乳腺癌骨髓微转移检测及特异性免疫治疗的实验研究
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摘要
目的:乳腺癌已成为女性发病率最高的恶性肿瘤,并呈逐年升高趋势,虽然采取以手术为主的综合治疗措施,但其远期生存率并没有获得明显提高,影响预后的主要因素是发生了远处器官的转移。据临床资料统计显示,约有25~30%腋淋巴结阴性的乳腺癌患者,于术后5年内出现远处转移而导致治疗失败,如果能在早期检测到微小转移灶,则对疾病的分期、治疗及改善预后具有重要意义。骨髓是乳腺癌最易发生转移的部位,骨髓微转移(bone marrow micrometastasis, BMM)的检测较腋窝淋巴结和外周血更能反映肿瘤细胞的全身播散情况,不仅能动态监测转移、观察疗效,并可成为预测乳腺癌预后的独立因素。因此,对于远处微小转移灶的早期检测及治疗成为提高乳腺癌患者远期生存率的主要途径之一。
造成乳腺癌患者治疗后复发和转移的主要原因是机体免疫力低下及肿瘤的免疫逃逸机制。因此,免疫治疗正逐渐成为肿瘤综合治疗的研究热点,有望成为肿瘤治疗的又一主要手段。免疫治疗中常用的活性细胞有:淋巴因子激活的杀伤细胞(LAK)、肿瘤浸润淋巴细胞(TIL)、细胞因子诱导的杀伤细胞(CIK)和特异性细胞毒T淋巴细胞(CTL)。有报道提出,利用树突状细胞(dendritic cell, DC)的高抗原递呈活性,将乳腺癌细胞抗原肽负载DC,从而激活机体免疫反应,可促进抗瘤效应和特异性。树突状细胞作为唯一的专职抗原递呈细胞(antigen processing cell, APC),处于免疫反应的中心地位。非成熟的DC捕获抗原后被激活成为成熟的DC,其细胞表面表达MHCⅠ类和Ⅱ类分子、共刺激分子B7-1(CD80)和B7-2(CD86)、黏附分子CD54(ICAM-1)和CD50(ICAM-3)等,并将抗原肽递呈给CD4+和CD8+T细胞,诱导其成为特异性细胞毒性T细胞(CTL),分泌细胞因子(如IL-12等),产生Th1型免疫应答而发挥抗肿瘤作用。
我们采用免疫磁珠(IMB)富集,联合扫描电镜(SEM)及激光共聚焦显微镜(LSM),检测了乳腺癌患者骨髓微转移(BMM);从乳腺癌患者腋窝区域引流淋巴结单个核细胞中,诱导培养DC和肿瘤抗原特异性CTL,通过体外和乳腺癌荷瘤裸鼠体内杀伤实验,以期寻找一种治疗乳腺
癌微转移的新途径。方法:
第一部分IMB联合SEM、LSM检测乳腺癌骨髓微转移选取2006年3月至10月我院收治的乳腺癌患者45例,健康志愿者4例(均签署了骨髓穿刺知情同意书)。研究对象均为女性,年龄30～67岁,平均49.8岁。术前在局部浸润麻醉下,行髂前上棘骨髓穿刺,抽取骨髓10~15ml,应用淋巴细胞分离液分离、收集有核细胞,IMB富集阳性对照、正常人对照、乳腺癌患者骨髓中转移的肿瘤细胞,将富集后细胞分成两等份,一份在SEM下采集肿瘤细胞图像,另一份用anti-CK-FITC标记细胞,在LSM下检测CK+细胞。将乳腺癌细胞株MDA-231制作成电镜标本,SEM下采集标准乳腺癌细胞的超微结构图象。
