Synergistic Antitumor And Antimetastatic Effects By Adenovirus-mediated Intratumoral Coexpression Of LIGHT And IL-12 In A Murine Mammary Carcinoma Model

Breast cancer is one of the leading causes of mortality among women throughout the world and it ranks second following lung cancer in cancer deaths. The breast cancer incidence has increased dramatically in China with 3% to 4% annual increments during the past two decades. It is reported that 50% of patients with early or localized breast cancer will develop advanced metastasized disease. Although much progress has been made in the detection and treatment of localized (nonmetastatic) breast cancers, there has been relatively modest progress in the treatment of advanced disease, and more than 40 000 women die every year as a result of metastatic breast cancer. There is an urgent need to develop new therapeutic approaches that are feasible, affordable, and less toxic for control both of the actual tumor load and the metastatic potential of the disease. One such experimental approach is gene therapy that emploies an immuologically mechanism that could lead to regression of the primary tumor as well as the residual microscopic metastatic foci.Strategies using cytokines to induce or augment the antitumor immune respose have been proven effective in a variety of animal models. A number of cytokines, including interleukin (IL-2, IL-6, IL-7 and IL-12), interferon-gammer (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), have beendemonstrated to be effective in augmenting either T-cell-dependent or inflammatory resposes that can lead to tumor injection. Importantly, mice that were challenged with cytokine gene-modified tumor cells often developed strong long-lasting protective immunity to unmodified tumor cells f6'71.LIGHT(also known as TNFSF14, which is an acronym for homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D fro HVEM, a receptor expressed by T lymphocyte) is a member of the TNF superfamily that was maily expressed on activated T cells , NK cells, immature dendritic cells (iDCs) and some tumor cell lines[8>9>10>111. LIGHT has three receptors, HVEM1121 , LT P RI9] and TR6tl3]. Early studies found that LIGHT can selectively induce the apoptosis of some tumor cells as other TNF superfamily members191, Following studies have proved that it can provide costimulatory signals to T cells and induce the maturation of DCs[ul . Therefore, LIGHT may be applied to anti-tumor immunity. Tamada K et al reported that LIGHT was a co-stimualtory molecular for CD28-independent T cell activation and preferentially induced the IFN-y and GM-CSF production, gene transfer of LIGHT to P815 tumor increased its immunogenicity, thus induced an antigen-specific cytolytic T-cell response and therapeutic immunity against established mouse P815 tumor. In vivo T cell subsets depletion studies showed that CD8+T cells played the major role in antitumor effects1'51. Recently, Fan ZS et al demonstrated that forced expression of LIGHT inside tumor tissues could lead to rapid rejection of tumor in a NK dependent fashion, the activated NK cells directly elicited tumor specific CTL proliferation and maturation in situ for the rejection of a well-established tumor1161. In a fibrosarcoma animal model, Yu P and coworkers revealed that constitutive expression of LIGHT upregulated the production of chemokines and the expression of adhension molecules in tumor tissues, resulting in the recruitment of naive T cells that were then effcientlyactivated and expanded inside the tumor, which greatly enhanced the host's resistance to progressive tumorigenesis and further led to the rejection of well-established parental tumors at local and distant sitesI17l Taking together, LIGHT can be a promising candidate for cancer gene therapy and the underlying mechanisms need to be extensively investigated in the future.IL-12, a heterodimer composed of p35 and p40 subunits, is produced primarily by dendritic cells, macrophages/monocytes, and neutrophils. Both subunits are necessary for the bioactivity of IL-12. IL-12 acts on T cells and NK cells by enhancing generation and activity of cytotoxic lymphocytes and inducing the production of Thl cytokines, especially IFN-y. IL-12 is also the the major cytokine resposible for the differentiation of T helper 1 cells, which are potent producers of IFN-y. In addition, by inducing the expression of IP-10 and Mig genes and proteins in the tumor[18'191, IL-12 can exert potent anti-angiogenic and anti-neoplastic effects in a variety of tumors in animal models'20"211. During the past few years, IL-12 has been demonstrated to be a key mediator of innate and adaptive immunity with potent antitumor and antimetastatic activity in many experimental animal models. In a phase I clinical trail, recombinat IL-12 also stimulated significant immunological activity in cancer patients1221. However, despite initial enthusiasm for recombinat IL-12 as a potential anti-tumor agent, severe systemic toxicities have been repeatedly reported in clinical trails, limiting its use[23J. In contrast to direct cytokine adminstration, IL-12 gene therapy using an adenoviral vector in animal cancer models has been shown to be as effective as protein exposure but avoids the systemic toxicity in human