Study On Micrometastasis In Bone Marrow And Specific Immunotherapy Of Breast Cancer

Objective: Breast cancer is become a most common malignant tumor among women in recent years, the incidence of breast cancer is higher year by year. All though comprehensive measures, including surgery are taken, there is not improvement for long-term survival rate significantly. The main reason is associated with metastasis to a remote organ. By clinical statistics, it is showed that, the treatment is failed on the axillary lymph nodes metastasis negative patients about 25~30%, and the remote metastasis appear in 5 years after surgery. If micrometastasis were detected in early phase, it is important for staging and treatment of breast cancer as well as improvement of the disease prognosis. Bone marrow is most likely place for micrometastasis occuring in breast cancer patients. Detection of bone marrow micrometastasis(BMM) is better than that of axilary lymph nodes and blood metastasis on judging if cancer cells spread all over the body. It not only help us supervise metastasis dynamically, observe the treatment effect, but also has been an independent factor of predicting the prognosis of breast cancer. Therefore, early detection and treatment of remote metastasis is one of the ways to increase the long-term survival rate of breast cancer patients.The main reasons for breast cancer recurrence and metastasis after treatment are decreasing of patient's immune function and tumor escaping from host immune surveillance. Therfore, immunotherapy will become another major method of cancer treatment. The effective cells using in immunotherapy include lymphakine active killer cells (LAK), tumor-infiltrating lympjocytes(TIL), cytokine induced killer cells(CIK), and specific cytotoxicity T lymphocytes(CTL). The researches have showed that dendritic cell(DC) can greatly process tumor antigen, DC loaded by tumor antigen can activate host immune, promote the activity and specificity of CTL to kill tumor cells. DC, as the professional antigen processing cells, play a center role in immune reaction. The im-matured DCs will be activated to matured DCs after captured antigen, which express MHC classⅠand classⅡmolecule,co-stimulatory molecule B7-1(CD80) and B7-2 (CD86),adhesion molecule CD54 (ICAM-1) and CD50 (ICAM-3) in the cell surface. After presenting the antigen peptide to CD4+ and CD8+ T cells, DCs induce the T cells become to specific cytotoxicity T cells,which can excrete cytokines and produce Th1 type immune response to educe the anti-tumor effect.In this research, we detected BMM of breast cancer patients by immunomagnetic bead (IMB) combined with scanning electron microscope (SEM) and laser scanning microscope (LSM). Meanwhile DCs and specific CTLs were induced from single-nucleus cells of axillary lymph nodes and analyzed by killing test in vitro and breast cancer model in nude mice. The aim is hoping to find a new method to treat BMM of breast cancer.Method: Part one: Immunomagnetic bead combined with SEM and LSM to detect micrometastasis in bone marrow of breast cancer patients.In this rsearch, 4 healthy volunteers and 45 breast cancer cases in my hospital were taken at random between March and October in 2006. All of whom signed on consent to bone marrow puncture. These subjects were all of female, aged in 30 and 67, with an average of 49.8. After partial anesthetics, bone marrow puncture was done in the iliac upper front spina, 10-15ml bone marrow was taken, centrifuged and delaminated in lymphocytic liner. Karyocytes were collected, positive control, normal person negative control and cancer cells in the bone marrow of the breast cancer patients were enriched with IMB, and were divided into two equal portions. The one part of tumor cells imaging was analyzed under SEM, the other cells were reacted with anti-CK-FITC and CK+ cells were detected under LSM. Electron microscope samples were made by using breast cancer cell line of MDA-231, and as standard tumor cells imaging was observed by SEM. Part two: Influence on DCs from axillary draining lymph nodes of breast cancer patients to the specific killing activity of CTLs to auto-breast tumor in vitro.One or two lymphonodes strictly with asepsis were taked in operation. Then it was separated single-nucleus cells (SNC) in albuginea rete. The SNC from lymphonodes were cultured in 10% FCS RPMI1640. After adherencing 2 hours, the attached cells were cultured with rhGM-CSF (1000U/ml), rhIL-4(200U/ml) and TNF-α(200U/ml) to be induced into DCs. The unattached cells were cultured with rhIL-2(200U/ml) induce into tumor draining lymph node cells (TDLNCs). Breast cancer cells separated from auto-breast tumor by enzyme digestion were made for the auto-breast cancer freeze-thawing antigen. DCs were stimulated by the auto-breast cancer