第二部分乳腺癌腋下淋巴结来源的DCs对自体CTLs体外特异性杀伤活性的影响术中严格无菌摘取乳腺癌引流淋巴结1~2枚,用机械法获取淋巴细胞悬液,并用淋巴细胞分离液分离单个核细胞,以含有10%胎牛血清的RPMI1640培养基重悬、培养2h,贴壁细胞以rhGM-CSF(1000U/ml), rhIL-4(200U/ml)和TNF-α(200U/ml)联合培养诱导DCs;非贴壁细胞,加入rhIL-2(200U/ml)培养为TDLNCs。用酶消化法将乳腺癌组织制备自体乳腺癌单细胞悬液,通过冻融法制备肿瘤抗原,负载DCs,将后者与TDLNCs共培养,以诱导肿瘤特异性CTLs。分别培养至第1天和第7天收获DCs,加入PE-CD1a、PE-CD83、FITC-CD86单克隆抗体,用流式细胞分析仪检测细胞表型;于第7天和第10天收获DC-TDLNCs,分别加入FITC-CD3单克隆抗体,FITC-CD4/PE-CD8二联抗体,检测其细胞表型。用非放射性细胞毒分析试剂盒,测定不同方法诱导的CTLs细胞对自体乳腺癌细胞和MCF-7细胞的体外杀伤活性,分析经诱导的DCs功能和CTLs杀伤特异性。
第三部分自体肿瘤特异性CTLs对裸鼠乳腺癌模型抑瘤作用的研究手术时分别无菌切取2例乳腺癌患者癌组织,剪成约1mm3大小组织块。将32只3~4周龄BALB/c裸鼠随机分成4组,每组8只,癌组织分别种植于裸鼠胸垫部皮下,建立裸鼠乳腺癌模型,移植瘤生长2周后,每5天注射一次不同的免疫效应细胞。(1)肿瘤特异性CTL组:每只裸鼠局部注射肿瘤特异性CTLs 0.2ml(5×106/ml);(2)非特异性CTL组:每只裸鼠局部注射未经自体肿瘤冻融抗原激活的非特异性CTLs 0.2ml(5×106/ml);(3)异体CTL组(种植另一患者癌组织):每只裸鼠局部注射非自体肿瘤特异性CTLs 0.2ml(5×106/ml);(4)空白对照组:每只裸鼠移植瘤模型局部注射生理盐水0.2ml。每5天用游标卡尺测量肿瘤长径(a)、短径(b),按体积(V)=πab2/6计算肿瘤体积,并计算肿瘤生长抑制率,30天后处死裸鼠取出瘤块。将肿瘤组织放入福尔马林溶液中固定、脱水、石蜡包埋、切片。经常规HE染色,镜下观察肿瘤细胞形态;免疫组化染色,观察T细胞和树突状细胞浸润情况。
结果:
1 4份骨髓有核细胞中掺入乳腺癌细胞株MDA-231的阳性对照组,在SEM、LSM下均可检测到肿瘤细胞;4份正常人骨髓有核细胞在SEM、LSM下均未检测到肿瘤细胞。
2 45例乳腺癌患者的骨髓标本中,有16例在SEM、LSM下同时检测到肿瘤细胞,BMM阳性检出率为35.6%。
3乳腺癌患者的BMM阳性率随着原发肿瘤的增大而增高,肿瘤直径≤2cm组、2~5cm组及>5cm组的BMM阳性率分别为11.1%、30.8%、70.0%,三者之间均有显著性差异, P=0.020。
4乳腺癌BMM阳性率随临床病理分期的增加而增高,临床Ⅰ期组BMM阳性率为20.0%、Ⅱ期组25.0%、Ⅲ期组87.5%,三者之间均有显著性差异, P=0.003。
5有腋淋巴结转移的乳腺癌患者之BMM阳性率为48.0%,无转移组为20.0%,两者之间无统计学差异,P=0.053。经分层研究发现,随着腋淋巴结转移数目的增加BMM阳性率增高,腋淋巴结转移数目≥4个组的BMM阳性率为58.8%,高于腋淋巴结转移数目为1~3个组(25.0%)及无腋淋巴结转移组(20.0%),并均有显著性差异,P=0.038。
6乳腺癌患者的BMM与患者的年龄、月经状况无关,P>0.05。
