Mechanism of antitumor effect on mouse hepatocellular carcinoma by intratumoral injection of OK-432, a streptococcal preparation

Abstract

Intratumoral (i.t.) injection of OK-432, a streptococcal preparation, into implanted tumors of mouse hepatocellular carcinoma (MIH-2) showed antitumor effect including tumor eradication. Intraperitoneal administration of same dose OK-432 did not exhibit tumor suppressive effect. In vitro cytotoxic test suggested that direct cytotoxic effect of OK-432 was not associated with antitumor activity by i.t.-OK-432 treatment. It was also found that Toll-like receptor 4 signaling was not involved in i.t.-OK-432 treatment. Three mice out of five, which had shown tumor eradication by i.t.-OK-432 treatment did not reject re-challenge of MIH-2 cells. Splenocytes from i.t.-OK-432 treated mice did not produce IFN-gamma by stimulation with MIH-2 cells in vitro, but produced abundant IFN-gamma by stimulation with OK-432. Immunofluorescence microscopy demonstrated that CD4+T cells, but not CD8+T cells, infiltrated to i.t.-OK-432 treated tumor tissue produced IFN-gamma. Tumor-infiltrating CD4+T cells from i.t.-OK-432 treated tumor tissue produced IFN-gamma by in vitro stimulation with OK-432 higher than those from untreated tumor tissue. IFN-gamma directly induced apoptosis of MIH-2 cells in vitro. Collectively, i.t.-OK-432 treatment induced priming of CD4+T cells to antigenecity of OK-432, and repetitive i.t.-OK-432 treatment induced IFN-gamma production from OK-432-sensitized CD4+T cells in tumor site, leading to apoptosis of MIH-2 cells susceptible to IFN-gamma.

Fig. 1

Intratumoral injection of OK-432 (i.t.-OK-432 treatment) produced antitumor activity against MIH-2 tumor, but intraperitoneal injection of same dose OK-432 did not. a MIH-2 cells (10⁶/mouse) were implanted subcutaneously in male C3H/HeN mice. Three days after the implantation, 0.1 mg of OK-432 dissolved in 0.1 ml of PBS was injected into tumor site. Treatment with OK-432 was performed every 2 days, six times totally. Open circles indicate untreated control (n = 10) and close circles i.t.-OK-432 treatment (n = 10). Tumor size (long diameter × short diameter: mm²) was measured once a week. ^* p < 0.0001 repeated measures ANOVA. b MIH-2 cells (10⁶/mouse) were implanted subcutaneously in male C3H/HeN mice. Three days after the implantation, 0.1 mg of OK-432 dissolved in 0.1 ml of PBS was injected into peritoneal cavity. Treatment with OK-432 was performed every 2 days, six times totally. Open circles indicate untreated control (n = 14) and close circles i.p.OK-432 treatment (n = 9). ^** p = 0.94 repeated measures ANOVA

Fig. 2

Tumor suppressive effect of i.t.-OK-432 treatment was not mediated by direct toxicity of OK-432. a MIH-2 cells (10⁶/mouse) were implanted to C3H/HeN mice (n = 10), MC38 cells (10⁶/mouse) to C57BL/6 mice (n = 4), and C26 cells (3 × 10⁵/mouse) to BALB/c mice (n = 5). i.t.-OK-432 treatment and measurement of tumor size were performed in the same way as described in the legend of Fig. 1a. b MIH-2 cells, MC38 cells and C26 cells were cultured on six-well culture plates in the absence or presence of OK-432 (3, 10, 30, and 100 μg/ml). Number of trypan-blue non-dyable cells was counted after 48-h incubation (n = 3). Numbers in parentheses were percentage of cells with each treatment when cell number of untreated control cells was expressed as 100%

Fig. 3

TLR4 signaling stimulated with OK-PSA was not involved in antitumor activity by i.t.-OK-432 treatment. a MIH-2 cells (10⁶/mouse) were implanted to male C3H/HeN mice or TLR4 deficient C3H/HeJ mice. i.t.-OK-432 treatment and measurement of tumor size were performed in the same way as described in the legend of Fig. 1a. Open circles indicate untreated control C3H/HeN mice, closed circles i.t.OK-432 treated C3H/HeN mice, open square untreated control C3H/HeJ mice, and closed square i.t.OK-432 treated C3H/HeJ mice (n = 5). b Intratumoral injection of OK-PSA (0.1 mg/mouse) was performed in the same way as described in the legend of Fig. 1a. Open circles indicate untreated control C3H/HeN mice, closed circles i.t.OK-PSA treated C3H/HeN mice (n = 10)

Fig. 4

Mice which showed MIH-2 tumor eradication by previous i.t.OK-432 treatment did not reject re-challenge of MIH-2 cells. Five mice which showed MIH-2 tumor eradication by previous i.t.OK-432 treatment were implanted with 10⁶ of MIH-2 cells (closed circle). As control, age-matched untreated 11 mice were also implanted with 10⁶ of MIH-2 cells (open circle). Tumor incidence and tumor size were measured once a week. Numbers in the parentheses are tumor incidence

Fig. 5

Interferon-gamma production by Tumor-infiltrating CD4⁺T cells. a Immuno-fluorescence microscopy—Upper tumor tissue treated with i.t.OK-432 was stained with FITC indicating CD4 and Rodamine indicating IFN-gamma; Lower tumor tissue treated with i.t.OK-432 was stained with FITC indicating CD8 and Rodamine indicating IFN-gamma. b Interferon-gamma production by tumor-infiltrating CD4⁺T cells stimulated with OK-432 in vitro: A tumor-infiltrating CD4⁺T cells from untreated mice, not stimulated in vitro; B tumor-infiltrating CD4⁺T cells from untreated mice, stimulated with OK-432 in vitro (1 μg/ml); C tumor-infiltrating CD4⁺T cells from OK-432-treated mice, not stimulated in vitro; D tumor-infiltrating CD4⁺T cells from OK-432-treated mice, stimulated with OK-432 in vitro (1 μg/ml)

Fig. 6

Active caspase-3 shown by FACS analysis. MIH-2 cells were incubated in the presence (solid line) or absence (dotted line) of 1 ng/ml of IFN-gamma for 72 h at 37 °C. Expression of active caspase-3 was examined using FACS analysis

Fig. 7

Treatment of mice receiving i.t.-OK-432 injection with anti-IFN-gamma mAb. MIH-2 cells were implanted to mice on day 0. Anti-IFN-gamma mAb (0.5 mg/mouse) was administered i.p. to mice receiving usual i.t.-OK-432 treatment on days −1, 6, 13 and 20. Rat IgG at the same dose was administered to mice as a control in the same way. Diamonds indicate the growth of MIH-2 tumor in untreated control mice, and squares do that in i.t.-OK-432-treated mice. Circles indicate the growth of MIH-2 tumor in i.t.-OK-432-treated mice administered with anti-IFN-gamma mAb, and triangles do that administered with control rat IgG (n = 5)