Metabolic Changes in Ovarian Cancer最新文献_第2页

Abstract A41: Ovarian cancer as an infectious disease. Targeting of mitochondrial activity to prevent and treat recurrent ovarian cancer 摘要:卵巢癌是一种感染性疾病。靶向线粒体活性预防和治疗复发性卵巢癌

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A41

M. Bazzaro

引用次数: 1

Abstract A20: Claudin-4 regulates ovarian tumor cell response to the microenvironment 摘要:Claudin-4调控卵巢肿瘤细胞对微环境的反应

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A20

D. Hicks, C. Breed, Patricia G. Webb, K. Neal, K. Behbakht, H. Baumgartner

Interaction of tumor cells with extracellular matrix proteins of the peritoneal lining plays a critical role in the unique metastatic process of ovarian cancer. Although peritoneal attachment is known to be a key step in the progression of ovarian cancer, the underlying molecular mechanisms driving adhesion and the downstream changes in cell behavior that lead to poor treatment outcomes are not well understood. The objective of this study was to investigate the potential role of the transmembrane protein claudin-4 in regulating ovarian tumor cell interaction with and response to extracellular matrix proteins. Adhesion, apoptosis (cleaved caspase-3), proliferation (DNA content), and scratch assays were performed with ovarian tumor cells (OVCAR3, PEO4) cultured on different proteins found in the extracellular matrix of the peritoneal mesothelium (type I collagen, type IV collagen, fibronectin, and laminin) or a nonphysiologic cell adhesive (Cell-Tak). Number of cells attached within one hour, percent cells positive for apoptosis at 24 hours post treatment, cell number over time, and percent wound closure at 8 hours was measured in response to claudin-4 disruption (DFYNP mimic peptide) or loss of claudin-4 expression (shRNA-mediated gene silencing). Immunofluorescence of phosphorylated focal adhesion kinase (pFAK) was performed to examine formation of focal adhesions in response to claudin-4 disruption or loss of expression. Proximity ligation assays, immunoprecipitation, and immunofluorescence were performed to examine interaction of claudin-4 with tubulin. Results from these studies showed that ovarian tumor cells preferentially attach to type I collagen compared to the other matrix proteins and that disruption of claudin-4 inhibited this attachment. Attachment to type I collagen made tumor cells more resistant to apoptosis, more proliferative, and more migratory compared to tumor cells cultured on the other matrix proteins. In the presence of type I collagen, disruption of claudin-4 restored tumor cell apoptotic response to paclitaxel, induced mitotic arrest, reduced proliferation rate, and inhibited migration. The size of pFAK-containing focal adhesions was significantly smaller and fewer adhesions were present in ovarian tumor cells cultured on type I collagen treated with the claudin-4 disrupting peptide or with loss of claudin-4 expression compared to cells that express high levels of claudin-4. Additionally, we observed a direct interaction of claudin-4 with both alpha and beta tubulin that was dependent on stage of cell cycle. In conclusion, we have demonstrated a novel role for claudin-4 in regulating ovarian tumor cell response to the tumor microenvironment to promote tumor survival and growth. These observations have important therapeutic implications for inhibiting the survival and deadly spread of ovarian tumors through blocking the biologic activity of claudin-4. Citation Format: Douglas A. Hicks, Christopher Breed, Patricia G. Webb,

肿瘤细胞与腹膜外基质蛋白的相互作用在卵巢癌独特的转移过程中起着关键作用。虽然已知腹膜附着是卵巢癌进展的关键步骤，但驱动粘附的潜在分子机制和导致不良治疗结果的细胞行为的下游变化尚不清楚。本研究的目的是探讨跨膜蛋白claudin-4在调节卵巢肿瘤细胞与细胞外基质蛋白相互作用和应答中的潜在作用。将卵巢肿瘤细胞(OVCAR3、PEO4)培养在腹膜间皮细胞外基质(I型胶原、IV型胶原、纤维连接蛋白和层粘连蛋白)或非生理性细胞粘接剂(cell - tak)上，进行粘附、凋亡(cleaved caspase-3)、增殖(DNA含量)和划痕实验。在claudin-4中断(DFYNP模拟肽)或claudin-4表达缺失(shrna介导的基因沉默)的反应中，我们测量了1小时内附着的细胞数量、处理后24小时凋亡阳性细胞的百分比、随时间变化的细胞数量以及8小时伤口愈合的百分比。磷酸化局灶黏附激酶(pFAK)的免疫荧光检测了在claudin-4破坏或表达缺失的情况下局灶黏附的形成。采用近端结扎法、免疫沉淀法和免疫荧光法检测了claudin-4与微管蛋白的相互作用。这些研究结果表明，与其他基质蛋白相比，卵巢肿瘤细胞更倾向于附着于I型胶原蛋白，而claudin-4的破坏抑制了这种附着。与在其他基质蛋白上培养的肿瘤细胞相比，与I型胶原蛋白的附着使肿瘤细胞更耐凋亡、更增殖、更迁移。在I型胶原存在的情况下，claudin-4的破坏恢复了肿瘤细胞对紫杉醇的凋亡反应，诱导有丝分裂停止，降低增殖率，抑制迁移。与表达高水平claudin-4的细胞相比，用claudin-4干扰肽处理或claudin-4表达缺失的I型胶原培养的卵巢肿瘤细胞中，含有pfak的局灶性粘连的大小明显更小，出现的粘连也更少。此外，我们观察到claudin-4与α和β微管蛋白的直接相互作用取决于细胞周期的阶段。总之，我们已经证明了claudin-4在调节卵巢肿瘤细胞对肿瘤微环境的反应以促进肿瘤生存和生长中的新作用。这些观察结果对于通过阻断claudin-4的生物活性来抑制卵巢肿瘤的生存和致命扩散具有重要的治疗意义。引文格式:Douglas A. Hicks, Christopher Breed, Patricia G. Webb, Kristin L. Neal, Kian Behbakht, Heidi K. Baumgartner。Claudin-4调节卵巢肿瘤细胞对微环境的反应。[摘要]。AACR会议论文集:解决卵巢癌研究和治疗中的关键问题;2017年10月1-4日;宾夕法尼亚州匹兹堡。费城(PA): AACR;临床肿瘤杂志，2018;24(15 -增刊):摘要11 - 20。