freeze-thawing antigen in order to load the tumor antigen, then, were co-cultured with TDLNCs to derivation tumor antigen specific CTLs. DCs suspension were harvested at the 1st day and the 7th day in vitro culturing and co-culturing with PE-CD1a,PE-CD83,FITC-CD86 MoAb, the cytophenotype was detected with flow cytometry(FCM). DC-TDLNCs were harvested at the 7th day and the 10th day and co-cultured with FITC-CD3 and FITC-CD4/PE-CD8 antibody, the cytophenotype of TDLNCs was detected. The cytotoxicity of the CTLs to auto-breast cancer cells and MCF-7 cells were determined with non-radioaction cytotoxic analytical reagent kite, to analysis the function of differentiated DCs and specificity of the CTLs.Part three: Study on suppress effect of specific CTLs for nude mice model of breast cancer in vivo.Fresh homobody breast cancer tissue was collected during operation. The tumor tissue was 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 had 8). The nude mice were injected every 5 days by different effective T cells. (1) Specific CTL group: each nude mouse was injected tumor specific CTLs 0.2ml; (2) Non-specific CTL group: each nude mouse was injected non-specific CTLs 0.2ml; (3) Varient CTL group: the nude mice were transplanted another patient's tumor tissue, and each nude mice was injected tumor specific CTLs 0.2ml; (4) Control group: each nude mouse was injected partly normal sodium 0.2ml. The concentration of effective cells was used for 5×106/ml. Maximal and minimal diameters of tumor tissue were measured every 5 days, and the tumor volume (V) was calculated as V =πab2/6, where"a"denotes the maximal diameter and"b"denotes minimal diameter. The inhibition parameter for tumor growth was calculated. After 30 days of implantation, the mice were sacrificed and the implanted tumors were collected. The tumor tissue was fixed in formalin solution, dehydrated, and imbedded in paraffin. The sections of tumor tissue were 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 study for the infiltration of T cells and DCs by immunohistochemistry (IHC).Results:1 Tumor cells were detected in the 4 positive control (normal person's BMM specimen was add in MDA-231) under SEM and LSM, while none were detected in the 4 negative control ones.2 From bone marrow specimen of 45 breast cancer patients, tumor cells were detected in 16 cases by SEM and LSM, BMM positive detection rate was 35.6%.3 BMM positive rate in breast cancer patients was increased as protopathic tumor became bigger. The BMM positive rate was 11.1%,30.8% and 70.0% respectively for the≤2cm,2~5cm and>5cm (in diameter). There are statistically significance among three groups, P=0.020.4 BMM positive rate in breast cancer patients was increased by the clinical TNM staging increased. BMM positive rate was 20.0%,25.0% and 87.5% respectively for StageⅠ,ⅡandⅢ. There are statistically significance among three groups, P=0.003. 5 Though the BMM positive rate (48.0%) of the axillary lymph nodes metastasis group was higher than that of the non-metastasis group (20.0%), there was no statistical difference between them, P = 0.053. But,by delamination research found that, with increased the number of the transferring axillary lymph nodes,BMM positive rate increased. BMM positive rate in the group whose transferred axillary lymph nodes were more than 4 was 58.8%, higher than the group whose transferred axillary lymph nodes between 1~3 (25.0%) and the non-metastasis group (20.0%), P=0.038.6 There was not relationship between the breast cancer BMM positive rate and patient's age and menstruation, P>0.05.7 BMM positive rate was increased with histological grade increasing. BMM positive rate of the Grade I group was 9.1%, lower than the Grade II group (33.3%) and Grade III group (61.5%), P=0.027. It was not relationship between the breast cancer BMM positive rate and the pathological typing of breast cancer, P>0.05.8 BMM positive rate in breast cancer patients was decreased with ER and PR expressions in tumor tissues intensified. BMM positive rate in ER and PR positive groups was 18.2% and 7.7% respectively, lower than ER and PR negative group (52.2%,46.9%), P<0.05. There was not relationship between BMM positive rate and C-erbB-2 and VEGF expressions in the tumor tissues, P>0.05.9 The mononuclear cells (TDLNCs) from axillary draining lymph nodes could be induced to typical maturated DCs in cell appearance after stimulating with cytokines (rhGM-CSF,rhIL-4 and TNF-α).10 The percentage with specific surface marker CD1a,CD83,CD86 on DCs induced from mononuclear cells in axillary draining lymph nodes before stimulation with antigen were 10.98±2.38,26.55±5.24,32.96±6.09 respectively; They were obviously heightened after cultured with rhGM-CSF and rhIL-4 and induced by autoallergic breast cancer freeze-thawing antigen and TNF-α, the