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• W3158326913 abstract "Cancer has become one of the greatest threats to human health, and new technologies are urgently needed to further clarify the mechanisms of cancer so that better detection and treatment strategies can be developed. At present, extensive genomic analysis and testing of clinical specimens shape the insights into carcinoma. Nevertheless, carcinoma of humans is a complex ecosystem of cells, including carcinoma cells and immunity-related and stroma-related subsets, with accurate characteristics obscured by extensive genome-related approaches. A growing body of research shows that sequencing of single-cell RNA (scRNA-seq) is emerging to be an effective way for dissecting human tumor tissue at single-cell resolution, presenting one prominent way for explaining carcinoma biology. This review summarizes the research progress of scRNA-seq in the field of tumors, focusing on the application of scRNA-seq in tumor circulating cells, tumor stem cells, tumor drug resistance, the tumor microenvironment, and so on, which provides a new perspective for tumor research. Cancer has become one of the greatest threats to human health, and new technologies are urgently needed to further clarify the mechanisms of cancer so that better detection and treatment strategies can be developed. At present, extensive genomic analysis and testing of clinical specimens shape the insights into carcinoma. Nevertheless, carcinoma of humans is a complex ecosystem of cells, including carcinoma cells and immunity-related and stroma-related subsets, with accurate characteristics obscured by extensive genome-related approaches. A growing body of research shows that sequencing of single-cell RNA (scRNA-seq) is emerging to be an effective way for dissecting human tumor tissue at single-cell resolution, presenting one prominent way for explaining carcinoma biology. This review summarizes the research progress of scRNA-seq in the field of tumors, focusing on the application of scRNA-seq in tumor circulating cells, tumor stem cells, tumor drug resistance, the tumor microenvironment, and so on, which provides a new perspective for tumor research. During the last three decades, carcinoma studies have largely discussed somatic gene-centric mutations (so-called oncogenes) that target their functional characteristics and biochemical activity.1Vogelstein B. Papadopoulos N. Velculescu V.E. Zhou S. Diaz Jr., L.A. Kinzler K.W. Cancer genome landscapes.Science. 2013; 339: 1546-1558Crossref PubMed Scopus (4345) Google Scholar,2Heyer J. Kwong L.N. Lowe S.W. Chin L. Non-germline genetically engineered mouse models for translational cancer research.Nat. Rev. Cancer. 2010; 10: 470-480Crossref PubMed Scopus (117) Google Scholar Thus far, multiple targeted therapies have been approved to treat multiple tumors, and more are in development or in early clinical trials. However, with the extensive use of targeted therapies, common themes of treating relapse and drug resistance have been proposed.3Ma C.X. Reinert T. Chmielewska I. Ellis M.J. Mechanisms of aromatase inhibitor resistance.Nat. Rev. Cancer. 2015; 15: 261-275Crossref PubMed Scopus (200) Google Scholar,4Lito P. Rosen N. Solit D.B. Tumor adaptation and resistance to RAF inhibitors.Nat. Med. 2013; 19: 1401-1409Crossref PubMed Scopus (374) Google Scholar Carcinoma refers to a selectively proliferating, invasive somatic mutant phenotype. There is an implicit concept that following the evolution path of carcinoma, genetically complex groups of different individual carcinoma cells may develop and interact in a dynamic manner with each other. Therefore, studying this potential intratumoral genetic heterogeneity is of great significance for the selection of anti-target treatment methods. Indeed, as next-generation sequencing (NGS) technology is emerging, sequencing of large amounts of DNA and RNA retrieved from carcinoma tissue can be conducted in depth, fine-grained studies of intra-tumor genetic heterogeneity can be carried out, and computational inferences of subclonality can be achieved.5Morganti S. Tarantino P. Ferraro E. D’Amico P. Duso B.A. Curigliano G. Next generation sequencing (NGS): A revolutionary technology in pharmacogenomics and personalized medicine in cancer.Adv. Exp. Med. Biol. 2019; 1168: 9-30Crossref PubMed Scopus (21) Google Scholar,6Garziera M. Roncato R. Montico M. De Mattia E. Gagno S. Poletto E. Scalone S. Canzonieri V. Giorda G. Sorio R. et al.New challenges in tumor mutation heterogeneity in advanced ovarian cancer by a targeted next-generation sequencing (NGS) approach.Cells. 