{"title":"Abstract A20: Claudin-4 regulates ovarian tumor cell response to the microenvironment","authors":"D. Hicks, C. Breed, Patricia G. Webb, K. Neal, K. Behbakht, H. Baumgartner","doi":"10.1158/1557-3265.OVCA17-A20","DOIUrl":"https://doi.org/10.1158/1557-3265.OVCA17-A20","url":null,"abstract":"Interaction of tumor cells with extracellular matrix proteins of the peritoneal lining plays a critical role in the unique metastatic process of ovarian cancer. Although peritoneal attachment is known to be a key step in the progression of ovarian cancer, the underlying molecular mechanisms driving adhesion and the downstream changes in cell behavior that lead to poor treatment outcomes are not well understood. The objective of this study was to investigate the potential role of the transmembrane protein claudin-4 in regulating ovarian tumor cell interaction with and response to extracellular matrix proteins. Adhesion, apoptosis (cleaved caspase-3), proliferation (DNA content), and scratch assays were performed with ovarian tumor cells (OVCAR3, PEO4) cultured on different proteins found in the extracellular matrix of the peritoneal mesothelium (type I collagen, type IV collagen, fibronectin, and laminin) or a nonphysiologic cell adhesive (Cell-Tak). Number of cells attached within one hour, percent cells positive for apoptosis at 24 hours post treatment, cell number over time, and percent wound closure at 8 hours was measured in response to claudin-4 disruption (DFYNP mimic peptide) or loss of claudin-4 expression (shRNA-mediated gene silencing). Immunofluorescence of phosphorylated focal adhesion kinase (pFAK) was performed to examine formation of focal adhesions in response to claudin-4 disruption or loss of expression. Proximity ligation assays, immunoprecipitation, and immunofluorescence were performed to examine interaction of claudin-4 with tubulin. Results from these studies showed that ovarian tumor cells preferentially attach to type I collagen compared to the other matrix proteins and that disruption of claudin-4 inhibited this attachment. Attachment to type I collagen made tumor cells more resistant to apoptosis, more proliferative, and more migratory compared to tumor cells cultured on the other matrix proteins. In the presence of type I collagen, disruption of claudin-4 restored tumor cell apoptotic response to paclitaxel, induced mitotic arrest, reduced proliferation rate, and inhibited migration. The size of pFAK-containing focal adhesions was significantly smaller and fewer adhesions were present in ovarian tumor cells cultured on type I collagen treated with the claudin-4 disrupting peptide or with loss of claudin-4 expression compared to cells that express high levels of claudin-4. Additionally, we observed a direct interaction of claudin-4 with both alpha and beta tubulin that was dependent on stage of cell cycle. In conclusion, we have demonstrated a novel role for claudin-4 in regulating ovarian tumor cell response to the tumor microenvironment to promote tumor survival and growth. These observations have important therapeutic implications for inhibiting the survival and deadly spread of ovarian tumors through blocking the biologic activity of claudin-4. Citation Format: Douglas A. Hicks, Christopher Breed, Patricia G. Webb, ","PeriodicalId":18646,"journal":{"name":"Metabolic Changes in Ovarian Cancer","volume":"1 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91485198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Abstract A56: Development of a cloud-based machine learning system (CLOBNET) to predict platinum resistance in high-grade serous ovarian cancer A56:基于云的机器学习系统(CLOBNET)的开发预测高级别浆液性卵巢癌铂耐药性

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.ovca17-a56

Veli-Matti Isoviita, Liina Salminen, Jimmy Azar, J. Hynninen, R. Lehtonen, Pia Röring, S. Grénman, A. Färkkilä, S. Hautaniemi

引用次数: 0

Abstract A37: Epigenetic modification of ovarian cancer immunogenicity A37:卵巢癌免疫原性的表观遗传修饰

