CDC50A阳性细胞在卵巢癌细胞系及原代细胞中干细胞特性的鉴定及相关研究
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摘要
研究背景
卵巢上皮癌(EOC)是致死率最高的妇科恶性肿瘤,其中75%的患者诊断时已为晚期(Ⅲ～Ⅳ)。在理想的肿瘤细胞减灭术的基础上辅助铂类药物为主的联合化疗后,约60-80%的患者发生肿瘤复发,晚期卵巢癌患者的中位生存时间仅为16-22个月,5年生存率约为30%。肿瘤细胞对于化疗药物产生耐受性被认为是造成这一现象的主要原因之一。近期研究的靶向药物及小分子化合物的临床实验均未能显著改善卵巢癌患者的预后。随着肿瘤基因组图谱的揭示,学者们对卵巢癌的研究主要致力于基因组及表观遗传学的变化对临床预后的影响。但是卵巢癌化疗耐药及肿瘤复发的机制仍不清楚,从而成为卵巢癌治疗中的最大障碍。
干细胞是机体内一类具有自我更新能力和多向分化潜能的特殊细胞。根据其来源和分化能力,主要分为,胚胎干细胞、骨髓干细胞、以及成体干细胞。随着肿瘤生物学的发展,上世纪学者们提出了肿瘤干细胞(CSCs)理论。大量研究发现肿瘤中存在的极少量的CSCs,其既拥有类似干细胞自我更新及分化的特性,又维持着肿瘤细胞的致瘤性及细胞异质性,在肿瘤中长期处于静息状态,可以逃避化疗药的杀伤,导致肿瘤的耐药,疾病进展及复发。早在1997年Bonnet等人分离出表型为CD34+CD38的白血病干细胞,首次在细胞水平上直观证实了肿瘤干细胞的存在。随后,CSCs理论在许多实体瘤如乳腺癌、前列腺癌、结肠癌、肺癌、胰腺癌等中得到验证,并建立了一系列CSCs分离、富集、鉴定及分子机制研究的方法。目前,CSCs的分离及富集方法主要包括：侧群细胞(side population, SP)的流式分选,根据特异性的CSCs表面标记蛋白的流式分选,以及肿瘤球形体(sphere)的培养等。
近年来,许多致力于寻找卵巢癌治疗新途径的学者们也将目光转移到肿瘤干细胞,借鉴在其它实体瘤的研究中的成熟经验,探索了卵巢癌干细胞研究的新领域。目前对于卵巢癌干细胞的分选仍然是根据在其它实体瘤干细胞研究中的结果,采用表面标记蛋白的流式分选技术,使用最多的标记物主要包括,CD133、CD117、CD44、 ALDH1、EPCAM、CD24、以及CD90等。虽然取得了一些研究进展,但由于这些标记物均来自于其它实体瘤干细胞的研究,其对卵巢癌干细胞的分离富集作用并不十分理想,仍然存在争论。因此,寻找卵巢癌特异性表面标记蛋白,成功分离并鉴定卵巢癌干细胞,对于未来卵巢癌的靶向治疗可能具有重大意义。
本课题在前期工作中,采用稳定同位素标记和以质谱为基础的定量蛋白质组学技术对SP和非SP细胞的膜蛋白进行比较蛋白质组学分析,成功筛选出卵巢癌干细胞表面标记候选蛋白——跨膜蛋白30A (transmembrane protein30A，简称TMEM30A或CDC50A),并在体外实验中对其表达阳性细胞的干细胞特性进行了初步鉴定。本研究采用流式细胞分选及无血清悬浮培养技术,通过体内和体外实验,在卵巢癌细胞系和卵巢癌患者的组织和腹水中验证CDC50A表面标记阳性(CDC50A+) EOC细胞的sphere形成能力,自我更新能力,分化能力及致瘤能力,检测其对顺铂的耐药性,并分析其表达与EOC患者临床预后的关系。并初步探讨雌激素在卵巢癌干细胞微环境中的作用及其对术后卵巢癌患者临床预后的影响。
研究方法
1、运用流式细胞学方法分析卵巢癌细胞系SKOV3, A2780, IGROV1, COC1, OVCAR3, ES2中CDC50A+细胞的比例。采用流式细胞仪分选CDC50A+及CDC50A+细胞,验证其在HG-DMEM/10%FBS培养基中体外培养后再次分化成其他亚群的能力。采用无血清悬浮培养的方法检测CDC50A+及CDC50A+细胞sphere形成能力及sphere传代能力,并运用免疫荧光及流式细胞学方法检测SKOV3sphere对CDC50A的富集作用。流式分选出CDC50A+及CDC50A+细胞,分别等量梯度接种于NOD/SCID小鼠皮下,观察成瘤速度,观察终点为两组成瘤率或成瘤大小出现明显统计学差异。待小鼠成瘤后,再次分选出两群细胞接种小鼠,观察成瘤情况。流式分析移植瘤中CDC50A+细胞的比例,检测CDC50A+及CDC50A+细胞体内分化能力。
2、收集EOC患者卵巢癌组织及腹水,分离卵巢癌细胞,运用流式细胞学方法分析原代卵巢癌细胞中CDC50A+细胞的比例。建立原代卵巢癌细胞sphere的培养方法,检测原代卵巢癌CDC50A+及CDC50A+细胞sphere形成能力及传代能力。运用免疫荧光及流式细胞学方法检测原代卵巢癌sphere对CDC50A的富集作用。流式分选出CDC50A+及CDC50A+细胞,分别等量梯度接种于NSG小鼠皮下,观察成瘤速度,观察终点为两组成瘤率或成瘤大小出现明显统计学差异。待小鼠成瘤后,再次分选出两群细胞接种小鼠,观察成瘤情况。流式分析移植瘤中CDC50A+细胞的比例,检测CDC50A+及CDC50A+细胞体内分化能力。
3、MTT法验证SKOV3及ES2细胞系中CDC50A+及CDC50A-细胞对顺铂耐药性差异,流式细胞学方法检测不同浓度的顺铂对SKOV3/CDC50A+细胞的富集作用。流式细胞学方法检测顺铂化疗对EOC患者卵巢癌组织中CDC50A+细胞的富集作用；建立NOD/SCID小鼠卵巢癌移植瘤模型,检测顺铂化疗对小鼠移植瘤中CDC50A+细胞的富集作用。
4、运用流式细胞学方法分析EOC患者卵巢癌原位灶及转移灶中CDC50A+细胞的比例,流式分析处于临床治疗不同阶段的EOC患者组织中CDC50A+细胞比例的分布情况,免疫组化分析76例EOC患者CDC50A蛋白的表达与临床预后的相关性。
5、采用不同浓度的雌激素作用CDC50A+细胞,检测雌激素在维持卵巢癌干细胞的生长及自我更新中的作用。结合临床资料,回顾性分析术后激素替代治疗对卵巢癌患者的复发及生存的影响。
研究结果
1、流式检测发现6种卵巢癌细胞株中均存在少量CDC50A+细胞群,其比例稳定维持在0.4%～2%之间。流式分选出CDC50A+细胞亚群,在细胞分化条件下体外培养2周,再次流式分析发现CDC50A+细胞分化出CDC50A细胞,其阳性率仍保持原来水平,而CDC50A细胞未能分化出CDC50A+细胞,表明CDC50A+细胞具有分化潜能。无血清悬浮培养出SKOV3sphere,流式分选出CDC50A+及CDC50A-细胞,分别悬浮培养,发现CDC50A+细胞形成sphere的个数明显多于CDC50A细胞,CDC50A+细胞形成的sphere能连续体外传代至少4代,而CDC50A细胞形成的sphere几乎不能传代。免疫荧光与流式分析均证实CDC50A+细胞在SKOV3sphere表达显著高于贴壁SKOV3细胞；体内动物实验进一步验证了CDC50A+细胞体内成瘤能力明显强于CDC50A细胞,102个CDC50A+细胞即能使NOD/SCID小鼠成瘤,而至少104个CDC50A细胞才能使NOD/SCID小鼠成瘤。CDC50A+细胞移植瘤能够在小鼠体内连续传代,而CDC50A细胞移植瘤不能传代。HE染色显示CDC50A+细胞移植瘤与第二代移植瘤的组织学类型保持一致。流式分析显示CDC50A+细胞移植瘤中CDC50A阳性率保持不变,而CDC50A细胞移植瘤中几乎无CDC50A+细胞,说明CDC50A+细胞在体内仍具有分化能力。
