单细胞测序技术在乳腺癌中的研究进展

乳腺癌是全球女性发病率最高的恶性肿瘤，也是女性癌症死亡的主要原因之一。越来越多的研究表明乳腺癌是一种高度异质性疾病，传统高通量测序技术对乳腺癌的发生发展、精确分型、诊断及治疗方面提供了重要参考，但仍有局限性，无法揭示肿瘤内异质性。与传统测序技术相比，单细胞测序技术在灵敏度、准确度和工作效率方面都得到极大的提高，该技术可以分析复杂混合细胞中单个细胞的分子生物学信息，应用在肿瘤异质性方面展现出极大的优越性。包括从单细胞层面解析乳腺癌转录组图谱、探索乳腺癌微环境异质性、探索乳腺癌转移与耐药发生机制、探索乳腺癌克隆异质性及其演化研究。本文对近年来单细胞测序技术在乳腺癌中的相关研究进展进行综述，旨在为乳腺癌单细胞测序技术研究提供理论参考。     

单细胞转录组测序和肿瘤精准医疗

单细胞测序技术是指以单个细胞为单位,通过高通量测序技术全方位分析单个细胞内的遗传信息,其已成为现代生物 学深度研究的一种强有力工具,使生物医药领域研究和临床应用转化研究达到了传统测序方法难以实现的极高精确度和灵敏度。 目前,单细胞转录组测序（又称“单细胞RNA测序”,scRNA-seq）已成为最广泛应用的单细胞测序技术。简要介绍scRNA-seq技术 的发展历程、技术原理和方法,系统阐述其应用在肿瘤异质性、肿瘤转移、肿瘤免疫、肿瘤耐药等基础领域的研究成果及其在肿瘤精 准医疗的临床转化与应用中的研究进展,单细胞测序技术的发展和应用必将推动肿瘤精准医疗迈向更高水平。     

肿瘤异质性-精准临床诊治的挑战

肿瘤异质性是肿瘤演化过程中出现的一个普遍而又至关重要的表现特征，是肿瘤形成及进一步演进的重要支撑点。尽管近几年，肿瘤异质性内在规律和形成机制等方面的研究有诸多新进展，但肿瘤异质性仍是实现临床精准诊治、克服耐药问题的重大挑战。近几年，随着高通量测序和单细胞测序技术的发展和不断完善，对肿瘤单个细胞或全基因组测序已逐步揭示肿瘤异质性的时空变异和分子机制。本篇文章对肿瘤异质性的复杂性、形成机制和对肿瘤演进的影响以及对临床诊治带来的挑战的最新研究进展进行综述。      

单细胞测序技术在乳腺癌研究中的应用进展

乳腺癌是全球女性发病率最高的恶性肿瘤,也是女性癌症相关致死的主要原因之一。乳腺癌是一种高度异质性的疾病,肿瘤间异质性和肿瘤内异质性使得精准分型治疗工作进展缓慢。传统高通量测序技术在乳腺癌研究中的应用已为揭示乳腺癌的发生、发展、驱动基因、精确分型、诊断及治疗方面提供了重要参考,但仍有局限性,无法揭示肿瘤内异质性。近年来兴起的单细胞测序技术在灵敏度、准确度和效率方面都得到了极大的提高,研究人员可以分析复杂混合细胞中的单个细胞的分子生物学信息,该技术在研究肿瘤异质性方面展现出极大的优越性。近年来,单细胞测序技术在乳腺癌研究中的应用已有多项重要研究成果发表,包括从单细胞层面解析乳腺癌转录组图谱、探索乳腺癌微环境异质性、探索乳腺癌耐药发生机制、探索乳腺癌克隆异质性及克隆演化研究,以及综合挖掘单细胞公共数据资源揭示乳腺癌异质性。本文对近年来单细胞测序技术在乳腺癌中的应用相关前沿重要研究进行综述并深入思考,旨在为乳腺癌研究者应用单细胞测序技术提供参考,促进单细胞测序技术在乳腺癌研究中的广泛应用。     

