Column-based device and method for retrieval of rare cells based on size, and uses thereof

Abstract

A device and method for retrieving cells of interest, in particular rare cells, were disclosed. Tumor-derived endothelial cell clusters (TECCs) retrieved using the disclosed device and method can be used for the diagnosis and prognosis of cancer.

1 . A method for detecting a tumor-derived endothelial cell cluster (TECC) in a blood sample of a subject, the method comprising:
 (a) obtaining a blood sample from a subject;   (b) filtering the sample by passing the sample at a flow-rate selected from the group consisting of at least about 0.05 mL/min, at least about 0.10 mL/min, at least about 0.15 mL/min, at least about 0.20 mL/min, at least about 0.25 mL/min, at least about 0.30 mL/min, at least about 0.40 mL/min, and at least about 0.50 mL/min through a sieve that comprises a plurality of pores having a diameter selected from the group consisting of at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 12 μm, at least about 14 μm, at least about 16 μm, at least about 18 μm and at least about 20 μm;   (c) lysing the cells retained on a surface of the sieve;   (d) contacting the lysed cell sample from step (c) with a reverse primer from a first primer pair, the reverse primer from the first primer pair being directed to a target RNA region, and a reverse transcriptase to effect reverse transcription of the RNA into cDNA;   (e) subsequently contacting the sample from step (d) with:
 (i) a forward primer from the first primer pair, the forward primer from the first primer pair being directed to a target cDNA region, 
 (ii) a reverse primer and a forward primer from a second primer pair, the reverse primer and forward primer from the second primer pair being directed to a target DNA region, and 
 (iii) a DNA polymerase to simultaneously amplify the target cDNA region and the target DNA region in a pre-amplification step; and 
   (f) analyzing the amplified target cDNA region and/or the amplified target DNA region;   wherein the target DNA region comprises a region that encodes one or more of an epithelial-mesenchymal transition marker, an epithelial marker, a tumor endothelial cell marker, an endothelial cell marker, a megakaryocyte or platelet-specific marker, or a hemotopoietic cell marker.   
     
     
         2 . The method of  claim 1 , further comprising: one or more of the following:
 (A) subjecting the sample from step (e) to a semi-nested PCR using the reverse primer in step (d) or the forward primer in step (e)(i), and a nested primer that binds within the amplified target cDNA region;   (B) (i) further subjecting the sample from step (e) to a nested PCR using a nested primer pair that binds within the amplified target DNA region; (ii) the steps (d) and (e) being conducted in the same reaction mixture; and (iii) the analysis in step (f) comprising analyzing the amplified target cDNA for one or more of the following: gene expression and mutations;   (C) the first or the second primer pair being selected from the group consisting of: (i) SEQ ID NO: 1 and SEQ ID NO: 2; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; (iii) SEQ ID NO: 5 and SEQ ID NO: 6; (iv) SEQ ID NO: 7 and SEQ ID NO: 8; (v) SEQ ID NO: 9 and SEQ ID NO: 10; (vi) SEQ ID NO: 11 and SEQ ID NO: 12; (vii) SEQ ID NO: 13 and SEQ ID NO: 14; (viii) SEQ ID NO: 15 and SEQ ID NO: 16; (ix) SEQ ID NO: 17 and SEQ ID NO: 18; (x) SEQ ID NO: 19 and SEQ ID NO: 20; (xi) SEQ ID NO: 21 and SEQ ID NO: 22; (xii) SEQ ID NO: 23 and SEQ ID NO: 24; (xiii) SEQ ID NO: 25 and SEQ ID NO: 26; (xiv) SEQ ID NO: 27 and SEQ ID NO: 28; (xv) SEQ ID NO: 29 and SEQ ID NO: 30; (xvi) SEQ ID NO: 31 and SEQ ID NO: 32; (xvii) SEQ ID NO: 33 and SEQ ID NO: 34; (xviii) SEQ ID NO: 52 and 53; and (xix) SEQ ID NO: 54 and 55; and   (D) the primer pair for semi-nested or nested PCR being selected from the group consisting of: (i) SEQ ID NO: 35 and SEQ ID NO: 2; (ii) SEQ ID NO: 3 and SEQ ID NO: 36; (iii) SEQ ID NO: 5 and SEQ ID NO: 37; (iv) SEQ ID NO: 38 and SEQ ID NO: 8; (v) SEQ ID NO: 39 and SEQ ID NO: 10; (vi) SEQ ID NO: 40 and SEQ ID NO: 12; (vii) SEQ ID NO: 41 and SEQ ID NO: 14; (viii) SEQ ID NO: 42 and SEQ ID NO: 16; (ix) SEQ ID NO: 17 and SEQ ID NO: 43; (x) SEQ ID NO: 44 and SEQ ID NO: 20; (xi) SEQ ID NO: 45 and SEQ ID NO: 22; (xii) SEQ ID NO: 46 and SEQ ID NO: 24; (xiii) SEQ ID NO: 25 and SEQ ID NO: 47; (xiv) SEQ ID NO: 27 and SEQ ID NO: 48; (xv) SEQ ID NO: 49 and SEQ ID NO: 30; (xvi) SEQ ID NO: 31 and SEQ ID NO: 50; (xvii) SEQ ID NO: 51 and SEQ ID NO: 34; (xviii) SEQ ID NO: 56 and SEQ ID NO: 57; and (xix) SEQ ID NO: 58 and SEQ ID NO: 55.   
     