2、从原代卵巢癌组织及腹水中成功分离出卵巢癌细胞,流式分析显示原代卵巢癌组织及腹水细胞中均存在CDC50A+细胞群。成功建立了原代卵巢癌细胞sphere培养体系,发现原代组织中CDC50A+细胞能形成sphere,并能连续传代,而CDC50A细胞几乎不能形成sphere;免疫荧光及流式细胞学检测均证实CDC50A+细胞在原代卵巢癌sphere中的表达显著增高。体内动物实验进一步验证了原代卵巢癌CDC50A+细胞体内成瘤能力明显强于CDC50A细胞,103个CDC50A+细胞仍能使NSG小鼠成瘤,而至少105个CDC50A细胞才能使NSG小鼠成瘤。CDC50A+细胞移植瘤能在小鼠体内连续传代,而CDC50A细胞移植瘤不能传代；HE染色显示原代CDC50A+细胞移植瘤与患者组织学类型保持一致。流式分析显示原代CDC50A+田胞移植瘤中CDC50A阳性率保持不变,而CDC50A细胞移植瘤中几乎无CDC50A+细胞,说明原代CDC50A+细胞在体内仍具有分化能力。
3、MTT法对细胞耐药性的检测发现,在SKOV3和ES2细胞系中CDC50A+细胞对顺铂的耐药性明显高于CDC50A细胞。流式细胞学检测发现,不同浓度的顺铂作用后的SKOV3细胞后,细胞总数减少,CDC50A阳性率却逐渐增高,而且CDC50A+细胞绝对数基本不变。分析同一患者先期化疗前后卵巢癌组织中CDC50A阳性率,发现顺铂化疗后CDC50A+细胞比例显著增高。动物实验显示,接受顺铂化疗后的小鼠移植瘤增长速度显著慢于未治疗组,而移植瘤中CDC50A阳性率却显著高于未治疗组,进一步证明了顺铂对CDC50A具有富集作用。
4、流式细胞学方法分析发现,EOC患者卵巢癌转移灶中CDC50A+细胞比例显著高于原位灶。对处于临床治疗不同阶段的EOC患者组织中CDC50A+细胞比例分析发现,CDC50A+细胞在初治手术患者中平均比例为1.47%,在先期化疗患者中为9.76%,而在复发的患者中为13.35%。免疫组化分析发现76例EOC患者中22例患者CDC50A蛋白高表达,54例患者CDC50A蛋白低表达或阴性表达。Logistic回归分析发现CDC50A蛋白表达和术后残余病灶是影响患者耐药的独立因素。Kaplan-Meier分析证实CDC50A蛋白高表达患者无进展生存时间(PFS)显著低于CDC50A蛋白低表达患者,中位无进展生存时间分别为10个月和17个月。Cox多元回归模型分析显示卵巢癌FIGO分期和CDC50A蛋白表达是影响PFS独立因素,CDC50A蛋白高表达患者发生卵巢癌复发的风险是低表达患者的2.85倍(95%CI1.32-6.16,p<0.01)。
5、采用不同浓度的雌激素作用CDC50A+细胞,发现其对sphere的生长及自我更新能力均无显著影响。回顾性病例分析显示：HRT不是PFS及OS的独立影响因素,而且不同HRT方案对EOC患者预后无明显影响。残余病灶大小及FIGO分期是影响无进展生存期的独立因素。FIGO分期还是影响总生存期的独立因素。
结论
1、CDC50A+细胞在六种卵巢癌细胞系中的表达稳定在0.4%～2%之间,符合干细胞比例。卵巢癌细胞系中CDC50A+细胞群具有体内外分化能力,sphere形成能力,自我更新能力等CSCs特性。CDC50A+细胞具有更强的肿瘤形成能力,并能在小鼠体内连续传代。
2、EOC患者原代卵巢癌细胞中能够分离出CDC50A+细胞群,该细胞群具有体内分化能力,sphere形成能力,自我更新能力等CSCs特性。原代卵巢癌细胞中的CDC50A+细胞具有更强的致瘤能力,且能在小鼠体内连续传代。
3、卵巢癌细胞系、原代卵巢癌细胞、以及动物实验均证实顺铂对于CDC50A+细胞具有富集作用。
4、EOC患者卵巢癌转移灶中CDC50A+细胞比例显著高于原位灶,表明CDC50A可能与卵巢癌的转移有关。CDC50A阳性细胞的比例在复发患者中最高,其次是先期化疗患者,初治手术患者的表达比例最低,进一步说明CDC50A可能与患者耐药及复发有关。临床分析表明,CDC50A蛋白的表达是患者耐药及复发的独立影响因素,高表达CDC50A的患者具有更高的复发风险。
5、通过体外实验发现：雌激素在维持CDC50A+卵巢癌干细胞的生长及自我更新中无显著作用,其并不是维持卵巢癌干细胞的重要因子。结合临床资料,采用回顾性病例分析发现术后激素替代治疗对卵巢癌患者的复发及生存无明显影响。
Background
Epithelial ovarian cancer (EOC) is the leading causes of gynecological cancer mortality, among which75%patients were diagnosed as advanced ovarian cancer. Under the treatment of primary cytoreductive surgery followed by chemotherapy based on platinum, about60～80%patients relapsed. The median survival time of advanced ovarian cancer patients was only16～22months, and the5-year survival rate is only30%. The chemotherapy resistance of the tumor cells is considered to be one of the main reasons for this phenomenon. A recent study of targeted drugs and small molecular compounds in clinical trials failed to improve the prognosis of patients with ovarian cancer. With the emergence of cancer genome map, many scholars are mainly engaged in the effects of the genome and epigenome changes on the clinical outcomes of ovarian cancer. But the phenotype mechanism of ovarian cancer chemotherapy resistance and rumor recurrence is still not clear, so it become the biggest obstacle for ovarian cancer therapy.
