| Immunotherapy is one of the main therapies to cancer now. The cytokine gene therapy plays a considerable role in cancer immunotherapy. The efficacy of the gene therapy critically depends on the genes'distribution in vivo. Therefore, to obtain a good response it is necessary these genes to migrate targetedly to cancer site. When cancer formed and enlarged, angiogenesis is essential, so cells of or relating to endothelial cells ( ECs ) may be mobilized and targetedly migrate to cancer. Some studies showed that mesenchymal stem cells ( MSCs ) could be induced to ECs. This research observed the feasiblility of MSCs induced to cells of or relating to ECs, and the induced cells as gene vectors carrying human gamma interferon ( hγIFN ) genes to inhibit cancer.The mononuclear cells ( MNCs ) were separated from the bone marrow of health donators by gradient centrifugation and then recultured in vitro to keep the adhering portions. The cell surface markers of third generation were tested by flow cytometer. CD29, CD44 and CD166 reacted positively, while CD14, CD45, CD34, CD133, VEGFR-2, CD31, CD106 and HLA-DR reacted negatively, which was consistent to the previous studies. Refering to the method of literatures, MSCs were cultured in vitro in the media with vascular endothelial growth factor ( VEGF165, 20 ng/ml ) for 2 weeks, and the surface markers of induced cells were CD34, CD133 and VEGFR-2 positive tested by flow cytometer, other markers were in common with MSCs, which proved the cells had been induced to EPCs. EPCs could regenerate in vitro. The gene of enhanced green fluorescent protein ( EGFP ) was used as reporting gene to observe the transfecting efficiency of adenovirual vetors with EGFP genes ( Ad5-EGFP ) to EPCs. When the multipotential of infection ( MOI ) was 150 pfu/EPC, the amount of EGFP positive cells were more than 95 percent in all EPCs two days after transfection, which proved transfecting efficiency was excellent. The transfection of adenovirus did not affect the proliferation of EPCs significantly. After EPCs were transfected by the adenovirual ventors which carried the genes of human gamma interforen ( Ad5-hγIFN ), hγIFN could be detected in the culturing media by enzyme linked immunosorbent assay ( ELISA ). the concentrations of hγIFN was above 1300 pg/ml on the second day of transfection, from day 6 to the day 14, the hγIFN was more than 1400 pg/ml, the genes in EPCs expressed efficiently and constantly.Three cancer cell lines ( Lovo colon cancer cells, SGC7901 gastric cancer cells, and H460 lung cancer cells ) were co-cultured with different concentrations of hβIFN and hγIFN respectively for four days. The viability of the cancer cells was tested by MTT assay, the cancer cells were inhibited in a concentration-depend fashon. High concentration of IFN ( hβIFN or hγIFN≥100 IU/ml or 200 IU/ml ) could inhibit the growth of cancer cells. The three kinds of cancer cells were stained with CFSE and then co-cultured with EPCs-hγIFN or EPCs-hβIFN respectively at a ratio of 5:1 for four days. After that, the cancer cells were counted. Compared with the cancer cells cultured alone, the growth of co-cultured cancer cells were inhibited ( P<0.05 ). The mean percentages of cancer cells co-cultured with EPCs-hβIFN to cancer cells cultured alone were as following: Lovo, 82.34%±6.12%; SGC7901, 77.89%±3.99%; H460, 76.98%±6.19%. The mean percentages of cancer cells co-cultured with EPCs-hγIFN to cancer cells cultured alone were as following: Lovo, 69.52%±3.78%; SGC7901, 79.36%±6.35%; H460, 77.87%±6.5%. The cancer cells co-cultured with EPCs growed as well as the cancer cells cultured alone.EPCs-EGFP were administered by caudal vein to nude mice with xenograft tumors ( n = 8 ). The tumors and the main organs of the mice were separated and sectioned after five and ten days respectively. The fluorescenct positive cells could be detected in the margin parts of the tumors with a fluorescent microscope. There were no fluorescent positive cells in other organs, which meant that EPCs could targetedly distribute to the cancer site.To study the antitumor efficacy of EPCs-hγIFN in vivo, the nude mice with xenograft tumor were divided into four groups. The mice of group one were injected intravenously ( IV ) PBS 100μl weekly as control ( n = 7 ); group two, EPCs-hγIFN 1×106 IV weekly ( n = 8 ); group three were injected subcutaneously ( SC ) hγIFN 10,000 IU three times a week ( n = 5 ); group four, EPCs-EGFP 1×106 IV weekly ( n = 5 ). Four weeks later, the tumors were separated and weighed. The tumors'growth were inhibited by EPCs-hγIFN IV compared with control ( mean tumors weight: 0.48±0.78g vs 2.47±2.58g,P<0.05 ), but not by hγIFN SC, or EPCs-EGFP IV. The survivals of each nude mouse were recorded since the treatment began till the mouse died. EPCs-hγIFN ( 1×106 IV weekly for four