     
         3 . The method of  claim 1 , wherein the endothelial cell marker is selected from the group consisting of PECAM1, VWF, and CDH5. 
     
     
         4 . The method of  claim 1 , wherein the leukocyte and megakaryocytic or platelet-specific marker is selected from the group consisting of PTPRC, ITGA2B, and GP1BA. 
     
     
         5 . A method of diagnosing a cancer in a subject, comprising
 (a) obtaining a blood sample from a subject;   (b) filtering the sample by passing the sample at a flow-rate selected from the group consisting of at least about 0.05 mL/min, at least about 0.10 mL/min, at least about 0.15 mL/min, at least about 0.20 mL/min, at least about 0.25 mL/min, at least about 0.30 mL/min, at least about 0.40 mL/min, and at least about 0.50 mL/min through a sieve that comprises a plurality of pores having a diameter selected from the group consisting of at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 12 μm, at least about 14 μm, at least about 16 μm, at least about 18 μm and at least about 20 μm;   and   (c) detecting and analyzing a sample from the subject for presence of one or more TECCs based on one or more morphological characteristics and/or expression of one or more target biomarkers expressed by TECCs,   wherein presence of one or more TECCs indicates that the subject has cancer.   
     
     
         6 . The method of  claim 5 , wherein detecting and analyzing a sample for the presence of TECCs further comprises (i) contacting the sample retained on a surface of the sieve with at least one antibody that is coupled to a detectable label to allow binding of the antibody to one or more target biomarkers expressed on the cells, wherein the target biomarkers is selected from the group consisting of PAI-1, Vimentin, FOXC1, keratin-8, keratin-18, VWF, PECAM-1, CD34, PSMA, CD105, CD309, CD144, and CD202B; and (ii) removing unbound antibody from the sample. 
     
     
         7 . The method of  claim 6 , wherein the detectable label is selected from the group consisting of a fluorescent group, a radioisotope, a stable isotope, an enzymatic group, a chemiluminescent group, and a biotinyl group. 
     
     
         8 . The method of  claim 5 , wherein the cancer is:
 (a) selected from the group consisting of carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, colon cancer, rectal cancer, breast cancer, prostate cancer, renal cell cancer, transitional cell carcinoma, lung cancer, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary, ovarian carcinoma, endometrial carcinoma, glioblastoma, neuroblastoma, Hodgkin lymphoma, and non-Hodgkin lymphomas;   (b) an invasive and/or metastatic cancer;   (c) a stage I cancer, stage II cancer, stage III cancer, or stage IV cancer; and/or   (d) early stage cancer.   
     
