TWI790399B - 循環腫瘤細胞資訊評估方法及其分析方法 - Google Patents
循環腫瘤細胞資訊評估方法及其分析方法 Download PDFInfo
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- TWI790399B TWI790399B TW108131164A TW108131164A TWI790399B TW I790399 B TWI790399 B TW I790399B TW 108131164 A TW108131164 A TW 108131164A TW 108131164 A TW108131164 A TW 108131164A TW I790399 B TWI790399 B TW I790399B
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Abstract
本發明揭露了一種循環腫瘤細胞資訊評估方法及其分析方法。主要來說,本發明透過結合負向篩選以及三維細胞培養的方式,得以篩選並培養出精確的循環腫瘤細胞樣本。據此,進一步根據該些精確的循環腫瘤細胞樣本,進行如腫瘤型態分析、腫瘤監控、預後評估、用藥指導或用藥成效分析等各種不同的評估方法或分析方法。
Description
本發明係一種循環腫瘤細胞資訊評估方法及其分析方法,尤指一種可透過特別的有核細胞類別篩選及數量分析技術來評估腫瘤未來發展可能性及狀況之循環腫瘤細胞資訊評估方法及其分析方法。
循環腫瘤細胞(Circulating Tumor Cells,CTCs)屬液態切片其中一項分類。常被用於評估血液檢體中的數目,並應用於臨床診斷、預後、治療成效評估或疾病監控等用途。但目前仍急需技術突破,來克服循環腫瘤細胞(Circulating Tumor Cells,CTCs)的部份先天特性,例如數目極為稀少及高度異質性(heterogeneity)造成應用上的困難。
循環腫瘤細胞(Circulating Tumor Cells,CTCs)作為樣本可應用於基礎研究、細胞擴增及細胞功能分析、腫瘤動物模式建立、分子生物研究(包含核酸、蛋白、基因體分析等)、臨床研究如腫瘤藥物耐性分析及臨床關聯性分析(包含預後、監控、治療成效評估等)。
有關篩選的技術,目前大致區分為純物理法(Physical method)以及生物化學法(Biochemical method)兩類。其中,物理法主要透過細胞的尺寸、密度或介電泳力來篩選循環腫瘤細胞(Circulating Tumor Cells,CTCs)。但是其可達到的精準度和純度會輸給生物化學法。
而目前主要的生物化學法必須使用到特殊的表面標記(Surface marker),用以區分循環腫瘤細胞(Circulating Tumor Cells,CTCs)和其他細胞。
但是,循環腫瘤細胞(Circulating Tumor Cells,CTCs)因異質性(heterogeneity)的緣故,時常導致其數量評估容易產生誤差。所謂的異質性包即含細胞型態、生理特性甚至是分子特性上的差異。
然而,習知技術中對於循環腫瘤細胞之於惡性轉移(Metastasis),其後續資訊評估仍然不足。其主要的原因在於,習知技術無法有效篩除不具有細胞活性的循環腫瘤細胞(Circulating Tumor Cells,CTCs),以致無法精確分析真正對惡性轉移(Metastasis)直接貢獻之循環腫瘤細胞(Circulating Tumor Cells,CTCs)。
例如,以純物理法(Physical method)來說,常見為利用循環腫瘤細胞(Circulating Tumor Cells,CTCs)粒徑大於血球細胞的特點,以特定篩孔大小之篩網分離兩種細胞。但已知循環腫瘤細胞(Circulating Tumor Cells,CTCs)之粒徑不一,依此方法會有漏失與白血球粒徑相仿的循環腫瘤細胞(Circulating Tumor Cells,CTCs)之風險。