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.ovca17-a37

P. Spiliopoulou, J. Walton, Suzanne Dowson, Alexander W. D. Binks, O. Maddocks, P. Adams, I. McNeish

引用次数: 0

Abstract A45: Treatment patterns and outcomes among platinum-refractory/resistant ovarian cancer patients A45:铂难治/耐药卵巢癌患者的治疗模式和结局

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A45

R. Parikh, S. Kurosky, M. Udall, Jane Chang, J. Cappelleri, J. Doherty, J. Kaye

Background: Real-world evidence on current treatment patterns and outcomes is limited for patients with platinum-refractory/resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer (PRROC). This study aimed to describe the treatment patterns and outcomes of patients with PRROC in the United States (US), the United Kingdom (UK), and Canada (CA). Methods: Physicians retrospectively reviewed medical records of females aged ≥18 years diagnosed with PRROC from January 2010 to June 2014. Follow-up data available through October 2016 were extracted. Patient characteristics, initial PRROC treatment regimens, and associated health care utilization were assessed descriptively; clinical outcomes were estimated using the Kaplan-Meier and Cox proportional-hazards methods. Results: Data were obtained on 392 US, 296 UK, and 82 CA patients. At initial diagnosis of epithelial ovarian, fallopian tube, or peritoneal cancer, 65.8% (US), 93.3% (UK), and 82.9% (CA) of patients had stage III/IV disease and 43.6% (US), 73.7% (UK), and 56.1% (CA) had high-grade tumors. Most patients were diagnosed with PRROC in 2013 or 2014 (US: 64.8%, UK: 72.3%, CA: 64.6%) and mean age at PRROC diagnosis was 57 years in the US and CA and 59 years in the UK. The proportion of patients with ECOG performance status (PS) ≤1 at PRROC diagnosis was 57.7% in the US, 80.1% in the UK, and 36.6% in CA. Most patients received systemic treatment after PRROC diagnosis (US 71.4%; UK 83.1%; CA 81.7%). Most of the patients received only one treatment line at the time of extraction (US: 64.3%, UK: 75.6%, CA: 70.2%). Bevacizumab ± chemotherapy (US 41.4%; UK 12.6%; CA 35.8%) and pegylated liposomal doxorubicin (PLD) monotherapy (US 18.6%; UK 50.0%; CA 34.3%) were the most common initial therapies. Common subsequent treatments varied between the countries, including topotecan, gemcitabine, PLD, paclitaxel. During initial treatment for PRROC, 80.7%, 59.8%, and 44.8% of patients had at least one office visit and 18.9%, 7.3%, and 19.4% of patients had at least one emergency department visit in the US, UK, and CA, respectively. Hospitalizations during initial treatment for PRROC were observed among 17.5% of patients in the US, 10.2% in the UK, and 14.9% in CA. Treatment toxicity was the most common reason for hospitalization (US 75.5%; UK 64.0%; CA 80.0%). Median progression-free survival (PFS; 95% confidence interval) was 6.4 (5.4-9.3), 8.0 (6.8-9.2), and 5.6 (4.9-6.2) months in the US, UK, and CA, respectively. The Cox proportional-hazards model showed that stage III/IV, high-grade tumors, and poorer PS were associated with shorter survival. Conclusions: Even though bevacizumab ± chemotherapy and PLD were the most common initial PRROC treatments in the three countries, relatively higher utilization of bevacizumab ± chemotherapy was observed in the US and CA than the UK, plausibly due to lack of bevacizumab reimbursement in the UK for the treatment of PRROC. Limited PFS and a high prevalence

