Potent antitumor effects of combination therapy with IFNs and monocytes in mouse models of established human ovarian and melanoma tumors

Abstract

Interferon-activated monocytes are known to exert cytocidal activity against tumor cells in vitro. Here, we have examined whether a combination of IFN-α2a and IFN-γ and human monocytes mediate significant antitumor effects against human ovarian and melanoma tumor xenografts in mouse models. OVCAR-3 tumors were treated i.t. with monocytes alone, IFN-α2a and IFN-γ alone or combination of all three on day 0, 15 or 30 post-tumor implantation. Mice receiving combination therapy beginning day 15 showed significantly reduced tumor growth and prolonged survival including complete regression in 40% mice. Tumor volumes measured on day 80 in mice receiving combination therapy (206 mm(3)) were significantly smaller than those of mice receiving the IFNs alone (1,041 mm(3)), monocytes alone (1,111 mm(3)) or untreated controls (1,728 mm(3)). Similarly, combination therapy with monocytes and IFNs of much larger tumor also inhibited OVCAR-3 tumor growth. Immunohistochemistry studies showed a large number of activated macrophages (CD31(+)/CD68(+)) infiltrating into OVCAR-3 tumors and higher densities of IL-12, IP10 and NOS2, markers of M1 (classical) macrophages in tumors treated with combination therapy compared to the controls. Interestingly, IFNs-activated macrophages induced apoptosis of OVCAR-3 tumor cells as monocytes alone or IFNs alone did not mediate significant apoptosis. Similar antitumor activity was observed in the LOX melanoma mouse model, but not as profound as seen with the OVCAR-3 tumors. Administration of either mixture of monocytes and IFN-α2a or monocytes and IFN-γ did not inhibit Lox melanoma growth; however, a significant inhibition was observed when tumors were treated with a mixture of monocytes, IFN-α2a and IFN-γ. These results indicate that monocytes and both IFN-α2a and IFN-γ may be required to mediate profound antitumor effect against human ovarian and melanoma tumors in mouse models.

Fig. 1

Intratumoral administration of IFNs-activated monocytes inhibited tumor growth and prolonged survival of animals in established OVCAR-3 ovarian tumors. a Mice were subcutaneously injected with 10 × 10⁶ of OVCAR-3 tumors and treated with monocytes (3 × 10⁷) or IFN-α2a and IFN-γ (20 ng each), or combination of both IFNs plus monocytes by i.t. injection on day 0, 15 or 30 post-tumor implantation. The mice receiving combination therapy showed significant inhibition of tumor growth compared to IFNs alone, monocytes alone and PBS-treated mice in the OVCAR-3 tumor model. b Kaplan–Meier survival curves of mice with OVCAR-3 tumors are shown. Each group contained 5 mice. CR represents complete regression. Tumor volumes were measured by Vernier caliper, and overall survival time was calculated based on the killing of mice when tumors size reached >2 cm in diameter. Experiment was repeated twice; bars, SD

Fig. 2

Combination therapy with monocytes and IFNs reduced LOX melanoma tumor size and prolonged survival. a Mice were injected s.c. with 0.5 × 10⁶ of LOX melanoma tumors and treated with monocytes (3 × 10⁷) or IFN-α2a (20 ng) plus IFN-γ (20 ng) or combination by i.t. injection on day 0, 3 or 9 post-tumor implantation. Tumor volume in mice receiving combination therapy on day 0 and on day 3 was significantly smaller than that of untreated control mice, IFNs alone or monocytes alone. b Kaplan–Meier survival curves of LOX tumor models were plotted. Each group contained 5 mice. Time (OST) was calculated based on the killing of mice when tumors reached >2 cm. The OST of the mice was significantly increased in the combination therapy group (treated on either day 0 or day 3). The mice that received combination treatment on day 9 did not show any significant prolongation in survival. Experiment was repeated twice; bars, SD. c Individual IFN-α2a (40 ng/mouse) or IFN-γ (40 ng/mouse) was administrated with monocytes on day 3 after tumor injection in LOX melanoma tumor model. The treatment of mice with IFN-α2a or IFN-γ with monocytes slightly delayed the tumor growth compared to the excipient treated control on day 18 after tumor injection. On the other hand, the mice receiving combination therapy with IFNs (both IFN-α2a and IFN-γ) with monocytes significantly reduced the tumor burdens on day 20 compared to individual treatments of IFN-α2a or IFN-γ combined with monocytes. d Kaplan–Meier survival curve of mice treated under (c)

Fig. 3

Activated macrophages are accumulated into OVCAR-3 tumors. The tumor samples were collected on day 17 (2 days after 15-day post-inoculation treatment) from the mice in Fig. 1a. The immunofluorescence microscopy of tumors from mice was done using antibodies specific for CD31 and CD68 counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). The representative images of a tumor from each group are shown (magnification ×400)

Fig. 4

IFNs activate differentiation of monocytes to classical (M1) macrophages in OVCAR-3 tumors. The tumor samples were collected on day 17 (2 days after 15-day post-inoculation treatment) from the mice in Fig. 1a, and the immunofluorescence microscopy was done using antibodies specific for IL-12, CXCL10 (IP-10) and NOS2, IL-10, and Arginase-1 (magnification ×400)

Fig. 5

Monocytes activated by IFNs induced apoptosis in OVCAR-3 tumors. a The tumor samples were collected on day 17 (2 days after 15-day post-inoculation treatment) from the mice in Fig. 1a and stained with H&E. Large number of macrophages (arrow heads) were accumulated around tumors (a). Tumor samples were also stained using the TUNEL. Apoptotic cells were assessed and measured by fluorescent microscopy (b). The numbers of apoptotic cells were counted in each group. The numbers of apoptotic cells in tumors harvested from combination therapy–treated mice were significantly higher (P < 0.005) compared to control, monocytes or IFNs-only-treated tumors (c) (per 400× field view)

Fig. 6

Combination therapy caused no toxicity to vital organs. a Vital organs such as liver, kidney, lung and spleen of treated mice were examined for hepatocellular, tubular, pulmonary and splenic necrosis. No histological changes were observed in these organs. b Body weight was observed before and after treatment with combination therapy group compared to the controls. No body weight changes were observed with combination therapy. Each group contained 5 mice