Susceptibility of breast cancer cells to an oncolytic matrix (M) protein mutant of vesicular stomatitis virus

Abstract

Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attractive candidates as oncolytic viruses for tumor therapies because of their capacity to selectively target cancer cells. The effectiveness of rM51R-M virus as an antitumor agent for the treatment of breast cancer was assessed by determining the ability of rM51R-M virus to infect and kill breast cancer cells in vitro and in vivo. Several human- and mouse-derived breast cancer cell lines were susceptible to infection and killing by rM51R-M virus. Importantly, non-tumorigenic cell lines from normal mammary tissues were also sensitive to VSV infection suggesting that oncogenic transformation does not alter the susceptibility of breast cancer cells to oncolytic VSV. In contrast to results obtained in vitro, rM51R-M virus was only partially effective at inducing regression of primary breast tumors in vivo. Furthermore, we were unable to induce complete regression of the primary and metastatic tumors when tumor-bearing mice were treated with a vector expressing interleukin (IL)-12 or a combination of rM51R-M virus and IL-12. Our results indicate that although breast cancer cells may be susceptible to VSV in vitro, more aggressive treatment combinations are required to effectively treat both local and metastatic breast cancers in vivo.

Figure 1

Single-cycle growth analysis of wt and M protein mutant viruses in non-tumorigenic and tumorigenic breast cancer cells. HME (a), HMLE (b) and HMLE-PR (c) cells were infected with rwt and rM51R-M viruses at a multiplicity of 10PFU per cell. A small aliquot of the supernatant was removed at the indicated times post-infection to determine the amount of progeny virus by plaque assay. Data are the average of two independent experiments.

Figure 2

Rates of host and viral protein synthesis in VSV-infected HME, HMLE and HMLE-PR cells. Cells were infected with rwt and rM51R-M viruses at a multiplicity of 10 or 0.1PFU per cell, or were mock infected as a control. Cells were labeled with a 15-min pulse of [³⁵S]methionine (100 μCi ml⁻¹) at 6-, 12- and 24-h post-infection. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and labeled proteins were quantitated by phosphorimaging. (a) Representative images of HME, HMLE and HMLE-PR cells infected with rwt and rM51R-M viruses at a MOI of 10PFU per cell. Positions of viral proteins are indicated on the left. (b) Rates of host protein synthesis (MOI = 10PFU per cell) were quantitated from images similar to that in panel (a). Results are shown as a percentage of the mock-infected control. Rates of viral protein synthesis were determined by quantitating labeled M proteins in cells infected at multiplicities of 10PFU per cell (c) and 0.1PFU per cell (d) and are expressed as the percentage of the rwt M protein in HME cells labeled at 12-h post-infection (MOI = 10PFU per cell). Data are the mean ± standard error of four experiments.

Figure 3

Viability of non-tumorigenic and tumorigenic breast cancer cells infected with rwt and rM51R-M viruses under singleand multiple-cycle infection conditions. HME, HMLE and HMLE-PR cells were infected with rwt and rM51R-M viruses at multiplicities of 10 (a) and 0.1PFU per cell (b). At 24- and 48-h post-infection, live cells were measured by MTT assay. Data are expressed as the percentage of the cell viability of mock-infected cells and represent the means ± s.d. of three experiments.

Figure 4

Sensitivity of human breast cancer cell lines to killing by VSV. MCF10A (a) and MCF7 (b) cells were infected with rwt and rM51R-M viruses at multiplicities of 10 and 0.1PFU per cell. At indicated times post-infection, live cells were measured by MTT assay. Data are expressed as the percentage of the cell viability of mock-infected cells and represent the means ± s.d. of three experiments.

Figure 5

Treatment of 4T1 tumors with M protein mutant VSV and IL-12. (a) 4T1 cells were infected with rwt and rM51R-M viruses at multiplicities of 10 and 0.1PFU per cell. Cell viability was measured at different times post-infection. Data are expressed as the cell viability of mock-infected cells. (b) 4T1 cells were injected subcutaneously in the flanks of BALB/c mice. Animals with palpable tumors were randomly separated into five experimental groups (5–10 animals per group) and were injected in the tumor with 1 × 10⁷ PFU of rM51R-M virus at days 1, 3 and 5, 50 μg of IL-12 plasmid at days 1 and 3, or PBS alone as a negative control (mock). Tumor volume was measured daily with calipers. Results are expressed as the change in tumor volume on treatment of mice with rM51R-M virus and/or IL-12. Data represent two separate experiments and are shown as the percentage of original tumor size on day 1 (mean ± s.e.). (c) Viral antigen staining in sections of 4T1 tumors from untreated mice or mice treated with rM51R-M virus and rM51R-M + IL-12.

Figure 6

IL-12 and IFNγ levels in the tumors and spleens of tumor-bearing mice treated with rM51R-M virus or IL-12 plasmid DNA. Tumors and spleens from tumor-bearing mice were harvested at days 7 and 14 post-treatment and assayed for the presence of IL-12 p40 (a and b) and IFNγ (c) by enzyme-linked immunosorbent assay (ELISA). Data represent the average ± s.d. of a total of 3–5 mice from two experiments.

Figure 7

Reduction of spontaneous metastases to the lungs of tumor-bearing mice. Lungs of treated mice were collected at day 14 post-treatment and examined for metastases. (a) Hematoxylin and eosin (H and E) staining of lung tissue from a mock-treated mouse. (b) Number of metastatic 4T1 cells in the lungs of mice treated with rM51R-M virus or IL-12. (c) Weight of lungs from tumor-bearing mice. Data is the average ± s.d. of 4–5 mice from two experiments.