Stem cells are known as a kind of special cells which has the ability of self-renewal and multi-directional differentiation. According to its origin and differentiation ability, they are mainly divides into embryonic stem cells, mesenchymal stem cells and adult stem cells. With the development of tumor biology, scholars put forward the theory of cancer stem cells (CSCs) in the last century. Lots of studies have found that there were a very small number of CSCs existed in tumors at quiescent state, which not only have the similar characteristics of stem cells, such as self-renewal and differentiation, but also maintained the tumorigenicity and cell heterogeneity of tumor cells. So it can escape from the toxic damage of chemotherapy drugs, and result in the drug resistance, progression and recurrence of tumor. Early in1997, Bonnet and his companies isolated the phenotype of CD34+CD38" leukemia stem cells, and directly confirmed the existence of cancer stem cells for the first time at the cellular level. After that, the theory of CSCs was identified in many solid tumors such as breast cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, and established a series of separation, enrichment, identification and molecular mechanism research methods of CSCs. At present, the separation and enrichment of CSCs method mainly includes:the sorting of side population cells (side population, SP) by flow cytometry, sorting according to the specific CSCs surface marker by flow cytometry, and the cultivation of the tumor sphere.
Recently, many scholars begin to research in the cancer stem cells to looking for the new treatment of ovarian cancer. They will study on the basis of the outcomes of other solid cancer stem cells, and may open a new chapter of ovarian cancer stem cell research. Sorting according to the specific CSCs surface marker by flow cytometry is still the main way to isolate ovarian cancer stem cells, and the present study mainly include the following surface markers:CD133, CD117, CD44, ALDH1, EPCAM, CD24, CD90and so on. Although some research progress has been made, because these markers are draw lessons from other solid tumor stem cells, the separation and enrichment of ovarian cancer stem cells is not very effective, and there is still a debate. Therefore, looking for a specific surface marker to successfully separate and identifiy the ovarian cancer stem cells, may has great value to targeted therapy of ovarian cancer.
In the early stage of our work, we used quantitative proteomic techniques based on the stable isotope labeling and mass spectrometry to compare the membrane protein of SP and SP cells, and successfully screened the ovarian cancer stem cell surface candidate protein-transmembrane protein30A (TMEM30A, also called CDC50A). So, on the basis of the previous study, in this research, we mainly identified the CSCs properties of CDC50A surface markers positive (CDC50A+) cells in vivo and in vitro by flow cytometry and serum-free suspension culture technology, which mainly included the ability of sphere formation, self-renewal, differentiation and tumorigenesis of CDC50A positive ovarian cancer cells. Besides, we also validated the cisplatin resistance of CDC50A positive cells, and analyzed the relationship between the expression of CDC50A protein and the clinical outcomes of EOC patients. At last, we demonstrated the role of estrogen played in the microenvironment of CSCs and discussed the effect of HRT on the prognosis of EOC patients.
Methods
1. The proportion of CDC50A positive cells in the ovarian cancer cell lines such as SKOV3, A2780, IGROV1, COC1,OVCAR3, ES2were analyzed by flow cytometry; CDC50A positive and CDC50A negative cells were sorted by Moflo fluorescent activated cell stream sorter and cultured in HG-DMEM/10%FBS medium, then we verified the differentiation capability of the two populations in vitro; The sphere formation and serial passage ability of the CDC50A positive and CDC50A negative cells were tested by serum-free suspension culture technology; The enrichment of CDC50A positive cells in SKOV3spheres were tested by immunofluorescence and flow cytometry; SKOV3cells were sorted into CDC50A positive and negative population, then serial dilutions of the two population cells were resuspended (1:1) in PBS:Matrigel and implanted subcutaneously into the scapular region of NOD/SCID mice respectively to identify the tumorigenesis capacity of two populations. When the transplanted tumors formed, sorting out of the two populations, inoculated into mice again to test the self renewal capacity in vivo; The proportion of CDC50A positive cells in transplanted tumors was analyzed by flow cytometry to test the differentiation ability of CDC50A positive and negative cells in vivo.
2. Tissues and ascites of the EOC patients were collected and the ovarian cancer cells were isolated; The proportion of CDC50A positive cells in the primary ovarian cancer cells were analyzed by flow cytometry; We established the primary tumor sphere culture system, and test the sphere formation capacity and serial passage ability of the primary CDC50A positive and CDC50A negative tumor cells; The enrichment of CDC50A positive cells in primary ovarian tumor spheres were tested by immunofluorescence and flow cytometry; Then, ovarian tumor cells were sorted into CDC50A positive and negative population, and serial dilutions of the two population cells were resuspended (1:1) in PBS:Matrigel and implanted subcutaneously into the scapular region of NSG mice respectively to identify the tumorigenesis capacity of two populations. When the transplanted tumors formed, sorting out of the two populations, inoculated into mice again to test the self renewal capacity in vivo; The proportion of CDC50A positive cells in transplanted tumors was analyzed by flow cytometry to test the differentiation ability of CDC50A positive and negative cells in vivo.
3. We tested the difference of cisplatin resistance to CDC50A positive and negative cells by MTT assay in vitro experiments; Then the flow cytometry analysis were used to detect the enrichment of SKOV3CDC50A positive cells with different concentrations of cisplatin; we used the same method to test the enrichment of primary CDC50A positive tumor cells sorted from EOC patient with cisplatin chemotherapy; Besides, we established the transplanted tumor model with NOD/SCID mice, and test the enrichment of CDC50A positive tumor cells with cisplatin chemotherapy.
4. We analyzed the proportion of CDC50A positive cells in primary tumors and metastasis of EOC patients respectively by flow cytometry; Then we analyzed the proportion of CDC50A positive cells in different EOC patients; Immunohistochemistry were used to analyzed CDC50A protein expression of76EOC patients and its correlation with clinical prognosis.
5. Different concentration of estrogen were added to culture of CDC50A positive cells, and demenstrated the role of it in maintaining the growth and the self-renewal of ovarian cancer stem cells. Then, in combination with clinical data, we analyzed the impact of postoperative hormone replacement therapy on the patients with ovarian cancer.
Results
1. We found that CDC50A positive population were exist in six ovarian cancer cell lines, and the proportion of CDC50A positive cells were ranged from0.4%to2%; We cultured CDC50A positive cells in vitro after sorting from SKOV3cell line, which could again differentiate to CDC50A negative cells and maintain its positive rate, but the CDC50A negative cells couldn't differentiate to CDC50A positive cell subsets, which indicated that CDC50A positive cells had the potential to differentiate to CDC50A negative cells; The SKOV3sphere were formed by serum-free suspension culture after one week, and compared to the CDC50A negative cells, CDC50A positive cells were more capable of generating spheres. Besides, CDC50A positive spheres can passage consecutively, while the CDC50A-spheres can't. Besides, we examined the expression of CDC50A protein in SKOV3spheres and attached cells by immunofluorescence, which showed that the majority spheroid cells stained for CDC50A, while the attached cells can't. Similarily, we found that the expression of CDC50A in spheroid cells was much higher than the attached cells by flow cytometry. The tumor formation experiment showed that1/3mice injected with1×104CDC50A negative cells can formed tumors, but no tumor formation detected following injection of1×103cells, While CDC50A positive cells were tumorigenic in mice even with1×102cells. We also found that only CDC50A positive cells were able to generate secondary tumors in NOD/SCID mice. The H&E staining and flow cytometry analysis showed that not only the histology of the first and second passage xenografts was similar, but also the frequency of CDC50A positive cells was maintained in xenografts obtained by serial in vivo passaging of CDC50A positive cells,which indicated that CDC50A positive cells also have the capacity of differentiation m vivo.