2019; 8: 584Crossref Google Scholar However, as impacted by practical and technical limitations, deep sequencing alone is insufficient to fully explore the genomic and transcriptomic heterogeneity of carcinoma. Individual cells are the basic substrates for the mechanisms of mutation and selection at work to evolve complex structures known as tumor blocks. For this reason, gaining insights into individual carcinoma cells under the individual condition and, overall, into dynamically related systems of interaction (carcinoma microenvironment) will indeed further clarify therapy-related resistance and biology of tumors generally. The quickly advancing method of single-cell RNA sequencing (scRNA-seq), by exhibiting its ability for characterizing the epigenome, transcriptome, and genome of one individual cell, profoundly presents insights into genetics and tumor biology, and it will enable us to understand the changes in the various stages of tumor progression to advanced metastatic disease.7Tang F. Barbacioru C. Wang Y. Nordman E. Lee C. Xu N. Wang X. Bodeau J. Tuch B.B. Siddiqui A. et al.mRNA-seq whole-transcriptome analysis of a single cell.Nat. Methods. 2009; 6: 377-382Crossref PubMed Scopus (1336) Google Scholar,8Navin N. Kendall J. Troge J. Andrews P. Rodgers L. McIndoo J. Cook K. Stepansky A. Levy D. Esposito D. et al.Tumour evolution inferred by single-cell sequencing.Nature. 2011; 472: 90-94Crossref PubMed Scopus (1595) Google Scholar In addition, the clinical application of scRNA-seq may profoundly alter the way we treat carcinoma. Much information has been gathered using the mentioned techniques, and, although numerous difficulties still exist, it is considered that the capacity exhibited by scRNA-seq will continuously drive innovation and yield methods to solve current issues that profoundly increase our understanding of the disease. In this review, we discuss the topic of scRNA-seq in the carcinoma field and highlight the use of emerging scRNA-seq approaches to circulating tumor cells (CTCs), tumor drug resistance, and the tumor microenvironment (TME). RNA sequencing (either cell collections or individual cells) is one effective approach to investigate gene expressing modes, involving the reverse transcribing of RNA to cDNA, followed by DNA sequencing under significant throughput. Genes exhibiting a significant expression state in the sample generate greater amounts of DNA sequence readings, cDNA and RNA, as opposed to those with a lower expressing state. For this reason, RNA sequencing presents a digital gene expressing state reading, of which DNA sequence readings have a number abiding by the expressing level of one gene inside the sample. The scRNA-seq idea is consistent with that mentioned, although an individual cell should first receive the isolation because of its tiny RNA content, which requires an effective amplifying procedure for producing enough cDNA as an attempt to sequence protein. Under conventional RNA sequencing, the more considerable amount of reads on the sequence of DNA of one gene represents the greater expressing state of such a gene in the cell.9Potter S.S. Single-cell RNA sequencing for the study of development, physiology and disease.Nat. Rev. Nephrol. 2018; 14: 479-492Crossref PubMed Scopus (81) Google Scholar, 10Crow M. Gillis J. Single cell RNA-sequencing: Replicability of cell types.Curr. Opin. Neurobiol. 