单细胞测序技术在恶性肿瘤研究中的应用进展

恶性肿瘤是严重威胁人类健康的公共卫生问题之一，具有多样性、复杂性和死亡率高等特点，已成为学术界关注的热点与难点。异质性为恶性肿瘤的主要特征之一，影响肿瘤发生、发展、转移和治疗等。随着人类基因组计划的完成和测序技术的不断发展，单细胞测序技术已应用于多个生物医学领域，且在分析肿瘤细胞亚群、揭示细胞异质性和研究肿瘤的发生、演变及耐药性等方面具有独特的优势，为探索恶性肿瘤的临床诊治和预后判断等提供新方法和新思路。本文主要对单细胞测序技术的发展及其在恶性肿瘤研究中的应用予以综述。      

2023年度肿瘤单细胞与空间多组学研究进展盘点

单细胞组学技术能从单细胞分辨率解析瘤内异质性和细胞间交互,空间组学技术则从空间维度探究异质性与肿瘤微环境。因此,单细胞与空间多组学技术在肿瘤研究中具有不可替代的优势。本文针对2023年单细胞与空间多组学技术在肿瘤细胞图谱、肿瘤异质性和微环境及临床应用与药物研发的研究进展进行盘点,并尝试分析目前仍面临的挑战,以期对本领域研究者提供帮助。     

免疫细胞单细胞测序对肿瘤免疫治疗疗效预测的意义

免疫检查点抑制剂及CAR-T细胞的基础及临床转化研究已成为肿瘤研究的热点之一。免疫治疗已在多种类型的肿瘤中应用,并可观察到持续的应答和显著的生存优势,但仍有部分患者并未受益,如何对免疫治疗疗效进行有效的预测,是亟待解决的问题。单细胞测序技术是在单个细胞水平上,对基因组、转录组、表观组进行高通量测序,揭示单个细胞的基因结构和基因表达状态,反映细胞间的异质性,破解多种类型肿瘤的免疫微环境及外周血循环免疫细胞的亚型及图谱,分析肿瘤细胞及肿瘤微环境的异质性,在发掘新的疾病诊断标志物、分辨细胞亚型、治疗靶标及在个体化治疗方面具有独特的优势。     

单细胞测序技术在多发性骨髓瘤研究及其精准医疗中的应用

由于多发性骨髓瘤(MM)细胞及其微环境的高度异质性,导致其诊断和治疗方面面临巨大挑战。单细胞测序（sc-Seq）技术是一种以单个细胞为测试对象,分析其细胞内遗传信息的研究工具,近年来sc-Seq 在MM研究及治疗中广泛应用,为其更精准的诊断及个体化治疗作出了重要贡献。本文论述了近年来sc-Seq 技术在MM细胞异质性、免疫微环境、生物标志物及其在精准诊疗等方面应用的研究进展,为MM的基础与临床诊疗研究提供了参考依据。     

单细胞测序技术在头颈部恶性肿瘤中的研究与发展

单细胞测序是针对单个细胞的遗传信息进行测序,能更好地认识细胞之间的差异,解决了普通测序带来的异质性问题。头颈部恶性肿瘤已经成为癌症相关疾病死亡的第六大病因,单细胞测序技术应用于揭示头颈部恶性肿瘤的异质性、描述头颈部恶性肿瘤的微环境、评估头颈部恶性肿瘤的侵袭、转移和评价治疗效果、评估治疗抵抗、探究循环肿瘤细胞的作用等方面,对头颈部恶性肿瘤发生发展的研究和个体化治疗方案的探索有着巨大的推动作用。本综述旨在概述单细胞测序的机理和意义,总结其在头颈部恶性肿瘤中的研究进展。     