2. To further demonstrated the properties of CDC50A+cells in primary tumor cells, we analyzed primary EOC specimens to find that CDC50A was present at variable expression in all primary tumors and ascite samples. Similar to cell lines, we isolated and inoculated the human ovarian tumor and ascite cells at a density of104cells/ml to low-attachment6-well plates in serum-free medium with cytokines. Spheres were observed about2weeks. In order to further demonstrate the sphere formation capacity of CDC50A+/Lin-cells, CDC50A+/Lin-and CDC50A7Lin-populations were sorted from three human ovarian tumors to inoculate in suspension culture at a density of10000cells/well. Compared to CDC50A-/Lin-cells, CDC50A+/Lin-cells were more capable of generating spheres and even with serial passage. Then, we found that CDC50A protein was expressed at a high level in spheres by immunofluorescence. Similarily, compared to the isolated tumor cells from human tissues, the proportion of CDC50A+/Lin-cells in spheres was much higher. To test if the CDC50A+/Lin-cells isolated from human EOC to generate tumors in mice, sorted cells were suspended in Matrigel and subcutaneously injected into NSG mice. We found that104CDC50A-/Lin-cells did not grow tumors, While104CDC50A+/Lin-cells generated tumors from3/8. Tumor generation required approximately～5months. Similarly, We found that only CDC50A+/Lin-cells were able to generate secondary tumors in NSG mice. Histological analysis of the xenograft tumor generated by CDC50A+/Lin-cells was identical to that seen in the primary tumor; Flow cytometry analysis showed that the frequency of CDC50A+/Lin-cells was maintained in CDC50A positive xenografts obtained by serial in vivo passaging.
3. Cell survival assay showed greater cisplatin resistance of CDC50A positive cells from SKOV3and ES2cell lines. Then we assayed the proportion of CDC50A positive cells after treatment with increasing doses of cisplatin. A clear, dose dependent decrease in the total number of viable cells were observed, While there was a significant increase in the proportion of CDC50A+cells, the most interesting thing is that the absolute number of CDC50A positive cells were maintained during the treatment of cisplatin, which suggested CDC50A positive cells played an important role in ovarian cancer cisplatin chemoresistance. Besides, we compared the proportion of CDC50A+/Lin-cells in the same patient with ovarian cancer before and after cisplatin chemotherapy, the result indicated that the chemotherepy can enrich the CDC50A+/Lin-cells in human ovarian tumors. To further demonstrated CDC50A positive cells are resistant to cisplatin in vivo, NOD/SCID mice were inoculated with SKOV3cells, and treated by saline or cisplatin for a month. Then we observed that cisplatin significantly inhibited the increase of the mean volumes of tumors in the treatment group. Besides, we identified that the proportion of CDC50A+/Lin-cells from the cisplatin treated tumors were much higher than the control tumors, which further demonstrated that the cisplatin can enrich the CDC50A+/Lin-cells.
4. Flow cytometry analysis showed that a higher proportion of CDC50A positive cells were observed in metastasis. During the primary ovarian tumors we collected, the patients with advanced stage serous adenocarcinoma were classified to three group: initial diagnosed patients, patients with neoadjuvant chemotherapy based on cisplatin and the relapsed patients. According to the analysis by flow cytometry, the average proportion of CDC50A positive cells was1.47%in tumors of initial diagnosed patients,9.76%in tumors of neoadjuvant chemotherapy patients and13.35%in tumors of relapsed patients. It is suggested that the expression of CDC50A may correlated with the cisplatin resistance and relapse. To further assess whether our experimental findings on CDC50A are relevant to human ovarian cancer patients in the clinic, we therefore scored a panel of76ovarian tumors for the presence of CDC50A expressing cells using immunohistochemistry. There were22patients with CDC50A high expression and54with low or negative expression. The Logistic regression analysis showed that CDC50A expression and resection status were the independent factor that impact on the platinum resistance of patients. Besides, Kaplan-Meier analysis demonstrated that patients with CDC50A high expression tumor cells have worse progression free survival (PFS) compared to the CDC50A low expression group. And the median survival time for PFS were10months for CDC50A high expression patients and17months for CDC50A low expression patients. Then we performed a Cox multivariate analysis of PFS, which showed that CDC50A expression was an independent prognostic factor, as well as tumor stage. The relative risk of relapse due to ovarian cancer was2.85for patients with CDC50A positive tumors compared to patients with CDC50A negative tumors (95%CI1.32-6.16, p<0.01), which indicated that the expression of CDC50A may predict poor clinical outcome of ovarian cancer patients.
5. We found that the growth and self-renewal ability of sphere had no significant influence under the different concentration of estrogen. According to the multivariate analysis, HRT did not significantly influence progression-free or overall survival. Similarly, different types of HRT had no significant effect on the prognosis of epithelial ovarian cancer patients. And the strongest independent variable in predicting both progression-free survival and overall survival was the FIGO stage of the disease.
Conclusion
1. The ratio of CDC50A positive cell subsets in six ovarian carcinoma cell lines is low, which is similar to the ratio of stem cells; The experiments in vitro and in vivo showed that CDC50A positive cells sorted from SKOV3cell lines have the ovarian cancer stem cell properties, such as serial sphere generation, self-renewal, differentiation and tumorigenesis capability.
2. we successful isolated the ovarian tumor cells from EOC patients, and identified the CDC50A positive cell subsets exist in a low proportion in all samples we collected. Similarly, we found the primary CDC50A positive tumor cells also have the ovarian cancer stem cell properties, such as serial sphere generation, self-renewal, differentiation and tumorigenesis capability.
3. The expriments of SKOV3, primary ovarian tumor cells and NOD/SCID mice all demonstrated that CDC50A positive cells are highly resistant to cisplatin chemotherapy in vitro and in vivo.
4. In the clinic, compared to primary EOC, higer proportion of CDC50A positive cells located in metastasis. Besides, compared to the diagnosed patients, the CDC50A positive cells are rich in the tumors of neoadjuvant chemotherapy patients and the relapsed patients. What' more, Logistic regression analysis and Cox multivariate analysis showed that CDC50A expression were the independent factors of cisplatin resistance as well as progression free survival in EOC patients, patients with CDC50A high expression have the high risk of tumor relapse.
5. This study found estrogen was not the important factor which played a role in the growth and self-renewal of the CSCs. Similarily, the postoperative HRT does not have a negative effect on the progression free and overall survival of epithelial ovarian cancer patients.