此外,循環腫瘤細胞(Circulating Tumor Cells,CTCs)之半衰期較短,進入血液循環後數小時內即可能死亡;儘有0.01%循環腫瘤細胞(Circulating Tumor Cells,CTCs)可成功轉移,亦即如果將這特性放至計數篩選的循環腫瘤細胞(Circulating Tumor Cells,CTCs),並不是全數的循環腫瘤細胞(Circulating Tumor Cells,CTCs)都具有細胞活性,而對腫瘤惡性轉移(Metastasis)有貢獻。
對此,現有的技術極需一套有效的篩除方式以及辨識方式,針對循環腫瘤細胞(Circulating Tumor Cells,CTCs)所給予的資訊進行進一步的應用。
為了解決先前技術中所提到的問題,本發明提供了一種循環腫瘤細胞資訊評估方法及其分析方法。
具體而言,本發明之該循環腫瘤細胞資訊評估方法主要包含步驟(a)~(e)。其中,步驟(a)會先提供一生物樣本,該生物樣本包含複數個非目標細胞及複數個目標細胞。接著步驟(b)篩除該複數個非目標細胞,使該複數個目標細胞存活。
而步驟(c)接著對該生物樣本進行一免疫螢光染色,辨別出該複數個目標細胞中的複數個循環腫瘤細胞。進一步,步驟(d)會進一步辨識該複數個循環腫瘤細胞為複數個移轉潛力細胞及複數個移轉表徵細胞個別之一生物參數。最後,步驟(e)將會依照該生物參數對該複數個移轉潛力細胞及該複數個移轉表徵細胞進行至少一第一生物檢測。以本發明來說,該生物樣本可以是周邊全血(Peripheral blood)。
而針對上述提及的循環腫瘤細胞資訊評估方法,本發明更提供了了一種循環腫瘤細胞資訊的分析方法。首先,步驟(A)會提供前述步驟(e)的該第一生物檢測之至少一結果參數。接著,步驟(B)則將該至少一結果參數用以進行腫瘤型態分析、腫瘤監控、預後評估、用藥指導、用藥成效分析或其組合。
據此,本發明得以解決循環腫瘤細胞異質性如細胞型態、生理特性、分子特性導致其數量評估時容易產生誤差的困境。更可避免篩選偏差(Selection bias)的問題導致循環腫瘤細胞數目低估的情形。此外,本發明更能
解決負向篩選固有之循環腫瘤細胞純度不足的問題,使後續基礎研究用途或臨床研究用途更加精準。
以上對本發明的簡述,目的在於對本發明之數種面向和技術特徵作一基本說明。發明簡述並非對本發明的詳細表述,因此其目的不在特別列舉本發明的關鍵性或重要元件,也不是用來界定本發明的範圍,僅為以簡明的方式呈現本發明的數種概念而已。
(a)~(e):步驟
(a1)~(a3):步驟
(b1)~(b2):步驟
(b2’):步驟
(c1)~(c3):步驟
(d1)~(d3):步驟
(A)~(B):步驟
圖1係本發明實施例循環腫瘤細胞資訊評估方法的流程圖。
圖2係本發明實施例步驟(a)後續可選的處理流程圖。
圖3係本發明實施例步驟(b)的詳細流程圖。
圖4係本發明實施例步驟(c)的詳細流程圖。
圖5係本發明實施例步驟(d)的詳細流程圖。
圖6係本發明實施例循環腫瘤細胞資訊用途的流程圖。
為能瞭解本發明的技術特徵及實用功效,並可依照說明書的內容來實施,茲進一步以如圖式所示的較佳實施例,詳細說明如後:請參照圖1,圖1係本發明實施例循環腫瘤細胞資訊評估方法的流程圖。如圖1所示,本實施例主要包含步驟(a)~(e)。其中,步驟(a)會先提供一生物樣本,該生物樣本包含複數個非目標細胞及複數個目標細胞。接著步驟(b)係以篩除該複數個非目標細胞,使該複數個目標細胞存活。
而步驟(c)接著對該生物樣本進行一免疫螢光染色,辨別該複數個目標細胞中的複數個循環腫瘤細胞。進一步,步驟(d)會進一步辨識該複數個循環腫瘤細胞為複數個移轉潛力細胞及複數個移轉表徵細胞個別之一生物參數。最後,步驟(e)將會依照該生物參數對該複數個移轉潛力細胞及該複數個移轉表徵細胞進行至少一第一生物檢測。以本發明來說,該生物樣本可以是周邊全血(Peripheral blood)。
在本實施例之步驟(a)中,該生物樣本係周邊全血(Peripheral blood)。在可能實施的前提下,該周邊全血(Peripheral blood)係取樣自滿足下列條件之人類(Homo sapiens):
(1)非癌症患者、被確診為新發或復發癌症且處於治療前、中或後的病患。
(2)所述癌症可以是肝癌、肺癌、大腸直腸癌、乳癌、鼻咽癌、攝護腺癌、食道癌、胰臟癌或頭頸部癌。
(3)所述癌症係依照第八版的美國癌症聯合委員會(AJCC)的基準判定其病症階段為I到IV之局部癌或轉移癌。