trails'24-251.One of the major advantages of adenovirus-mediated gene transfer compared with the administration of recombinant proteins is the quicker achievement of steady-state levels of circulating protein1261, besides, adenovirus-mediated genetransfer has a much higher efficiency compared with other gene delievery systerms. Therefore, adenovirus has been a widely used vector to carry functional moleculars for gene therapy.The 4T1 tumors established in the mouse mammary gland closely resemble human breast cancer in their immunogenicity from BALB/c mice, this cells are considered very weakly immunogenic with relative antigenic property1271, after subcutaneous (s.c.) inoculation in the abdominal mammary fatpad, the primary tumor grows into a nodule with the histology of a high-grade breast cancer and sheds spontaneous systemic metastases to the lungs, liver, lymph nodes, bone marrow, and central nervers systerm. Metastatic growth in the lungs is usually the main cause of death of mice, and surgical removel of the primary tumor once it becomes palpable does't affect metastases growth. Therefore, this model is suitable for testing the effects of experimental therapies on metastatic disease1281.In the present study, we employed the aggressive, tumorigenic, and noimmunogenic 4T1 mouse mammary carcinoma model to evaluate the synergistic antitumor and antimetastatic effects by adenovirus-mediated introtumoral coexpression of LIGHT and IL-12, the related immunological mechanisms were also investigated.The main work can be divided into two parts:Part I: Characterization of Ad LIGHT and Ad IL-12We used Ad LIGHT and Ad IL-12 to infect 4T1 cells, RT-PCR, ELISA and flow cytometry analysis were performed to prove that LIGHT and IL-12 can be effectively expressed in 4T1 cells both in mRNA and protein level. To test whether the adenovirus mediated expression of LIGHT and IL-12 were bioactive, we observedtheir ability to stimulate the proliferation of T lymphocytes. The results show that, compared with the negative control, 4T1 cells that infected Ad LIGHT or Ad IL-12 alone can effectively stimulate the proliferation of T lymphocytes and IFN-y production, but only when they were used together that, a much stronger proliferation and more production of IFN-y were achieved, which indicated that LIGHT and IL-12 may have synergistic costimulation activity on T cells.Part II: Synergistic antitumor and antimetastatic effects by adenovirus-mediated intratumoral coexpression of LIGHT and IL-12 in a murine mammary carcinoma modelSeveral studies have revealed the impotant role of LIGHT in rejecting established tumors, while many studies have proven that IL-12 has antitumor and antimetastatic activity in many experimental animal models. Here we hypothesized that the combined use of Ad LIGHT and Ad IL-12 (Ad LIGHT/IL-12) gene therapy may elicit more significant antitumor effects in a high risk metastatic breast cancer model (4T1 mammary carcinoma model).6-8 week female BALB/c mice were subcutaneously inoculated with 4T1 cells, when the tumor nodules were visible and palpable, 50 mice were randomly devided into five groups (each contained 10 mice) and treated with PBS (blank control), Ad LacZ (blank virus control), Ad LIGHT, Ad IL-12, Ad LIGHT/IL-12, respectively. The tumor size was measured every other day and survival period of tumor-bearing mice was observed. Five days after the last adenovirus injection, splenocytes were prepared from tumor-bearing mice for the the induction of cytokines and CTLs. The CTL and NK activity were further determined by a standard LDH release assay[29]. Tumorswere removed for histological examination and semi-quantative RT-PCR was performed to characterize the IP-10 and Mig expression from the mRNA extracted from the tumor tissues. To test whether the combined therapy can induce a protective immunity against the parent 4T1 cells, the tumor free mice that received the combined therapy were rechallenge with 4T1 cells or CT26 colon cancer cells, respectively. The tumor occurrence and survial time were observed.The results show that the most potent and effetive inhibition of tumor growth was achieved after combined therapy of Ad LIGHT and Ad IL-12 compared with mice received therapy of PBS, Ad LacZ , Ad LIGHT or Ad IL-12 alone. In the negative control group, tumor grew progressively and the tumor-bearing mice became weak around 30 days, all the 10 mice from PBS or Ad LacZ group eventually died around 40 days after tumor inoculation. Tumor-bearing mice that received injection of either Ad LIGHT or Ad IL-12 alone showed a significantly reduced tumor growth, but only 28.6% or 42.9% of the mice turned tumor free and survived more than 90 days. After the combined therapy of Ad LIGHT and Ad IL-12, the tumor growth was greatly retarded and no tumor diameter was more than 6 mm, more importanly, 100% of these mice survived healthily with complete tumor eradication and consequently acquired a strong protective immunity against parent 4T1 tumor cells. In addition, these tumor free mice also developed some no-specific ^protective immunity because 40% of the tumor free mice resisited CT26 colon cancer cells challenge and survived more than 90 days, In contrast, the naive mice challenged with either 4T1 or CT26 cells showed