卵巢上皮癌(EOC)是致死率最高的妇科恶性肿瘤,其中75%的患者诊断时已为晚期(Ⅲ～Ⅳ)。在理想的肿瘤细胞减灭术的基础上辅助铂类药物为主的联合化疗后,约60-80%的患者发生肿瘤复发,晚期卵巢癌患者的中位生存时间仅为16-22个月,5年生存率约为30%。肿瘤细胞对于化疗药物产生耐受性被认为是造成这一现象的主要原因之一。近期研究的靶向药物及小分子化合物的临床实验均未能显著改善卵巢癌患者的预后。随着肿瘤基因组图谱的揭示,学者们对卵巢癌的研究主要致力于基因组及表观遗传学的变化对临床预后的影响。但是卵巢癌化疗耐药及肿瘤复发的机制仍不清楚,从而成为卵巢癌治疗中的最大障碍。
干细胞是机体内一类具有自我更新能力和多向分化潜能的特殊细胞。根据其来源和分化能力,主要分为,胚胎干细胞、骨髓干细胞、以及成体干细胞。随着肿瘤生物学的发展,上世纪学者们提出了肿瘤干细胞(CSCs)理论。大量研究发现肿瘤中存在的极少量的CSCs,其既拥有类似干细胞自我更新及分化的特性,又维持着肿瘤细胞的致瘤性及细胞异质性,在肿瘤中长期处于静息状态,可以逃避化疗药的杀伤,导致肿瘤的耐药,疾病进展及复发。早在1997年Bonnet等人分离出表型为CD34+CD38的白血病干细胞,首次在细胞水平上直观证实了肿瘤干细胞的存在。随后,CSCs理论在许多实体瘤如乳腺癌、前列腺癌、结肠癌、肺癌、胰腺癌等中得到验证,并建立了一系列CSCs分离、富集、鉴定及分子机制研究的方法。目前,CSCs的分离及富集方法主要包括：侧群细胞(side population, SP)的流式分选,根据特异性的CSCs表面标记蛋白的流式分选,以及肿瘤球形体(sphere)的培养等。
近年来,许多致力于寻找卵巢癌治疗新途径的学者们也将目光转移到肿瘤干细胞,借鉴在其它实体瘤的研究中的成熟经验,探索了卵巢癌干细胞研究的新领域。目前对于卵巢癌干细胞的分选仍然是根据在其它实体瘤干细胞研究中的结果,采用表面标记蛋白的流式分选技术,使用最多的标记物主要包括,CD133、CD117、CD44、 ALDH1、EPCAM、CD24、以及CD90等。虽然取得了一些研究进展,但由于这些标记物均来自于其它实体瘤干细胞的研究,其对卵巢癌干细胞的分离富集作用并不十分理想,仍然存在争论。因此,寻找卵巢癌特异性表面标记蛋白,成功分离并鉴定卵巢癌干细胞,对于未来卵巢癌的靶向治疗可能具有重大意义。
本课题在前期工作中,采用稳定同位素标记和以质谱为基础的定量蛋白质组学技术对SP和非SP细胞的膜蛋白进行比较蛋白质组学分析,成功筛选出卵巢癌干细胞表面标记候选蛋白——跨膜蛋白30A (transmembrane protein30A，简称TMEM30A或CDC50A),并在体外实验中对其表达阳性细胞的干细胞特性进行了初步鉴定。本研究采用流式细胞分选及无血清悬浮培养技术,通过体内和体外实验,在卵巢癌细胞系和卵巢癌患者的组织和腹水中验证CDC50A表面标记阳性(CDC50A+) EOC细胞的sphere形成能力,自我更新能力,分化能力及致瘤能力,检测其对顺铂的耐药性,并分析其表达与EOC患者临床预后的关系。并初步探讨雌激素在卵巢癌干细胞微环境中的作用及其对术后卵巢癌患者临床预后的影响。
研究方法
1、运用流式细胞学方法分析卵巢癌细胞系SKOV3, A2780, IGROV1, COC1, OVCAR3, ES2中CDC50A+细胞的比例。采用流式细胞仪分选CDC50A+及CDC50A+细胞,验证其在HG-DMEM/10%FBS培养基中体外培养后再次分化成其他亚群的能力。采用无血清悬浮培养的方法检测CDC50A+及CDC50A+细胞sphere形成能力及sphere传代能力,并运用免疫荧光及流式细胞学方法检测SKOV3sphere对CDC50A的富集作用。流式分选出CDC50A+及CDC50A+细胞,分别等量梯度接种于NOD/SCID小鼠皮下,观察成瘤速度,观察终点为两组成瘤率或成瘤大小出现明显统计学差异。待小鼠成瘤后,再次分选出两群细胞接种小鼠,观察成瘤情况。流式分析移植瘤中CDC50A+细胞的比例,检测CDC50A+及CDC50A+细胞体内分化能力。
2、收集EOC患者卵巢癌组织及腹水,分离卵巢癌细胞,运用流式细胞学方法分析原代卵巢癌细胞中CDC50A+细胞的比例。建立原代卵巢癌细胞sphere的培养方法,检测原代卵巢癌CDC50A+及CDC50A+细胞sphere形成能力及传代能力。运用免疫荧光及流式细胞学方法检测原代卵巢癌sphere对CDC50A的富集作用。流式分选出CDC50A+及CDC50A+细胞,分别等量梯度接种于NSG小鼠皮下,观察成瘤速度,观察终点为两组成瘤率或成瘤大小出现明显统计学差异。待小鼠成瘤后,再次分选出两群细胞接种小鼠,观察成瘤情况。流式分析移植瘤中CDC50A+细胞的比例,检测CDC50A+及CDC50A+细胞体内分化能力。
3、MTT法验证SKOV3及ES2细胞系中CDC50A+及CDC50A-细胞对顺铂耐药性差异,流式细胞学方法检测不同浓度的顺铂对SKOV3/CDC50A+细胞的富集作用。流式细胞学方法检测顺铂化疗对EOC患者卵巢癌组织中CDC50A+细胞的富集作用；建立NOD/SCID小鼠卵巢癌移植瘤模型,检测顺铂化疗对小鼠移植瘤中CDC50A+细胞的富集作用。
4、运用流式细胞学方法分析EOC患者卵巢癌原位灶及转移灶中CDC50A+细胞的比例,流式分析处于临床治疗不同阶段的EOC患者组织中CDC50A+细胞比例的分布情况,免疫组化分析76例EOC患者CDC50A蛋白的表达与临床预后的相关性。
5、采用不同浓度的雌激素作用CDC50A+细胞,检测雌激素在维持卵巢癌干细胞的生长及自我更新中的作用。结合临床资料,回顾性分析术后激素替代治疗对卵巢癌患者的复发及生存的影响。
研究结果
1、流式检测发现6种卵巢癌细胞株中均存在少量CDC50A+细胞群,其比例稳定维持在0.4%～2%之间。流式分选出CDC50A+细胞亚群,在细胞分化条件下体外培养2周,再次流式分析发现CDC50A+细胞分化出CDC50A细胞,其阳性率仍保持原来水平,而CDC50A细胞未能分化出CDC50A+细胞,表明CDC50A+细胞具有分化潜能。无血清悬浮培养出SKOV3sphere,流式分选出CDC50A+及CDC50A-细胞,分别悬浮培养,发现CDC50A+细胞形成sphere的个数明显多于CDC50A细胞,CDC50A+细胞形成的sphere能连续体外传代至少4代,而CDC50A细胞形成的sphere几乎不能传代。免疫荧光与流式分析均证实CDC50A+细胞在SKOV3sphere表达显著高于贴壁SKOV3细胞；体内动物实验进一步验证了CDC50A+细胞体内成瘤能力明显强于CDC50A细胞,102个CDC50A+细胞即能使NOD/SCID小鼠成瘤,而至少104个CDC50A细胞才能使NOD/SCID小鼠成瘤。CDC50A+细胞移植瘤能够在小鼠体内连续传代,而CDC50A细胞移植瘤不能传代。HE染色显示CDC50A+细胞移植瘤与第二代移植瘤的组织学类型保持一致。流式分析显示CDC50A+细胞移植瘤中CDC50A阳性率保持不变,而CDC50A细胞移植瘤中几乎无CDC50A+细胞,说明CDC50A+细胞在体内仍具有分化能力。
