201122479 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種捕捉體液樣本或 + a 3血清樣本中之腫瘤 細胞的套組及方法。特定言之，接 本發明之套組及方法 來捕捉循環性腫瘤細胞。 【先前技術】 癌症為累積性的多基因突變之結果，該等突變使致癌基 因活化及/錢射卩驢因失活。癌症仍為全球死亡率= 主要原因。儘管在診斷及治療方面已有進步，但在過去多 年的總存活率並未㈣顯著改善。仍有需要實現更㈣之 腫瘤診斷方式。 大多數癌症死亡並非由於原發性腫瘤所致。實際上，死 亡由轉移（亦即多個分布廣泛之腫瘤群^，由自身自原始 腫瘤部位脫離且常常行經身體到達遠端部位的惡性細胞： 成）而引<。若能儘早谓測到原發性腫瘤，則手術、韓射 、化學療法或彼等治療之某種組合常可將其消除。令人遺 撼的是，轉移群料交難偵測及消除，無法成功地治 療所有轉移群《。因&，就臨床觀點^，轉 然癌症進程中之決定性因素。此外，轉移能力為一可 ^ =徵化惡、性腫瘤之性質。癌轉移包含以下—系列複雜的連 續事件：1.自原發地點擴展至周圍組織；2.穿透至體腔及 血管中；3.釋方文腫瘤細胞以經由循帛系統輸送至遠端部位 ;4•在停滯部位處再侵入組織；纟，5適應新環境以促進 腫瘤細胞存活、血管形成及腫瘤生長。基於癌症及癌轉移 145593.doc 201122479 之複雜性以及多年來在治療癌症患者方面之挫折，已有 眾多嘗試來開發針對轉移及早期復發之發展情況的偵測 測試。 循環性腫瘤細胞（CTC)為自原發性腫瘤或其在周邊血液 中循環之轉移灶脫出之癌細胞。雖然轉移為造成大多數癌 症死亡之直接原因，但CTC可構成轉移之起因且可指出疾 病之擴散程度。當CTC極其稀少（每毫升至多數個CTC)時 ，鑑別CTC可指示癌症，或甚至在出現明顯臨床症狀之前 指示癌前期生長。在一個多世紀前已對偵測周邊血液中之 LTC首次表現出潛在關注，但接著便有所減退，因為樣本 中之CTC數目極少，所以由習知方法難以偵測到。對於鑑 別及特徵化在血液中循環之稀少腫瘤細胞而言，其挑戰在 於開發一種結合高敏感性與高特異性之方法，從而能夠將 腫瘤細胞與正常上皮細胞及白血球加以區分。循環性腫瘤 細胞之偵測可有助於癌症預後、微小殘留疾病之診斷、腫 瘤對抗癌藥物之敏感性評估及抗癌療法之個人化 (personalization)。CTC之高度敏感性與特異性鑑別亦可應 用於侵襲性癌症之早期診斷及篩選。 基於DNA或RNA之腫瘤特異性異常之PCR擴增的分子技 術已有助於隱蔽（隱藏）性腫瘤細胞之偵測。已發展出能夠 在一百萬個正常細胞中以常規方式偵測—個腫瘤細胞的基 於PCR之測試，來鑑別各種類型癌瘤中之循環性腫瘤細胞 。舉例而言’ Smith B.等人開發出逆轉錄酶_聚合酶鏈反應 來偵測周邊血液中之黑素瘤細胞（Lancet，338. 1236 14li593.doc 201122479 測）。然而，料方法可^法有效地自 活腫瘤細胞。因為細胞 ，我擴〜 ^ Μ - ^/r Μ- 萃取期間遭到 破壞，所以此方法jj且磁·5* m 忐阻礙了對細胞形態及表型之分析，詳言 之，因而可能既無法區別自正 吊、、且織直接脫出之物質盘自 腫瘤脫出之物質，亦盔法f+ …' 丌…、法對相同細胞中之若干相關變化進 行偵測。 免疫螢光顯微術能夠 刃刀析細胞形態且能夠對可鑑別之推 定腫瘤細胞直接進彳千# m α 计數使用適當抗體對細胞作免疫標 記來進㈣測。然而，因為迄今尚不存在針對腫瘤之抗體 ，所以已使㈣隸抗原來㈣CTC。此外，料偵測結 腸直腸癌患者體内之CTC團鎮，亦已開發並評估使用免疫 細胞化學標記之免疫磁性細胞分離(Clinical Cancer earch，2001，第 7卷，第 4〇8〇_4〇85 頁）。W0 2006050352 提供-種改良之細胞黏附基質（「CAMj )及一種改良之分 離乾細胞（諸如來自血液或其他组織液樣本之腫瘤細胞、 月。兒、，田胞及血管生成細胞）的細胞分離裝置。此外，w〇 2009051734揭示一種捕捉循環、非造血性腫瘤細胞之裝置 ，其包括結合腫瘤特異性結合劑之微流通道及在該通道中 產生0.1至20 dyn/cm2之連續、單向剪應力的泵。然而，上 述技術均無法有效地偵測CTC。 顯而易見的是，尤其在癌症早期，極其需要一種在繼發 性腫瘤形成之前鑑別在循環中具有轉移潛力之彼等細胞的 方法及/或套組。 【發明内容】 145593.doc 201122479 本發明之一目的在於提供一種捕捉體液樣本或含血清樣 本中之腫瘤細胞的方法，其包含以下步驟： (a) 使類内皮（endothelium-like)細胞或類上皮（epithelium-like)細胞附著於固體支撐物上； (b) 用一或多種發炎誘導劑誘導（a)之細胞的發炎反應； (c) 使白血球附著於（a)之細胞上；及 (d) 將體液樣本或含血清樣本添加至固體支撐物，藉此 ’白也球及類内皮細胞或類上皮細胞捕捉體液樣本或含血 清樣本中所含之腫瘤細胞。 本發明之另一目的在於提供一種捕捉體液樣本或含血清 樣本中之腫瘤細胞的套組’其包含固體支撐物；（b)附 著於該固體支撐物上之類内皮細胞或類上皮細胞；（c)附著 於（b)之細胞上的白血球；及（d) 一或多種發炎誘導劑。 【實施方式】 本發明係關於一種捕捉體液樣本或含血清樣本中之腫瘤 細胞的方法及套組。藉由利用白血球可捕捉樣本中活的含 核異源細胞之概念，可達成對體液樣本或含血清樣本中之 腫瘤細胞的偵測及確認。本發明之套組及方法可捕捉活腫 瘤細胞，而無法捕捉非活的腫瘤細胞或細胞片段，從而可 藉由進一步培養所捕捉之腫瘤細胞來進一步鑑別該腫瘤之 種類。測試樣本中之未知腫瘤細胞難以鑑別，因為其稀少 且具有各種尺寸及抗原決定基。本發明之套組及方法可輕 易地鑑別樣本是否含有腫瘤細胞且收集此等腫瘤細胞以供 進一步鑑別，從而可發現癌症之存在以及轉移與早期復發 145593.doc 201122479 之發展情況。 在一態樣中’本發明提供一種捕捉體液樣本或含血清樣 本中之腫瘤細胞的方法，其包含以下步驟： (a) 使類内皮細胞或類上皮細胞附著於固體支撐物上； (b) 用一或多種發炎誘導劑誘導（a)之細胞的發炎反應； (c) 使白血球附著於（a)之細胞上；及 (d) 將體液樣本添加至固體支撐物，藉此，白血球及類 内皮細胞或類上皮細胞捕捉體液樣本或含血清樣本中所含 之腫瘤細胞。 在一貫施例中，該方法可進一步包含在使用該方法之前 ，預處理體液樣本或含血清樣本以使該樣本處於生理條件 下的步驟。在另-實施例中，該方法可進一步包含在使用 該方法之前，將生物樣本預處理成體液樣本或含血清樣本 之步驟。在另-實施例中，該方法亦可進一步包含在步驟 (d)之後收集所捕捉之腫瘤細胞的步驟。 在另一態樣中，本發明提供一種捕捉體液樣本或含企清 樣本中之腫瘤細胞的套組，其包含（a)固體支撐物，附 著於该固體支撐物上之類内皮細胞或類上皮細胞，⑷附著 . 於（b)之細胞上的白血球，及（d)—或多種發炎誘導劑。 ' 根據本發明’「固體支撐物」或「固體載體」意謂任何 固相材料，上面可附著、接合或固定本發明捕捉方法中所 用之細胞。固體支撐物涵蓋諸如「膜」、「樹脂」、「固相_ 、「表面」及「支稽物」之術語。在一實施例中固體支 樓物為親水性的。特定言之，可將固體支樓物之表面修飾 145593.doc 201122479 成親水性表面。修飾方法可為(但不限於)氧電漿處理、水 電漿處理及化學處理。此支撑物可由以下有機聚合物構成 :諸如琐化纖維素膜、耐綸膜（nyl〇n membr_)、聚苯乙 稀、聚乙烯、聚丙烯、聚氟乙烯、聚氧乙烯及聚丙烯醯胺 ’以及其共聚物及接枝聚合物。固體支擇物亦可為無機物 ’諸如玻璃、二氧化碎、逆相二氧化梦或生物晶片。固體 支樓物之組態可呈珠粒、纯!#、柄ws . 琛體粒子、顆粒、凝膠或表面 之形式。表面可為平面、實質上平面或非平面的。固體支 撐物之組態可呈孔（weU)、凹陷（depressi〇n)或其他貯器 (container)、容器（vessel)或位置之形式。固體支撐物較佳 為皮氏培養皿（petri dish)或盤，其較佳具有複數個可進行 檢定之孔。《較佳為纽微量滴定盤。在另—實施例中 ，支撑物表面可具有電#。該（該等）電極可债測所捕捉之 腫瘤細胞的存在及其量。使用電極價測細胞在此項技術中 為已知的。美國專利第6,716,633號提供一種血細胞偵測器 ’其包括具有單-孔口之孔口區'將第一也液試樣供應至 孔口區t之第一供應區、將第二血液試樣供應至孔口區中 之第二供應區，以及在孔口之相對側上提供之第一電極及 第一電極，當使第一血液試樣及第二血液試樣選擇性地通 過孔口時，第一電極及第二電極用於偵測第一血液試樣及 第二血液試樣中之每一者的阻抗變化，該專利以引用的方 式併入本文中。在一實施例中，電極可由氧化銦錫（IT0) 、碳奈米官、矽或氧化鈦構成。藉由改變電流頻率，電極 可產生介電泳力（DEP力）。DEP力可提供能夠捕捉細胞或 14：)593.doc 201122479 將細胞移走之力。在具有電極之區域上捕捉到值定細胞且 該區域經證實為定量計數區域。 根據本發明，「類内皮細胞或類上皮細胞」係指内皮細 胞或上皮細胞及其類似細胞。類内皮細胞或類上皮細胞較 佳為血管細胞、淋巴管細胞、口腔上皮細胞或人類臍靜脈 内皮細胞。 根據本發明’「發炎誘導劑」係指可誘導發炎反應之試 劑。發炎誘導劑較佳為細胞因子、生長因子、表面蛋白或 介白素。發炎誘導劑較佳為腫瘤壞死因子或介白素。發炎 誘導劑更佳為腫瘤壞死因子-α、介白素_1(ΙΙΜ)、IL_2、 IL-6、IL-8、IL-Ιβ、干擾素-γ(ΐΡΝ-γ)、干擾素_a(IFN_a)或 TNF-a。 根據本發明’「白血球」係指保護身體免受傳染病與外 來物質侵害之免疫系統細胞。在一實施例中，白血球包括 嗜中性球（neutrophil)、嗜酸性球（eosin〇phil)、嗜鹼性球 (basophil)、淋巴細胞、單核細胞及巨噬細胞。白血球較佳 為嗜中性球及巨嗔細胞。在發炎期間，白血球向發炎部位 遷移且其附著外來物質以殺死此等物質。根據本發明，白 血球之特徵為附著活的含核異源細胞。根據本發明誘導發 炎反應之後，白血球移動至類内皮細胞或類上皮細胞且附 著於其上。在本發明之一實施例中，本發明方法中所用之 白血球可原始存在於體液樣本中。在本發明之另一實施例 中，可額外添加白血球且將其應用於本發明。 根據本發明，「體液樣本」或「含jk清樣本」可來源於 145593.doc 201122479 任何生物來源，諸如生理流體，包括全血、腹水、唾液、 叔液、滑液、腹膜液、羊膜液、腦脊髓液、漿液、脊髓液 及可能含有腫瘤細胞之其他身體組份。若樣本含有腫瘤細 胞，則在執行本發明之套組及方法時，白血球可捕捉腫瘤 細胞，因為該等腫瘤細胞為含核異源細胞且可由白血球鑑 別及識別。根據本發明之一實施例，因為本發明之一目的 在於捕捉活腫瘤細胞且在本發明之方法及套組中使用活細 胞，所以在供本發明之方法及套組使用之前，可預處理體 液樣本或含血清樣本以使該樣本處於生理條件下。根據本 發明之另一實施例’該方法可進一步包含將生物樣本預處 理成含血清樣本之步驟。根據本發明，生物樣本為任何種 類之身體樣本；生物樣本較佳為骨髓抽取物、骨髓均質物 、淋巴組織均質物或組織均質物。生物樣本較佳為含有組 織之溶液。較佳將樣本預稀釋10倍以上。在一實施例中， 本發明中所用之樣本體積在約5 mL至約3〇 mL之範圍内。 樣本體積較佳為約5 mL至約25 mL、約5 mL至約20 mL·、 約5 mL至約15 mL、約5 mL至約1〇 mL、約1〇 mL至約25 mL、約15 mL至約25 mL、約1〇 mL至約20 mL或15 mL至 20 mL 〇 根據本發明，體液樣本或含血清樣本中之腫瘤細胞為自 腫瘤組織剝奪或分泌且接著進入體液樣本中之腫瘤細胞。 