great tumor burden and completely died around 40 days after the tumor inoculation. Lymphocytes from the mice with combined therapy showed a much higher NK and CTL activity, suggesting that augmentation of tumor-specific and nonspecific immunity both participated in the enhanced antitumor immune respose induced by Ad LIGHT and Ad IL-12 combined therapy. It was noted that the production of IL-2 andIFN-y from lymphotes derived from mice treated with Ad LIGHT, Ad IL-12 , Ad LIGHT/IL-12 was significantly higher than control groups. The IL-2 and IFN-y levels in mice with combined treatment were highest among those groups. This suggested that the lymphocytes of the mice that received combined treatment were inclined to produce more Thl (IL-2, IFN-y) cytokines, which may play an important role in the immune respose against established tumors. These results suggested that adenovirus mediated coexpression of LIGHT and IL-12 may elicit more potent antitumor immune respose in 4T1 mammary carcinoma model.Histological examination of tumor tissues showed that the most severe necrosis was observed from the tumor-bearing mice that received combined therapy of Ad LIGHT and Ad IL-12, accompaning with obvious inflitration of inflammatory cells (neutrophils, lymphocytes, monocytes) inside and around the tumors. Tumor necrosis and inflammatory cell infiltration were markedly observed in mice treated with Ad-LIGHT or Ad IL-12 alone, whereas relatively less necrosis and infiltration were present in the tumors of PBS or Ad LacZ treated group.IP-10 and Mig are two important chemokines that have been reported to possess antiangiogenic and antineoplastic activity^30'311, they may also modulate a variety of other functions such as lymphocytes activation, differentiation and effector function132*331. We used semiquantative RT-PCR to examine the expression of the two chemokines in tumor tissues after Ad LIGHT and Ad IL-12 injection. We found IP-10 and Mig mRNA to be strongly expressed in tumor tissues of Ad LIGHT/IL-12 treated group and relatively lowly expressed from tissues of Ad LIGHT or Ad IL-12 treated group. Little or no IP-10 and Mig mRNA was detected from the tumor tissues of Ad PBS or LacZ treated group. Thus, the higher expression of IP-10 and Mig may contribute to the reduction of new blood vessels and further the retardation of tumor progression1341. Besides, CCL21, another important chemokine was also induced toexpress in tumor tissues of the mice from Ad LIGHT or Ad LIGHT/IL-12 treated group.The 4T1 tumor has a high risk of systemic and disseminated metastases, especially to the lung, which may be the main cause of death. Here we tried to study whether the combined therapy can reduce the tumor metastase to the lung. Because tumor nodules on the surface of the lung can't be easily visualized at the early stage of metastases, lungs removed from the sacrificed mice of each group were minced and single cell suspenssions were prepared and cultured in a medium containing 6-thioguanine for clonogenic growth. The 6-thioguanine resist clonogenic metastatic 4T1 tumor cells in the lung were then enumerated1281. At the late stage, lungs from the tumor-bearing mice were removed for histological examination or injected with India ink to visualize individual tumor nodules on the surface1351. The results showed that, compared to those of the mice treated with PBS or Ad LacZ, the number of colonies of metastatic 4T1 cells in the mice treated with Ad LIGHT or Ad IL-12 alone reduced significantly, no 4T1 cell colonies were found in the mice that received combined therapy of Ad LIGHT and Ad IL-12. These results were further proved by histological examination of the lungs and enumeration of the tumor nodules on the lung surface, which strongly suggested that the Ad LIGHT plus Ad IL-12 therapy greatly reduced the tumor metastases to the lung of tumor-bearing mice.To investagate the resposibility of T cells and NK cells in the antitumor immune respose induced by Ad LIGHT plus Ad IL-12 therapy. BALB/c nude mice and the mice depleted of NK cells were used to test the therapeutic effects of the combined therapy. We found that the antitumor effects were partially abrogated both in BALB/c nude and NK depleted mice, which suggested that both NK cells and T cells are required for the induction of antitumor immune respose after the combined therapy of tumor-bearing mice.In conclusion, here we demonstrated that, in a highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model, the combined intratumoral therapy of Ad LIGHT and Ad IL-12 led to significantly suppression of the tumor growth and lung metastases, increased tumor necrosis and inflammatory cell infiltration, as compared to either Ad LIGHT or Ad IL-12 alone. This enhanced antitumor and antimetastatic effects of the combined strategy were associated with a relatively higher NK and CTL activity, accompaning with more production of Thl cytokines from the spelocytes derived from the tumor-bearing mice. Besides, the combined therapy also induced a long protective immune respose against parent tumor cells rechallenge. These collectively data suggested that the combined immunotherapy by intratumoral injection of Ad LIGHT and Ad IL-12 can elicit a more potent antitumor and antimetastatic immune respose.