7 BMM阳性率随乳腺癌组织学分级增加而升高,组织学分化Ⅰ级组BMM阳性率为9.1%,Ⅱ级组为33.3%,Ⅲ级组为61.5%,三者之间有显著性差异,P=0.027。BMM与乳腺癌的病理类型无关,P>0.05。
8乳腺癌患者的BMM阳性率随肿瘤组织ER、PR蛋白表达的增强而降低,ER、PR阳性组BMM阳性率分别为18.2%、7.7%,低于ER、PR阴性组(52.2%、46.9%),P<0.05。乳腺癌患者的BMM与肿瘤组织C-erbB-2、VEGF的表达无关(P>0.05)。
9乳腺癌患者腋下引流淋巴结中的单个核细胞(TDLNCS)用细胞因子(rhGM-CSF、rhIL-4和TNF-α)诱导培养后,可以形成具有成熟DC形态特点的细胞。
10诱导前的TDLNCS中,DCs特异性表面标志物CD1a、CD83和CD86百分含量分别为10.98±2.38、26.55±5.24和32.96±6.09,经与rhGM-CSF、rhIL-4共同培养,并经自体肿瘤冻融抗原加TNF-α诱导后,DC-Ag-TDLNC细胞中CD1a、CD83和CD86百分含量明显增加,分别为50.17±5.68、60.48±16.46、56.22±16.38,P<0.01。
11诱导前的TDLNCs中,CD3+和CD8+细胞含量分别为73.93±2.18和32.78±3.21;诱导后DC-Ag-TDLNC组中CD3+和CD8+细胞含量分别为82.67±2.79和62.54±2.51,诱导后CD3+、CD8+T细胞含量均明显升高, P<0.01。诱导前TDLNCs中CD4+细胞含量为27.3±2.58;诱导后DC-Ag-TDLNC组中CD4+细胞含量为17.49±4.21;DC-TDLNC组中CD4+细胞含量为19.49±2.12。三组细胞相比较,CD4+细胞含量无明显变化,P>0.05。
12 DC-Ag-TDLNC细胞对自体乳腺癌细胞的杀伤率为67.64%;DC-TDLNC和TDLNC对自体乳腺癌细胞的杀伤率分别为31.25%、26.36%。DC-Ag-TDLNC、DC-TDLNC和TDLNC之间相比,对自体乳腺癌细胞的杀伤率逐渐降低,P<0.001;DC-TDLNC和TDLNC组间比较P<0.05。三组细胞对MCF-7细胞的杀伤率无明显差异,P>0.05。
13人乳腺癌组织在裸鼠体内的移植瘤成活率为100%。
14肿瘤特异性CTLs、非特异性CTLs及异体CTLs对裸鼠移植瘤的生长均有抑制作用,从移植瘤的第15天到第30天,各治疗组移植瘤体积均小于空白对照组,且都有显著性差异。对移植瘤生长的抑制作用以自体特异性CTL组最高,治疗15天后肿瘤体积平均为82.70±2.09mm3,小于非特异性CTL组(96.15±5.35mm3)和异体CTL组(96.93±4.51mm3),P =0.000;自体特异性CTL组抑瘤率(47.62%),明显高于非特异性CTL组(30.44%)和异体CTL组(24.69%)抑瘤率, P =0.000;
15移植瘤标本经HE染色证实为乳腺浸润性导管癌。免疫组化表明,治疗组瘤组织可见大量淋巴细胞和树突状细胞浸润。
结论:
1 IMB联合SEM、LSM检测原发性乳腺癌BMM,阳性检出率为35.6%。