腫瘤細胞較佳為循環腫瘤細胞。根據本發明，在捕捉腫瘤 細胞之後，可由此項技術中已知之方法進一步收集該等腫 瘤細胞。舉例而言，用完全DMEM十之廣效性抗生素及抗 145593.doc •10· 201122479 真菌溶液充分洗蘇腫瘤細胞或其片段（洗條6-7次）以達到與 標準細胞培養條件相容之無®程度。接著，將細胞或月段 切割成1至2 _3小片。將此等小片接種於預塗有 PolyHEMA且含2mL完全培養基之6孔盤中。將腫瘤片段培 養2天，且接著回收培養基並以單獨培養基或含藥物培養 基置換，歷時24小時。所用藥物為拓撲異構酶-I抑制劑代 謝物SN-38(7-乙基]〇_羥基喜樹鹼（7_灿叫〇· hydr〇xycamptotecin ;其為喜樹鹼（campt〇thecin)之活性代 4t物）10-5 mM/l ,該濃度與將喜樹驗靜脈投與人類後於 腫瘤中所觀測到的濃度類似。在此時段結束時，進行洗脫 以清除藥物，且歷經72小時添加培養基。 本發明之套組為用於實施本發明方法之套組。根據本發 明，該套組包括（a)固體支撐物，（b)附著於該固體支撐物 上之類内皮細胞或類上皮細胞，（c)附著於（b)之細胞上的 白血球，及（d)—或多種發炎誘導劑。將該套組之（a)、（b) 、（c)及（d)之上述材料中之每一者置放於獨立貯器（諸如小 瓶、管及其類似物）中。亦即，各貯器包含—種欲在檢定 中使用之獨立材料。套組可進一步包括一組使用該套組之 說明書以指導捕捉體液樣本中之腫瘤細胞的預定檢定。 在本發明之套組及方法中，可誘導附著於固體支撐物之 類内皮細胞或類上皮細胞以產生發炎反應。藉由添加一或 多種發炎誘導劑來誘導發炎反應。在誘導發炎後’本發明 之套組及方法中所用之白血球將向炎性細胞遷移且附著於 炎性細胞。由於白企球將捕捉活的含核異源細胞，因此在 145593.doc 11 201122479 添加體液樣本後，白血球將捕挺樣品中之腫瘤細胞。然而 ，白血球不會捕捉樣本中之非活性細胞、非活性細胞片段 及其他污染物。藉由使用本發明之套組及方法，可獲得體 液樣本或含血清樣本中之活腫瘤細胞，且對其進行培養後 ’可使用此項技術中已知之方法（諸如即時PCR或流動式細 跑測量術）進一步鑑別腫瘤細胞種類（Barrett DL, Jensen RH, King EB, Dean PN, Mayall BH. Flow cytometry of human gynecologic specimens using log chromomycin A: iluorescence and log 9〇" light scatter. J Histochem Cytochem 27(1): 573-5 78, 1979 ; Darzynkiewicz Z:201122479 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a kit and method for capturing tumor cells in a body fluid sample or a + a serum sample. In particular, the kits and methods of the invention are used to capture circulating tumor cells. [Prior Art] Cancer is the result of cumulative multi-gene mutations that inactivate carcinogenic genes and/or inactivated money. Cancer remains the global mortality rate = the main reason. Despite advances in diagnosis and treatment, overall survival has not improved significantly over the past several years. There is still a need to achieve more (4) tumor diagnosis methods. Most cancer deaths are not due to primary tumors. In fact, death is caused by metastasis (i.e., multiple widely distributed tumor groups, from malignant cells that are detached from the original tumor site and often travel through the body to the distal site:). If a primary tumor is detected as early as possible, surgery, Korean injection, chemotherapy, or some combination of these treatments can often be eliminated. What is embarrassing is that the transfer of group materials is difficult to detect and eliminate, and it is impossible to successfully treat all transfer groups. Because of &, the clinical point of view ^, the decisive factor in the progression of cancer. In addition, the metastatic ability is a property that can be used to catalyze evil and sexual tumors. Cancer metastasis includes the following - a series of complex continuous events: 1. Expanding from the primary site to the surrounding tissue; 2. Penetrating into the body cavity and blood vessels; 3. Release the tumor cells to the distal site via the circulatory system; 4 • Re-invade the tissue at the stagnant site; 纟, 5 adapt to the new environment to promote tumor cell survival, angiogenesis and tumor growth. Based on the complexity of cancer and cancer metastasis and the frustrations in treating cancer patients over the years, there have been numerous attempts to develop detection tests for the development of metastasis and early relapse. Circulating tumor cells (CTCs) are cancer cells that are detached from a primary tumor or a metastases circulating in peripheral blood. Although metastasis is the direct cause of death for most cancers, CTC can constitute the cause of the metastasis and can indicate the extent of the disease. When CTC is extremely rare (per milliliter to most CTCs), identifying CTCs can indicate cancer, or even indicate precancerous growth before significant clinical symptoms appear. The potential for LTC detection in peripheral blood has been shown for the first time more than a century ago, but then it has subsided because the number of CTCs in the sample is extremely small, so it is difficult to detect by conventional methods. The challenge for identifying and characterizing rare tumor cells circulating in the blood is to develop a method that combines high sensitivity with high specificity to distinguish tumor cells from normal epithelial cells and white blood cells. Circulating tumor cell detection can contribute to cancer prognosis, diagnosis of minimal residual disease, sensitivity assessment of tumor anticancer drugs, and personalization of anticancer therapies. The high sensitivity and specificity of CTC can also be applied to the early diagnosis and screening of invasive cancers. Molecular techniques for PCR-based amplification of tumor-specific abnormalities based on DNA or RNA have contributed to the detection of concealed (hidden) tumor cells. PCR-based assays capable of detecting tumor cells in a conventional manner in one million normal cells have been developed to identify circulating tumor cells in various types of cancer. For example, Smith B. et al. developed a reverse transcriptase-polymerase chain reaction to detect melanoma cells in peripheral blood (Lancet, 338. 