在本實施例中,該周邊全血(Peripheral blood)之取樣時之前3-5毫升(ml)之血液必須拋棄,避免上皮細胞的汙染。之後,取樣作為生物樣本的周邊全血(Peripheral blood)收集於含有抗凝血劑(如三鉀乙二胺四乙酸(Tripotassium EDTA))的真空採血管(Vacutainer tube)中,並儲存於溫度攝氏4度下,並於6小時內完成血液前處理(步驟(a)並進入培養步驟(步驟(b)))。
本實施例之步驟(a)後更可包含詳細的步驟(a1)-(a3)。請參照圖2,圖2係本發明實施例步驟(a)圖2係本發明實施例步驟(a)後續可選的處理流程圖。步驟(a1)-(a3)於本實施例可用以取代步驟(b)、與步驟(b)任意組合,抑或完成後再
執行步驟(b),據以增加篩除複數個非目標細胞的效率。在本實施例中,所述複數個非目標細胞係指血球細胞(包含紅血球或白血球等)或不具細胞活性的循環腫瘤細胞(Circulating Tumor Cells,CTCs);而所述複數個目標細胞則為具細胞活性的循環腫瘤細胞(Circulating Tumor Cells,CTCs)。
首先,步驟(a1)係將作為該生物樣本之周邊全血(Peripheral blood)先以紅血球裂解液將其中之該複數個非目標細胞裂解後,離心去除該生物樣本的上清液。
本實施例中,該複數個非目標細胞可以包含紅血球(Erythrocyte)。每公升之該紅血球裂解液包含8.26公克的氯化銨(NH4Cl)、1.19公克的碳酸氫鈉(NaHCO3)、200微升(μl),濃度0.5M且酸鹼度(pH值)8的乙二胺四乙酸(Ethylenediaminetetraacetic acid)。該紅血球裂解液之最終酸鹼度(pH值)為7.3。
在步驟(a1)實施的過程中,本實施例係先將全血之該周邊全血(Peripheral blood)與該紅血球裂解液以1:10的比例混合作用,作用時間不超過10分鐘,並離心去除該生物樣本的上清液。完成步驟(a1)後,以磷酸鹽緩衝生理鹽水(Phosphate buffered saline,PBS)打散該細胞,再執行步驟(a2),以低轉速去除血小板(Platelet)。
接著執行步驟(a3),去除該生物樣本中的部份白血球(Leukocytes)。本發明並不限制去除部份白血球(Leukocytes)的方式,可利用一般的商業組件(Kit)進行去除,亦可以將處理完之該生物樣本以一白血球辨識抗體辨識該生物樣本中的白血球(Leukocytes),再以可辨識該白血球辨識抗體之一磁珠複合體結合該白血球(Leukocytes)與該白血球辨識抗體,並分離
出其餘未被該磁珠複合體結合的該複數個目標細胞。本實施例中之該白血球辨識抗體為表面抗原分化簇45受體(CD45)抗體。
基於步驟(a3)的要旨在於去除該生物樣本中的部份白血球(Leukocytes)。更進一步來說,本實施例之步驟(a3)係以免疫磁珠負向篩選法(Immunomagnetic bead-based negative selection)去除大多數的白血球(Leukocytes)。
因此,本實施例之步驟(a3)其詳細作法係將步驟(a2)處理完之該生物樣本以表面抗原分化簇45受體(CD45)抗體辨識該生物樣本中的白血球(Leukocytes),再以可辨識表面抗原分化簇45受體(CD45)抗體之磁珠複合體結合抗體與白血球(Leukocytes),最後於磁場中分離出其餘未被磁珠複合體結合的複數個目標細胞,進而達到將大多數的白血球(Leukocytes)去除的功效。基於此前提,步驟(a3)雖無法保證去除所有的白血球(Leukocytes),但至少可依照操作需求決定要去除多少種類或數量的白血球(Leukocytes),本發明並不加以限制。
無論(a1)-(a3)的步驟流程如何與步驟(b)作結合,甚至直接執行步驟(b),應皆屬於本發明的範圍中。本發明之步驟(b)係篩除該複數個非目標細胞,使該複數個目標細胞存活。請參照圖3,圖3係本發明實施例步驟(b)的詳細流程圖。在本實施例的步驟(b)中,詳細的步驟更包含(b1)-(b2)以及額外可選擇的步驟(b2’)。