背景:对于铂难治/耐药的上皮性卵巢癌、输卵管癌或原发性腹膜癌(proroc)患者，目前的治疗模式和结果的真实证据有限。本研究旨在描述美国(US)、英国(UK)和加拿大(CA) proroc患者的治疗模式和结果。方法:回顾性分析2010年1月至2014年6月诊断为proroc的年龄≥18岁女性的病历。提取截至2016年10月的随访数据。描述性地评估患者特征、初始proroc治疗方案和相关的医疗保健利用;使用Kaplan-Meier和Cox比例风险法估计临床结果。结果:获得了392例美国、296例英国和82例CA患者的数据。在最初诊断上皮性卵巢癌、输卵管癌或腹膜癌时，65.8%(美国)、93.3%(英国)和82.9% (CA)的患者为III/IV期疾病，43.6%(美国)、73.7%(英国)和56.1% (CA)的患者为高级别肿瘤。大多数患者在2013年或2014年被诊断为proroc(美国:64.8%，英国:72.3%，CA: 64.6%)，美国和CA的平均proroc诊断年龄为57岁，英国为59岁。在PRROC诊断时，ECOG表现状态(PS)≤1的患者比例在美国为57.7%，在英国为80.1%，在CA为36.6%。大多数患者在PRROC诊断后接受了全身治疗(美国71.4%;英国83.1%;CA 81.7%)。大多数患者在拔牙时只接受一种治疗(美国:64.3%，英国:75.6%，CA: 70.2%)。贝伐单抗±化疗(US 41.4%;英国12.6%;CA 35.8%)和聚乙二醇化脂质体多柔比星(PLD)单药治疗(US 18.6%;英国50.0%;CA(34.3%)是最常见的初始治疗方法。常见的后续治疗因国家而异，包括拓扑替康、吉西他滨、PLD、紫杉醇。在PRROC的初始治疗期间，美国、英国和英国分别有80.7%、59.8%和44.8%的患者至少有一次办公室就诊，18.9%、7.3%和19.4%的患者至少有一次急诊科就诊。美国17.5%的患者在接受proroc初始治疗期间住院，英国10.2%，中国14.9%。治疗毒性是最常见的住院原因(美国75.5%;英国64.0%;CA 80.0%)。中位无进展生存期(PFS;95%可信区间)分别为6.4(5.4-9.3)、8.0(6.8-9.2)和5.6(4.9-6.2)个月。Cox比例风险模型显示，III/IV期、高级别肿瘤和较差的PS与较短的生存期相关。结论:尽管在这三个国家，贝伐单抗±化疗和PLD是最常见的初始PRROC治疗方法，但在美国和英国，贝伐单抗±化疗的使用率相对高于英国，这可能是由于英国缺乏贝伐单抗治疗PRROC的报销。有限的PFS和由于初始治疗观察到的治疗毒性而住院的高流行率表明，继续需要更有效和可耐受的治疗策略。引文格式:Rohan Parikh, Samantha Kurosky, Margarita Udall, Jane Chang, Joseph C. Cappelleri, Jim P. Doherty, James A. Kaye。铂难治/耐药卵巢癌患者的治疗模式和结局[摘要]。AACR会议论文集:解决卵巢癌研究和治疗中的关键问题;2017年10月1-4日;宾夕法尼亚州匹兹堡。费城(PA): AACR;临床肿瘤杂志，2018;24(15 -增刊):1 - 5。

{"title":"Abstract A45: Treatment patterns and outcomes among platinum-refractory/resistant ovarian cancer patients","authors":"R. Parikh, S. Kurosky, M. Udall, Jane Chang, J. Cappelleri, J. Doherty, J. Kaye","doi":"10.1158/1557-3265.OVCA17-A45","DOIUrl":"https://doi.org/10.1158/1557-3265.OVCA17-A45","url":null,"abstract":"Background: Real-world evidence on current treatment patterns and outcomes is limited for patients with platinum-refractory/resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer (PRROC). This study aimed to describe the treatment patterns and outcomes of patients with PRROC in the United States (US), the United Kingdom (UK), and Canada (CA). Methods: Physicians retrospectively reviewed medical records of females aged ≥18 years diagnosed with PRROC from January 2010 to June 2014. Follow-up data available through October 2016 were extracted. Patient characteristics, initial PRROC treatment regimens, and associated health care utilization were assessed descriptively; clinical outcomes were estimated using the Kaplan-Meier and Cox proportional-hazards methods. Results: Data were obtained on 392 US, 296 UK, and 82 CA patients. At initial diagnosis of epithelial ovarian, fallopian tube, or peritoneal cancer, 65.8% (US), 93.3% (UK), and 82.9% (CA) of patients had stage III/IV disease and 43.6% (US), 73.7% (UK), and 56.1% (CA) had high-grade tumors. Most patients were diagnosed with PRROC in 2013 or 2014 (US: 64.8%, UK: 72.3%, CA: 64.6%) and mean age at PRROC diagnosis was 57 years in the US and CA and 59 years in the UK. The proportion of patients with ECOG performance status (PS) ≤1 at PRROC diagnosis was 57.7% in the US, 80.1% in the UK, and 36.6% in CA. Most patients received systemic treatment after PRROC diagnosis (US 71.4%; UK 83.1%; CA 81.7%). Most of the patients received only one treatment line at the time of extraction (US: 64.3%, UK: 75.6%, CA: 70.2%). Bevacizumab ± chemotherapy (US 41.4%; UK 12.6%; CA 35.8%) and pegylated liposomal doxorubicin (PLD) monotherapy (US 18.6%; UK 50.0%; CA 34.3%) were the most common initial therapies. Common subsequent treatments varied between the countries, including topotecan, gemcitabine, PLD, paclitaxel. During initial treatment for PRROC, 80.7%, 59.8%, and 44.8% of patients had at least one office visit and 18.9%, 7.3%, and 19.4% of patients had at least one emergency department visit in the US, UK, and CA, respectively. Hospitalizations during initial treatment for PRROC were observed among 17.5% of patients in the US, 10.2% in the UK, and 14.9% in CA. Treatment toxicity was the most common reason for hospitalization (US 75.5%; UK 64.0%; CA 80.0%). Median progression-free survival (PFS; 95% confidence interval) was 6.4 (5.4-9.3), 8.0 (6.8-9.2), and 5.6 (4.9-6.2) months in the US, UK, and CA, respectively. The Cox proportional-hazards model showed that stage III/IV, high-grade tumors, and poorer PS were associated with shorter survival. Conclusions: Even though bevacizumab ± chemotherapy and PLD were the most common initial PRROC treatments in the three countries, relatively higher utilization of bevacizumab ± chemotherapy was observed in the US and CA than the UK, plausibly due to lack of bevacizumab reimbursement in the UK for the treatment of PRROC. Limited PFS and a high prevalence","PeriodicalId":18646,"journal":{"name":"Metabolic Changes in Ovarian Cancer","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89539464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Abstract A29: Effects of lysophosphatidic acid on ovarian cancer metastatic dissemination 摘要:溶血磷脂酸对卵巢癌转移传播的影响