1236 14li593.doc 201122479). However, the method can effectively activate tumor cells. Because of the cell, I expanded ~ ^ Μ - ^/r Μ - during the extraction, it was destroyed, so this method jj and magnetic · 5 * m 忐 hindered the analysis of cell morphology and phenotype, in detail, it may not be possible The difference between the material that is directly detached from the hang, and the material that is directly detached from the tumor, is also detected by the method of the helmet f+ ...' 丌..., the method detects some related changes in the same cell. Immunofluorescence microscopy can be used to analyze the cell morphology and to directly identify the tumor cells that can be identified by using the appropriate antibody to immunolabel the cells (4). However, since there have been no antibodies against tumors so far, (iv) has been given antigens to (4) CTCs. In addition, the detection of CTC clusters in patients with colorectal cancer has also been developed and evaluated for immunomagnetic cell separation using immunocytochemical markers (Clinical Cancer earch, 2001, Vol. 7, No. 4〇8〇_4〇) 85 pages). W0 2006050352 provides a cell separation device with an improved cell adhesion matrix ("CAMj") and an improved isolated stem cell (such as tumor cells, cells, cells, and angiogenic cells from blood or other tissue fluid samples) Furthermore, WO 2009051734 discloses a device for capturing circulating, non-hematopoietic tumor cells comprising a microfluidic channel bound to a tumor-specific binding agent and a continuous, unidirectional shear stress of 0.1 to 20 dyn/cm 2 in the channel. However, none of the above techniques are effective in detecting CTCs. It is apparent that, especially in the early stages of cancer, there is a great need for a method for identifying cells that have metastatic potential in circulation prior to secondary tumor formation and/or [Invention] 145593.doc 201122479 An object of the present invention is to provide a method for capturing tumor cells in a body fluid sample or a serum-containing sample, comprising the steps of: (a) endothelium-like cells Or epithelium-like cells attached to a solid support; (b) induced with one or more inflammatory inducing agents (a) (c) attaching a white blood cell to the cell of (a); and (d) adding a body fluid sample or a serum-containing sample to the solid support, thereby using 'white ball and endothelial-like cells or epithelial-like cells The cell captures a tumor cell sample or a tumor cell contained in a serum-containing sample. Another object of the present invention is to provide a kit for capturing a tumor fluid sample or a tumor cell in a serum-containing sample, which comprises a solid support; (b) attached to An endothelial cell or epithelial cell such as a solid support; (c) a white blood cell attached to the cell of (b); and (d) one or more inflammatory inducing agents. [Embodiment] The present invention relates to a body fluid capturing Method and kit for tumor cells in a sample or serum sample. By using white blood cells to capture the concept of living nuclear-containing heterologous cells in a sample, detection of tumor cells in a body fluid sample or serum-containing sample can be achieved. It is confirmed that the kit and method of the present invention can capture living tumor cells and cannot capture non-live tumor cells or cell fragments, thereby further culturing the captured tumor cells To further identify the type of tumor. Unknown tumor cells in the test sample are difficult to identify because they are rare and have various sizes and epitopes. The kit and method of the present invention can easily identify whether a sample contains tumor cells and collect such Tumor cells for further identification, such that the presence of cancer and the development of metastasis and early recurrence 145593.doc 201122479 can be found. In one aspect, the invention provides a method of capturing a tumor sample or a tumor cell in a serum-containing sample, It comprises the steps of: (a) attaching endothelial-like cells or epithelial cells to a solid support; (b) inducing inflammatory response of the cells of (a) with one or more inflammatory inducing agents; (c) attaching white blood cells to And (d) adding a body fluid sample to the solid support, whereby the white blood cells and the endothelial-like cells or epithelial cells capture the tumor cells contained in the body fluid sample or the serum-containing sample. In a consistent embodiment, the method can further comprise the step of pretreating the body fluid sample or serum-containing sample to subject the sample to physiological conditions prior to use of the method. In another embodiment, the method can further comprise the step of pretreating the biological sample into a body fluid sample or a serum containing sample prior to using the method. In another embodiment, the method may further comprise the step of collecting