2乳腺癌BMM与原发肿瘤大小及临床病理TNM分期有关,肿瘤的侵袭转移能力随着原发肿瘤增大而增强,临床分期越晚,出现骨髓转移几率越高。但肿块小、无淋巴结转移的Ⅰ期乳腺癌患者,亦存在约20%的BMM,提示乳腺癌是一种全身性疾病,转移及播散很早即可发生。
3虽然乳腺癌BMM与腋淋巴结转移与否无明显相关性,但进一步分层研究发现,随着腋淋巴结转移数目的增加BMM阳性率增高,说明血行播散与淋巴结转移是乳腺癌转移的两条相对独立的途径,当腋窝淋巴结出现较多转移时,癌细胞可能通过淋巴导管再次入血从而增加了转移机会。
4乳腺癌BMM阳性率随组织学分级增加而升高,说明分化程度低的肿瘤细胞具有更强的增殖、侵袭、转移能力,对这部分病人应加强化疗等综合性治疗,密切关注全身转移情况。
5乳腺癌BMM阳性率随ER、PR蛋白表达增强而降低,说明分化愈差的肿瘤细胞,ER、PR丢失愈多,可能具有更强的增殖、侵袭、转移能力。
6本研究未发现乳腺癌BMM与患者的年龄、月经状况、病理类型及肿瘤组织中C-erbB-2、VEGF的表达有关。
7乳腺癌患者腋下引流淋巴结来源的单个核细胞,在rhGM-CSF、rhIL-4刺激活化和自体乳腺癌细胞冻融抗原及TNF-α作用下,可以诱导培养出具有典型细胞学形态特征和高表达DC特异性表面标志物的成熟DCs,并具有较强的抗原提呈功能,可以促进TDLNCs增殖、分化为肿瘤抗原特异性CTLs。
8肿瘤抗原特异性CTLs对自体乳腺癌细胞有较高的体外杀伤效应,而对异体乳腺癌细胞不具有特异性杀伤效应。未经自体乳腺癌细胞冻融抗原刺激诱导的DCs虽也可以使TDLNCs增殖、分化为CTLs,但此CTLs不具有针对自体乳腺癌细胞的特异性杀伤效应,其对自体乳腺癌细胞的杀伤活性与以rhIL-2培养的TDLNCs相同。
9自体肿瘤特异性CTLs对裸鼠体内自体移植瘤有较高的抑制作用。癌组织内有DCs和大量淋巴细胞浸润。
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.
造成乳腺癌患者治疗后复发和转移的主要原因是机体免疫力低下及肿瘤的免疫逃逸机制。因此,免疫治疗正逐渐成为肿瘤综合治疗的研究热点,有望成为肿瘤治疗的又一主要手段。免疫治疗中常用的活性细胞有:淋巴因子激活的杀伤细胞(LAK)、肿瘤浸润淋巴细胞(TIL)、细胞因子诱导的杀伤细胞(CIK)和特异性细胞毒T淋巴细胞(CTL)。有报道提出,利用树突状细胞(dendritic cell, DC)的高抗原递呈活性,将乳腺癌细胞抗原肽负载DC,从而激活机体免疫反应,可促进抗瘤效应和特异性。树突状细胞作为唯一的专职抗原递呈细胞(antigen processing cell, APC),处于免疫反应的中心地位。非成熟的DC捕获抗原后被激活成为成熟的DC,其细胞表面表达MHCⅠ类和Ⅱ类分子、共刺激分子B7-1(CD80)和B7-2(CD86)、黏附分子CD54(ICAM-1)和CD50(ICAM-3)等,并将抗原肽递呈给CD4+和CD8+T细胞,诱导其成为特异性细胞毒性T细胞(CTL),分泌细胞因子(如IL-12等),产生Th1型免疫应答而发挥抗肿瘤作用。
我们采用免疫磁珠(IMB)富集,联合扫描电镜(SEM)及激光共聚焦显微镜(LSM),检测了乳腺癌患者骨髓微转移(BMM);从乳腺癌患者腋窝区域引流淋巴结单个核细胞中,诱导培养DC和肿瘤抗原特异性CTL,通过体外和乳腺癌荷瘤裸鼠体内杀伤实验,以期寻找一种治疗乳腺
