The Anti-tumor Effects And Mechanisms Of IP10, ITAC And Dual Functional Molecule In Murine 4T1 Mammary Carcinoma Model

1. The anti-tumor effects and mechanisms of IP10 in murine 4T1 mammary carcinoma model.In launching an immune response against tumors, it is necessary for T lymphocytes and other leukocytes to be recruited to the sites of tumors. This directed movement is mediated by the superfamily of small (8-10 kDa) chemotactic cytokines, termed chemokines. In addition to their role in leukocyte trafficking, chemokines also modulate leukocyte adhesion molecule expression, angiogenesis and so on. Chemokine gene transfer represents a promising approach in the treatment of malignancies.The plasmid pcDNA3-IP10 was constructed and stably transfected into a mammary carcinoma cell line 4T1 cells (4T1-IP10). Mice were injected with 4T1-IP10 cells, 4T1-pcDNA3 or parental 4T1 cells. The results showed that the growth of tumors formed by 4T1-IP10 cells was significantly inhibited with lighter tumor weight. The mice bearing 4T1-IP10 tumors resulted in a longer live span. Accumulated lymphocytes were found in 4T1-IP10 tumors. Immunohistochemistry assay showed that profound T lymphocytes infiltrated in 4T1-IP10 tumors. Moreover, both CXCR3~+CD4~+ and CXCR3~+CD8~+ T cells increased significantly in 4T1-IP10 tumors. Importantly, the proliferative response to 4T1 cells and IFN-γ secretion of the lymphocytes isolated from 4T1-IP10 tumors enhanced significantly, verifying that recruitment followed by functional augment of tumor specific lymphocytes is pivotal in IP10-mediated anti-tumor effects. The cellular immune response was investigated in tumor bearing mice. After stimulation with 4T1 cells in vitro, splenocytes from the mice inoculated with 4T1-IP10 cells exhibited vigorous specific proliferative response and cytotoxic activity. The experimental lung metastatic model demonstrated that the mice inoculated with 4T1-IP10 cells inhibited the disseminated metastases as indicated by the reduced metastatic forci on the surface of the lungs with lighter lung weight. Histological analysis also showed that few metastases were found in the micetreated with 4T1-IP10 cells, indicating IP10 expressed in tumors can protect mice from challenge wild-type 4T1 tumor cells.IP10 has been demonstrated to have angiostatic activity. In the present study, with a dose-dependent manner IP10 could inhibit the growth of vascular endothelial cells. Compared to bFGF alone, regrowth from the wound edge into the denuded zone 2 days after injury was inhibited 80% by IP10. Administration of recombinant bFGF into avascular cornea generated a robust neovascularization response from the limbus within 10 days. The growth of vascular endothelial cells in 4T1-IP10-conditioned medium was dramatically suppressed compared to those both in 4T1-conditioned medium and in RPMI1640. H&E staining of tumor sections showed that significant necrotic tumor tissues were found in 4T1-IP10 tumors, suggesting angiostatic effect of IP10. On the contrary, multiple and big vessels were found in controls. 2. The anti-tumor effects and mechanisms of ITAC in murine 4T1 mammary carcinoma model.The plasmid pcDNA3-ITAC was constructed and stably transfected into a mammary carcinoma cell line 4T1 cells (4T1-ITAC). The BALB/c and nude mice were injected with 4T1-ITAC cells, 4Tl-pcDNA3 or parental 4T1 cells, which were demonstrated to grow at the same rate in vitro. The results showed that the growth of tumors formed by 4T1- ITAC cells was significantly retarded and the mean volume of these tumors was smaller. The mice bearing 4T1-ITAC tumors resulted in a longer live span. However, tumor growth was slower in BALB/c mice than that in nude mice, indicating T lymphocytes may participate in ITAC-mediated tumor growth inhibition. The effects of ITAC on the infiltration of lymphocytes were investigated. The results showed that the number of lymphocytes detected in 4T1-ITAC tumors was three times large than that in controls. FACS analyses showed that accumulated CXCR3~+ lymphocytes infiltrated in 4T1-ITAC tumors. Compared to controls, the infiltrated lymphocytes from 4T1-ITAC tumors exhibited enhanced Ag-specific proliferative response and cytotoxic activity against 4T1 cells. The IFN-γ secretion was also increased, suggesting that lymphocytes infiltrated in 4T1-ITAC tumors are highly activated. The cellular immune response was examined in tumor bearing mice. After stimulation with 4T1 cells in vitro, splenocytes from the mice inoculated with 4T1-ITAC cells exhibited vigorous specific proliferative response and cytotoxic activity. The experimental lung metastatic model demonstrated that the mice inoculated with 4T1-ITAC cells inhibited the disseminated metastases as indicated bythe reduced metastatic forci on the surface