2、从原代卵巢癌组织及腹水中成功分离出卵巢癌细胞,流式分析显示原代卵巢癌组织及腹水细胞中均存在CDC50A+细胞群。成功建立了原代卵巢癌细胞sphere培养体系,发现原代组织中CDC50A+细胞能形成sphere,并能连续传代,而CDC50A细胞几乎不能形成sphere;免疫荧光及流式细胞学检测均证实CDC50A+细胞在原代卵巢癌sphere中的表达显著增高。体内动物实验进一步验证了原代卵巢癌CDC50A+细胞体内成瘤能力明显强于CDC50A细胞,103个CDC50A+细胞仍能使NSG小鼠成瘤,而至少105个CDC50A细胞才能使NSG小鼠成瘤。CDC50A+细胞移植瘤能在小鼠体内连续传代,而CDC50A细胞移植瘤不能传代；HE染色显示原代CDC50A+细胞移植瘤与患者组织学类型保持一致。流式分析显示原代CDC50A+田胞移植瘤中CDC50A阳性率保持不变,而CDC50A细胞移植瘤中几乎无CDC50A+细胞,说明原代CDC50A+细胞在体内仍具有分化能力。
3、MTT法对细胞耐药性的检测发现,在SKOV3和ES2细胞系中CDC50A+细胞对顺铂的耐药性明显高于CDC50A细胞。流式细胞学检测发现,不同浓度的顺铂作用后的SKOV3细胞后,细胞总数减少,CDC50A阳性率却逐渐增高,而且CDC50A+细胞绝对数基本不变。分析同一患者先期化疗前后卵巢癌组织中CDC50A阳性率,发现顺铂化疗后CDC50A+细胞比例显著增高。动物实验显示,接受顺铂化疗后的小鼠移植瘤增长速度显著慢于未治疗组,而移植瘤中CDC50A阳性率却显著高于未治疗组,进一步证明了顺铂对CDC50A具有富集作用。
4、流式细胞学方法分析发现,EOC患者卵巢癌转移灶中CDC50A+细胞比例显著高于原位灶。对处于临床治疗不同阶段的EOC患者组织中CDC50A+细胞比例分析发现,CDC50A+细胞在初治手术患者中平均比例为1.47%,在先期化疗患者中为9.76%,而在复发的患者中为13.35%。免疫组化分析发现76例EOC患者中22例患者CDC50A蛋白高表达,54例患者CDC50A蛋白低表达或阴性表达。Logistic回归分析发现CDC50A蛋白表达和术后残余病灶是影响患者耐药的独立因素。Kaplan-Meier分析证实CDC50A蛋白高表达患者无进展生存时间(PFS)显著低于CDC50A蛋白低表达患者,中位无进展生存时间分别为10个月和17个月。Cox多元回归模型分析显示卵巢癌FIGO分期和CDC50A蛋白表达是影响PFS独立因素,CDC50A蛋白高表达患者发生卵巢癌复发的风险是低表达患者的2.85倍(95%CI1.32-6.16,p<0.01)。
5、采用不同浓度的雌激素作用CDC50A+细胞,发现其对sphere的生长及自我更新能力均无显著影响。回顾性病例分析显示：HRT不是PFS及OS的独立影响因素,而且不同HRT方案对EOC患者预后无明显影响。残余病灶大小及FIGO分期是影响无进展生存期的独立因素。FIGO分期还是影响总生存期的独立因素。
结论
1、CDC50A+细胞在六种卵巢癌细胞系中的表达稳定在0.4%～2%之间,符合干细胞比例。卵巢癌细胞系中CDC50A+细胞群具有体内外分化能力,sphere形成能力,自我更新能力等CSCs特性。CDC50A+细胞具有更强的肿瘤形成能力,并能在小鼠体内连续传代。
2、EOC患者原代卵巢癌细胞中能够分离出CDC50A+细胞群,该细胞群具有体内分化能力,sphere形成能力,自我更新能力等CSCs特性。原代卵巢癌细胞中的CDC50A+细胞具有更强的致瘤能力,且能在小鼠体内连续传代。
3、卵巢癌细胞系、原代卵巢癌细胞、以及动物实验均证实顺铂对于CDC50A+细胞具有富集作用。
4、EOC患者卵巢癌转移灶中CDC50A+细胞比例显著高于原位灶,表明CDC50A可能与卵巢癌的转移有关。CDC50A阳性细胞的比例在复发患者中最高,其次是先期化疗患者,初治手术患者的表达比例最低,进一步说明CDC50A可能与患者耐药及复发有关。临床分析表明,CDC50A蛋白的表达是患者耐药及复发的独立影响因素,高表达CDC50A的患者具有更高的复发风险。
5、通过体外实验发现：雌激素在维持CDC50A+卵巢癌干细胞的生长及自我更新中无显著作用,其并不是维持卵巢癌干细胞的重要因子。结合临床资料,采用回顾性病例分析发现术后激素替代治疗对卵巢癌患者的复发及生存无明显影响。
Background
Epithelial ovarian cancer (EOC) is the leading causes of gynecological cancer mortality, among which75%patients were diagnosed as advanced ovarian cancer. Under the treatment of primary cytoreductive surgery followed by chemotherapy based on platinum, about60～80%patients relapsed. The median survival time of advanced ovarian cancer patients was only16～22months, and the5-year survival rate is only30%. The chemotherapy resistance of the tumor cells is considered to be one of the main reasons for this phenomenon. A recent study of targeted drugs and small molecular compounds in clinical trials failed to improve the prognosis of patients with ovarian cancer. With the emergence of cancer genome map, many scholars are mainly engaged in the effects of the genome and epigenome changes on the clinical outcomes of ovarian cancer. But the phenotype mechanism of ovarian cancer chemotherapy resistance and rumor recurrence is still not clear, so it become the biggest obstacle for ovarian cancer therapy.
