 overnight at 2–8 °C. NOTE: If you must thaw the Cultrex BME on the day of the experiment, roll the vial between your hands and try to avoid warming up the BME too much as this will cause gelling. Immediately place the thawed vial of BME on ice.

3. Pre-chill on ice: tubes, cells, medium, syringes, needles, and all material that could come into contact with the BME to avoid polymerization.

4. Pellet the cells, or finely minced biopsy material, for 5 minutes in a tabletop centrifuge at room temperature. NOTE: You can prepare a tube of cells or tissue for multiple injections. Finely chopped biopsy material must fit in an 18 gauge needle.

5. Decant the supernate, tap the tip of the tube with your finger to disperse the pellet, and place the tube on ice.

Keep everything on ice for all future steps:

6. Note the total volume and add an equal amount of thawed Cultrex BME, Type 3 on ice. NOTE: Make up about 15% extra material as some is lost in the syringe and you want to be sure to have enough for each animal. Generally, use 6-8 mice per data point. NOTE: Avoid introducing bubbles into the Cultrex BME. As Cultrex BME is a viscous extract, try to manipulate it gently and always mix it slowly by inversion. After thawing and diluting the cells, mix well by inversion a few times.

7. Mix gently to avoid cell/tissue damage and bubbles. Load a pre-cooled syringe and immediately inject the cells/tissue into the animal.

8. To avoid settling of the cells in the syringe and gelling of Cultrex BME, Type 3, it is recommended to load each syringe with enough cells-BME volume to inject no more than two mice. NOTE: Between injections, we recommend inverting the syringe and trying to work as quickly as possible. Use a 25 gauge needle if possible as a smaller bore will produce less leaking. At this point, you do not need to worry about sterility. When injecting subcutaneously, use the full length of the needle so that the injected material is more distant from the entry hole.

9. As a general rule, use 100 to 300 μL per injection for a final concentration of 7–10 mg/mL of Cultrex BME, Type 3.

10. Hold the syringe in place for 10–20 seconds to allow the Cultrex BME, Type 3 to begin to gel. NOTE: Do not reuse the needle or the syringe as some gelling may have occurred. For the larger bore needle, wait longer for the gelling to occur. There will be a “bump” where the tumor cells are injected.

11. Gently remove the needle from the syringe with rotation to seal the hole. Some leaking may occur. NOTE: If female mice are used, they can be grouped in cages. Male mice will need to be caged individually.

12. Monitor tumor growth with calipers, measuring 1–3 times a week.

13. Once the cells begin to grow, they degrade the Cultrex BME, Type 3, and the initial “bump” of BME that appears right after the injection is replaced by the actual tumor.

14. Tumor growth may be observable as early as 10 days or could take up to 1 month.