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A29

Y. Klymenko, Brandi Bos, L. Campbell, Elizabeth A. Loughran, Yueying Liu, Oleg Kim, K. Nephew, M. Stack

引用次数: 0

Abstract A25: Tissue transglutaminase interacts with Frizzled 7 in ovarian cancer stem cells A25:组织转谷氨酰胺酶与卵巢癌干细胞中的卷曲7相互作用

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A25

S. Condello, L. Sima, C. Ivan, H. Cardenas, G. Schiltz, Rama K. Mishra, D. Matei

引用次数: 0

Abstract A61: Ascites-derived and tissue-derived ovarian cancer cell primary 3D cultures aimed for personalized medicine 摘要A61:腹水来源和组织来源的卵巢癌细胞原代3D培养用于个性化医疗

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A61

Y. Nanki, A. Hirasawa, H. Nomura, A. Okubo, Manabu Itoh, T. Akahane, T. Chiyoda, F. Kataoka, E. Tominaga, D. Aoki

Objective: Ovarian cancer is a poor-prognosis gynecologic disease with over 220,000 diagnoses each year worldwide and a 5-year survival rate less than 40%. In this study, we present a method for isolation and characterization of ovarian cancer cells from patient-derived ascites and tissue samples by using three-dimensional (3D) cell culture. Materials and Methods: Ascites and tissue samples were obtained from primary ovarian, peritoneal, and fallopian tube cancer patients intraoperatively. Samples were isolated and cultured within 6 hours after collection. After isolation, cells were resuspended in Matrigel and were placed at the center of each well. Optimized medium is added to each well. NanoCulture Plate LH96 (Low-Binding, MH pattern, 96-wells, Medical and Biological Laboratories Co., Ltd., Japan) were used for 3D cell culture. Plates were incubated at 37°C. The cultures were examined for metabolically active cells by ATP assay and medium changed on days 0, 1, 4, 7, 10, and 14. Results: We successfully obtained ascites-derived primary cell cultures within 1-7 days from 100% (3/31) of the ascites samples and 62% (5/8) from tissue-derived primary cell cultures. Spheroids-like structures were formed in 30% (1/3) of ascites samples and 50% (4/8) of tissue samples. The tumorigenicity and invasiveness of the cells were demonstrated using new 3D spheroid model cultured in vitro by NanoCulture Plate LH96. Conclusion: Primary ascites and tissue culture of ovarian cancer cells can successfully be cultured by 3D cell culture plates. We may apply this method for drug sensitivity testing; therefore, 3D cell culture has a potential to evaluate the sensitivity of candidate chemotherapeutic drug for individual patients. Citation Format: Yoshiko Nanki, Akira Hirasawa, Hiroyuki Nomura, Aki Okubo, Manabu Itoh, Tomoko Akahane, Tatsuyuki Chiyoda, Fumio Kataoka, Eiichiro Tominaga, Daisuke Aoki. Ascites-derived and tissue-derived ovarian cancer cell primary 3D cultures aimed for personalized medicine. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A61.

目的:卵巢癌是一种预后不良的妇科疾病，全球每年有超过22万例诊断，5年生存率不到40%。在这项研究中，我们提出了一种通过三维(3D)细胞培养从患者来源的腹水和组织样本中分离和表征卵巢癌细胞的方法。材料与方法:术中获取原发性卵巢、腹膜和输卵管癌患者的腹水和组织样本。样品在采集后6小时内分离培养。分离后，细胞在Matrigel中重悬，并置于每孔的中心。每口井都添加了优化的介质。采用纳米培养板LH96 (Low-Binding, MH pattern, 96-wells, Medical and Biological Laboratories Co.， Ltd, Japan)进行三维细胞培养。37℃孵育。通过ATP法检测培养物的代谢活性细胞，并在第0、1、4、7、10和14天更换培养基。结果:我们在1-7天内成功获得100%(3/31)的腹水来源的原代细胞培养，62%(5/8)的组织来源的原代细胞培养。30%(1/3)的腹水样本和50%(4/8)的组织样本形成球状结构。利用纳米培养板LH96体外培养新的三维球体模型，证实了细胞的致瘤性和侵袭性。结论:三维细胞培养板可成功培养卵巢癌细胞原代腹水和组织培养。本方法可用于药敏试验;因此，3D细胞培养有可能评估候选化疗药物对个体患者的敏感性。引文格式:南木良子、平泽明、野村博之、大久保明、伊藤真昭、赤韩知子、千代田达之、片冈文雄、富永英一郎、青木大辅。腹水来源和组织来源的卵巢癌细胞原代3D培养旨在个性化医疗。[摘要]。AACR会议论文集:解决卵巢癌研究和治疗中的关键问题;2017年10月1-4日;宾夕法尼亚州匹兹堡。费城(PA): AACR;临床肿瘤杂志，2018;24(15 -增刊):摘要nr A61。

