IP10,ITAC及其双功能分子诱导的及其双功能分子诱导的 抗肿瘤免疫及机制研究抗肿瘤免疫及机制研究 Anti-tumor immunity and the mechanisms induced by IP10, ITAC and their dual functional molecule Research Background Anti-tumor immunotherapy Anti-angiogenesis Anti-tumor Strategies Surgery Radiation therapy chemotherapy ? molecules Biologic Therapeutics Goals of anti-tumor immunotherapy：effector cells activation and recruitment. Directed movement of lymphocytes mediated by chemokines. Chemokines, a superfamily of cytokines, act by way of G-protein-coupled receptors . Research Background Research Background cancer Angiogenesis Inflammation Infection -Co-receptor of HIVReproductive Hematopoiesis Thymus development Immunomodulator graft rejection chemokines Chemokines enhance anti-tumor immunity Th1 Immature APC Apoptosis cells maturation SLC CTL CTL Tumor cell IP10, ITAC MIP, CXCL16 Killing Migration IP10, ITAC SDF-1 Angiogenesis of chemokines in tumor development VECproliferation VEC migration IL-8, IP10, ITAC Recruitment of effector cells Anti-angiogenesis Enhancing anti-tumor efficacy ? chemokines BRAK MIP-1, MIP-2 IP10, ITAC PF4 SDF-1 BCA-1 CXC superfamily non-ELR chemokine Similar structure Common receptor CXCR3 Chemotactic activity for activated T cells Anti-angiogenesis candidates Recruitment of effector cells Anti-angiogenesis Breast cancer One of the common malignancies for women One of the leading causes of death for women is metastatic breast cancer Two therapeutic strategies: enhancing anti-tumor immunity and anti-angiogenesis Tumor cells derived chemokines Control lymphocytes infiltration in tumors Regulate angiogenesis As autocrine or paracrine growth factors Control activity of tumor cells itself IP10, ITAC IP10, ITAC The expression of chemokines in human breast cancer biopsy and tumor cell lines Part I IP10 and ITAC expression in human breast cancer biopsies * P 0.05 IP10ITAC 0.0 0.2 0.4 0.6 0.8 1.0 inner tumor outer tumor IP10(ITAC)/GAPDH * * GAPDH IP10 ITAC inner tumor outer tumor Lane 1,GAPDH; lane 2, PF4; lane 3, MIG; lane 4, IP10; lane5, ITAC; lane 6,IL-8; lane 7,MCP-1,; lane 8,MCP-2; lane 9,MCP-3; lane 10, Frak; lane 11, MIP-2; lane 12, MIP-1a; lane 13, MIP-1b; lane 14,BCA-1; lane 15, LARC; lane 16,BRAK; lane 17, SDF-1. Chemokines expression in breast cancer cell lines 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 4T1 MCF-7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 4T1 cell line BALB/c mice derived mammary carcinoma cell line Growth characteristics parallel invasive human mammary carcinoma Spontaneous metastasis to lung and lymph node Resistant to hormone-therapy 4T1 mammary carcinoma model IP10-mediated anti-tumor effects vImmunomodulator vAnti-angiogenesis References Dorsey, et al., Cancer Res, 2002. Huang, et al., Int J cancer, 2004. Pertle, et al., J Immunol, 2001. Arenberg, et al., Cancer Immunother, 2001. ? Anti-tumor effects of ITAC vInduction of Th1-type response vThe most potent agonist of CXCR3 with the highest receptor affinity vInduction of CXCR3 internalization vPromoting T cell-endothelial cell contact vInhibition of CCR3-mediated Th2-type response B Construction and identification of pcDNA3-IP10 A CCCATGGGCGGCCGCCTGCAGACCAGGTCTATAAGAATCCGGCCGCC AAGGCATCCACTGTAACCACAGAAGGTAGCGTGGAGTGTGCACGTGA CTTTTAGAGACAAATATTTTTCATAGCCAAGTAGAACAGTTTAGATG TATCGGACCTTCTAGAAAGTTCTGCAGCCTGGTAATACGTGGCTGCA TGTTCCAAGACAGCAGAGGGTCAGGTTCCTGGCACAGAGTTCTTATT GGAGGGCTCACAGTCAGACGTTCCCAGGATGTCACATGTTTTGACGC CTTAAAAAATTCTTTTTTTCTATTGCCTGCATTATGAGGCGAGCTTG CTTGGATCTGGGGTCCAGGCACCTTTGTCGTTTATGAGCCTTCATAG TAACAATCACTTCAACTTTGTCGCAGCCGTTACTCGGGTAAATTACA GAAGCTTTCTCGATCTCTGCCATTTTGACGGCTTTCATCCCGGGGCC GATGCAAAGACAGCGCCCCTGTTTGAACATAAGGAAGCCTTGAGCAG CTGTGGCCCAAGCCGCTGCAGCTCTCTGCC 340bp M 1 2 Fig A. M. Marker; 2. Undigested; 3. Digested with EcoRI/XhoI. DNA sequencing GGCAGAGAGCTGCAGCGGCTGCTGAGACGAACAGGAAGGTCACA GCCATAGCCCTGGCTGCGATCATCTGGGCCACAGCTGCTCAAGG CTTCCTTATGTTCAAACAGGGGCGCTGTCTTTGCATCGGCCCCGG GATGAAAGCCGTCAAAATGGAGAGATCGAGAAGCTTGAAATCATC CCTGCGAGCCTATCCTGCCCACGTGTTGAGATCATTGCCACGATG AAAAAGAATGATGAGCAGAGATGTCTGAATCCGGAATCTAAGACC ATCAAGAATTTAATGAAAGCGTTTAGCCAAAAAAGGTCTAAAAGGG CTCCTCTCGAGGATCCGGCTGTGGAATGTGTGTCAGTTAGGGTGT GGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCAT GCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTC CCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGC AACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCC GCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTT ATTTATGCAGAGGCCGAGGCCGCCT AB 580bp 1 2 M Fig A. 1. Digested with EcoRI/XhoI; 2. Undigested; M, Marker. Construction and identification of pcDNA3-ITAC DNA sequencing IP10 and ITAC expression in transfected 4T1 cells A 4T14T1-pcDNA34T1-IP10 0.0 0.2 0.4 0.6 0.8 1.0 IP10/GAPDH * B 4T14T1-pcDNA34T1-ITAC 0.0 0.2 0.4 0.6 0.8 1.0 ITA