首先,步驟(b1)係提供一培養槽,使該培養槽形成一三維細胞培養槽。更精確來說,步驟(b1)在部份可實施的樣態中,係以一親水凝膠均勻滴加於一培養槽內,形成足以覆蓋該培養槽之一薄層,使該培養槽形成一三維細胞
培養槽。而本實施例所述之該三維細胞培養槽,最佳可採用三維球狀體細胞培養(Three-dimensional spheroid cell culture)技術。
步驟(b2)將該複數個目標細胞以一時間培養於該三維細胞培養槽內。本實施例中,該培養的時間為八天。且該三維細胞培養槽中的培養液包含表皮生長因數(Epidermal growth factor,EGF)、成纖維細胞生長因數(Fibroblast Growth Factor,FGF)以及營養補充劑等組合。
在該複數個目標細胞培養完成八天後,亦可額外進行步驟(b2’),將該複數個目標細胞進行一第二生物檢測。本實施例中,步驟(b2’)之第二生物檢測為基因體資訊分析,所述基因體資訊分析主要為癌症相關基因的資訊分析。更進一步來說,將培養八天後的該複數個目標細胞自培養槽中取出,然後萃取其核酸。所述核酸可以是核糖核酸(RNA)、去氧核醣核酸(DNA)或其組合,本發明並不加以限制。
然本實施例中,該核酸係以總核醣核酸(Total RNA)實施之;進一步來說,本實施例採用皮可普兒核醣核酸分離套組(PicoPureTM RNA Isolation Kit)進行萃取。
接著將該核酸作為樣本分析,獲得其生物資訊。以本發明論之,所述生物資訊係藉由分析該核酸中的至少一目標核酸區域而獲得。而由於本實施例之核酸採用總核醣核酸(Total RNA)且需要分析特定與癌症相關的至少一目標分析基因,因此係先進行互補去氧核醣核酸(cDNA)合成作業,接著使用即時聚合酶連鎖反應系統(Real-time polymerase chain reaction system)進行癌症相關的至少一目標分析基因之基因體資訊分析。在本實施例中,相關的目標分析基因可包含:ALDH1、CDH1、CDH2、JUP、KRT19、MRP1、MRP2、MRP4、
MRP5、MRP7、NANOG、OCT3/4、PROM1、SNAI1、SOX2、TWIST1、VIM或其組合,本發明並不加以限制。
在本實施例中,可將如B2M基因等管家基因(Housekeeping gene)作為內部控制組(Internal control)以計算目標分析基因之相對表現等級(Relative expression level)。一旦該目標分析基因的相對表現等級大於或等於所有分析之生物檢體該目標分析基因的中值(Median value)時,即將之列入高表現群組(High expression group);反之,該目標分析基因的相對表現等級小於或等於所有分析之生物檢體該目標分析基因的中值(Median value)時,即將之列入低表現群組(Low expression group)。
當然,上述的實施例雖採用中值(Median value)作為相對表現等級的判定;然實際上,依據各種不同生物資訊之檢測條件和所需不同,本發明更可衍生出更多的實施例,採用平均值或接收者操作特徵曲線(Receiver Operating Characteristic curve,ROC curve)等,計算出最適之截止值(Cut-off value)。
接著執行步驟(c),對該生物樣本進行一免疫螢光染色,辨別該複數個目標細胞中的複數個循環腫瘤細胞。更精確來說,本實施例之複數個目標細胞可能包含了許多未篩除完全的複數個非目標細胞或是已經不具活性的循環腫瘤細胞。為了達到辨識何者為本實施例所需具有活性的循環腫瘤細胞,因此透過步驟(c)來實現。
請參照圖4,圖4係本發明實施例步驟(c)的詳細流程圖。本實施例之步驟(c)以圖4中詳細的步驟(c1)-(c3)執行。
首先,執行步驟(c1),將含有該複數個目標細胞之全數或均分為複數份後製成至少一細胞塗片。其中,該至少一細胞塗片的數量可依照後續欲執行的生物分析種類決定。例如,本實施例欲鑑別移轉潛力細胞及移轉表徵細胞兩種細胞,則應製作為兩片細胞塗片,用以分別進行免疫螢光染色,本發明並不加以限制。