{"title":"Abstract A61: Ascites-derived and tissue-derived ovarian cancer cell primary 3D cultures aimed for personalized medicine","authors":"Y. Nanki, A. Hirasawa, H. Nomura, A. Okubo, Manabu Itoh, T. Akahane, T. Chiyoda, F. Kataoka, E. Tominaga, D. Aoki","doi":"10.1158/1557-3265.OVCA17-A61","DOIUrl":"https://doi.org/10.1158/1557-3265.OVCA17-A61","url":null,"abstract":"Objective: Ovarian cancer is a poor-prognosis gynecologic disease with over 220,000 diagnoses each year worldwide and a 5-year survival rate less than 40%. In this study, we present a method for isolation and characterization of ovarian cancer cells from patient-derived ascites and tissue samples by using three-dimensional (3D) cell culture. Materials and Methods: Ascites and tissue samples were obtained from primary ovarian, peritoneal, and fallopian tube cancer patients intraoperatively. Samples were isolated and cultured within 6 hours after collection. After isolation, cells were resuspended in Matrigel and were placed at the center of each well. Optimized medium is added to each well. NanoCulture Plate LH96 (Low-Binding, MH pattern, 96-wells, Medical and Biological Laboratories Co., Ltd., Japan) were used for 3D cell culture. Plates were incubated at 37°C. The cultures were examined for metabolically active cells by ATP assay and medium changed on days 0, 1, 4, 7, 10, and 14. Results: We successfully obtained ascites-derived primary cell cultures within 1-7 days from 100% (3/31) of the ascites samples and 62% (5/8) from tissue-derived primary cell cultures. Spheroids-like structures were formed in 30% (1/3) of ascites samples and 50% (4/8) of tissue samples. The tumorigenicity and invasiveness of the cells were demonstrated using new 3D spheroid model cultured in vitro by NanoCulture Plate LH96. Conclusion: Primary ascites and tissue culture of ovarian cancer cells can successfully be cultured by 3D cell culture plates. We may apply this method for drug sensitivity testing; therefore, 3D cell culture has a potential to evaluate the sensitivity of candidate chemotherapeutic drug for individual patients. Citation Format: Yoshiko Nanki, Akira Hirasawa, Hiroyuki Nomura, Aki Okubo, Manabu Itoh, Tomoko Akahane, Tatsuyuki Chiyoda, Fumio Kataoka, Eiichiro Tominaga, Daisuke Aoki. Ascites-derived and tissue-derived ovarian cancer cell primary 3D cultures aimed for personalized medicine. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A61.","PeriodicalId":18646,"journal":{"name":"Metabolic Changes in Ovarian Cancer","volume":"295 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79227247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Abstract A32: Single-cell RNA-seq analysis of primary tumor and corresponding metastatic lesion in high-grade serous ovarian cancer A32:高级别浆液性卵巢癌原发肿瘤及相应转移灶的单细胞RNA-seq分析

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A32

A. Shih, A. Menzin, J. Whyte, J. Lovecchio, A. Liew, H. Khalili, K. Onel, P. Gregersen, Annette Lee

Ovarian cancer is highly curable when diagnosed early as localized disease. Most women come to medical attention, however, with metastatic disease. For these women, cure rates are quite low; only 30% of patients with late-stage high-grade serous ovarian cancer (HGSOC) will live more than 5 years. Although initially sensitive to platinum-based chemotherapy, in most cases, drug resistance develops and a progressive disease course ensues. Therefore, in order to improve prognosis and overall survival, there is an urgent need to understand the basis of drug resistance and to identify new therapeutic targets. Previous studies have stressed the significant role that tumor heterogeneity and microenvironment have in clinical outcome. It is our goal to understand the genomics of metastatic lesions as compared to primary lesions, to identify the genetic drivers of metastasis and drug resistance, which we can then functionally investigate in order to develop novel therapies. Corresponding primary and metastatic tumor tissue samples from women with HGSOC were analyzed by single-cell RNA-seq. Isolated cells from each paired tissue sample were processed for next-gen sequencing using the BioRad droplet digital SEQ Single Cell Isolator and the Illumina SureCell Whole Transcriptome Analysis 3’ library prep kit, Normalization of expression, clustering of cells and gene expression markers defining each cluster was done by using the Seurat package in R. To identify specific tumor cell subsets in intra- and inter-patient analyses, a graph-based clustering using the principal components of the most variable expressed genes and T-distributed stochastic neighbor embedding (tSNE) analyses was performed. Overall, we have found that while there is considerable heterogeneity among primary tumor cells from different patients, the expression profiles of metastatic lesions from different patients are remarkably similar, and are distinct from the primary lesions. As one example, by single-cell RNA-seq paired analysis of HGSOC primary tumor and corresponding metastatic lesions from 2 patients (primary fallopian and primary ovarian), we identified several cell clusters based on gene expression of common cellular markers. Further analysis identified significant expression of CD24, EPCAM, and KRT18 in epithelial cells of primary tumors while elevated CD44 expression was found in the T and B cell clusters of the metastatic lesions. Published studies have suggested elevated CD44 as a prognostic marker of poor overall survival. Whether elevated CD44 expression influences survival in our patients remains to be determined since clinical response data are not yet available. Additional analysis of gene expression profiles in other cell clusters is in progress. Our ability to study patient-derived primary tumor and corresponding metastatic lesions using high-throughput single-cell analysis represents an unprecedented unique opportunity to study ovarian cancer without a priori knowledge of t