2019; 56: 69-77Crossref PubMed Scopus (5) Google Scholar, 11Ziegenhain C. Vieth B. Parekh S. Reinius B. Guillaumet-Adkins A. Smets M. Leonhardt H. Heyn H. Hellmann I. Enard W. Comparative analysis of single-cell RNA sequencing methods.Mol. Cell. 2017; 65: 631-643.e4Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar It is noteworthy that the current scRNA-seq approach can determine the expression levels of all genes. In summary, existing scRNA-seq technologies overall have a general workflow as follows: a sample preparing process, an individual cell capturing process, a reverse transcribing process, an amplifying process, a library preparing process, and a sequencing and analyzing process (Figure 1).12Haque A. Engel J. Teichmann S.A. Lönnberg T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.Genome Med. 2017; 9: 75Crossref PubMed Scopus (190) Google Scholar The sample preparing process is critical for producing reliable individual-cell transcriptome information. A key step in the sample preparation process, especially in terms of dense tissue and three-dimensional-like organ models, refers to individual-cell dissociation, usually done based on enzymatic conditions with mild mechanical agitation for limiting noise in background and extreme cell lysis. Moreover, proteolytic enzymes (e.g., trypsin, collagenase, and free enzymes) and digestion times are required to be rigorously selected for maximizing individual cell production and minimizing apoptosis. If a cell received full dissociation, it can receive the isolation to an individual cell by drawing upon various cell capturing technologies (e.g., the plate-related cell isolating process to droplet-based methods). Maintaining large numbers of isolated living cells shows critical significance for improving data quality. In addition, the approach of cell capturing commonly depends on the target sample nature. Common single-cell separation technologies include limiting dilution, micromanipulation, flow-activated cell sorting (FACS), and laser capture microdissection. Limiting dilution is done with pipette dilution to isolate individual cells. When using this method, usually when the concentration is diluted to 0.5 cells per aliquot, one can only complete about one third of the preparation wells, so this method is not very effective. Micromanipulation is a classic method of extracting cells from early embryos or uncultured microorganisms.13Brehm-Stecher B.F. Johnson E.A. Single-cell microbiology: Tools, technologies, and applications.Microbiol. Mol. Biol. Rev. 2004; 68: 538-559Crossref PubMed Scopus (0) Google Scholar,14Guo F. Li L. Li J. Wu X. Hu B. Zhu P. Wen L. Tang F. Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.Cell Res. 2017; 27: 967-988Crossref PubMed Scopus (111) Google Scholar Microscopically guided capillary straws have been used to extract individual cells from suspensions. However, these methods are time-consuming and have a low throughput. Therefore, this method is not often used. Recently, FACS has been a commonly used technology to isolate a high purity of single cells. When the target cells express very low levels of markers, FACS is also the preferred method. In this way, the cells are first labeled with fluorescent monoclonal antibodies. These antibodies have an ability to identify specific surface markings and are able to classify different groups. In addition, negative selection can be used for unstained populations. In this case, based on the predetermined fluorescence parameters, an electric charge is applied to the cell of interest using an electrostatic deflection system, and the cell is magnetically isolated.15Julius M.H. Masuda T. Herzenberg L.A. Demonstration that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter.Proc. Natl. Acad. Sci. USA. 1972; 69: 1934-1938Crossref PubMed Google Scholar The potential limitations of these techniques include the need for a large initial amount of cells (it is difficult to isolate cells from a low input of less than 10,000) and the need for monoclonal antibodies against the target protein of interest. For laser capture microdissection, cells are isolated from solid samples using a laser system assisted by a computer system.16Nichterwitz S. Chen G. Aguila Benitez J. Yilmaz M. Storvall H. Cao M. Sandberg R. Deng Q. Hedlund E. Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling.Nat. Commun. 