the captured tumor cells after step (d). In another aspect, the invention provides a kit for capturing a body fluid sample or a tumor cell in a serum-containing sample, comprising: (a) a solid support attached to an endothelial cell or epithelial such as the solid support Cells, (4) attached to white blood cells on cells of (b), and (d) - or a variety of inflammatory inducers. "Solid support" or "solid support" according to the invention means any solid phase material on which the cells used in the capture method of the invention can be attached, ligated or immobilized. Solid supports cover terms such as "film", "resin", "solid phase", "surface" and "subject". In one embodiment the solid support is hydrophilic. Specifically, the surface of the solid support can be modified to 145593.doc 201122479 into a hydrophilic surface. Modification methods can be, but are not limited to, oxygen plasma treatment, water plasma treatment, and chemical treatment. The support may be composed of the following organic polymers: such as trigonal cellulose film, nylon film (nyl〇n membr_), polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyoxyethylene, and polypropylene decylamine. 'And its copolymers and graft polymers. The solid support may also be an inorganic material such as glass, oxidized granules, reverse phase oxidizing dreams or biochips. The configuration of the solid slab can be in the form of beads, pure!#, shank ws. steroid particles, granules, gel or surface. The surface can be planar, substantially planar or non-planar. The configuration of the solid support can be in the form of a hole (weU), a depression (depressi〇n) or other container, vessel or position. The solid support is preferably a petri dish or tray, which preferably has a plurality of pores that can be assayed. "It is better to use a microtiter plate. In another embodiment, the surface of the support may have electricity #. The (these) electrodes can measure the presence and amount of tumor cells captured. The use of electrode valence cells is known in the art. US Patent No. 6,716,633 provides a blood cell detector that includes an orifice region having a single orifice that supplies a first liquid sample to a first supply zone of an orifice region t, and supplies a second blood sample a second supply region to the aperture region, and a first electrode and a first electrode provided on opposite sides of the aperture, when the first blood sample and the second blood sample are selectively passed through the aperture The first electrode and the second electrode are for detecting a change in impedance of each of the first blood sample and the second blood sample, which is incorporated herein by reference. In one embodiment, the electrode may be comprised of indium tin oxide (IT0), carbon nanotubes, tantalum or titanium oxide. By changing the current frequency, the electrode can generate dielectrophoretic force (DEP force). The DEP force can provide the ability to capture cells or 14:) 593.doc 201122479 The ability to remove cells. A valued cell was captured on the area with the electrode and the area was confirmed to be a quantitative count area. According to the present invention, "endothelial cells or epithelial cells" refer to endothelial cells or epithelial cells and the like. Endothelial cells or epithelial cells are preferably vascular cells, lymphatic cells, oral epithelial cells or human umbilical vein endothelial cells. The "inflammation inducing agent" according to the present invention means a test agent which can induce an inflammatory reaction. The inflammatory inducing agent is preferably a cytokine, a growth factor, a surface protein or a mesogen. The inflammatory inducing agent is preferably tumor necrosis factor or interleukin. The inflammatory inducing agent is preferably tumor necrosis factor-α, interleukin-1 (ΙΙΜ), IL_2, IL-6, IL-8, IL-Ιβ, interferon-γ (ΐΡΝ-γ), interferon_a ( IFN_a) or TNF-a. According to the present invention, "white blood cells" refers to cells of the immune system that protect the body from infectious diseases and foreign substances. In one embodiment, the white blood cells include neutrophil, eosin〇phil, basophil, lymphocytes, monocytes, and macrophages. The white blood cells are preferably neutrophils and giant scorpion cells. During inflammation, white blood cells migrate to the inflamed site and attach foreign substances to kill them. According to the present invention, leukocytes are characterized by attachment of living nuclear-containing heterologous cells. After the induction of the inflammatory response according to the present invention, the white blood cells move to and are attached to the endothelial-like or epithelial-like cells. In one embodiment of the invention, the white blood cells used in the methods of the invention may be originally present in the body fluid sample. In another embodiment of the invention, white blood cells may be additionally added and applied to the present