Time-dependent cytotoxic drugs selectively cooperate with IL-18 for cancer chemo-immunotherapy

Abstract

Background: Time-dependent chemotherapeutic agents can selectively target tumor cells in susceptible phases of the cell cycle however a fraction of tumor cells in non-vulnerable cell cycle phases remain drug-resistant. Immunotherapy represents a promising approach to overcome the limitation of phase-specific drugs and improve their clinical efficacy. Here, we investigated the potential use of anticancer chemotherapeutic drugs in combination with IL-18, a cytokine with strong immunostimulatory properties.

Methods: Four chemotherapeutic drugs commonly used in ovarian cancer were first tested for the ability to increase the immunogenicity and killing of the murine ovarian cancer cell line ID8 in vitro. Chemotherapeutric agents with measured time-dependent immune-enhancing effects were then tested for antitumor effectiveness in vivo in combination with IL-18 immunotherapy using the ID8-Vegf ovarian cancer model.

Results: Paclitaxel or topotecan exposure alone mediated incomplete, time-dependent killing against the murine ovarian cancer cell line ID8 in vitro, whereas carboplatin or gemcitabine mediated comprehensive, dose-dependent killing. In the plateau phase of the time-dependent killing by topotecan or paclitaxel, drug-resistant ID8 cells were more immunogenic with elevated expression of MHC-I and Fas, and increased sensitivity to CTL and Fas agonistic antibody in vitro. Moreover, the antitumor effectiveness of time-dependent agents in vivo was significantly improved with the addition of IL-18 through a T cell-dependent mechanism, while the effectiveness of drugs without significant phase specificity were not.

Conclusions: Tumor immunotherapy with IL-18 can significantly augment the killing fraction of phase-specific chemotherapeutic drugs and provide survival benefit. The safety profile of IL-18 and its positive interactions with select anticancer chemotherapeutic agents strongly supports the clinical investigation of this combinatorial approach.

Figure 1

Topotecan and paclitaxel upregulate MHC-I and Fas on ID8 cells and exert their cytotoxic effects in a phase-specific manner. (A) Cytotoxic effects of paclitaxel, gemcitabine, topotecan, carboplatin or control sodium azide on ID8 tumor cells. ID8 cells were incubated with the chemotherapeutic agents for 6 hours. Survival fraction 42 hours later versus concentration (IC₅₀ is used as unit) is shown. Curves are sigmoidal and for the same time of exposure (6 hours) they plateau at a level that depends on the cell cycle specificity. For topotecan and paclitaxel the plateau level is > 30% and > 20% respectively, indicating significant phase specificity. For carboplatin and gemcitabine the sigmoidal curve plateaus at a level < 2.5%. The sigmoidal curves represent the fit of the obtained data to a logistic regression dose response curve. The killing curve of sodium azide (chemical, no interfering with the cell cycle) was used as a negative control. (B) Upregulation of MHC-I and Fas on viable ID8 cells treated with topotecan (left) or paclitaxel (right). Cells were exposed to the drugs for 6 hours, washed and incubated in drug free media for 42 hours before harvesting and staining with MHC-I and Fas antibodies. Isotype control (Red); untreated (Blue); Drug concentration inducing approximately 50% killing (IC₅₀; Green); Drug concentration corresponding to the plateau of the dose response curve (10 fold IC₅₀; Brown). All the histograms depict Annexin-V negative (non apoptotic) cells. (C) Dot plot diagrams depicting the upregulation of MHCI and Fas in non-apoptotic tumor cells exposed to Topotecan (upper) and Paclitaxel (lower) at IC₅₀ or 10 fold IC₅₀ for 6 hours 2 days before. (D) Growth curve of sorted MHC-I positive ID8cells following treatment with topotecan, paclitaxel and carboplatin, as indicated. Error bars represent interquartile range (25-75%).

