Male or female C57BL/6 mice, 6-8 weeks old, were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences. CMT93 is a mouse colon cancer cell line from an induced carcinoma of mouse rectum[14], which was purchased from ATCC in America. Plasmid pCMV/MCP3 containing mouse MCP3 gene and plasmid pCDNA/YT4 containing mouse CCR1 (C-C chemokine receptor 1) gene were presented kindly by Dr. Ji Ming Wang in NCI in America. MCP-3 RT-PCR primers were presented kindly by Dr. Ji Ming Wang as well.

According to the LipofectAMINE (Gibco) kit protocol. Briefly, (1)2 μg plasmid DNA and 15 μL LipofectAMINE are added to 200 μL DMEM without FCS, mix gently, and incubate at 37 °C for 30 min. (2) Wash the CMT93 cells in 6 well plate with FCS-free DMEM. (3) Add 0.8 mL of serum-free DMEM to the tube containing the DNA and lipofectAMINE complexes, mix gently and overlay the rinsed cells. (4) Incubate the cells with complexes for 5 h at 37 °C in CO₂ incubator. (5) Add 1mL of DMEM containing 200 mL·L^-1 serum, incubate overnight. (6) Replace the medium with fresh DMEM. (7) Digest the cells and plate them to the 100 mL bottles, incubate 24 h. (8) Replace the medium with fresh DMEM containing 800 μg/mL G418 and select the positive clones.

The Total RNA from cells was extracted with RNeasy Mini kit (QIAGEN) according to the manufacture’s instructions. Total of 0.5 μg RNA was used for RT-PCR. For mouse MCP-3, sense primer 5’-TCTGTGCCTGCTGCTCATAG-3’ (nucleotide 73-92) and antisense primer 5’-CTTTGGAGTTGGGGTTTTCA-3’ (nucleotide 321-340) were used to yield a 268-base pair product. For mouse β-actin, sense primer 5’-TGTGATGGTGGGAATGGGTCGG-3’ and antisense primer 5’-TTTGATGTCACGCACGATTTCC3’ were purchased from STRAATAGENE to amplify a 514-base pair fragment. RT-PCR was performed with high fidelity ProSTRAR^TM HF single-tube RT-PCR system (STRATAGEBE), consisting of a 15-min reverse transcription at 37 °C, 1 min of inactivation of Moloney murine leukemia virus reverse transcriptase at 95 °C, 40 cycles of denaturing at 95 °C (30 s), annealing at 60 °C (30 s), and extension at 68 °C (2 min), as well as a final extension for 10 min at 68 °C.

Chemotaxis assays were performed using 48-well chemotaxis charmbers (Neuro probe, Cabin John, MD, USA). As described previously[15]. First 27-29 μL cell culture supernatants from CMT93 and its gene transfectants were placed in the wells of the lower compartment of the chamber. 50 μL cells (1 × 10⁶/mL in binding medium, BM: RPMI1640 containing 10 mL·L^-1 BSA, 25 mmol·L^-1 HEPES) were plated in the wells of the upper compartment. The upper and lower compartments were separated by a polycarbonate filter (Osmonics, Livermore, CA, USA; 10 μm pore-size) which was pre-coated with 50 μg·mL^-1 collagen type I (Collaborative Biomedical Products, Bedford, MA, USA). After incubation at 37 °C for 5 h, the filter was removed, stained, and the cells migrated across the filter were counted under light microscope after coding the samples. The results were expressed as chemotaxis index (CI), which represents the fold increase in the number of migrated cells in response to chemoattractants over the spontaneous cell migration in response to control medium.

As previously reported[16], Balb/c mice were inoculated subcutaneously on the back with 5 × 10⁶ CMT93/MCP-3 cells (8 mice), or CMT93/mock cells (7 mice) or CMT93 cells (5 mice). Tumor sizes were assessed for every 5 days by measuring perpendicular diameters with caliper (0.002 cm). The tumor sizes from each groups were expressed as mean square (long diameter × short diameter). On day 14, one mice in each groups was selected randomly and sacrificed for histologic evaluation. The other mice were observed and the tumor metastasis was measured.

Tissues were removed from the site of tumor cell inoculation 14 days after injection, or from the drainage lymph node of the site of tumor cell inoculation. Then fixed in 100 mL·L^-1 formalin, blocked in paraffin, sectioned at 4-6 um, and stained with hematoxylin and eosin. The infiltrating leukocytes in tumor tissue were observed and evaluated, and the tumor metastasis in the drainage lymph node was determined.