卵巢癌在早期诊断为局部疾病时，治愈率很高。然而，大多数女性就诊时都伴有转移性疾病。对这些妇女来说，治愈率相当低;只有30%的晚期高级别浆液性卵巢癌(HGSOC)患者能存活5年以上。虽然最初对铂类化疗敏感，但在大多数情况下，会产生耐药性，并导致病程进展。因此，为了改善预后和总体生存，迫切需要了解耐药的基础，寻找新的治疗靶点。既往研究强调肿瘤异质性和微环境在临床预后中的重要作用。我们的目标是了解转移性病变与原发性病变的基因组学，确定转移和耐药性的遗传驱动因素，然后我们可以对其进行功能研究，以开发新的治疗方法。通过单细胞RNA-seq分析HGSOC女性相应的原发和转移性肿瘤组织样本。使用BioRad液滴数字SEQ单细胞隔离器和Illumina SureCell全转录组分析3’文库准备试剂盒对每个配对组织样本分离的细胞进行处理，进行下一代测序，使用r中的Seurat包进行表达归一化，细胞聚类和定义每个聚类的基因表达标记。利用可变表达基因的主成分和t分布随机邻居嵌入(tSNE)分析进行了基于图的聚类。总的来说，我们发现，虽然不同患者的原发肿瘤细胞存在相当大的异质性，但不同患者的转移灶的表达谱却非常相似，并且与原发灶不同。例如，通过对2例患者(原发输卵管和原发卵巢)的HGSOC原发肿瘤和相应转移灶的单细胞RNA-seq配对分析，我们根据常见细胞标志物的基因表达确定了几个细胞簇。进一步分析发现，CD24、EPCAM和KRT18在原发肿瘤上皮细胞中显著表达，而CD44在转移灶的T细胞群和B细胞群中表达升高。已发表的研究表明，CD44升高是总生存率差的预后标志。由于尚未获得临床反应数据，CD44表达升高是否影响患者的生存仍有待确定。其他细胞群基因表达谱的进一步分析正在进行中。我们利用高通量单细胞分析研究患者来源的原发肿瘤和相应的转移性病变的能力，为研究卵巢癌提供了前所未有的独特机会，而无需先验地了解肿瘤和间质细胞的相互关系。患者来源样本的单细胞评估可以提供了解高级别浆液性卵巢癌中常见的化疗耐药所需的关键信息。引文格式:Andrew Shih, Andrew Menzin, Jill Whyte, John Lovecchio, Anthony Liew, Houman Khalili, Kenan Onel, Peter Gregersen, Annette Lee。高级别浆液性卵巢癌原发肿瘤及相应转移灶的单细胞RNA-seq分析。[摘要]。AACR会议论文集:解决卵巢癌研究和治疗中的关键问题;2017年10月1-4日;宾夕法尼亚州匹兹堡。费城(PA): AACR;临床肿瘤杂志，2018;24(15 -增刊):A32。

{"title":"Abstract A32: Single-cell RNA-seq analysis of primary tumor and corresponding metastatic lesion in high-grade serous ovarian cancer","authors":"A. Shih, A. Menzin, J. Whyte, J. Lovecchio, A. Liew, H. Khalili, K. Onel, P. Gregersen, Annette Lee","doi":"10.1158/1557-3265.OVCA17-A32","DOIUrl":"https://doi.org/10.1158/1557-3265.OVCA17-A32","url":null,"abstract":"Ovarian cancer is highly curable when diagnosed early as localized disease. Most women come to medical attention, however, with metastatic disease. For these women, cure rates are quite low; only 30% of patients with late-stage high-grade serous ovarian cancer (HGSOC) will live more than 5 years. Although initially sensitive to platinum-based chemotherapy, in most cases, drug resistance develops and a progressive disease course ensues. Therefore, in order to improve prognosis and overall survival, there is an urgent need to understand the basis of drug resistance and to identify new therapeutic targets. Previous studies have stressed the significant role that tumor heterogeneity and microenvironment have in clinical outcome. It is our goal to understand the genomics of metastatic lesions as compared to primary lesions, to identify the genetic drivers of metastasis and drug resistance, which we can then functionally investigate in order to develop novel therapies. Corresponding primary and metastatic tumor tissue samples from women with HGSOC were analyzed by single-cell RNA-seq. Isolated cells from each paired tissue sample were processed for next-gen sequencing using the BioRad droplet digital SEQ Single Cell Isolator and the Illumina SureCell Whole Transcriptome Analysis 3’ library prep kit, Normalization of expression, clustering of cells and gene expression markers defining each cluster was done by using the Seurat package in R. To identify specific tumor cell subsets in intra- and inter-patient analyses, a graph-based clustering using the principal components of the most variable expressed genes and T-distributed stochastic neighbor embedding (tSNE) analyses was performed. Overall, we have found that while there is considerable heterogeneity among primary tumor cells from different patients, the expression profiles of metastatic lesions from different patients are remarkably similar, and are distinct from the primary lesions. As one example, by single-cell RNA-seq paired analysis of HGSOC primary tumor and corresponding metastatic lesions from 2 patients (primary fallopian and primary ovarian), we identified several cell clusters based on gene expression of common cellular markers. Further analysis identified significant expression of CD24, EPCAM, and KRT18 in epithelial cells of primary tumors while elevated CD44 expression was found in the T and B cell clusters of the metastatic lesions. Published studies have suggested elevated CD44 as a prognostic marker of poor overall survival. Whether elevated CD44 expression influences survival in our patients remains to be determined since clinical response data are not yet available. Additional analysis of gene expression profiles in other cell clusters is in progress. Our ability to study patient-derived primary tumor and corresponding metastatic lesions using high-throughput single-cell analysis represents an unprecedented unique opportunity to study ovarian cancer without a priori knowledge of t","PeriodicalId":18646,"journal":{"name":"Metabolic Changes in Ovarian Cancer","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73779939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer A23:卵巢癌调节性T细胞中Neuropilin-1的表达