invention. According to the present invention, a "body fluid sample" or "jk-containing sample" may be derived from any biological source such as physic fluids, including whole blood, ascites, saliva, tert fluid, synovial fluid, peritoneal fluid, amniotic fluid, Cerebrospinal fluid, serum, spinal fluid, and other body components that may contain tumor cells. If the sample contains tumor cells, the leukocytes can capture tumor cells when performing the kits and methods of the present invention because the tumor cells are nuclear-containing heterologous cells and can be identified and identified by white blood cells. In accordance with an embodiment of the present invention, since one of the objects of the present invention is to capture living tumor cells and to use living cells in the methods and kits of the present invention, the body fluids can be pretreated prior to use in the methods and kits of the present invention. The sample or serum-containing sample is placed under physiological conditions. According to another embodiment of the invention, the method may further comprise the step of pre-treating the biological sample into a serum-containing sample. According to the present invention, the biological sample is a body sample of any kind; the biological sample is preferably a bone marrow aspirate, a bone marrow homogenate, a lymphoid tissue homogenate or a tissue homogenate. The biological sample is preferably a solution containing a tissue. Preferably, the sample is pre-diluted by more than 10 times. In one embodiment, the sample volume used in the present invention is in the range of from about 5 mL to about 3 〇 mL. The sample volume is preferably from about 5 mL to about 25 mL, from about 5 mL to about 20 mL, from about 5 mL to about 15 mL, from about 5 mL to about 1 mL, from about 1 mL to about 25 mL, about 15 From about 25 mL to about 25 mL, from about 1 mL to about 20 mL, or from 15 mL to 20 mL. According to the present invention, the tumor cells in the body fluid sample or the serum-containing sample are tumors that are deprived or secreted from the tumor tissue and then enter the body fluid sample. cell. The tumor cells are preferably circulating tumor cells. According to the present invention, after tumor cells are captured, the tumor cells can be further collected by methods known in the art. For example, fully wash the tumor cells or fragments thereof (washing strips 6-7 times) with complete DMEM and broad-acting antibiotics and anti-145593.doc •10·201122479 fungal solution to achieve compatibility with standard cell culture conditions. No degree of ®. Next, the cells or sections are cut into 1 to 2 _3 pieces. These pieces were inoculated into 6-well plates pre-coated with PolyHEMA and containing 2 mL of complete medium. Tumor fragments were cultured for 2 days, and then the medium was recovered and replaced with a medium alone or with a drug-containing medium for 24 hours. The drug used is the topoisomerase-I inhibitor metabolite SN-38 (7-ethyl) hydrazine-hydroxycamptothecin (7_can be 〇 hydr〇xycamptotecin; it is camptothecin (campt〇thecin) The active agent 4t) 10-5 mM/l, which is similar to the concentration observed in the tumor after administration of the Camptothecin to humans. At the end of this period, elution is performed to remove the drug, and after 72 cycles. The medium is added in hours. The kit of the invention is a kit for carrying out the method of the invention. According to the invention, the kit comprises (a) a solid support, (b) an endothelial cell attached to the solid support or Epithelial cells, (c) white blood cells attached to the cells of (b), and (d) - or a plurality of inflammatory inducing agents. (a), (b), (c) and (d) of the set Each of the above materials is placed in a separate reservoir (such as a vial, tube, and the like). That is, each reservoir contains a separate material to be used in the assay. The kit may further include a set The kit instructions are used to guide the predetermined assay for capturing tumor cells in a body fluid sample. And methods for inducing adhesion to endothelial cells or epithelial cells such as solid supports to produce an inflammatory response. Inflammation response is induced by the addition of one or more inflammatory inducing agents. The set and method of the invention after induction of inflammation The white blood cells used in the migration will migrate to the inflammatory cells and attach to the inflammatory cells. Since the white ball will capture the living nuclear-containing heterologous cells, after the body fluid sample is added at 145593.doc 11 201122479, the white blood cells will trap the tumor cells in the sample. However, white blood cells do not capture inactive cells, inactive cell fragments, and other contaminants in the sample. By using the kits and methods of the present invention, live tumor cells or live tumor cells in serum-containing samples can be obtained, and After culturing, 'can be used to further identify tumor cell types using methods known in the art (such as real-time PCR or flow-type traverse measurements) (Barrett DL, Jensen RH, King EB, Dean PN, Mayall BH. Flow cytometry of Human gynecologic specimens using log chromomycin A: iluorescence and log 9〇" light scatter. J Histochem Cytoche m 27(1): 573-5 78, 1979 ; Darzynkiewicz Z:
Acridine orange as a molecular probe in studies of nucleic acids in situ. Flow Cytometry and Sorting, Melamed MR, Mullaney PF，Mendelsohn ML (編），John Wiley & Sons, New York, 1979，第 285-316 頁；Frost JK，Tyrer HW， Pressman NJ, Albright CD, Vansickel MH, Gill GW: Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters. J Histochem Cytochem 27545-551，1979 ;及 Frost JK，Tyrer HW，Acridine orange as a molecular probe in studies of nucleic acids in situ. Flow Cytometry and Sorting, Melamed MR, Mullaney PF, Mendelsohn ML (ed.), John Wiley & Sons, New York, 1979, pp. 285-316; Frost JK , Tyrer HW, Pressman NJ, Albright CD, Vansickel MH, Gill GW: Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters. J Histochem Cytochem 27545-551, 1979; and Frost JK, Tyrer HW,
Pressman NJ, Adams LA, Vansickel MH, Albright CD, Gill GW, Tiffany SM: Automatic cell identification and enrichment in lung cancer. Light scatter and two fluorescence parameters. J Histochem Cytochem 27557-559, 1979)〇 實例 145593.doc 12 201122479 實例1腫瘤細胞於上皮細胞上之附著情況 將具有經明膠修飾之表面的玻璃盤置於皮氏培養皿之底 部’且接著將約5〇,000個人類胚腎293Τ(ΗΕΚ-293Τ)内皮細 胞或人類臍靜脈内皮細胞（HUVEC)接種於該玻璃盤中。將 DMEM及]VI199培養基添加至皮氏培養皿中之後，在37eC、 5% C〇2下培養細胞24小時。隨後，自皮氏培養皿中移除 DMEM及Ml 99培養基，且用PBS緩衝液洗滌該等皮氏培養 皿。隨後’將新鮮DMEM及M199培養基添加至皮氏培養皿 中。將50 ng發炎誘導劑（諸如介白素及腫瘤壞死 因子-a(TNF-a))添加至皮氏培養皿中以誘導HEK-293T及 HUVEC之發炎反應，歷時24小時。由酶聯結免疫檢定 (ELISA)量測介白素-6(IL-6)之表現量。若il-6之表現量大 於30 ng/ml ’則出現發炎反應。或者’可由流動式細胞測 量術偵測IL-6之表現量。將附著有螢光素異硫氰酸鹽 (FITC)之ICAM抗體添加至皮氏培養皿中以捕提由IL_丨及 TNFa誘導且由内皮細胞表現之;[cam-1 (細胞間黏附分子υ '。由流動式細胞測量術量測FITC螢光素。若ICAM-1之表 現量大於10 ng/ml，則出現發炎反應。 用綠色螢光蛋白（GFP)轉染腫瘤細胞，亦即肝癌細胞 hepG2。將含GFP之hepG2細胞添加至皮氏培養皿中。震盪 W分鐘後，添加15 ml PBS緩衝液進行洗滌。洗滌3次後， 分別使用光學顯微鏡及螢光顯微術來檢查該等皮氏培養 ΤϊΤΙ. 〇 圖1係關於光學顯微鏡之照片’其展示在使用IL_ip誘導 145593.doc 13 201122479 上皮細胞上之發炎反應的情況下（圖1(B))或在未使用IL_ip 誘導上皮細胞上之發炎反應的情況下（圖1(A))，腫瘤細胞 於上皮細胞上之附著情況。在圖1(A)中，洗滌後，無 hepG2細胞附著於上皮細胞；而在圖丨（B)中，洗條後，可 觀察到hepG2細胞附著於上皮細胞。 圖2係關於光學顯微鏡之照片，其展示在使用IL_丨β(圖 2(A))及TNF-α(圖2(B))誘導上皮細胞上之發炎反應後，腫 瘤細胞於上皮細胞上之附著情況。在圖2(A)與圖2(B)中， 洗/滌後’均可觀察到hepG2細胞附著於上皮細胞。在由α-ΐ β 或 TNF-a誘 導發炎 反應後 ，上皮 細胞可 捕捉腫 瘤細胞 。 實例2在誘導上皮細胞上之發炎反應後，腫瘤細胞於白 血球（嗜中性球）上之附著情況 將具有經明膠修飾之表面的玻璃盤置於皮氏培養皿之底 部’且接著將约50,〇〇〇個人類胚腎293Τ(ηΕΚ_293Τ)内皮細 胞或人類臍靜脈内皮細胞（HUVEC)接種於該玻璃盤中。將 0河£]\4及]\(1199培養基添加至皮氏培養皿中之後，在37。〇、 5% C〇2下培養細胞24小時。隨後，自皮氏培養皿中移除 DMEM及M199培養基，且用PBS緩衝液洗滌該等皮氏培養 皿。隨後，將新鮮DMEM&M199培養基添加至皮氏培養皿 中。將50 ng發炎誘導劑（諸如介白素_ip(IL_ip)及腫瘤壞死 因子-a(TNF-a))添加至皮氏培養皿中以誘導即尺別丁及 HUVEC之發炎反應，歷時24小時。由酶聯結免疫檢定 (ELISA)量測介白素-6(IL_6)之表現量。若IL_6之表現量大 於30 ng/m卜則出現發炎反應.或者，可由流動式細胞測 145593.doc 14 201122479 量術偵測江-6之表現量。將附著有營光素異硫氰酸鹽 (FITC)之ICAM抗體添加至皮氏培養里中以捕捉由lL_u ™Fa誘導且由内皮細胞表現之ICAM-1(細胞間黏附分子υ 。由流動式細胞測量術量測FITC螢光素。若ΚΑΜ]之表 現量大於10 ng/m卜則出現發炎反應。 將嗜中性球添加至皮氏培養贩甲且其附著於發炎性 HEK-293T及HUVEC細胞。用、綠色螢光蛋白（GFp)轉染腫瘤 細胞，亦即肝癌細胞hepG2。將含GFP之hepG2細胞添加至 皮氏培養皿中。震盪1 〇分鐘後，添加丨5 m丨pBs緩衝液進 行洗務。洗滌3次後，分別使用光學顯微鏡及螢光顯微術 來檢查該等皮氏培養皿。 圖3係關於光學顯微鏡之照片，其展示在使(圖 3(B))及TNF-a(圖3(C))誘導上皮細胞上之發炎反應後，腫 瘤細胞於白血球（嗜中性球）上之附著情況。圖3(A)為未添 加白血球之對照組。在圖3(B)及圖3(c)中，hepG2細胞附 著於°耆中性球且叢集在一起；而在圖3(A)中，hepG2細胞 隨機分布於皮氏培養Jffll中。 實例3在紅血球存在下腫瘤細胞之捕捉情況 將具有經明膠修飾之表面的玻璃盤置於皮氏培養孤之底 部’且接著將約50,000個人類胚腎293T(HEK-293T)内皮細 胞或人類臍靜脈内皮細胞（HUVEC)接種於該玻璃盤中》將 DMEM及M199培養基添加至皮氏培養皿中之後，在37°C、 5% C〇2下培養細胞24小時。隨後，自皮氏培養皿中移除 DMEM及M199培養基，且用PBS缓衝液洗滌該等皮氏培養 145593.doc -15- 201122479 孤。隨後，將新鮮DMEM及M199培養基添加至皮氏培養皿 中。將50 ng發炎誘導劑（諸如介白素及腫瘤壞死 因子-a(TNF-a))添加至皮氏培養皿中以誘導HEK-293T及 HUVEC之發炎反應’歷時24小時。由酶聯結免疫檢定 (ELISA)量測介白素-6(IL-6)之表現量。若IL_6之表現量大 於30 ng/ml，則出現發炎反應。或者，可由流動式細胞測 量術偵測IL-6之表現量。將附著有螢光素異硫氰酸鹽 (FITC)之ICAM抗體添加至皮氏培養皿中以捕捉由化」及 TNFa誘導且由内皮細胞表現之ICAM](細胞間黏附分子1} 。由流動式細胞測量術量測FITC螢光素。若之表 現量大於10 ng/ml，則出現發炎反應。 將嗜中性球添加至皮氏培養皿令且其附著於發炎性 HEK-293T及HUVEC細胞。對紅血球的存在是否影響白血 球捕捉腫瘤細胞進行評估。收集含有hepG2細胞之全血測 試樣本。分別對該等A液樣本不進行稀釋，或稀釋1〇倍及 1〇〇〇倍。接著將瑞氏染色劑（wright，s灿⑴添加至血液樣 本中以標記其中所含之hePG2細胞。染色後，將A液樣本 添加至皮氏培養皿中。隨後，使用光學顯微鏡來檢查該等 皮氏培養皿。 此外，收集含有hepG2細胞 列瑪像本且將其；I 30分鐘。對血液樣本不進行稀釋或稀釋1_倍。接著網 氏染色劑添加至灰液樣本中以標記其中所含之—細 。染色後’將血液樣本添加至皮氏培養皿中。隨後，使 光學顯微鏡來檢查該等皮氏培養亚。 145593.doc •16· 201122479 圖4係關於光學顯微鏡之照片，其展示在未進行稀釋或 進行稀釋（10倍或1，000倍）之情況下，全血樣本中活腫瘤細 胞或死腫瘤細胞之捕捉情況。圖4(A)及圖4(B)分別展示在 稀釋10倍及1，000倍之情況下含有活hepG2細胞之全血樣本 的照片。圖4(C)展示在未進行稀釋之情況下含有活hepG2 細胞之全血樣本的照片，且圖4(D)展示在稀釋】，〇〇〇倍之情 況下含有死hePG2細胞之全血樣本的照片。此證實紅血球 的存在並不影響嗜中性球捕捉腫瘤細胞。 【圖式簡單說明】 圖1係關於光學顯微鏡之照片，其展示在使用1 β誘導 上皮細胞上之發炎反應（(B))或未使用IL_丨β誘導上皮細胞 上之發炎反應（(A))的情況下，腫瘤細胞於上皮細胞上之附 著情況。在圖中，「a」表示腫瘤細胞（hepG2細胞）且「b」 表示上皮細胞。 圖2係關於光學顯微鏡之照片，其展示在使用比_1(3(圖 2(A))及TNF-α(圖2(B))誘導上皮細胞上之發炎反應後，腫 瘤細胞於上皮細胞上之附著情況。在圖中，「a」表示腫瘤 細胞（hepG2細胞）且「b」表示上皮細胞。 圖3係關於光學顯微鏡之照片，其展示在使用IL_丨β(圖 3(B))及TNF-α(圖3(C))誘導上皮細胞上之發炎反應後，腫 瘤細胞於白血球（嗜中性球）上之附著情況。圖3(A)為未添 加白血球之對照組。在圖中’「a」表示腫瘤細胞（hepG2 細胞）；「b」表示上皮細胞；且「^」表示白血球（嗜中性 球）。 145593.doc •17· 201122479 圖4(A)及圖4(B)分別展示在稀釋1 0倍及1,000倍之情況下 含有活hepG2細胞之全血樣本的照片。圖4(C)展示在未進 行稀釋之情況下含有活hepG2細胞之全血樣本的照片，且 M 4(D)展示在稀釋1，000倍之情況下含有死hepG2細胞之全 血樣本的照片。在圖中，「a」表示腫瘤細胞（hepG2細胞） :「b」表示上皮細胞；「c」表示白血球（嗜中性球）；且「d 」表示紅血球。 