After gene transfection and the following G418-resistant clone selection, we detected MCP-3 mRNA expression in MCP-3 gene transfected CMT93 cells by RT-PCR using one-step kit. In contrast, the wild type CMT93 cells and the mock transfected CMT93 cells did not express MCP-3 (Figure 1).

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Figure 1 MCP-3 mRNA expression of CMT93 or its gene transfectants (RT-PCR). Lane 1: 100bp DNA ladder marker; Lane 2:CMT93+Primer MCP-3; Lane 3: CMT93 + Primer β-actin; Lane 4: CMT93/Mock + Primer MCP-3; Lane 5: CMT93/Mock + Primer β-actin; Lane 6: CMT93/MCP-3 + Primer MCP-3; Lane 7: CMT93/MCP-3 + Primer β-actin.

After the detection of MCP-3 mRNA expression, we tested the biological function of MCP-3 secreted in the culture supernatant. Chemotaxis analyses revealed that the RBL/2H3 cells transfected with CCR1, which is one of the receptors of MCP-3, migrated when supernatants from MCP-3 transfected CMT93 (CMT93/ MCP-3) cells were present in the lower wells of the chemotaxis chamber (Figure 2, 3D). No increased cell migration occurred when supernatants from mock transfected CMT93 (CMT93/ mock) or wild type CMT93 cells were present in the lower wells of the chemotaxis chamber (Figure 2, Figure 3C, 3B). These results showed that not only the transfected MCP-3 gene expressed but also the expressed protein possessed biological function.

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Figure 2 Chemotactic activity of MCP-3 in the supernatant from CMT93 cells or its gene transfectants. P < 0.05 vs CMT93 or CMT93/mock group.

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Figure 3 Chemotactic activity of MCP-3 in supernatant from CMT93 or its transfectants (Wright staining showed the chemoattracted target cells × 25). A: medium; B: CMT93 supernatant. The chemoattracted cells were not increased significantly; C: CMT93/Mock supernatant. The chemoattracted cells were not increased significantly; D: CMT93/MCP-3 supernatant. The chemoattracted cells were increased significantly.

To examine whether MCP-3 secretion retard tumor growth, we injected MCP-3 transfectants subcutaneously into Balb/c mice to test their tumorigenicity. As showed in Figure 4, all mice had tumor growth on day 5 after tumor cell inoculation. But tumors from CMT93/MCP-3 grew more slowly than that from mock transfectants or wild type CMT93 (P < 0.05, Table 1). These results suggested though MCP-3 secretion could not inhibit tumor formation completely; it retarded tumor growth.

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Figure 4 The tumor growth curves of CMT93 or its gene transfectants

MCP-3 gene transfer profoundly altered leukocyte recruitment in tumors grown in syngeneic Balb/c mice. On day 14 after tumor inoculation, all the mice showed tumor growths of about (0.6-0.8) × (0.6-0.8) cm² in size, which allowed a histologic study. Histologic results showed that tumor derived from MCP-3 gene transfected CMT93 cells contained much more infiltrating leukocytes, but tumor derived from the wild type CMT93 cells contained less leukocytes (Figure 5). Infiltrating leukocytes were located in the cancer nests mainly. The tumors derived from mock transfectants contained less leukocytes as well (data not shown). These results suggested that the effect of MCP-3 gene transfer in retarding tumor growth maybe related to the infiltration of leukocytes which were chemoattracted by MCP-3.

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Figure 5 Histopathology of the tumors derived from CMT93 or CMT93/MCP3 (HE staining × 200). A: tumor derived from CMT93 cells; few infiltrated immune cells were found; B:tu-mor derived from CMT93/MCP-3 cells, the infiltrated immune cells were increased.

On day 30, tumors in all 4 mice inoculated with CMT93 cells into the backs metastasized, two to inguinal drainage lymph node, other two to cervical drainage lymph node. Of tumors in 5 mice inoculated with CMT93/mock cells in the backs, 4 metastasized to inguinal drainage lymph node, and the other one did not metastasize. Tumors in all 7 mice inoculated with CMT93/MCP-3 did not metastasize. Histological study showed a metastatic lymph node (Figure 6A). In another metastasis lesion, tumor cells went out of the lymph node to invade the muscle tissue (Figure 6B). The percentage of tumor metastasis of MCP-3 gene transfectants was significantly lower than that of wild type or mock transfected CMT93 tumor cells (Table 2, P < 0.05).

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Figure 6 Histopathology of tumor derived from CMT93 me-tastasized to the local drainage lymph node(HE staining × 200). A: up-left is the lymph tissue,down-right is the tumor tissue; B: tumor infiltrated to the muscle tissue.