接著執行步驟(c2),將該至少一細胞塗片固定後,如欲染色該至少一細胞中的非細胞膜表面抗原,則以一表面活性劑對該至少一細胞塗片中的該複數個目標細胞進行穿孔。本實施例中該至少一細胞塗片的固定方式係以福馬林(Formalin)為之,而該表面活性劑則選用曲拉通X-100(C14H22O(C2H4O)n,Triton X-100)。換言之,當染色的標的屬於非細胞膜表面抗原時,自然需要透過特別的手段使細胞穿孔;然本步驟可依據使用者操作或分析標的的不同改變,本發明並不加以限制。
待該複數個目標細胞穿孔完成後,執行步驟(c3),對該至少一細胞塗片進行該免疫螢光染色。本發明並不限制免疫螢光染色所選用的一級抗體或二級抗體,僅讓操作者可依照染色的標的進行改變。
如以本實施例而言,步驟(c3)進行免疫螢光染色後,可依據其結果進一步辨識出具有活性的複數個循環腫瘤細胞以及其他殘存未被篩除完全的該複數個非目標細胞。針對這些未被篩除完全的該複數個非目標細胞,甚至是已經不具活性的複數個循環腫瘤細胞,辨識其之目標細胞標誌可包含辨別白血球的表面抗原,如表面抗原分化簇4受體(CD4)、表面抗原分化簇8受體(CD8)、表面抗原分化簇14受體(CD14)、表面抗原分化簇11b受體(CD11b)、表面抗原分化簇34受體(CD34)、表面抗原分化簇45受體(CD45)、表面抗原分化簇
68受體(CD68)或其組合。而針對未篩除完全之該紅血球的部份,其細胞標誌可包含表面抗原分化簇235a受體(CD235a)進行辨識。上述技術的主要目的在於辨識先前步驟(a)-(b)步驟中未被完全篩除之白血球(Leukocytes)、紅血球或是已經不具活性的其他細胞,避免被誤判為欲辨識的循環腫瘤細胞(Circulating Tumor Cells,CTCs)。
而本實施例中,針對該複數個循環腫瘤細胞進行免疫螢光染色之一級抗體,其目標細胞標誌為上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)之細胞標誌以及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)之細胞標誌。
本實施例以上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)以及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)之細胞標誌作為免疫螢光染色的標的係在於兩者細胞間的上皮細胞間質轉化(Epithelial-to-Mesenchymal Transition,EMT)現象為腫瘤惡性轉移(Metastasis)的重要程序,可做為腫瘤惡性轉移(Metastasis)後續檢測分析或監控評估之用。
換言之,以本實施例分析上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)以及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)之數量或其基因資訊,即可獲得更多與腫瘤有關的資訊,進一步進行各種評估和用途。
綜上,本實施例選用上皮細胞黏附因子(epithelial cell adhesion molecule,EpCAM)、泛角蛋白(CKs)、E-鈣粘蛋白(E-cadherin)、封閉蛋白(Claudin)、外週膜蛋白-1(Zonula Occludens protein-1,ZO-1)、橋粒斑蛋白
(desmoplakin)、黏蛋白Muc-1(Mucoprotein Muc-1)、乙型卡特林因子(beta-catenin)、跨膜蛋白-1(syndecan-1)或其組合之細胞標誌,作為上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)之免疫螢光染色抗體對抗標的。
至於間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)細胞標誌的部份,本實施例選用鋅指蛋白SNAI1(Zinc finger protein SNAI1,Snail)、鋅指蛋白SNAI2(Zinc finger protein SNAI2,Slug)、基質金屬蛋白酶(MMPs)、波形蛋白(vimentin)、纖連蛋白(fibronectin)、甲型平滑肌肌動蛋白(alpha-SMA)、凝血栓蛋白(thrombospondin)、第一型胞漿素原活化抑制酶(plasminogen activator inhibitor-1,PAI-1)、乙型轉化生長因數(TGF-β)或其組合作為間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)的免疫螢光染色抗體對抗標的。
