Testing oncolytic myxoma virus in immunocompetent mouse model for cancer therapy

Abstract

Oncolytic viruses (OVs) have emerged as a class of novel cancer immunotherapeutic. Members of both DNA and RNA viruses developed as OVs for treating diverse types of human cancers. Preclinical research assessing immunotherapeutic efficacy is an essential step toward further development of these OVs. Mice tumor model systems are widely used in preclinical oncolytic viral therapies for evaluating the treatment regimens’ efficacy. However, choosing the most appropriate model for a study can be challenging. Here, we described a simple method of establishing subcutaneous tumors in immunocompetent mice for pre-clinical studies of oncolytic myxoma virus (MYXV).

1. Introduction

Oncolytic viruses (OVs) preferentially infect and kill cancer cells while sparing their normal cellular counterparts. Members from diverse families of viruses have been developed as OV[1–3]. Overall, OVs mediate antitumor activity through multiple mechanisms such as direct cytolytic effects (also known as oncolysis) on infected cancer cells, releasing of tumor-associated antigens, activation of the antitumor innate and adaptive immune response, recruitment of activated immune cells into the tumor microenvironment (TME) [4, 5]. Moreover, these antitumor activities can be further potentiated by making engineered OVs expressing different types of transgenes [6–8].

Oncolytic myxoma virus (MYXV) is a member of the Leporipoxvirus genus of the Poxviridae family of DNA viruses. The natural hosts of MYXV are rabbits of the Sylvilagus genus, such as the American brush rabbits and Brazilian tapeti, where the virus has co-evolved and only causes benign cutaneous lesions. In stark contrast to Sylvilagus rabbits, MYXV causes a lethal disease called myxomatosis in the Oryctolagus cuniculus (European rabbit) species [9, 10]. Because of the extreme lethality against European rabbits, MYXV was released in the 1950s to control the feral European rabbit populations in Australia and Europe [11, 12]. During this time, it was demonstrated that MYXV was nonpathogenic for all nonrabbit animals, including humans [12, 13]. However, the virus can infect and kill most cancer cell types originating from humans, mice, or other species [14–16]. Thus, the very restrictive natural host tropism of MYXV ensures its safety for oncolytic virotherapy in humans and preclinical cancer models without modifications. The oncolytic activity of MYXV has been tested in multiple animal models for cancers, including melanoma, gliomas, gallbladder cancer, pancreatic cancer, lung cancer, and hematological malignancies [14, 17–20]. These preclinical studies demonstrated the efficacy of oncolytic MYXV against diverse types of cancers.

Different types of preclinical models for determination of the oncolytic efficiency exist: syngeneic mouse tumor model, cell line derived xenografts, patient-derived xenografts, genetically engineered mouse model, humanized mouse model, and more recently, tumor spheroids/organoids [21, 22]. However, syngeneic mouse models are the most used in immuno-oncology [23]. These models are established by transplanting immortalized mouse cell lines or tumor tissues to the same immunocompetent mouse strain, which enables researchers to assess the impact of the oncolytic virus in the context of a complete immune response [24]. These models are easy to develop, are practical for screening many virus candidates, understanding their mechanisms of action, and can reflect the tumor microenvironment comprehensively. At the same time, the risk of tissue rejection is reduced [25]. Transplantable tumor cells may be injected either orthotopically (in the tissue/organ of the origin) or ectopically (not in the origin site), usually by subcutaneous injection in the hind flanks of the mice. Choosing the most appropriate tumor model for a specific research objective is essential for a successful study. Here, we describe a method for evaluating the oncolytic efficacy of MYXV using a subcutaneous tumor model in mice (Fig. 1). This method can be used for both immunocompetent and immunodeficient allografts or xenografts.

Fig. 1:

Diagram of experimental setup to test oncolytic MYXV in a B16-F10 immunocompetent murine melanoma tumor model. Bilateral flank tumor models are established by subcutaneously injecting C57BL/6 mice with 1×10⁶ B16-F10 cells in the right and left flanks. After tumors reach a volume of 50–100 mm³, virus is injected intratumorally only into one of the flanks. Measure tumors volume using a caliper. Euthanize mice when tumors reach the maximum volume or meet any other criteria based on the IACUC protocol.

2. Materials

2.1. General Reagents

1. 1x Phosphate buffer saline (PBS)

2. Fetal Bovine serum (FBS)

3. Dulbecco’s Modified Eagle (DMEM) medium

4. 1x Trypsin + 0.25% Ethylenediaminetetraacetic acid (EDTA)

5. 1x L-glutamine

6. 1x Penicillin and streptomycin solution

7. Cancer cells: In this protocol, we used murine B16F10 melanoma cells (ATCC, CRL-6475).

8. Complete DMEM media for growing and maintaining B16F10 cells: 1x DMEM supplemented with 10% FBS, 1x L-glutamine, and 1x penicillin and streptomycin

9. Oncolytic MYXV: In this protocol, we used a wild-type MYXV construct expressing reporter green fluorescence protein (GFP) under a poxvirus synthetic early/late promoter and TdTomato (TdT) Red protein under a poxvirus late promoter (vMyx-GFP-TdT) [26]. Virus stock is purified using sucrose gradient method [27].