Stem cells are known as a kind of special cells which has the ability of self-renewal and multi-directional differentiation. According to its origin and differentiation ability, they are mainly divides into embryonic stem cells, mesenchymal stem cells and adult stem cells. With the development of tumor biology, scholars put forward the theory of cancer stem cells (CSCs) in the last century. Lots of studies have found that there were a very small number of CSCs existed in tumors at quiescent state, which not only have the similar characteristics of stem cells, such as self-renewal and differentiation, but also maintained the tumorigenicity and cell heterogeneity of tumor cells. So it can escape from the toxic damage of chemotherapy drugs, and result in the drug resistance, progression and recurrence of tumor. Early in1997, Bonnet and his companies isolated the phenotype of CD34+CD38" leukemia stem cells, and directly confirmed the existence of cancer stem cells for the first time at the cellular level. After that, the theory of CSCs was identified in many solid tumors such as breast cancer, prostate cancer, colon cancer, lung cancer, pancreatic cancer, and established a series of separation, enrichment, identification and molecular mechanism research methods of CSCs. At present, the separation and enrichment of CSCs method mainly includes:the sorting of side population cells (side population, SP) by flow cytometry, sorting according to the specific CSCs surface marker by flow cytometry, and the cultivation of the tumor sphere.
Recently, many scholars begin to research in the cancer stem cells to looking for the new treatment of ovarian cancer. They will study on the basis of the outcomes of other solid cancer stem cells, and may open a new chapter of ovarian cancer stem cell research. Sorting according to the specific CSCs surface marker by flow cytometry is still the main way to isolate ovarian cancer stem cells, and the present study mainly include the following surface markers:CD133, CD117, CD44, ALDH1, EPCAM, CD24, CD90and so on. Although some research progress has been made, because these markers are draw lessons from other solid tumor stem cells, the separation and enrichment of ovarian cancer stem cells is not very effective, and there is still a debate. Therefore, looking for a specific surface marker to successfully separate and identifiy the ovarian cancer stem cells, may has great value to targeted therapy of ovarian cancer.
In the early stage of our work, we used quantitative proteomic techniques based on the stable isotope labeling and mass spectrometry to compare the membrane protein of SP and SP cells, and successfully screened the ovarian cancer stem cell surface candidate protein-transmembrane protein30A (TMEM30A, also called CDC50A). So, on the basis of the previous study, in this research, we mainly identified the CSCs properties of CDC50A surface markers positive (CDC50A+) cells in vivo and in vitro by flow cytometry and serum-free suspension culture technology, which mainly included the ability of sphere formation, self-renewal, differentiation and tumorigenesis of CDC50A positive ovarian cancer cells. Besides, we also validated the cisplatin resistance of CDC50A positive cells, and analyzed the relationship between the expression of CDC50A protein and the clinical outcomes of EOC patients. At last, we demonstrated the role of estrogen played in the microenvironment of CSCs and discussed the effect of HRT on the prognosis of EOC patients.
Methods
1. The proportion of CDC50A positive cells in the ovarian cancer cell lines such as SKOV3, A2780, IGROV1, COC1,OVCAR3, ES2were analyzed by flow cytometry; CDC50A positive and CDC50A negative cells were sorted by Moflo fluorescent activated cell stream sorter and cultured in HG-DMEM/10%FBS medium, then we verified the differentiation capability of the two populations in vitro; The sphere formation and serial passage ability of the CDC50A positive and CDC50A negative cells were tested by serum-free suspension culture technology; The enrichment of CDC50A positive cells in SKOV3spheres were tested by immunofluorescence and flow cytometry; SKOV3cells were sorted into CDC50A positive and negative population, then serial dilutions of the two population cells were resuspended (1:1) in PBS:Matrigel and implanted subcutaneously into the scapular region of NOD/SCID mice respectively to identify the tumorigenesis capacity of two populations. When the transplanted tumors formed, sorting out of the two populations, inoculated into mice again to test the self renewal capacity in vivo; The proportion of CDC50A positive cells in transplanted tumors was analyzed by flow cytometry to test the differentiation ability of CDC50A positive and negative cells in vivo.
2. Tissues and ascites of the EOC patients were collected and the ovarian cancer cells were isolated; The proportion of CDC50A positive cells in the primary ovarian cancer cells were analyzed by flow cytometry; We established the primary tumor sphere culture system, and test the sphere formation capacity and serial passage ability of the primary CDC50A positive and CDC50A negative tumor cells; The enrichment of CDC50A positive cells in primary ovarian tumor spheres were tested by immunofluorescence and flow cytometry; Then, ovarian tumor cells were sorted into CDC50A positive and negative population, and serial dilutions of the two population cells were resuspended (1:1) in PBS:Matrigel and implanted subcutaneously into the scapular region of NSG mice respectively to identify the tumorigenesis capacity of two populations. When the transplanted tumors formed, sorting out of the two populations, inoculated into mice again to test the self renewal capacity in vivo; The proportion of CDC50A positive cells in transplanted tumors was analyzed by flow cytometry to test the differentiation ability of CDC50A positive and negative cells in vivo.
3. We tested the difference of cisplatin resistance to CDC50A positive and negative cells by MTT assay in vitro experiments; Then the flow cytometry analysis were used to detect the enrichment of SKOV3CDC50A positive cells with different concentrations of cisplatin; we used the same method to test the enrichment of primary CDC50A positive tumor cells sorted from EOC patient with cisplatin chemotherapy; Besides, we established the transplanted tumor model with NOD/SCID mice, and test the enrichment of CDC50A positive tumor cells with cisplatin chemotherapy.
4. We analyzed the proportion of CDC50A positive cells in primary tumors and metastasis of EOC patients respectively by flow cytometry; Then we analyzed the proportion of CDC50A positive cells in different EOC patients; Immunohistochemistry were used to analyzed CDC50A protein expression of76EOC patients and its correlation with clinical prognosis.
5. Different concentration of estrogen were added to culture of CDC50A positive cells, and demenstrated the role of it in maintaining the growth and the self-renewal of ovarian cancer stem cells. Then, in combination with clinical data, we analyzed the impact of postoperative hormone replacement therapy on the patients with ovarian cancer.
Results
1. We found that CDC50A positive population were exist in six ovarian cancer cell lines, and the proportion of CDC50A positive cells were ranged from0.4%to2%; We cultured CDC50A positive cells in vitro after sorting from SKOV3cell line, which could again differentiate to CDC50A negative cells and maintain its positive rate, but the CDC50A negative cells couldn't differentiate to CDC50A positive cell subsets, which indicated that CDC50A positive cells had the potential to differentiate to CDC50A negative cells; The SKOV3sphere were formed by serum-free suspension culture after one week, and compared to the CDC50A negative cells, CDC50A positive cells were more capable of generating spheres. Besides, CDC50A positive spheres can passage consecutively, while the CDC50A-spheres can't. Besides, we examined the expression of CDC50A protein in SKOV3spheres and attached cells by immunofluorescence, which showed that the majority spheroid cells stained for CDC50A, while the attached cells can't. Similarily, we found that the expression of CDC50A in spheroid cells was much higher than the attached cells by flow cytometry. The tumor formation experiment showed that1/3mice injected with1×104CDC50A negative cells can formed tumors, but no tumor formation detected following injection of1×103cells, While CDC50A positive cells were tumorigenic in mice even with1×102cells. We also found that only CDC50A positive cells were able to generate secondary tumors in NOD/SCID mice. The H&E staining and flow cytometry analysis showed that not only the histology of the first and second passage xenografts was similar, but also the frequency of CDC50A positive cells was maintained in xenografts obtained by serial in vivo passaging of CDC50A positive cells,which indicated that CDC50A positive cells also have the capacity of differentiation m vivo.