单细胞多组学测序技术进展及其在肿瘤研究中的应用

近年来，随着单细胞测序技术在肿瘤研究领域的广泛应用，单细胞多组学技术应运而生，且已公开发表了多种检测策略。单细胞多组学技术可在同一细胞中捕获分析两种以上的组学信息，包括转录组、基因组、表观基因组、蛋白组等。该技术突破了以往单细胞检测的单一性，可更全面、多角度地整合分析肿瘤细胞特征，为系统观察肿瘤细胞异质性、各类生物学特性及机制，研究肿瘤细胞间或与其他类型细胞互作，发现鉴定新的细胞亚型提供前所未有的优势。尽管目前单细胞多组学技术仍然处于技术探索及高通量开发阶段，但可预见广泛应用单细胞多组学分析技术是肿瘤研究发展的必然趋势。本文主要介绍多种单细胞多组学技术并综述目前多组学技术在肿瘤研究中的一些开创性研究，展望单细胞多组学技术的未来，以期为肿瘤研究提供有价值的参考。     

单细胞转录组测序及其在乳腺癌中的应用

乳腺癌占全部女性癌症的17.1%,位于中国女性癌症发病率首位。尽管目前根据乳腺癌分子分型采用的个体化治疗策略使乳腺癌治疗取得了阶段性突破,但是肿瘤异质性、耐药、转移等问题依然阻碍我们进一步提升临床疗效。单细胞转录组测序是一项能在单细胞水平上进行无偏倚、高通量、高分辨率全转录组学分析的新技术。该技术能以单细胞分辨率分析肿瘤内部基因表达特征,对认识乳腺癌具有不可替代的作用。就单细胞测序技术在肿瘤异质性、肿瘤微环境、转移、耐药等方面的应用进行综述,以期更加深刻地了解该技术并为乳腺癌研究提供全新思路。     

神经母细胞瘤研究前沿及思考

神经母细胞瘤（neuroblastoma,NB）是儿童最常见的颅内实体肿瘤,发现时常常已是晚期,易发生转移,高危NB患儿即使接受高强度治疗,仍可出现复发或转移,治疗效果差。目前NB的发病机制及治疗等已成为儿童实体恶性肿瘤研究的热点、难点。近年来,随着单细胞测序等各种技术手段的进步,NB的发病机制、治疗手段等相关研究取得了诸多进展。本文将对近年来NB的研究进展进行文献综述,阐述了NB的起源、分子遗传学调控机制及免疫学调控机制研究成果,并介绍NB单克隆抗体、肿瘤疫苗、过继细胞疗法及小分子抑制剂的最新进展。     

Detection of hypoxic cells in murine tumors using the comet assay: comparison with a conventional radiobiological assay.

The comet (single-cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/ C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H (r=0.85) and in EMT6/KU/balb/c (r=0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors.     

Detection of Hypoxic Cells in Murine Tumors Using the Comet Assay: Comparison with a Conventional Radiobiological Assay

The comet (single-cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H ( r = 0.85) and in EMT6/KU/balb/c ( r = 0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors.     

Control of cell death (apoptosis) by diethylstilbestrol in an estrogen-dependent kidney tumor

The role of cell death as a determinant for tumor growth and regression was studied using an estrogen-dependent, transplantable kidney tumor designated H301. H301 cells were injected s.c. into diethylstilbestrol(DES)-treated male Syrian hamsters and developed solid tumors of 1-2 g within 2-3 weeks. Upon withdrawal of estrogen the tumors regressed by 80-90% within 4 days. Mitoses, necrotic areas and single-cell death indicated by small, condensed cell residues, were counted in hematoxylin and eosin stained histological sections of the tumors. Coincident with tumor regression after DES withdrawal, mitotic activity decreased by approximately 90%; the rate of single-cell death increased (by approximately 2-fold at its maximum). The incidence of necrotic areas was not affected by DES withdrawal. DES re-treatment resulted in reduction of single-cell death by 80% within 8 h. Mitotic activity increased within 24 h to the level observed before DES withdrawal. Again, the incidence of necrotic areas did not change. As a result, tumors re-grew to their previous size within 2 days after resumption of DES treatment. These results led to the following conclusions: (i) DES treatment inhibits and DES withdrawal enhances single-cell death of H301 tumor cells. (ii) Both this functional property and its morphology characterize single-cell death in the tumors as apoptosis. (iii) Estrogen-dependent cell death determines, in addition to mitosis and necrosis, the growth rate of H301 tumors. (iv) This experimental model may provide a useful tool to study the interaction of potential anti-tumor drugs with apoptosis in neoplasia.     

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.