完成步驟(c1)-(c3)後,執行步驟(d),進一步辨識該複數個循環腫瘤細胞為複數個移轉潛力細胞及複數個移轉表徵細胞個別之一生物參數。而在本實施例中,生物參數為細胞數目。
請參照圖5,圖5係本發明實施例步驟(d)的詳細流程圖。在本實施例中,步驟(d)的詳細步驟為圖5中之步驟(d1)-(d3),且該複數個移轉潛力細胞即為前述之上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)。而該複數個移轉表徵細胞為前述之間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)。
首先,步驟(d1),將該複數個移轉潛力細胞之細胞標誌呈現陽性且該複數個白血球細胞之細胞標誌呈現陰性之該複數個目標細胞歸類為該複數
個移轉潛力細胞。接著執行步驟(d2),將該複數個移轉表徵細胞之細胞標誌呈現陽性且該複數個白血球細胞之細胞標誌呈現陰性之該複數個目標細胞歸類為該複數個移轉表徵細胞。接著依據上述資訊,執行步驟(d3),計算該複數個移轉潛力細胞及該複數個移轉表徵細胞的數目。
最後,執行步驟(e),對該複數個移轉潛力細胞及該複數個移轉表徵細胞進行至少一第一生物檢測。在本實施例中,該至少一生物檢測更可以加入一個體臨床參數進行評估。該個體臨床參數包含年齡、腫瘤部位、癌期、治療方式、首次治療評估結果、存活狀態或其組合,發明並不加以限制。
而本實施例步驟(e)所指之該至少一第一生物檢測可以是透過螢光顯微鏡觀測系統進行上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)兩者族群數目之計量。
抑或計量完畢後再透過生物統計學(Biostatistics)上針對上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)兩者族群數目的曼-惠特尼U檢驗(Mann-Whitney U test),或以Cox迴歸分析(Cox Regression)進行存活分析等。P值(P-Value)則以0.05作為統計學上的顯著差異。
更進一步來說,本實施例可以依據上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)個別甚至總體數量,進行不同臨床條件的統計學測試變化,本發明並不加以限制。
最後,請參照圖6,圖6係本發明實施例循環腫瘤細胞資訊用途的流程圖。圖6的實施例中為循環腫瘤細胞資訊的用途。首先,步驟(A)係先提供步驟(e)的該第一生物檢測之至少一結果參數,接著再以步驟(B)將該至少一結果參數用以進行腫瘤型態分析、腫瘤監控、預後評估、用藥指導、用藥成效分析或其組合。
以本實施例而言,由於生物檢測之至少一結果參數係由上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs)及間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)的分離純度和精準的數據進行統計和計算分析。因此,將本實施例生物檢測最後所產出之至少一結果參數作為腫瘤惡性轉移(Metastasis)個體的後續包含腫瘤型態分析、腫瘤監控、預後評估、用藥指導、用藥成效分析等在內的準確度十分優異。
惟以上所述者,僅為本發明之較佳實施例而已,當不能以此限定本發明實施之範圍,即依本發明申請專利範圍及說明內容所作之簡單變化與修飾,皆仍屬本發明涵蓋之範圍內。
(a)~(e):步驟
Claims (11)