of the lungs with lighter lung weight. Histological analysis also showed that no obvious metastases were found in the mice treated with 4T1-ITAC cells.The angiostatic activity of ITAC was also evaluated. Compared to bFGF alone, regrowth from the wound edge into the denuded zone 2 days after injury was inhibited 56% by ITAC. The growth of vascular endothelial cells in 4T1-ITAC-conditioned medium was suppressed.3. Designing and construction of dual functional molecule containing N-terminal, N-Loop region of ITAC and C-terminal of IP10.Based on the studies in the second and third parts, stastical analyses showed that ITAC has stronger chemotactic activity for lymphocytes than IP10, and that the significant proliferative response and cytotoxic activity as well as IFN-γ secretion were detected in the lymphocytes of the mice bearing 4T1-ITAC tumors compared to that of 4T1-IP10 tumors. However, IP10 suppressed the proliferation of the vascular endothelial cells markedly compared to ITAC. The growth of vascular endothelial cells cultured in 4T1-IP10 cells conditioned medium was inhibited dramatically rather than in that of 4T1-ITAC cells. Combining with previous reports, we hypothesized that combining N-terminal and N-LOOP region of ITAC with C-terminal of IP10 will produce a dual functional molecule with distinct activity compared to IP10 or ITAC, which was named ITIP.Analyzed with InsightII work station, ITIP could form a stable dimentional structure. The homogeneity of amino acid with IP10 is 66%. The similarity in structure with IP10 is high as determined by RSM value 1.08. The coding gene of ITIP was amplified and subcloned into the pcDNA3 plasmid. The pcDNA3-ITIP was stablely transfected into 4T1 cells (4T1-ITIP). The increased transcription of ITIP was only detectable in 4T1-ITIP cells. Accumulated lymphocytes chemotaxed to the supernatants of 4T1-ITIP cells, which could be significantly blocked by CXCR3 antibody, indicating 4T1-ITIP cells can express and secret functional ITIP.4. The anti-tumor effects and mechanisms of dual functional molecule in murine 4T1 mammary carcinoma model.The effects of ITIP on the growth of 4T1 cells in vivo were observed. After inoculation of 5×10~4 tumor cells, 7/10 of the mice inoculated with 4T1-ITIP cells were tumor free compared to 4T1-IP10 (4/10), 4T1-ITAC (3/10), parental 4T1 cells (10/10) and 4T1-pcDNA3 cells (6/6) and 4T1-IP10+ITAC (6/6). After inoculation of1×10~5 tumor cells in nude mice, the growth of 4T1-ITIP tumors was as slow as 4T1-IP10 tumors compared to other groups, implificating that ITIP has angiostatic function involved in the tumor growth inhibition.The mechanisms of ITIP-mediated anti-tumor effects were explored. FACS analysis showed that large amounts of CXCR3-expressing lymphocytes infiltrated in 4T1-ITIP tumors. More importantly, the lymphocytes infiltrated in 4T1-ITIP tumors showed antigen-dependent expansion in situ. The effector function of lymphocytes in ITAC-conditioned tumor microenvironment was examined. After stimulation with 4T1 cells in vitro, the lymphocytes infiltrated in 4T1-ITIP tumors showed vigorous proliferative response and cytotoxic activity. The IFN-γ secretion was also increased markedly in the infiltrated lymphocytes of 4T1-ITIP tumors.The cellular immune response was investigated in the mice inoculated with 4T1-ITIP cells. After specific stimulation, the splenocytes from the mice inoculated with 4T1-ITIP cells exhibited significant proliferative response to 4T1 cells. Splenocytes from the mice inoculated with 4T1-ITIP cells showed vigrous cytotoxic activity against 4T1 cells. Another characteristic of T cell activation is cytokine production following stimulation. The splenocytes from the mice inoculated with 4T1-ITIP cells secreted more IFN-γ and TNF-α but less IL-4 compared to controls, indicating that the presence of ITIP increased the ability of the lymphocytes to proliferate in response to the tumor and a shift toward a type-1 cytokine response was occurred. Moreover, the growth of vascular endothelial cells in 4T1-ITIP-conditioned medium was inhibited markedly compared to RPMI1640, which was comparative to that of 4T1-IP10 cells.In summary, antiangiogenesis and recruitment followed by functional augment of tumor specific lymphocytes both are involed in the IP10 or ITAC-mediated anti-tumor effects. Importantly, ITAC can significantly enhance anti-tumor immune response, and IP10 can obviously inhibit the growth of endothelial cells. The dual functional molecule constructed by combining the N-terminal, N-LOOP region of ITAC with the C-terminal of IP10 was demonstrated to have anti-tumor effects. Recruiting CXCR3-expressing lymphocytes, inducing enhanced cellular immune response and antiangiogenesis might be involved in ITIP-mediated anti-tumor effects.