2016; 7: 12139Crossref PubMed Scopus (107) Google Scholar When successfully captured, a single cell is cleaved and treated to produce a first strand of cDNA by the reverse transcribing process, with a second strand synthesizing process and a PCR amplifying process conducted subsequently. When PCR amplifying and library preparing processes are conducted, the samples are sequenced. The data from sequencing are then analyzed, rather than simply quantitative analysis of the gene expressing state, for including deep studies on cellular heterogeneity, pedigree transformation, and relationships between cells.12Haque A. Engel J. Teichmann S.A. Lönnberg T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.Genome Med. 2017; 9: 75Crossref PubMed Scopus (190) Google Scholar,17Wu Y. Zhang K. Tools for the analysis of high-dimensional single-cell RNA sequencing data.Nat. Rev. Nephrol. 2020; 16: 408-421Crossref PubMed Scopus (2) Google Scholar,18Luecken M.D. Theis F.J. Current best practices in single-cell RNA-seq analysis: A tutorial.Mol. Syst. Biol. 2019; 15: e8746Crossref PubMed Scopus (218) Google Scholar Individual-cell gene expressing profiling has quickly turned out to be one of the normal analysis-related techniques. It has received extensive studies in numerous subjects. Herein, this technique’s use in carcinoma is largely discussed (Figure 2). We comprehensively reviewed the application of single-cell sequencing in cancer and found that this technique mainly plays an important role in the aspects of CTCs, carcinoma stem cells (CSCs), the TME, and tumor drug resistance, so we review previous research mainly around these four aspects. CTCs receive detection in most epithelial carcinomas and stand for carcinoma cells that have been obtained when passing via the bloodstream.19Nolan J. Nedosekin D.A. Galanzha E.I. Zharov V.P. Detection of apoptotic circulating tumor cells using in vivo fluorescence flow cytometry.Cytometry A. 2019; 95: 664-671Crossref PubMed Scopus (8) Google Scholar, 20Xu L. Mao X. Grey A. Scandura G. Guo T. Burke E. Marzec J. Abdu S. Stankiewicz E. Davies C.R. et al.Noninvasive detection of clinically significant prostate cancer using circulating tumor cells.J. Urol. 2020; 203: 73-82Crossref PubMed Scopus (6) Google Scholar, 21Troncarelli Flores B.C. Souza E Silva V. Ali Abdallah E. Mello C.A.L. Gobo Silva M.L. Gomes Mendes G. Camila Braun A. Aguiar Junior S. Thomé Domingos Chinen L. Molecular and kinetic analyses of circulating tumor cells as predictive markers of treatment response in locally advanced rectal cancer patients.Cells. 2019; 8: 641Crossref Google Scholar Such detection is currently thought to be key in mediating blood-borne transmission of carcinoma, whereas CTCs may not be easy to detect in the analysis of primary or metastatic tumor populations. The elements that cause primary tumors to produce CTCs are unknown, including parts of the carcinoma cells that are injected in an active manner in the bloodstream intravenously and those that are passively shed due to damage to the tumor’s vascular system. Compared with ordinary cells in the blood, CTC is extremely rare. There are fewer CTCs that can cause distant metastasis, which indicates that a large number of CTCs die in the blood, and only a small part is related to metastasis. However, emerging scRNA-seq brings new hope for CTC detection. We summarize the applications of scRNA-seq techniques in CTC-related aspects to date and present them in Table 1.Table 1Application of scRNA-seq in CTCsTumorApplication of scRNA-seqResearch achievementReferenceBreast cancerscRNA-seq was performed on the CTC clusters and on the individual CTCs in breast cancer patients and the date was matched in a single blood samplein mouse models, albumin knockout eliminated the formation of CTC clusters and inhibited lung metastasis; in breast cancer patients, both the abundance of CTC clusters and the elevation of tumor protein levels predict adverse outcomes22Aceto N. Bardia A. Miyamoto D.T. Donaldson M.C. Wittner B.S. Spencer J.A. Yu M. Pely A. Engstrom A. Zhu H. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1106) Google ScholarBreast cancerscRNA-seq of CTCs isolated from blood samples of patients with metastatic estrogen receptor (ER)+ breast cancer was performed to compare the progression of bone and internal organsthe cellular pathway activated by CTCs includes the androgen receptor (AR) signal pathway; expression of the AR gene and its structural active splicing variants AR-V7 increased significantly23Aceto N. Bardia A. Wittner B.S. Donaldson M.C. O’Keefe R. Engstrom A. Bersani F. Zheng Y. Comaills V. Niederhoffer K. et al.AR expression in breast cancer CTCs associates with bone metastases.Mol. Cancer Res. 2018; 16: 720-727Crossref PubMed Scopus (21) Google ScholarBreast cancerindividual CTC-associated WBCs and corresponding cancer cells in each CTC-WBC cluster were isolated and identified from breast cancer patients and mouse models and scRNA-seq was carried outthe association between neutrophils and CTCs promotes development of the cell cycle in the bloodstream and expands the metastatic potential of CTCs24Szczerba B.M. Castro-Giner F. Vetter M. Krol I. Gkountela S. Landin J. Scheidmann M.C. Donato C. Scherrer R. Singer J. et al.Neutrophils escort circulating tumour cells to enable cell cycle progression.Nature. 2019; 566: 553-557Crossref PubMed Scopus (256) Google ScholarBreast cancerHydro-Seq, a scalable fluid dynamic scRNA-seq bar code technology, was used on 666 CTC samples taken from 21 breast cancer patientsdrug therapy targets for breast cancer were identified and individual cells expressing tumor stem cell (CSCs) and epithelial/mesenchymal cell state transition markers were tracked; transcriptome analysis of these cells provides insight into the targeted therapy and process of tumor metastasis25Cheng Y.H. Chen Y.C. Lin E. Brien R. Jung S. Chen Y.T. Lee W. Hao Z. Sahoo S. Min Kang H. et al.Hydro-Seq enables contamination-free high-throughput single-cell RNA-sequencing for circulating tumor cells.Nat. Commun. 2019; 10: 2163Crossref PubMed Scopus (56) Google ScholarBreast cancerscRNA-seq was used to extract CTCs from concentrated CTCs and carryover PBMCstranscriptome analysis identified two types of CTC, one rich in estrogen reactivity and increased proliferation, and the other rich in decreased proliferation and EMT; immune avoidance was enhanced in the CTC population with EMT characteristics26Brechbuhl H.M. Vinod-Paul K. Gillen A.E. Kopin E.G. Gibney K. Elias A.D. Hayashi M. Sartorius C.A. Kabos P. Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.Mol. Carcinog. 2020; 59: 1129-1139Crossref PubMed Scopus (6) Google ScholarPancreatic cancersingle CTCs were isolated by epitope-independent microfluidic capture in a mouse model of pancreatic cancer and then scRNA-seq was performedboth mouse and human pancreas CTCs highly express interstitial-derived extracellular matrix (ECM) protein, including SPARC, and knockout in cancer cells can inhibit cell migration and invasion27Ting D.T. Wittner B.S. Ligorio M. Vincent Jordan N. Shah A.M. Miyamoto D.T. Aceto N. Bersani F. Brannigan B.W. Xega K. et al.Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.Cell Rep. 2014; 8: 1905-1918Abstract Full Text Full Text PDF PubMed Scopus (260) Google ScholarPancreatic cancera mouse model of xenotransplantation from highly metastatic pancreatic ductal adenocarcinoma (PDAC) patients was established and scRNA-seq was performed on circulating tumor cells isolated from human histocompatibility leukocyte antigen (HLA)isolated CTCs are highly tumorigenic and have the potential of metastasis; the expression profile of a CTC is different from that of the matched primary and metastatic tumors, and it is characterized by low expression of genes related to cell cycle and extracellular matrix28Dimitrov-Markov S. Perales-Patón J. Bockorny B. Dopazo A. Muñoz M. Baños N. Bonilla V. Menendez C. Duran Y. Huang L. et al.Discovery of new targets to control metastasis in pancreatic cancer by single-cell transcriptomics analysis of circulating tumor cells.Mol. Cancer Ther. 2020; 19: 1751-1760Crossref PubMed Scopus (6) Google ScholarHepatocellular carcinomaa sequential combination of image flow cytometry and high-density scRNA-seq was described for the identification of CTCs in hepatocellular carcinoma (HCC) patientsthe genome-wide expression profile of CTCs