Advertisement

Reovirus therapy of lymphoid malignancies

Tommy Alain, Kensuke Hirasawa, Kelly J. Pon, Sandra G. Nishikawa, Stefan J. Urbanski, Yvonna Auer, Joanne Luider, Anita Martin, Randal N. Johnston, Anna Janowska-Wieczorek, Patrick W. K. Lee and Anna E. Kossakowska

Article Figures & Data

Figures

  • Fig. 1.

    Effect of reovirus on lymphoma cell lines in vitro.

    (A) Reovirus-induced cytopathic effects in various lymphoma cell lines in vitro. Established human Burkitt lymphoma (Raji, CA46, Daudi, Ramos, and ST486) and diffuse large B-cell NHL (OCY-LY1, OCY-LY2, OCY-LY8, and OCY-LY10) cell lines were exposed to reovirus at an MOI of 20 PFUs per cell, and photomicrographs were taken at 96 hours after infection. Cytopathic effects are apparent in all diffuse large B-cell NHL as well as the Raji and CA46 cell lines, but not in Daudi, Ramos, or ST486 cells. Original magnification × 200. (B) Remaining viability. Viability of the same cell lines at 96 hours after infection was assessed by staining the cells with 0.25% trypan blue; unstained (viable) cells from 3 independent wells were counted by means of a hemocytometer. As expected, there is good correlation between cell death and manifestation of the cytopathic effects shown in Figure 1A. ■ indicates control; and ░, reovirus.

  • Fig. 2.

    Reovirus replication in lymphoma cell lines in vitro.

    (A) Reovirus protein synthesis in infected Burkitt lymphoma and diffuse large B-cell NHL cell lines. Established human Burkitt lymphoma (Raji, CA46, Daudi, Ramos, and ST486) and the diffuse large B-cell NHL (OCY-LY1, OCY-LY2, OCY-LY8, and OCY-LY10) cell lines were infected with reovirus. The cells were pulse-labeled with [35S]-methionine for 6 hours at 66 and 90 hours after infection for the Burkitt lymphomas and diffuse large B-cell lymphomas, respectively. Following labeling, the cells were harvested and lysed, and reovirus proteins were immunoprecipitated from part of the lysate by means of rabbit polyclonal antireovirus antibodies. Immunoprecipitates were then analyzed by SDS-PAGE. Reovirus protein synthesis is apparent in all diffuse large B-cell lymphomas as well as in the Raji and CA46 cell lines, but not the Daudi, Ramos, or ST486 cell lines. The 3 size classes of reovirus proteins (λ, μ, and ς) are indicated on the left. (B) Viral progeny. Established Burkitt lymphoma and diffuse large B-cell NHL cell lines were infected with reovirus. At 0 and 96 hours after infection, the cells were harvested and freeze-thawed 3 times, and the virus titer in the lysate was determined by plaque assay on L929 cells. Error bars indicate standard deviations of the means from 3 separate wells.

  • Fig. 3.

    Effect of reovirus on lymphoid tumors in SCID/NOD mice.

    (A-B) Intratumoral reovirus therapy of lymphoid tumors in SCID/NOD mice. SCID/NOD mice were subcutaneously implanted with 1 × 107 cells of either Raji (A) or Daudi (B) Burkitt lymphoma cell lines. Following the establishment of a palpable mass, the tumors received (on day 0) a single intratumoral injection of 1 × 107 PFUs of live reovirus (n = 8; ●) or UV-inactivated virus (n = 7; ○). Tumor growth was followed for a period of 30 days and measured 2-dimensionally with a caliper. (C) Intravenous administration of reovirus to SCID/NOD mice bearing Raji tumors. Animals bearing palpable subcutaneous Raji tumors were subjected (on day 0) to a single intravenous injection of 1 × 107 PFUs (n = 7) or 5 × 107 PFUs (n = 7) of reovirus (● and ▴, respectively). Control animals (n = 7) received saline injection (○). Tumor growth was followed for a period of 20 days and measured 2-dimensionally with a caliper. (D) H&E staining and IH of reovirus antigens in Raji tumors after intravenous reovirus treatment. H&E-stained section (original magnification × 400) shows necrosis of tumor cells 20 days after live reovirus treatment. IH-stained section (original magnification × 400) of remaining tumor cells stains positively for reovirus proteins (brown) while control tumor shows no staining.

  • Fig. 4.

    Effect of reovirus on primary human lymphoid neoplasms.

    CLL indicates chronic lymphocytic leukemia; DLCBL, diffuse large B-cell lymphoma; BL, Burkitt lymphoma; and PBL, peripheral blood lymphocytes from healthy donors. (A) Reovirus replication in normal cells (lymphocytes, CD34+ cells) and primary human lymphoid neoplasms. Normal lymphocytes, normal bone marrow stem cells, and cell suspensions from primary lymphoma tumors were infected with reovirus. The cells were labeled with [35S]-methionine from 24 to 48 hours after infection. The cells were then lysed, immunoprecipitated with antireovirus antibody, and analyzed by SDS-PAGE. (B) Flow cytometry analysis. Representative histograms of viable (7AAD) CLL cells or Burkitt lymphoma cells after infection with 20 MOI of reovirus and staining with CD5-phycoerythrin (PE) and CD20–fluorescein isothiocyanate (FITC) (for CLL), or CD10-PE and CD20-FITC (for Burkitt lymphoma) are shown on the left. CLL cells coexpressing CD5 and CD20 were counted by means of Flow Count beads, and the results for 5 CLL samples are shown on the right. Burkitt lymphoma cells coexpressing CD10 and CD20 were counted similarly, and the results are shown. (C) Immunohistochemistry (IH) of reovirus antigens in virus-treated Raji and CLL cells. Cells were prepared and infected with reovirus as described in “Materials and methods.” Positive (brown) staining is observed in both Raji and CLL cells infected with reovirus, but not in the controls.

Tables

  • Table 1.

    Susceptibility to reovirus infection of ex vivo specimens of human non-Hodgkin lymphomas and chronic lymphocytic leukemias

    DiagnosisLymphoma typeReovirus-susceptible samples, no.Reovirus-resistant samples, no.
    CLL150
    NHLMantle cell10
    NHLBurkitt lymphoma10
    NHLDiffuse large B-cell20
    NHLSmall lymphocytic11
    NHLFollicular, grade I10
    NHLFollicular, grade II04
    NHLFollicular, gade III01
  • Table 2.

    Susceptibility of clonogenic CD34+ cells to reovirus infection

    CellsControlInfectedP
    CFU-GM71.5 ± 15.556.0 ± 3.5.0989
    BFU-Es37.2 ± 20.826.8 ± 0.4.3560
    CFU-GEMM13.8 ± 2.512.8 ± 2.5.3390
    • As described in “Materials and methods,” 1000 CD34+ cells per milliliter for CFU-GMs and BFU-Es, and 2000 CD34 cells per milliliter for CFU-GEMMs, were plated in methylcellulose clonogenic assays in duplicate. Data represent the means ± standard deviation (SD) of 2 separate experiments.