癌微转移的新途径。方法:
第一部分IMB联合SEM、LSM检测乳腺癌骨髓微转移选取2006年3月至10月我院收治的乳腺癌患者45例,健康志愿者4例(均签署了骨髓穿刺知情同意书)。研究对象均为女性,年龄30～67岁,平均49.8岁。术前在局部浸润麻醉下,行髂前上棘骨髓穿刺,抽取骨髓10~15ml,应用淋巴细胞分离液分离、收集有核细胞,IMB富集阳性对照、正常人对照、乳腺癌患者骨髓中转移的肿瘤细胞,将富集后细胞分成两等份,一份在SEM下采集肿瘤细胞图像,另一份用anti-CK-FITC标记细胞,在LSM下检测CK+细胞。将乳腺癌细胞株MDA-231制作成电镜标本,SEM下采集标准乳腺癌细胞的超微结构图象。
第二部分乳腺癌腋下淋巴结来源的DCs对自体CTLs体外特异性杀伤活性的影响术中严格无菌摘取乳腺癌引流淋巴结1~2枚,用机械法获取淋巴细胞悬液,并用淋巴细胞分离液分离单个核细胞,以含有10%胎牛血清的RPMI1640培养基重悬、培养2h,贴壁细胞以rhGM-CSF(1000U/ml), rhIL-4(200U/ml)和TNF-α(200U/ml)联合培养诱导DCs;非贴壁细胞,加入rhIL-2(200U/ml)培养为TDLNCs。用酶消化法将乳腺癌组织制备自体乳腺癌单细胞悬液,通过冻融法制备肿瘤抗原,负载DCs,将后者与TDLNCs共培养,以诱导肿瘤特异性CTLs。分别培养至第1天和第7天收获DCs,加入PE-CD1a、PE-CD83、FITC-CD86单克隆抗体,用流式细胞分析仪检测细胞表型;于第7天和第10天收获DC-TDLNCs,分别加入FITC-CD3单克隆抗体,FITC-CD4/PE-CD8二联抗体,检测其细胞表型。用非放射性细胞毒分析试剂盒,测定不同方法诱导的CTLs细胞对自体乳腺癌细胞和MCF-7细胞的体外杀伤活性,分析经诱导的DCs功能和CTLs杀伤特异性。
第三部分自体肿瘤特异性CTLs对裸鼠乳腺癌模型抑瘤作用的研究手术时分别无菌切取2例乳腺癌患者癌组织,剪成约1mm3大小组织块。将32只3~4周龄BALB/c裸鼠随机分成4组,每组8只,癌组织分别种植于裸鼠胸垫部皮下,建立裸鼠乳腺癌模型,移植瘤生长2周后,每5天注射一次不同的免疫效应细胞。(1)肿瘤特异性CTL组:每只裸鼠局部注射肿瘤特异性CTLs 0.2ml(5×106/ml);(2)非特异性CTL组:每只裸鼠局部注射未经自体肿瘤冻融抗原激活的非特异性CTLs 0.2ml(5×106/ml);(3)异体CTL组(种植另一患者癌组织):每只裸鼠局部注射非自体肿瘤特异性CTLs 0.2ml(5×106/ml);(4)空白对照组:每只裸鼠移植瘤模型局部注射生理盐水0.2ml。每5天用游标卡尺测量肿瘤长径(a)、短径(b),按体积(V)=πab2/6计算肿瘤体积,并计算肿瘤生长抑制率,30天后处死裸鼠取出瘤块。将肿瘤组织放入福尔马林溶液中固定、脱水、石蜡包埋、切片。经常规HE染色,镜下观察肿瘤细胞形态;免疫组化染色,观察T细胞和树突状细胞浸润情况。
结果:
1 4份骨髓有核细胞中掺入乳腺癌细胞株MDA-231的阳性对照组,在SEM、LSM下均可检测到肿瘤细胞;4份正常人骨髓有核细胞在SEM、LSM下均未检测到肿瘤细胞。