10. Mouse strain: C57BL/6 mice 6–8 weeks old.

11. Trypan blue

2.2. Instruments and Materials

1. Water bath at 37°C

2. Cell counter or hemocytometer

3. Biosafety cabinet for cell culture

4. Incubator for cell culture

5. Tabletop centrifuge

6. Sterile syringes or insulin syringes (0.5 – 1 ml)

7. Vortex mixer

8. Microcentrifuge tubes (1.5 ml)

9. Centrifuge tubes (15 ml or 50 ml)

10. Sonicator for virus

11. Cell culture flasks/dishes

12. Digital caliper

13. Inverted microscope for cell culture

3. Methods

As an immunocompetent cancer model system, the murine B16F10 melanoma tumor derived from C57BL/6 mice is widely used for preclinical evaluation of anticancer therapies [28–31]. Here, to assess the therapeutic efficacy of oncolytic MYXV both in the treated tumor and the nontreated tumor, a bilateral flank model of B16F10 melanoma is established to study the local and abscopal effect. Once tumors are generated and treated with intratumoral injections of MYXV, both the treated and the untreated tumors are measured, as a readout for OV efficacy. Furthermore, these tumors can be analyzed for infiltration of active immune cells or histology (these methods are not described in this chapter).

C57BL/6 mice 6–8 weeks old are used for the experiments described in this protocol. Before any animal studies the protocol must be approved by the Institutional Animal Care and Use Committee (IACUC). After receiving mice from an approved vendor (Charles River or The Jackson Laboratory), mice are first acclimated in the animal facility for at least 1 week. The number of cancer cells implanted in the mice depends on the cancer model and cancer cell types. For subcutaneous tumors, 1×10⁵ to 1×10⁶ cancer cells are usually injected in one of the flanks. Before the cancer implantation, cryopreserved cancer cell lines should be grown in appropriate medium for few passages and check for mycoplasma contamination, proliferation and viability of cells.

3.1. Tumor implantation

• 1Maintain the B16-F10 cells in complete DMEM media (see Note 1).
• 2Two days prior to implantation, split cells and expand into several T175 cm² flasks or T150 cm² dishes to achieve 60–80% confluency on the day of implantation. The number of flasks/dishes depends on the number of mice to be implanted with cancer cells.
• 3On the day of implantation, remove growth media from the cells in culture and wash with 1x PBS.
• 4Aspirate PBS, add 3 ml of trypsin and incubate at 37 °C until the cells detach.
• 5Once the cells have lifted off, add 7 ml of 2% FBS containing growth medium and pipet up and down to get single cell suspension (see Note 2).
• 6Collect cells from all the flasks/dishes into a 50 ml conical tube and pellet the cells by centrifugation for 5 min at 250 × g at room temperature (see Note 3).
• 7Aspirate medium, wash cells by adding 10 ml of 1% PBS into the tube, pipette gently to resuspend the pellet.
• 8Count the viable cells on a hemocytometer or an automated cell counter using Trypan blue (see Note 4).
• 9Centrifuge the cell suspension for 5 min at 250 × g at room temperature.
• 10Aspirate PBS, resuspend cells in fresh 1% PBS in a volume that will give cells concentration of 1 ×10⁷ cells / ml. Count the viable cells again, to confirm desired cell concentration. This final cell suspension is stored on ice throughout the process of implantation (see Note 5).
• 11Transfer cells in sterile Eppendorf tubes.
• 12Use 0.5 – 1 ml insulin syringe to pull up and inject 1 × 10⁶ cells into the back flanks of the mice. To do this, by index finger and thumb pinch and pull skin of the mice away from its body. Inject smoothly into the pouch created by the fingers, this creates a bubble of cells under the skin. It is important to avoid spread of the cells beneath the skin. Repeat the injection on the other side of the flank and for all the mice (see Note 6).
• 13Place the mice back in the cage and allow them to recover.
• 14Monitor mice every two/three days by palpation until the tumor emerges. Typically, after 7–10 days post inoculation of 1 × 10⁶ tumor cells it is possible to measure tumor volume using a caliper.
• 15Using caliper to measure tumor progression is a simple way; width (W) and length (L) of the tumor is measured, then volume can be estimated by the formula: π/6 × W² × L or simply 1/2 × W² × L. These formulas derived from oblate spheroid volume formula [32] (see Note 7).

3.2. Intratumoral injection of tumors with virus

• 16While tumors reach a volume of around 50–100 mm³ randomize mice in among the different treatment cohorts, initially it is important that all groups to have same average of tumor volumes for a precise comparison.
• 17Thaw out the purified virus stock on ice and sonicate briefly for 30 seconds to break any clumps. Prepare a diluted virus stock with a titer between 1×10⁷–1×10⁸ FFU (Foci Forming Unit) in 50 μl of 1x PBS. However, the optimum dose of the virus to have a therapeutic effect should be determined by injecting different amounts of virus (see Note 8).
• 18For intratumoral injection in one of the tumors, hold the mice firmly, then directly inject the needle into the tumor and administer the virus. It has shown using multi side needle increase the outcome of the therapy comparing with conventional needles [33]. If using the conventional needles, it is better to inject the virus in multiple sites of the tumor.
• 19To improve therapy, it is recommended to repeat virus administration multiple times. Repeat steps 17 and 18 every 2–3 days for 3 additional times.
• 20Measure both tumors volume on every other day until the mice reach the maximum tumor volume permitted by the animal welfare regulations. Compare the tumor volumes of the treated group with the control mice which only had received PBS or unmodified viruses, or in case mice received tumor cells on both flanks, compare them with each other. Survival rates can be determined through this experiment, as well (see Note 9).