percentage of CD1a,CD83,CD86 were 50.17±5.68,60.48±16.46,56.22±16.38 respectively, the percentage increased markedly after induced, P<0.01.11 The percentage of CD3+ and CD8+ T cells in TDLNCs were 73.93±2.18 and 32.78±3.21 before stimulation with tumor antigen. and were 82.67±2.79 and 62.54±2.51 after stimulation with rhIL-2 and Ag-DCs. The percentage of CD3+ and CD8+ T cell could be increased by the Ag-DCs, P<0.01. The percentage of CD4+ T cells in TDLNCs was 27.3±2.58 before stimulation with tumor antigen,however the percentage in DC-Ag-TDLNCs and DC-TDLNCs were 17.49±4.21 and 19.49±2.12.The percentage of CD4+ T cells wasn't heighten after induced by comparison,P>0.05.12 The ratio of the CTLs activated by the DCs loaded with auto-breast cancer freeze-thawing antigen, and cytotoxicity(67.64%) for auto-breast cancer cells was higher than the CTLs activated by the DCs no-loaded the auto-breast cancer freeze-thawing antigen (31.25%) and by the TDLNCs (26.36%) , P<0.001, The ratio of the CTLs activated by the DCs no-loaded with the auto-breast cancer freeze-thawing antigen and the cytotoxicity for auto-breast cancer cells was higher than the TDLNCs, P<0.05. There was no significant difference of cytotoxicity in the three group cells for MCF-7 cells, P>0.05.13 The success rate of implanting human breast tumor tissues on nude mice was 100%.14 Every group treated with different effective T cells could inhibite the growth of implanted carcinoma. The autos specific CTL group shown the strongest inhibition effect, whose average gross tumor volume was 82.70±2.09mm3 after treated for 15 days. It was lower than non-specific CTL group (96.15±5.35mm3) and variant CTL group (96.93±4.51mm3), P =0.000. The tumor inhibition rate of autos specific CTL group (47.62%)was significantly higher than non-specific CTL group (30.44%) and variant CTL group (24.69%),P =0.000.15 It was confirmed that mammary infiltrating ductal carcinoma by HE stained, and generous lymphocytes and DCs infiltrate in the tumor tissue after treated with different effective T cells by IHC stained.Conclusions:1 BMM positive detection rate in 45 primary breast cancer patients was 35.6% detected by IMB combined with SEM and LSM.2 BMM in breast cancer patients relates to the size of the primary tumor and clinical pathological TNM staging. The ability of the tumor to invade and transfer strengths is stronger with the bigger of the primary tumor. The later the clinical staging,the more chances for BMM. But BMM may occur in Stage I breast cancer patients for 20% in whom the tumor is small and the axillary lymph nodes do not transfer. It indicates that breast cancer is a general disease. Its metastasis and spread may occur on an early phase of the disease.3 Though breast cancer BMM has nothing to do with the axillary lymph nodes metastasis, further delamination research found that,with the rise in the number of transferred axillary lymph nodes BMM positive rate increased, which shows that vascular spread and axillary lymph nodes metastasis are two relatively independent means of breast cancer metastasis. When many axillary lymph nodes transfer,cancer cells may increase their chances of metastasis by reentering blood through lymphoducts.4 Breast cancer BMM positive rate increased as histological grading increased, which indicates that low-differentiated cancer cells are much able to reproduce, invade and transfer.5 Breast cancer BMM positive rate relates to ER and PR protein expressions in tumor tissues. Breast cancer BMM positive rate decreased with ER and PR protain expressions intensified,which indicates that the lower tumor cells differentiate, the lower ER and PR express, and they could be of the capacity of infiltrating and metastasis.6 No relationship was found between breast cancer BMM positive detection rate of the 45 cases and patient's age, menstruation, the pathological typing or the C-erbB-2 and VEGF expressions in the tumor tissues.7 The mononuclear cells from axillary draining lymph nodes can be induced to typical maturated DCs in cell appearance after being stimulated by cytokines (rhGM-CSF,rhIL-4 and TNF-α), which highly express specific surface markers and possess stronger angtigen presentation capacity, and can stimulate TDLNCs to proliferate and differentiate into CTLs which possess highly killing effect.8 The tumor antigen specific CTLs possess stronger killing capacity to autoallergic breast cancer cells, however they have no the stronger capacity to other line of tumor cells. Autos specific CTLs are of higher inhibiting the growing of transplantation tumor in nude mice. DCs and massive lymphocytes are shown to infiltrate in transplanted tissue.9 Auto-tumor specific CTLs have a greater inhibition to implanted tumor, generous lymphocyte infiltrate and DCs are shown to infiltrate in tumor tissue.