2 45例乳腺癌患者的骨髓标本中,有16例在SEM、LSM下同时检测到肿瘤细胞,BMM阳性检出率为35.6%。
3乳腺癌患者的BMM阳性率随着原发肿瘤的增大而增高,肿瘤直径≤2cm组、2~5cm组及>5cm组的BMM阳性率分别为11.1%、30.8%、70.0%,三者之间均有显著性差异, P=0.020。
4乳腺癌BMM阳性率随临床病理分期的增加而增高,临床Ⅰ期组BMM阳性率为20.0%、Ⅱ期组25.0%、Ⅲ期组87.5%,三者之间均有显著性差异, P=0.003。
5有腋淋巴结转移的乳腺癌患者之BMM阳性率为48.0%,无转移组为20.0%,两者之间无统计学差异,P=0.053。经分层研究发现,随着腋淋巴结转移数目的增加BMM阳性率增高,腋淋巴结转移数目≥4个组的BMM阳性率为58.8%,高于腋淋巴结转移数目为1~3个组(25.0%)及无腋淋巴结转移组(20.0%),并均有显著性差异,P=0.038。
6乳腺癌患者的BMM与患者的年龄、月经状况无关,P>0.05。
7 BMM阳性率随乳腺癌组织学分级增加而升高,组织学分化Ⅰ级组BMM阳性率为9.1%,Ⅱ级组为33.3%,Ⅲ级组为61.5%,三者之间有显著性差异,P=0.027。BMM与乳腺癌的病理类型无关,P>0.05。
8乳腺癌患者的BMM阳性率随肿瘤组织ER、PR蛋白表达的增强而降低,ER、PR阳性组BMM阳性率分别为18.2%、7.7%,低于ER、PR阴性组(52.2%、46.9%),P<0.05。乳腺癌患者的BMM与肿瘤组织C-erbB-2、VEGF的表达无关(P>0.05)。
9乳腺癌患者腋下引流淋巴结中的单个核细胞(TDLNCS)用细胞因子(rhGM-CSF、rhIL-4和TNF-α)诱导培养后,可以形成具有成熟DC形态特点的细胞。
10诱导前的TDLNCS中,DCs特异性表面标志物CD1a、CD83和CD86百分含量分别为10.98±2.38、26.55±5.24和32.96±6.09,经与rhGM-CSF、rhIL-4共同培养,并经自体肿瘤冻融抗原加TNF-α诱导后,DC-Ag-TDLNC细胞中CD1a、CD83和CD86百分含量明显增加,分别为50.17±5.68、60.48±16.46、56.22±16.38,P<0.01。
11诱导前的TDLNCs中,CD3+和CD8+细胞含量分别为73.93±2.18和32.78±3.21;诱导后DC-Ag-TDLNC组中CD3+和CD8+细胞含量分别为82.67±2.79和62.54±2.51,诱导后CD3+、CD8+T细胞含量均明显升高, P<0.01。诱导前TDLNCs中CD4+细胞含量为27.3±2.58;诱导后DC-Ag-TDLNC组中CD4+细胞含量为17.49±4.21;DC-TDLNC组中CD4+细胞含量为19.49±2.12。三组细胞相比较,CD4+细胞含量无明显变化,P>0.05。
12 DC-Ag-TDLNC细胞对自体乳腺癌细胞的杀伤率为67.64%;DC-TDLNC和TDLNC对自体乳腺癌细胞的杀伤率分别为31.25%、26.36%。DC-Ag-TDLNC、DC-TDLNC和TDLNC之间相比,对自体乳腺癌细胞的杀伤率逐渐降低,P<0.001;DC-TDLNC和TDLNC组间比较P<0.05。三组细胞对MCF-7细胞的杀伤率无明显差异,P>0.05。