145593.doc -18-Pressman NJ, Adams LA, Vansickel MH, Albright CD, Gill GW, Tiffany SM: Automatic cell identification and enrichment in lung cancer. Light scatter and two fluorescence parameters. J Histochem Cytochem 27557-559, 1979) 〇 Example 145593.doc 12 201122479 Example 1 Attachment of Tumor Cells to Epithelial Cells A glass dish with a gelatin-modified surface was placed at the bottom of the Petri dish and then about 5,000 human embryonic kidney 293 (Τ-293Τ) endothelial cells were added. Or human umbilical vein endothelial cells (HUVEC) were inoculated into the glass dish. After DMEM and]VI199 medium were added to the Petri dish, the cells were cultured at 37 ° C, 5% C 2 for 24 hours. Subsequently, DMEM and Ml 99 medium were removed from the Petri dish and the Petri dishes were washed with PBS buffer. Subsequently, fresh DMEM and M199 medium were added to the Petri dish. 50 ng of an inflammatory inducing agent such as interleukin and tumor necrosis factor-a (TNF-a) was added to a Petri dish to induce an inflammatory response of HEK-293T and HUVEC for 24 hours. The amount of interleukin-6 (IL-6) was measured by enzyme-linked immunoassay (ELISA). An inflammatory response occurs if the expression of il-6 is greater than 30 ng/ml'. Alternatively, the amount of IL-6 expression can be detected by flow cytometry. ICAM antibody to which luciferin isothiocyanate (FITC) is attached is added to a Petri dish to capture and be induced by IL_丨 and TNFa and expressed by endothelial cells; [cam-1 (intercellular adhesion molecule) υ '. FITC luciferin is measured by flow cytometry. If the expression of ICAM-1 is greater than 10 ng/ml, an inflammatory reaction occurs. Transfecting tumor cells with green fluorescent protein (GFP), ie liver cancer Cell hepG2. Add GFP-containing hepG2 cells to the Petri dish. After shaking for W minutes, add 15 ml of PBS buffer for washing. After washing 3 times, examine them by optical microscopy and fluorescence microscopy, respectively. Petri culture ΤϊΤΙ. Figure 1 is a photograph of an optical microscope which is shown to induce epithelial cells in the absence of inflammatory response on IL_ip-induced 145593.doc 13 201122479 epithelial cells (Fig. 1 (B)) or without IL_ip In the case of an inflammatory reaction (Fig. 1(A)), the adhesion of tumor cells to epithelial cells. In Fig. 1 (A), after washing, no hepG2 cells adhere to epithelial cells; ), after washing the strip, hepG2 cells can be observed to adhere to Skin cells. Figure 2 is a photograph of an optical microscope showing the use of IL_丨β (Fig. 2(A)) and TNF-α (Fig. 2(B)) to induce inflammatory responses on epithelial cells. Adhesion on epithelial cells. In Fig. 2(A) and Fig. 2(B), hepG2 cells were observed to adhere to epithelial cells after washing/diluting. Inflammation was induced by α-ΐβ or TNF-a. After that, the epithelial cells can capture the tumor cells. Example 2 After inducing the inflammatory response on the epithelial cells, the attachment of the tumor cells to the white blood cells (neutrophils) placed the glass disk with the gelatin-modified surface in the Petri culture. The bottom of the dish 'and then inoculate about 50, 〇〇〇 personal embryonic kidney 293 Τ (ηΕΚ_293Τ) endothelial cells or human umbilical vein endothelial cells (HUVEC) in the glass dish. 0 rivers]]\4 and]\( After the 1199 medium was added to the Petri dish, the cells were cultured for 24 hours at 37 ° C, 5% C 2 . Subsequently, DMEM and M199 medium were removed from the Petri dish and washed with PBS buffer. Petri dish. Subsequently, fresh DMEM & M199 medium was added to Pepi's culture. In the dish, 50 ng of inflammatory inducing agents (such as interleukin _ip (IL_ip) and tumor necrosis factor-a (TNF-a)) were added to the Petri dish to induce the inflammatory response of the amphibians and HUVEC. The expression of interleukin-6 (IL_6) was measured by enzyme-linked immunoassay (ELISA) for 24 hours. If the expression of IL_6 is greater than 30 ng/m, an inflammatory reaction may occur. Alternatively, it may be measured by flow cytometry 145593. .doc 14 201122479 Quantitative detection of the performance of Jiang-6. ICAM antibody with camphorin isothiocyanate (FITC) was added to the Petri culture to capture ICAM-1 (intercellular adhesion molecule 诱导 induced by lL_u TMFa and expressed by endothelial cells. Cytometry measures FITC luciferin. If the expression is greater than 10 ng/m, the inflammatory response occurs. Neutrophils are added to the Petri culture and attached to the inflammatory HEK-293T and HUVEC. Cells were transfected with green fluorescent protein (GFp), ie hepatocellular carcinoma cell hepG2. HepG2 cells containing GFP were added to the Petri dish. After shaking for 1 minute, 丨5 m丨pBs buffer was added. Washing was performed. After washing 3 times, the Petri dishes were examined using optical microscopy and fluorescence microscopy, respectively. Figure 3 is a photograph of an optical microscope, which is shown in Figure 3 (B) and TNF. -a (Fig. 3(C)) The adhesion of tumor cells to leukocytes (neutrophils) after induction of an inflammatory response on epithelial cells. Figure 3 (A) is a control group without white blood cells added. In B) and in Figure 3(c), hepG2 cells are attached to the 耆-neutral sphere and clustered together; and in Figure 3(A) , hepG2 cells were randomly distributed in the Petri culture Jffll. Example 3 Capture of tumor cells in the presence of red blood cells Place a glass dish with a gelatin-modified surface at the bottom of the Petri culture ' and then about 50,000 human embryos Renal 293T (HEK-293T) endothelial cells or human umbilical vein endothelial cells (HUVEC) were seeded in the glass dish. After adding DMEM and M199 medium to the Petri dish, at 37 ° C, 5% C 〇 2 The cells were cultured for 24 hours. Subsequently, DMEM and M199 medium were removed from the Petri dish, and the Petri culture 145593.doc -15-201122479 was orphaned with PBS buffer. Subsequently, fresh DMEM and M199 medium were added to In a Petri dish. Add 50 ng of inflammatory inducing agents (such as interleukin and tumor necrosis factor-a (TNF-a)) to the Petri dish to induce the inflammatory response of HEK-293T and HUVEC. The amount of interleukin-6 (IL-6) measured by enzyme-linked immunoassay (ELISA). If the expression of IL_6 is greater than 30 ng/ml, an inflammatory reaction occurs. Alternatively, it can be detected by flow cytometry. Measure the amount of IL-6. Will be attached ICAM antibody of photo-isothiocyanate (FITC) is added to a Petri dish to capture ICAM induced by TNFa and expressed by endothelial cells (intercellular adhesion molecule 1). Flow cytometry The FITC luciferin was measured, and if the expression amount was more than 10 ng/ml, an inflammatory reaction occurred. The neutrophil was added to the Petri dish and attached to the inflammatory HEK-293T and HUVEC cells. Whether the presence of red blood cells affects leukocyte-captured tumor cells is assessed. A whole blood sample containing hepG2 cells was collected. These A liquid samples were not diluted or diluted 1〇 and 1〇〇〇, respectively. Next, Wright's stain (wright, scan (1) is added to the blood sample to label the hePG2 cells contained therein. After staining, the sample A is added to the Petri dish. Then, using an optical microscope to check these Petri dish. In addition, the hepG2 cell containing the Lehma image is collected and taken; I 30 minutes. The blood sample is not diluted or diluted 1 times. Then the mesh stain is added to the gray liquid sample to mark the Including - fine. After dyeing, add blood samples to the Petri dish. Then, optical microscopy is used to check the Petri culture. 145593.doc •16· 201122479 Figure 4 is a photograph of an optical microscope. Shows the capture of live or dead tumor cells in whole blood samples without dilution or dilution (10x or 1,000x). Figure 4(A) and Figure 4(B) show A photograph of a whole blood sample containing live hepG2 cells diluted 10 times and 1,000 times. Figure 4 (C) shows a photograph of a whole blood sample containing live hepG2 cells without dilution, and Figure 4 ( D) shown in the dilution], A photograph of a whole blood sample containing dead hePG2 cells in the case of 〇〇〇倍. This confirms that the presence of red blood cells does not affect the neutrophil capture of tumor cells. [Simplified Schematic] Fig. 1 is a photograph of an optical microscope. The adhesion of tumor cells to epithelial cells in the case where inflammatory response ((B)) on epithelial cells was induced by 1β or inflammatory reaction ((A)) on epithelial cells was not induced using IL_丨β. In the figure, "a" indicates tumor cells (hepG2 cells) and "b" indicates epithelial cells. Fig. 2 is a photograph of an optical microscope showing the use ratio _1 (3 (Fig. 2(A)) and TNF- α (Fig. 2(B)) shows the adhesion of tumor cells to epithelial cells after inducing an inflammatory response on epithelial cells. In the figure, "a" indicates tumor cells (hepG2 cells) and "b" indicates epithelial cells. 3 series photographs of optical microscopy showing tumor cells in leukocytes after inflammatory response induced on IL_丨β (Fig. 3(B)) and TNF-α (Fig. 3(C)) Attachment on the sexual ball) Figure 3 (A) is the pair without white blood cells added In the figure, 'a' indicates tumor cells (hepG2 cells); "b" indicates epithelial cells; and "^" indicates white blood cells (neutrophils). 145593.doc •17· 201122479 Figure 4(A) and Figure 4 (B) shows photographs of whole blood samples containing live hepG2 cells diluted 10 and 1,000 times, respectively. Figure 4 (C) shows whole blood samples containing live hepG2 cells without dilution. Photograph, and M 4 (D) shows a photograph of a whole blood sample containing dead hepG2 cells diluted 1,000 times. In the figure, "a" indicates tumor cells (hepG2 cells): "b" indicates epithelial cells; "c" indicates white blood cells (neutrophils); and "d" indicates red blood cells. 145593.doc -18-