Figure 2

Tumor cells surviving topotecan or paclitaxel are sensitized to effector T cells. (A) In vitro exposure to paclitaxel or topotecan enhances the sensitivity of ID8-E6E7 cells to activated E7-specific T cells. Bars show cytotoxicity after exposure to 2 different doses of paclitaxel and topotecan (IC₅₀ and 10 fold IC₅₀) for the same E:T ratio (20:1). Experiments were performed twice with similar results. (B) Treatment of ID8 cells with topotecan or paclitaxel sensitizes them to Fas agonistic antibody. ID8 cells (untreated or treated with topotecan or paclitaxel at IC₅₀ as described) were incubated with the Fas agonistic antibody and recombinant protein G or with isotype matched antibody and recombinant protein G for 24 hours, harvested, stained with trypan blue and the viable cells were counted (hemocytometer). The bars show the mean level of cytotoxicity and standard errors of three independent experiments. (C) The addition of Fas agonistic antibody to paclitaxel (upper left), carboplatin (upper right), topotecan (lower left) or gemcitabine (lower right) in vitro targets the resistant ID8 tumor cells and shifts the plateau phase of the dose-response curve for paclitaxel and topotecan downwards. The sigmoid curves represent the fit of the obtained data to a logistic regression dose response curve.

Figure 3

IL-18 induces activation of T effector cells, restricts ascites accumulation and prolongs survival of ID8-Vegf tumor bearing mice. (A) IL-18 does not have a direct cytotoxic effect on ID8 tumor cells. ID8 tumor cells (5 × 10⁵) were cultured for 48 hours in media containing IL-18 in a wide range of concentrations (0-1000 ng/ml), harvested, and viable cells counted after staining with Trypan blue. Results are means ± SEM of 3 experiments. (B) IL-18 induces activation of T effector cells. CD69 expression on CD3⁺ CD8⁺ cells isolated from the spleen of ID8-Vegf tumor bearing mice treated with saline or IL-18 (10 μg/day s.c for 20 days, starting 10 days after the tumor challenge). The bars are mean ± SEM of the CD69 expression in CD3⁺ CD8⁺ cells from spleens of 5 mice treated with IL-18 as above and 5 mice treated with saline. (C) Antitumor properties of IL-18 in C57BL/6 mice. IL-18 significantly restricts ascites accumulation as depicted by increase in the animal weight. The asterisks show data points were the difference between the groups is significant (Student's test, P < 0.05). (D) IL-18 significantly prolongs the survival of ID8-Vegf tumor bearing mice (P < 0.05). Control n = 9, IL-18 treated n = 9.

Figure 4

IL-18 improves only the antitumor effect of time-dependent drugs. (A) Combination therapy with IL-18 improves the survival benefit offered by paclitaxel and topotecan alone and restricts ascites accumulation (left). C57BL/6 mice were injected i.p. with 5 × 10⁶ ID8-Vegf cells on day 0 and subsequently treated with the indicated types of treatment. Mice treated with the combination of paclitaxel plus IL-18 had a significantly prolonged survival compared to the respective monotherapies (p < 0.01; control n = 23, IL-18 alone n = 22, paclitaxel n = 14, IL-18 plus paclitaxel n = 13). Combination therapy significantly restricted ascites accumulation (right) compared to the respective monotherapies as measured by the increase in the animal weight. The asterisks show data points where the difference in ascites accumulation is significant (Student's test, P < 0.001). (B) The combination of topotecan plus IL-18 also significantly prolonged the survival comparing to the respective monotherapies (p < 0.01; control n = 10, IL-18 alone n = 10, topotecan alone n = 9, IL-18 plus paclitaxel n = 10). Combination therapy significantly restricted ascites accumulation (right). The addition of IL-18 to carboplatin (C) or gemcitabine (D) did not significantly improve survival (left) or restrict ascites accumulation (right). IL-18/carboplatin experiment: Control n = 9, IL-18 n = 9, carboplatin n = 9, IL-18 plus carboplatin n = 9. IL-18/gemcitabine experiment: Control n = 10, IL-18 n = 10, gemcitabine n = 9, IL-18 plus gemcitabine n = 10.

Figure 5

The antitumor effect of combination IL-18/chemotherapy is T cell dependent. C57BL/6 (A) or C57SCID (B) mice were injected s.c. in the flank with ID8-Vegf cells and subsequently treated as described in "Materials and Methods." Mice with no treatment, topotecan alone, IL-18 alone or their combination were sacrificed when control tumors reached a size of approximately 600 to 800 mm³ ; all tumors were excised and weighed. Results are medians ± SEM: interquartile range (25%-75%; n = 10). The asterisk indicates the statistically significant difference between experimental and control groups. In the C57BL/6 mice, the topotecan/IL-18 combination treatment significantly restricted the tumor weight relative to the control group (Student's test, p < 0.05), while independent monotherapies did not. In the SCID mice the effect of the combinatorial therapy on the tumor growth was lost with no significant differences between the groups.