weeks, n = 7 ) could prolonged the survival of mice compared with control group ( PBS IV weekly for four weeks, n = 7 ) ( P<0.05 ). The median survival of EPCs-hγIFN group was 35 days ( 95% CI: 33.83～36.17 days), while the control group was 32 days ( 95% CI: 26.87～37.13 days ). Plasma hγIFN concentrations of mice injected EPCs-hγIFN ( 1×106 IV, n = 3 ) or hγIFN (10,000 IU SC, n = 3 ) were detected using ELISA. Mean hγIFN concentration in plasma was 486.36±52.12 pg/ml one day after EPCs-hγIFN injected, 185.77±22.59 pg/ml three days after the injection. Mean hγIFN concentration in plasma of mice was 1116.38±87.19 pg/ml one hour after hγIFN was injected, 21.04±5.15 pg/ml one day after the injection, and no hγIFN could be detected two days after the injection. Unlike hγIFN SC, EPCs-hγIFN IV could not get higher peak concentrations but a better antitumor efficacy, which proved the importance of targetedly distribution of EPCs-hγIFN.To many advanced cancer patients, especially aged and weak patients, the objectives of therapy are not complete response, but the promotion of the quality of life or the prolongation of survival. To these patients, it is valuabe to take another therapy with less adverse reaction after the chemotherapy which had controlled the cancer, instead of constant chemotherapy. In this study, EPCs-hγIFN were given followed by CPT-11, comparing with C225, to test their efficacy as maintenance therapy. And the efficacy of EPCs-hγIFN combined with C225 as maintenance therapy was also observed.In vitro, Lovo cells were cultured in five groups. Cells were cultured alone as control ( group one ), cells cultured with CPT-11 1st day ( group two), or with CPT-11 1st day + hγIFN from 2nd to 4th day ( group three ) or C225 ( group four )/and C225 ( group five) from 2nd to 4th day. The viability of cancer cells was measured with MTT assay. The viability percentage of co-cultured cancer cells to the control group were as below: group two, 92.77%±2.72%; group three, 82.44%±4.57%; group four, 85.16%±2.96%; group five, 80.68%±9.14%. Cancer cells co-cultured were significantly inhibited compared with the control ( P<0.05 ). The differences between group two and group three, group four and group five were also significant ( P<0.05 ). The differences among group three, group four and group five were insignificant. In vivo, the nude mice with xenograft tumor were divided into five groups too. Mice of group one were treated with PBS 100μl IV weekly as control ( n = 9 ); mice of other four groups were all intraperitoneally injected ( IP ) CPT-11 50 mg/kg twice in the first week, followed by treatment with PBS 100μl IV weekly ( group two, n = 9 ); or EPCs-hγIFN 1×106 IV weekly ( group three, n = 10 ); or C225 0.5mg IP twice a week ( group four, n = 10 ); or EPCs-hγIFN 1×106 IV weekly + C225 0.5mg IP twice a week ( group five, n = 10 ). After 4 weeks, The mean tumor volumes were as below: group one, 2024.28±1048.40 mm3; group two, 764.94±720.14 mm3; group three, 233.851±86.97 mm3; group four, 186.95±133.43 mm3; group five, 163.9±173.39 mm3. The differences between group one and other four groups were significant ( P<0.05 ). The differences between group two and group three, group four, group five were also significant( P<0.05 ). The differences between group three and group four, group five were insignificant ( P>0.05 ). The survivl were recorded from the treatment began till the mouse died. Median survivals were as below: group one, 34.2 days ( 95%CI: 32.8～35.6 days ); group two, 39.4 days ( 95% CI: 37.1～41.8 days ); group three, 44.5 days ( 95% CI: 42.9～46.1 days ); group four, 48.5 days ( 95% CI: 46.5～50.4 days ); group five, 51.3 days ( 95% CI: 49.8～52.8 days ). The differences of survival were tested by Kaplan-meier. The four treatment groups could signicantly survive longer than group one ( P<0.01 ). Paired comparison analysis showed the survival of group three, group four and group five were all longer than group two ( P<0.01 ). The survival of group four and group five were longer than group three ( P<0.01 ). The difference between group four and group five was insignificant ( P>0.05 ).In conclusion, MSCs could be induced to EPCs in vitro, which had low immunogenicity and could migrate targetedly to tumor site after injected intravenously. Carried the gene of hγIFN, EPCs could inhibte the growth of tumors. These results indicated that EPCs could be used as vectors, carrying genes to regulate the tumors micrenvironment and inhibite tumors'growth. EPCs-hγIFN used in maintenance therapy after chemotherapy could inhibit the growth of tumors and prolong the survival of nude mice. Combined with C225, the efficacy was more effective. EPCs carrying immune genes may be an ideal model as maintenance therapy following chemotherapy.
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