13人乳腺癌组织在裸鼠体内的移植瘤成活率为100%。
14肿瘤特异性CTLs、非特异性CTLs及异体CTLs对裸鼠移植瘤的生长均有抑制作用,从移植瘤的第15天到第30天,各治疗组移植瘤体积均小于空白对照组,且都有显著性差异。对移植瘤生长的抑制作用以自体特异性CTL组最高,治疗15天后肿瘤体积平均为82.70±2.09mm3,小于非特异性CTL组(96.15±5.35mm3)和异体CTL组(96.93±4.51mm3),P =0.000;自体特异性CTL组抑瘤率(47.62%),明显高于非特异性CTL组(30.44%)和异体CTL组(24.69%)抑瘤率, P =0.000;
15移植瘤标本经HE染色证实为乳腺浸润性导管癌。免疫组化表明,治疗组瘤组织可见大量淋巴细胞和树突状细胞浸润。
结论:
1 IMB联合SEM、LSM检测原发性乳腺癌BMM,阳性检出率为35.6%。
2乳腺癌BMM与原发肿瘤大小及临床病理TNM分期有关,肿瘤的侵袭转移能力随着原发肿瘤增大而增强,临床分期越晚,出现骨髓转移几率越高。但肿块小、无淋巴结转移的Ⅰ期乳腺癌患者,亦存在约20%的BMM,提示乳腺癌是一种全身性疾病,转移及播散很早即可发生。
3虽然乳腺癌BMM与腋淋巴结转移与否无明显相关性,但进一步分层研究发现,随着腋淋巴结转移数目的增加BMM阳性率增高,说明血行播散与淋巴结转移是乳腺癌转移的两条相对独立的途径,当腋窝淋巴结出现较多转移时,癌细胞可能通过淋巴导管再次入血从而增加了转移机会。
4乳腺癌BMM阳性率随组织学分级增加而升高,说明分化程度低的肿瘤细胞具有更强的增殖、侵袭、转移能力,对这部分病人应加强化疗等综合性治疗,密切关注全身转移情况。
5乳腺癌BMM阳性率随ER、PR蛋白表达增强而降低,说明分化愈差的肿瘤细胞,ER、PR丢失愈多,可能具有更强的增殖、侵袭、转移能力。
6本研究未发现乳腺癌BMM与患者的年龄、月经状况、病理类型及肿瘤组织中C-erbB-2、VEGF的表达有关。
7乳腺癌患者腋下引流淋巴结来源的单个核细胞,在rhGM-CSF、rhIL-4刺激活化和自体乳腺癌细胞冻融抗原及TNF-α作用下,可以诱导培养出具有典型细胞学形态特征和高表达DC特异性表面标志物的成熟DCs,并具有较强的抗原提呈功能,可以促进TDLNCs增殖、分化为肿瘤抗原特异性CTLs。
8肿瘤抗原特异性CTLs对自体乳腺癌细胞有较高的体外杀伤效应,而对异体乳腺癌细胞不具有特异性杀伤效应。未经自体乳腺癌细胞冻融抗原刺激诱导的DCs虽也可以使TDLNCs增殖、分化为CTLs,但此CTLs不具有针对自体乳腺癌细胞的特异性杀伤效应,其对自体乳腺癌细胞的杀伤活性与以rhIL-2培养的TDLNCs相同。
9自体肿瘤特异性CTLs对裸鼠体内自体移植瘤有较高的抑制作用。癌组织内有DCs和大量淋巴细胞浸润。
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.
引文
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1 袁 芃,徐兵河.乳腺癌骨髓微转移的检测及意义.国外医学肿瘤分册, 2002, 29(4) : 283~286
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