Myxoma virus oncolysis of primary and metastatic B16F10 mouse tumors in vivo

Abstract

Myxoma virus (MV) is a rabbit-specific poxvirus, whose unexpected tropism to human cancer cells has led to studies exploring its potential use in oncolytic therapy. MV infects a wide range of human cancer cells in vitro, in a manner intricately linked to the cellular activation of Akt kinase. MV has also been successfully used for treating human glioma xenografts in immunodeficient mice. This study examines the effectiveness of MV in treating primary and metastatic mouse tumors in immunocompetent C57BL6 mice. We have found that several mouse tumor cell lines, including B16 melanomas, are permissive to MV infection. B16F10 cells were used for assessing MV replication and efficacy in syngeneic primary tumor and metastatic models in vivo. Multiple intratumoral injections of MV resulted in dramatic inhibition of tumor growth. Systemic administration of MV in a lung metastasis model with B16F10LacZ cells was dramatically effective in reducing lung tumor burden. Combination therapy of MV with rapamycin reduced both size and number of lung metastases, and also reduced the induced antiviral neutralizing antibody titres, but did not affect tumor tropism. These results show MV to be a promising virotherapeutic agent in immunocompetent animal tumor models, with good efficacy in combination with rapamycin.

Figure 1. Permissiveness of myxoma virus (MV) in selected murine tumor cell lines

(a) Single-step growth analysis of vMyxlac was evaluated on B16F10 (closed triangles), 4T1 (open circles), and MT1A2 (open squares) murine tumor cells. All growth analyses were performed in trip-licate. (b) Early viral protein expression was determined by the expression of M-T7, and late protein expression by the expression of Serp-1 at the indicated time points by Western blot analysis of cellular super-natants. M indicates mock infection. (c) The levels of whole Akt protein and of phosphorylated Akt (at serine 473, S473 and threonine 308, T308) were detected in cellular lysates by Western blot analysis. (d) The effect of Akt inhibitors on MV replication in B16F10 cells. The cells were either mock-treated or pre-incubated with the indicated inhibitors Akt X (10 μmol/l; squares) or Akt VIII (5 μmol/l; closed triangles) for 4 hours prior to vMyxgfp infection. Ffu, focus forming units.

Figure 2. Effectiveness of myxoma virus (MV) treatment in a primary tumor model of B16F10

Pre-infected cells do not form tumors. (a) B16F10 cells were pre-infected with increasing doses of vMyxgfp in vitro prior to subcutaneous injection, and their ability to form tumors was evaluated over time by caliper measurement of tumor growth. The cells were either treated with no virus (closed squares) or 10⁷ focus forming units (ffu) (closed diamonds) of MV for 1 hour prior to injection. (b) Dose–response relating to pre-treatment of cells that were untreated (closed squares), or received either 10⁵ ffu (closed triangles) or 10⁶ ffu (closed circles) of MV 1 hour prior to injection. (c) Treatment of small tumors with vMyxgfp. The recipient mice were injected with B16F10 cells, and then either left untreated (closed squares), or treated with 3 × 10⁸ ffu of vMyxgfp intratumorally daily. The tumors were monitored for size by caliper measurement. (d) Deposition of extracellular matrix prevents dissemination of vMyxgfp. Tumors were sectioned and stained with the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI), for fibrin (MSB), the viral antigen M-T7,or stained with hemotoxylin and eosin (H&E). Magnification, ×50. MSB, Martius yellow-brilliant crystal scarlet-soluble blue.

Figure 3. Myxoma virus expressed enhanced green fluorescence protein (EGFP) localization within the lungs after systemic virus delivery

B16F10LacZ cells were injected intravenously (IV) into recipient C57BL6 mice. Seven days later, vMyx135KO virus was injected IV. Twenty four hours later, and the lung removed. (a) The expression of virally expressed EGFP was visualized under a fluorescent microscope, and (b) the tumor was visualized from the expression of brown melanin. The tumor is outlined for clarity.

Figure 4. Systemic administration of vMyx135KO virus prevents development of lung metastases of B16F10LacZ cells

B16F10LacZ cells were injected intravenously (IV) into recipient C57BL6 mice. Some of the cells were pre-infected with vMyx135KO virus 1 hour prior to injection into mice. Mice were infected with vMyx135KO virus on day 3 [myxoma virus (MV) 1× IV] or on days 1, 3, and 8 (MV 3× IV). At 14 days after injection, the lungs were collected and stained with an X-gal staining solution. The next day, (a) the stained lungs were first photographed, and then the lobes were separated and each lung was separately observed under a dissecting microscope; and (b) the stained surface metastases were counted. The P values result from an unpaired t-test.

Figure 5. Rapamycin augments systemic myxoma virus treatment of B16F10-LacZ lung metastases

(a) Schematic representation of treatment schedule; (b) The stained lungs were photographed, the lobes were separated, and each lung was separately observed under a dissecting microscope and (c) the number of stained surface metastases counted. The results represent those from 9–14 animals per group. (d) Measurement of the mean size of tumors. Fixed lungs were placed between two slides, and the diameters of individual tumors were measured using OpenLab software. The measurements from each lung were compiled and analyzed. The results are from six animals per treatment. (e) Estimation of tumor burden. One lobe from each animal lung was removed prior to fixation and homogenized. The extent of β-galactosidase activity was determined using a colorimetric assay. The results are representative of six animals per group. The P values result from an unpaired t-test. Ffu, focus forming units. IP, intraperitoneal; IV, intravenous.

Figure 6. Rapamycin suppresses the antibody response to systemic injection of myxoma virus

(a) Western blot analysis. Protein lysate was tested from B16F10 cells that had been infected with a high multiplicity of infection (MOI of 3) of vMyx135KO virus for 48 hours. Mouse serum was diluted 1:500; each lane represents the serum from a single mouse. The results are representative of at least three independent experiments. Arrows represent bands of interest. (b) Viral neutralization assay. Mouse or rabbit sera were pre-incubated with vMyxlac at an MOI of 0.001. This mixture was then used to infect baby green monkey kidney (BGMK) cells, and the effect on viral titer was assessed by evaluating foci formation after 48 hours. The results represent one rabbit serum, and sera from five to six mice per group, and are presented as a percentage of the foci formed by virus untreated with serum.