using this method shows heterogeneity of CTCs and helps to detect known oncogenic drivers of HCC, such as IGF229D’Avola D. Villacorta-Martin C. Martins-Filho S.N. Craig A. Labgaa I. von Felden J. Kimaada A. Bonaccorso A. Tabrizian P. Hartmann B.M. et al.High-density single cell mRNA sequencing to characterize circulating tumor cells in hepatocellular carcinoma.Sci. Rep. 2018; 8: 11570Crossref PubMed Scopus (0) Google ScholarVesicular rhabdomyosarcomascRNA-seq was performed on the CTCs from a child with vesicular rhabdomyosarcomaCTCs are easily detected at diagnosis, and their levels decrease with the success of treatment and can be detected in the blood of patients without radiological evidence of dominant metastasis30Hayashi M. Zhu P. McCarty G. Meyer C.F. Pratilas C.A. Levin A. Morris C.D. Albert C.M. Jackson K.W. Tang C.M. Loeb D.M. Size-based detection of sarcoma circulating tumor cells and cell clusters.Oncotarget. 2017; 8: 78965-78977Crossref PubMed Scopus (20) Google ScholarMultiple myelomasingle CTC RNA sequencing was used for classification of multiple myeloma (MM) and quantitative evaluation of genes related to prognosisCTCs provide the same genetic information as bone marrow multiple myeloma cells, and in some cases are even more sensitive than bone marrow biopsy to reveal mutations31Lohr J.G. Kim S. Gould J. Knoechel B. Drier Y. Cotton M.J. Gray D. Birrer N. Wong B. Ha G. et al.Genetic interrogation of circulating multiple myeloma cells at single-cell resolution.Sci. Transl. Med. 2016; 8: 363ra147Crossref PubMed Scopus (68) Google ScholarProstate cancersingle CTCs isolated from the blood of patients with metastatic prostate cancer and single prostate cancer cell line LNCaP cells added to the blood of healthy donors were analyzed by mRNA sequencingthe CTC RNA of patients with prostate cancer showed obvious signs of degradation, and the transcriptional characteristics of prostate cancer tissues could be easily detected by a single-CTC RNA sequencing32Cann G.M. Gulzar Z.G. Cooper S. Li R. Luo S. Tat M. Stuart S. Schroth G. Srinivas S. Ronaghi M. et al.mRNA-seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.PLoS ONE. 2012; 7: e49144Crossref PubMed Scopus (104) Google Scholar–the single-cell expression profiles of publicly available circulating tumor cells were collatedCTCs that span cancer exist in an almost perfect continuum of EMT; comprehensive analysis of CTC transcripts also highlighted the reverse gene expression patterns between PD-L1 and major histocompatibility complex (MHC), which are associated with cancer immunotherapy33Iyer A. Gupta K. Sharma S. Hari K. Lee Y.F. Ramalingam N. Yap Y.S. West J. Bhagat A.A. Subramani B.V. et al.Integrative analysis and machine learning based characterization of single circulating tumor cells.J. Clin. Med. 2020; 9: 1206Crossref Google Scholar–longitudinal isolated CTCs from animals treated with the BET inhibitor JQ1 for more than 4 days were analyzed by scRNA-seqthe details of the evolution of CTCs over time are revealed, which prove the change of CTCs as a biomarker of drug response and are helpful for future research to understand the role of CTCs in metastasis34Hamza B. Ng S.R. Prakadan S.M. Delgado F.F. Chin C.R. King E.M. Yang L.F. Davidson S.M. DeGouveia K.L. Cermak N. et al.Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.Proc. Natl. Acad. Sci. USA. 2019; 116: 2232-2236Crossref PubMed Scopus (21) Google Scholar Open table in a new tab Aceto et al.22Aceto N. Bardia A. Miyamoto D.T. Donaldson M.C. Wittner B.S. Spencer J.A. Yu M. Pely A. Engstrom A. Zhu H. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar used in vivo flow cytometry and scRNA-seq for studying CTCs in cases subjected to metastatic breast carcinoma (BC) and inside mouse tumor systems. They found that in mouse models, oligonucleotides from primary tumor cells take up one rare but significantly metastatic subset of CTCs in comparison with an individual circulating BC cell. scRNA-seq of the human breast CTC cluster determined that plakoglobin is the critical medium of aggregation of tumor cells, showing the expression inside one heterogeneous mode in the primary tumor. The expression inhibition of plakoglobin in a mouse model inhibited the forming process of CTC clusters and reduced transfer diffusion. In cases subjected to BC, according to the process to match CTC clusters inside one individual blood sample and individual-CTC resolution RNA sequencing, the cell-linking component plakoglobin exhibited high differential expression. For this reason, CTC clusters cover primary tumor cells’ multicellular clusters, which bind jointly by exploiting intercellular globin adhesion, significantly facilitating metastasis and carcinoma spread.22Aceto N. Bardia A. Miyamoto D.T. Donaldson M.C. Wittner B.S. Spencer J.A. Yu M. Pely A. Engstrom A. Zhu H. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar Subsequently, some research has suggested scRNA-seq application in detecting CTCs. Miyamoto et al.35Miyamoto D.T. Zheng Y. Wittner B.S. Lee R.J. Zhu H. Broderick K.T. Desai R. Fox D.B. Brannigan B.W. Trautwein J. et al.RNA-seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.Science. 2015; 349: 1351-1356Crossref PubMed Scopus (392) Google Scholar established scRNA-seq profiles of 77 intact CTCs isolated from 13 patients by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of androgen receptor (AR) gene mutations and splicing variants. According to the retrospective study on CTCs from cases advancing through the treating process exploiting one AR inhibiting element, as opposed to untreated cases, activation of noncanonical Wnt signaling is indicated. The ectopic expressing state exhibited by Wnt5a in prostate carcinoma cells downregulates the antiproliferation-related influence exerted by the AR inhibiting process, while its suppressing process in drug-resistant cells recovers partial sensitivity, a correlation also evident in an established mouse model. For this reason, the single-cell analyzing process on prostate CTCs suggests heterogeneity in signaling channels capable of causing treatment failure.35Miyamoto D.T. Zheng Y. Wittner B.S. Lee R.J. Zhu H. Broderick K.T. Desai R. Fox D.B. Brannigan B.W. Trautwein J. et al.RNA-seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.Science. 2015; 349: 1351-1356Crossref PubMed Scopus (392) Google Scholar In addition, Szczerba et al.24Szczerba B.M. Castro-Giner F. Vetter M. Krol I. Gkountela S. Landin J. Scheidmann M.C. Donato C. Scherrer R. Singer J. et al.Neutrophils escort circulating tumour cells to enable cell cycle progression.Nature. 2019; 566: 553-557Crossref PubMed Scopus (256) Google Scholar isolated and characterized individual CTC-associated white blood cells (WBCs), as well as corresponding carcinoma cells in the respective CTC-WBC cluster, from cases with BC and from mouse models. scRNA-seq was used for indicating a relationship of CTCs to neutrophils in most of the mentioned cases. Under the comparative process of the transcriptome profiles of CTCs displaying an association with neutrophils against those of CTCs independently, they found several genes with differential expression, outlining cell cycle progression and accelerating metastasis.24Szczerba B.M. Castro-Giner F. Vetter M. Krol I. Gkountela S. Landin J. Scheidmann M.C. Donato C. Scherrer R. Singer J. et al.Neutrophils escort circulating tumour cells to enable cell cycle progression.Nature. 2019; 566: 553-557Crossref PubMed Scopus (256) Google Scholar Interestingly, Cheng et al.25Cheng Y.H. Chen Y.C. Lin E. Brien R. Jung S. Chen Y.T. Lee W. Hao Z. Sahoo S. Min Kang H. et al.Hydro-Seq enables contamination-free high-throughput single-cell RNA-sequencing for circulating tumor cells.Nat. Commun. 2019; 10: 2163Crossref PubMed Scopus (56) Google Scholar developed Hydro-Seq, a technique for scalable hydrodynamic scRNA-seq barcoding as an attempt to conduct CTC analysis with large throughput. The high cell-capture efficiency and contamination removal capability of Hydro-Seq successfully carried out scRNA-seq of 666 CTCs from 21 BC pat" @default.
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• W3158326913 title "Single-cell RNA sequencing in cancer: Applications, advances, and emerging challenges" @default.
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