2. To further demonstrated the properties of CDC50A+cells in primary tumor cells, we analyzed primary EOC specimens to find that CDC50A was present at variable expression in all primary tumors and ascite samples. Similar to cell lines, we isolated and inoculated the human ovarian tumor and ascite cells at a density of104cells/ml to low-attachment6-well plates in serum-free medium with cytokines. Spheres were observed about2weeks. In order to further demonstrate the sphere formation capacity of CDC50A+/Lin-cells, CDC50A+/Lin-and CDC50A7Lin-populations were sorted from three human ovarian tumors to inoculate in suspension culture at a density of10000cells/well. Compared to CDC50A-/Lin-cells, CDC50A+/Lin-cells were more capable of generating spheres and even with serial passage. Then, we found that CDC50A protein was expressed at a high level in spheres by immunofluorescence. Similarily, compared to the isolated tumor cells from human tissues, the proportion of CDC50A+/Lin-cells in spheres was much higher. To test if the CDC50A+/Lin-cells isolated from human EOC to generate tumors in mice, sorted cells were suspended in Matrigel and subcutaneously injected into NSG mice. We found that104CDC50A-/Lin-cells did not grow tumors, While104CDC50A+/Lin-cells generated tumors from3/8. Tumor generation required approximately～5months. Similarly, We found that only CDC50A+/Lin-cells were able to generate secondary tumors in NSG mice. Histological analysis of the xenograft tumor generated by CDC50A+/Lin-cells was identical to that seen in the primary tumor; Flow cytometry analysis showed that the frequency of CDC50A+/Lin-cells was maintained in CDC50A positive xenografts obtained by serial in vivo passaging.
3. Cell survival assay showed greater cisplatin resistance of CDC50A positive cells from SKOV3and ES2cell lines. Then we assayed the proportion of CDC50A positive cells after treatment with increasing doses of cisplatin. A clear, dose dependent decrease in the total number of viable cells were observed, While there was a significant increase in the proportion of CDC50A+cells, the most interesting thing is that the absolute number of CDC50A positive cells were maintained during the treatment of cisplatin, which suggested CDC50A positive cells played an important role in ovarian cancer cisplatin chemoresistance. Besides, we compared the proportion of CDC50A+/Lin-cells in the same patient with ovarian cancer before and after cisplatin chemotherapy, the result indicated that the chemotherepy can enrich the CDC50A+/Lin-cells in human ovarian tumors. To further demonstrated CDC50A positive cells are resistant to cisplatin in vivo, NOD/SCID mice were inoculated with SKOV3cells, and treated by saline or cisplatin for a month. Then we observed that cisplatin significantly inhibited the increase of the mean volumes of tumors in the treatment group. Besides, we identified that the proportion of CDC50A+/Lin-cells from the cisplatin treated tumors were much higher than the control tumors, which further demonstrated that the cisplatin can enrich the CDC50A+/Lin-cells.
4. Flow cytometry analysis showed that a higher proportion of CDC50A positive cells were observed in metastasis. During the primary ovarian tumors we collected, the patients with advanced stage serous adenocarcinoma were classified to three group: initial diagnosed patients, patients with neoadjuvant chemotherapy based on cisplatin and the relapsed patients. According to the analysis by flow cytometry, the average proportion of CDC50A positive cells was1.47%in tumors of initial diagnosed patients,9.76%in tumors of neoadjuvant chemotherapy patients and13.35%in tumors of relapsed patients. It is suggested that the expression of CDC50A may correlated with the cisplatin resistance and relapse. To further assess whether our experimental findings on CDC50A are relevant to human ovarian cancer patients in the clinic, we therefore scored a panel of76ovarian tumors for the presence of CDC50A expressing cells using immunohistochemistry. There were22patients with CDC50A high expression and54with low or negative expression. The Logistic regression analysis showed that CDC50A expression and resection status were the independent factor that impact on the platinum resistance of patients. Besides, Kaplan-Meier analysis demonstrated that patients with CDC50A high expression tumor cells have worse progression free survival (PFS) compared to the CDC50A low expression group. And the median survival time for PFS were10months for CDC50A high expression patients and17months for CDC50A low expression patients. Then we performed a Cox multivariate analysis of PFS, which showed that CDC50A expression was an independent prognostic factor, as well as tumor stage. The relative risk of relapse due to ovarian cancer was2.85for patients with CDC50A positive tumors compared to patients with CDC50A negative tumors (95%CI1.32-6.16, p<0.01), which indicated that the expression of CDC50A may predict poor clinical outcome of ovarian cancer patients.
5. We found that the growth and self-renewal ability of sphere had no significant influence under the different concentration of estrogen. According to the multivariate analysis, HRT did not significantly influence progression-free or overall survival. Similarly, different types of HRT had no significant effect on the prognosis of epithelial ovarian cancer patients. And the strongest independent variable in predicting both progression-free survival and overall survival was the FIGO stage of the disease.
Conclusion
1. The ratio of CDC50A positive cell subsets in six ovarian carcinoma cell lines is low, which is similar to the ratio of stem cells; The experiments in vitro and in vivo showed that CDC50A positive cells sorted from SKOV3cell lines have the ovarian cancer stem cell properties, such as serial sphere generation, self-renewal, differentiation and tumorigenesis capability.
2. we successful isolated the ovarian tumor cells from EOC patients, and identified the CDC50A positive cell subsets exist in a low proportion in all samples we collected. Similarly, we found the primary CDC50A positive tumor cells also have the ovarian cancer stem cell properties, such as serial sphere generation, self-renewal, differentiation and tumorigenesis capability.
3. The expriments of SKOV3, primary ovarian tumor cells and NOD/SCID mice all demonstrated that CDC50A positive cells are highly resistant to cisplatin chemotherapy in vitro and in vivo.
4. In the clinic, compared to primary EOC, higer proportion of CDC50A positive cells located in metastasis. Besides, compared to the diagnosed patients, the CDC50A positive cells are rich in the tumors of neoadjuvant chemotherapy patients and the relapsed patients. What' more, Logistic regression analysis and Cox multivariate analysis showed that CDC50A expression were the independent factors of cisplatin resistance as well as progression free survival in EOC patients, patients with CDC50A high expression have the high risk of tumor relapse.
5. This study found estrogen was not the important factor which played a role in the growth and self-renewal of the CSCs. Similarily, the postoperative HRT does not have a negative effect on the progression free and overall survival of epithelial ovarian cancer patients.
引文
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