Metabolic Changes in Ovarian Cancer

Pub Date : 2018-08-01 DOI: 10.1158/1557-3265.OVCA17-A23

A. Cillo, T. Bruno, F. Modugno, R. Edwards, D. Vignali

Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naive CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD

调节性T细胞(Treg)是CD4+ T细胞的一个亚群，它抑制自身免疫反应，但也阻止肿瘤和慢性病毒感染的清除。在卵巢癌中，肿瘤浸润Treg的频率较高与预后不良有关，但在这种情况下，对Treg存活和抑制活性的机制的了解有限。我们的研究小组最近在小鼠癌症模型中描述了一个通过嗜神经蛋白-1 (NRP1)作用于Treg的信号轴，该信号轴促进Treg的存活和抑制功能。NRP1基因敲除小鼠Treg导致肿瘤生长减少和生存增加，强调了NRP1+ Treg在抑制抗肿瘤免疫中的重要性。鉴于NRP1+ Treg在小鼠肿瘤模型中的重要性，我们试图探索NRP1在卵巢癌患者肿瘤浸润性Treg中的表达模式。材料和方法:卵巢肿瘤通过匹兹堡大学健康科学组织库获得，卵巢腹水通过Magee妇女研究所获得。外周血通过红十字会从健康的献血者处获得。采用密度梯度离心分离外周血单个核细胞(PBMC)，先机械破坏肿瘤，分离肿瘤浸润淋巴细胞，然后用50 ug/mL Liberase DL在37℃下处理15分钟。流式细胞术分析Treg时，首先对血液和肿瘤细胞进行表面标记物染色，然后进行活/死区分、固定和渗透，最后进行细胞内标记物染色。细胞在希尔曼癌症中心流动核心的LSR Fortessa上进行分析。为了进行功能测试，从腹水中选择Treg，并测定其体外抑制幼稚CD8+ T细胞的能力。采用Wilcoxon秩和检验评估组间差异，双侧alpha值小于5%。结果:共对12例健康供体和20例卵巢癌患者进行了研究。通过流式细胞术检测健康供者外周血CD4+CD25+FOXP3+ Treg和卵巢癌肿瘤或卵巢癌患者腹水的肿瘤浸润淋巴细胞，评估细胞内和表面NRP1的表达。细胞内NRP1在健康供者Treg中表达的中位数为1.8%(四分位数范围[IQR]: 0.69%至4.8%)，而在健康供者Treg中表达的中位数为66% (IQR: 28%至90%;结论和未来方向:细胞内和表面NRP1在肿瘤浸润的Treg和来自腹水的Treg上的表达比来自健康供者外周血的Treg更频繁。虽然目前纳入本研究的患者数量较少，但也有证据表明，恶性组织中细胞内NRP1在Treg上的表达高于良性组织。此外，来自腹水的Treg能够在体外抑制CD8细胞。未来的研究将评估NRP1对人类Treg的阻断是否会限制Treg的存活或抑制能力。注:本摘要未在会议上发表。引文格式:Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali。Neuropilin-1在卵巢癌调节性T细胞中的表达。[摘要]。AACR会议论文集:解决卵巢癌研究和治疗中的关键问题;2017年10月1-4日;宾夕法尼亚州匹兹堡。费城(PA): AACR;临床肿瘤杂志，2018;24(15 -增刊):摘要nr - A23。

{"title":"Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer","authors":"A. Cillo, T. Bruno, F. Modugno, R. Edwards, D. Vignali","doi":"10.1158/1557-3265.OVCA17-A23","DOIUrl":"https://doi.org/10.1158/1557-3265.OVCA17-A23","url":null,"abstract":"Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naive CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD","PeriodicalId":18646,"journal":{"name":"Metabolic Changes in Ovarian Cancer","volume":"97 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89915502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0