The oncolytic effect of oHSV2 on 4T1 tumor cells is independent of the cell cycle, but oHSV2 increases the NK ratio in vivo.

<p>A) An cell cycle assay as described in the Methods section. Representative images of flow cytometry from the different groups are depicted. B) 4T1 cells were treated with oHSV2 or different doses of DOX for 24h. Cell cycle specificity analysis was performed using flow cytometry. C) The percentage of NK and Treg cells after oHSV2 or DOX treatment was assayed. Statistical analysis was performed using an unpaired Student’s t test: *, p<0.05; **, p<0.01; and ***, p<0.001.</p

Anticancer effect of DOX, oHSV1 and oHSV2 in 4T1 breast tumors.

<p>The mice bearing 4T1 tumors were treated with DOX, oHSV1 or oHSV2 as described in the Materials and Methods section. <b>A</b>) The tumor volume was measured every 4 days following treatments. The data are presented as the mean ± SEM (n = 12), p<0.001. <b>B</b>) The median survival times for the 3 groups are illustrated in Kaplan–Meier survival curves (n = 12). Median survival: Control, 28 days; DOX, 34 days, p = 0.0034; oHSV2, 34 days, p = 0.0009; and oHSV1, 31 days, p = 0.0043. <b>C</b>) The weight of the mice was measured every 4 days following treatments. The data are presented as the mean ± SEM (n = 12). ***, p<0.001 and ns, no significant differences. D) Schematic of the experimental design. Each spot represents one treatment.</p

Oncolytic spectrum of oHSV2.

<p><b>A</b>) oHSV2 and oHSV1 were used to infect human tumor cells, including BGC823, HT29, Krause, T47D, U2OS, CNE2Z, HuH7, PG and TSU, at the indicated MOIs and times. <b>B</b>) oHSV2 and oHSV1 were used to infect mouse tumor cells, including 4T1, GL261, TC-1, B16F10 and B16R, at the indicated MOIs and times. A and B were observed with an inverted phase contrast microscope at 100× objective magnification. <b>C</b>) U2OS cells were infected by oHSV2 at MOI = 1, and typical syncytia were observed at 100×, 200× and 400× objective magnification.</p

The cell viability of cancer cells was examined.

<p><b>A</b>) The 4T-1 cells were treated with oHSV1 or oHSV2 at the indicated MOIs for the indicated times. <b>B</b>) The B16R cells were treated with oHSV1 or oHSV2 at the indicated MOIs for the indicated times. <b>C</b>) The B16F10 cells were treated with oHSV1 or oHSV2 at the indicated MOIs for the indicated times. <b>D</b>) The 4T-1 cells were treated with oHSV2 of different MOIs for 24h. DOX was used as a positive control. Each value represents the mean ± SED of three independent samples.</p

<i>In vitro</i> comparison of oHSV2 with oHSV1.

<p>Both oHSV1 and oHSV2 induces necrosis in cancer cells. A) Flow cytometry analysis of cancer cell lines after oHSV2 or oHSV1 infection at the indicated MOIs for 24 h. B) The necrosis rates of the cancer cell lines were measured after oHSV2 or oHSV1 infection. Each value represents the mean ± SED of three independent samples. ns, no significant differences.</p

The primers used for the construction of pH2d34.5 and pdICP47H2 are shown, and the genome coordinates for the genomic sequences are indicated.

<p>The restriction sites used for the construction of the shuttle plasmids are indicated by bold italics.</p

Average CPE rates of cell lines. Cell lines were infected with oHSV1 or oHSV2 at MOI = 1 for 24 h.

<p>10 fields of vision under the microscope were calculated and each CPE rate were calculated in the formula: CPE rate  =  number of CPE cells/number of total cells. The total CPE cells and total cells in the 10 microscopic fields were showed next to the percentages. The CPEs were observed with an inverted phase contrast microscope at 200× objective magnification.</p