- 一種循環腫瘤細胞資訊評估方法,包含:(a)提供一生物樣本,該生物樣本包含複數個非目標細胞及複數個目標細胞;(b)篩除該複數個非目標細胞,並利用一三維細胞培養技術使該複數個目標細胞存活,其中更包含步驟:(b1)以紅血球裂解液、離心及免疫磁珠負向篩選法(Immunomagnetic bead-based negative selection)篩除該複數個非目標細胞;(b2)提供一培養槽,該培養槽包含一薄層,該薄層覆蓋該培養槽並隔開該生物樣本,使該培養槽形成一三維細胞培養槽;以及(b3)將該複數個目標細胞以一時間培養於該三維細胞培養槽內;(c)對該生物樣本進行一表面抗原分化簇45受體(CD45)免疫螢光染色、一移轉潛力免疫螢光染色及一移轉表徵免疫螢光染色,使該複數個目標細胞中的該複數個循環腫瘤細胞透過該表面抗原分化簇45受體(CD45)免疫螢光染色、該移轉潛力免疫螢光染色及該移轉表徵免疫螢光染色呈現複數個白血球細胞、複數個移轉潛力細胞以及複數個移轉表徵細胞之細胞標誌的陰/陽性;(d)進一步辨識該複數個移轉潛力細胞及該複數個移轉表徵細胞個別之一生物參數,其中更包含步驟: (d1)將該複數個移轉潛力細胞之細胞標誌呈現陽性且該複數個白血球細胞之表面抗原分化簇45受體(CD45)呈現陰性的該複數個目標細胞定義為該複數個移轉潛力細胞;(d2)將該複數個移轉表徵細胞之細胞標誌呈現陽性且該複數個白血球細胞之表面抗原分化簇45受體(CD45)呈現陰性的該複數個目標細胞定義為該複數個移轉表徵細胞;(d3)分析該複數個移轉潛力細胞及該複數個移轉表徵細胞個別之該生物參數;以及(e)依照該生物參數對該複數個移轉潛力細胞及該複數個移轉表徵細胞進行至少一第一生物檢測;其中,該生物樣本為周邊全血(Peripheral blood),且取樣後的前3-5毫升(ml)之該周邊全血(Peripheral blood)必須拋棄;該複數個移轉潛力細胞之細胞標誌包含上皮細胞黏附因子(epithelial cell adhesion molecule,EpCAM)、泛角蛋白(CKs)或其組合;該至少一第一生物檢測更加入一個體臨床參數進行評估。
- 如請求項1所述之循環腫瘤細胞資訊評估方法,步驟(d)中,該複數個移轉潛力細胞為上皮型循環腫瘤細胞(Epithelial circulating Tumor Cells,E-CTCs);該複數個移轉表徵細胞為間質型循環腫瘤細胞(Mesenchymal circulating Tumor Cells,M-CTCs)。
- 如請求項1所述之循環腫瘤細胞資訊評估方法,步驟(d)中,辨識該複數個移轉表徵細胞之細胞標誌包含鋅指蛋白SNAI1(Zinc finger protein SNAI1,Snail)、鋅指蛋白SNAI2(Zinc finger protein SNAI2,Slug)、基質金屬蛋白酶(MMPs)、波形蛋白(vimentin)、纖連蛋白(fibronectin)、甲型平滑肌肌動蛋白(alpha-SMA)、凝血栓蛋白(thrombospondin)、第一型胞漿素原活化抑制酶(plasminogen activator inhibitor-1,PAI-1)、乙型轉化生長因數(TGF-β)或其組合。
- 如請求項1所述之循環腫瘤細胞資訊評估方法,在步驟(b2)後,更進行:(b2’)將該複數個目標細胞進行一第二生物檢測。
- 如請求項4所述之循環腫瘤細胞資訊評估方法,其中該第二生物檢測為一基因體資訊分析,該基因體資訊分析包含:培養該複數個目標細胞;萃取該複數個目標細胞的核酸;以及將該核酸作為樣本分析,得到一生物資訊。
- 如請求項5所述之循環腫瘤細胞資訊評估方法,其中核酸包含核糖核酸(RNA)、去氧核醣核酸(DNA)或其組合。
- 如請求項5所述之循環腫瘤細胞資訊評估方法,其中該生物資訊係藉由分析該核酸中的至少一目標核酸區域而獲得。
- 如請求項1所述之循環腫瘤細胞資訊評估方法,步驟(d)中之該生物參數為細胞數目。
- 如請求項1所述之循環腫瘤細胞資訊評估方法,其中步驟(e)之該至少一第一生物檢測為細胞計量分析。
- 如請求項9所述之循環腫瘤細胞資訊評估方法,其中步驟(e)之該至少一生物檢測更加入一個體臨床參數進行評估。
- 一種循環腫瘤細胞資訊的分析方法,包含:(A)提供如請求項1-9任一項所述之該步驟(e)的該第一生物檢測之至少一結果參數;以及(B)將該至少一結果參數用以進行腫瘤型態分析、腫瘤監控、預後評估、用藥指導、用藥成效分析或其組合。
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