The Study Of The Tumor-inhibition Of Auto-tumor Specific CTL On Breast Cancer Model In Nude Mice

Objective: Dendritic cell (DC) is the most powerful dedicated antigen processing cell (APC). After capturing tumor antigen, DC migrates to tumor-draining lymph nodes (TDLNs) and presents tumor antigen to T cells, inducing it to be tumor specific cytotoxic T lymphocytes (CTLs) and kill tumor cells. TDLNs exist in tumor drainage region, which contain abundant lymphocytes and monocytes. We adopt inducing and cultivating TDLNs of breast cancer patients in vitro, stimulate those by DC which load tumor antigen, and make those to be tumor specific CTLs, then transfer tumor specific CTLs in nude mice of human homobody breast cancer. To study the tumor inhibition effects of tumor specific CTL on tumor-baring mice.Methods: Two to three lymph nodes without visually metastasis from breast cancer tumor drainage region were removed during operation. Meanwhile, a piece of fresh homobody tumor tissue and another patient's were cut. And get lymphocyte suspension by mechincal method, then centrifugate, induce and cultivate with rhGM-CSF,rhIL-4 and TNF-α. The morphologic changes of TDLNCs were observed periodically. Lymphocytes were collected and analyzed by flow cytometry (FCM), which were induced to be DC and TDLNC. DCs stimulated by the autoallergic breast cancer freeze-thawing antigen and T lymph cells were co-cultured to derivation tumor antigen specific CTL. The tumor tissue were scrapped into small pieces about 1 mm3. They were subcutaneously implanted in breast mat of 32 BALB/C nude mice aged 3 to 4 weeks to establish tumor-baring nude mice model. After two week, the mice were randomized into 4 groups (each has 8). The nude mice were injected every 5 days.①specific CTL group (each nude mice was injected antigen specific CTL5×106/ml 0.2ml)②non-specific CTL group(each nude mice was injected non-specific CTL5×106/ml 0.2ml)③varient CTL group: the nude mice were transplanted another patient's tumor tissue (each nude mice was injected antigen specific CTL5×106/ml 0.2ml)④control group (each nude mice was injected partly normal sodium 0.2ml). The maximal and minimal diameters were measured every 5 days, the tumor volume (V) was calculated as V =πab2/6, where"a"denotes the maximal diameter and"b"denotes minimal diameter. The tumor growth inhibition parameter was calculated and statistically analyzed. After 30 days of implantation, the mice were sacrificed and the implanted tumor was collected,The tumor tissue was fixed in formalin solution, dehydrated, and imbedded in paraffin. The sections were routinely stained by hematoxylin-eosin (HE). The morphological changes and apoptotic statu of the tumor cells were observed under light microscope. Part of the sections was selected by experienced pathologists for immunohistochemistry (IHC) to study the infiltration of T cell and DC. Results: 1 The percentage of CD1a,CD83,CD86 positive expresion in DC from mononuclear cells in axillary draining lymph node culturing in vitro first day was 10.98±2.3 ,26.55±5.24 and 32.96±6.09, respectivly. After cultured with rhGM-CSF and rhIL-4, breast cancer freeze-thawing antigenic and TNF-α, the percentage of CD1a,CD83,CD86 was 50.17±5.68,60.48±16.46 and 56.22±16.38, respectivly, P<0.01. 2 The percentage of CD3+ and CD8+ T cells in TDLNC was 73.93±2.18 and 32.78±3.21, however, they were much higher in DC-Ag-TDLNC, 82.67±2.79 and 62.54±2.51, respectively, P < 0.01. DCs and TDLNs in vitro were co-cultured by cytokines and proliferated bulkly. The percentage of CD3+ and CD8+ T cells in DC-TDLNC was 81.97±2.12 and 60.59±2.55, P<0.01. 3 The success rate of implanting human breast tumor cells on nude mice was 100%. 4 Every treated group could inhibite the growing of the implanted carcinoma. The most significant inhibition of the three group was autos specific CTL group, whose average gross tumor volume post-treated 15days was 82.70±2.09mm3. It was higher than non-specific CTL group (96.15±5.35mm3) and variant CTL group (96.93±4.51mm3),the difference was of significance (P =0.000). The tumor inhibition rate of autos specific CTL group (47.62%)was significantly greater than non-specific CTL group (30.44%) and variant CTL group (24.69%),the difference was of significance (P =0.000). 5 The HE stained implanted tumor sections under light microscope was confirmed mammary infiltrating ductal carcinoma, generous lymphocyte infiltrate in the tumor tissue which were treated. On IHC of every treated groups, the dark brown stained DC and massive lymphocytes were shown to infiltrate in tumor tissue.Conclusion: 1 Typical DCs can be induced from axillary draining lymph nodes after being stimulated with cytokine (rhGM-CSF,rhIL-4 and TNF-α). 2 The DCs possess stronger antigen processing function and can stimulate TDLNC to proliferate and differentiate to antigen specific CTL. 3 Autos specific CTL is of higher inhibiting the growing of transplantation tumor in nude mice, which provids theroy for immunotherapy of breast carcinoma. 4 The transplanted tumor tissue is confirmed of coincidence with human breast cancer, DC and massive lymphocytes are shown to infiltrate in transplanted tissue. 5 The success rate of implanting human breast tumor cells on nude mice is high, we succeed in establishing human breast carcinoma model in nude mice, which reserve biological features of human breast carcinoma, it may be an available model for feather research of breast carcinomaIn summary, this study, using cultured of TDLNCs in vitro, has obtained large amount of powerful tumor killing lymphocytes. It provides theoretical basis for the cellular immunotherapy on esophageal carcinoma.