     
         9 . A method for monitoring and/or predicting the response to treatment of a cancer patient, the method comprising
 (a) obtaining a blood sample from a cancer patient after a cancer treatment;   (b) filtering the sample by passing the sample at a flow-rate selected from the group consisting of at least about 0.05 mL/min, at least about 0.10 mL/min, at least about 0.15 mL/min, at least about 0.20 mL/min, at least about 0.25 mL/min, at least about 0.30 mL/min, at least about 0.40 mL/min, and at least about 0.50 mL/min through a sieve that comprises a plurality of pores having a diameter selected from the group consisting of at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 12 μm, at least about 14 μm, at least about 16 μm, at least about 18 μm, and at least about 20 μm;   and   (c) detecting and analyzing a sample to determine the number of TECCs in the sample based on one or more morphological characteristics and/or expression of one or more target biomarkers expressed by TECCs,   wherein a reduction in the number of TECCs compared to the number of TECCs in a baseline sample obtained from the patient prior to treatment indicates that the patient is responding positively to the treatment.   
     
     
         10 . The method of  claim 9 , wherein detecting and analyzing a sample for the presence of TECCs further comprises (i) contacting the sample retained on a surface of the sieve with at least one antibody that is coupled to a detectable label to allow binding of the antibody to one or more target biomarkers expressed on the cells, wherein the target biomarkers is selected from the group consisting of PAI-1, Vimentin, FOXC1, keratin-8, keratin-18, VWF, PECAM-1, CD34, PSMA, CD105, CD309, CD144, and CD202B; and (ii) removing unbound antibody from the sample. 
     
     
         11 . The method of  claim 10 , wherein the detectable label is selected from the group consisting of a fluorescent group, a radioisotope, a stable isotope, an enzymatic group, a chemiluminescent group, and a biotinyl group. 
     
     
         12 . The method of  claim 9 , wherein the cancer is:
 (a) selected from the group consisting of carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, colon cancer, rectal cancer, breast cancer, prostate cancer, renal cell cancer, transitional cell carcinoma, lung cancer, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary, ovarian carcinoma, endometrial carcinoma, glioblastoma, neuroblastoma, Hodgkin lymphoma, and non-Hodgkin lymphomas;   (b) an invasive and/or metastatic cancer;   (c) a stage I cancer, stage II cancer, stage III cancer, or stage IV cancer; and/or   (d) early stage cancer.   
     
     
         13 . A method for predicting the response to treatment of a cancer patient, the method comprising
 (a) obtaining a blood sample from the cancer patient;   (b) filtering the sample by passing the sample at a flow-rate selected from the group consisting of at least about 0.05 mL/min, at least about 0.10 mL/min, at least about 0.15 mL/min, at least about 0.20 mL/min, at least about 0.25 mL/min, at least about 0.30 mL/min, at least about 0.40 mL/min, and at least about 0.50 mL/min through a sieve that comprises a plurality of pores having a diameter selected from the group consisting of at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 12 μm, at least about 14 μm, at least about 16 μm, at least about 18 μm, and at least about 20 μm;   and   (c) detecting and analyzing the sample to determine the number of TECCs present in the sample based on one or more morphological characteristics and/or expression of one or more target biomarkers expressed by TECCs,   wherein an equal or higher number of TECCs compared to the number of TECCs in a sample obtained before treatment from a control patient or a group of control patients that have responded positively to the treatment, indicates that the cancer patient will respond positively to the treatment, and   wherein a lower number of TECCs compared to the number of TECCs in a sample obtained before treatment from a control patient or a group of control patients that have responded positively to the treatment, indicates that the cancer patient will respond negatively to the treatment.   
     
     
         14 . The method of  claim 13 , wherein detecting and analyzing a sample for the presence of TECCs further comprises (i) contacting the sample retained on a surface of the sieve with at least one antibody that is coupled to a detectable label to allow binding of the antibody to one or more target biomarkers expressed on the cells, wherein the target biomarkers is selected from the group consisting of PAI-1, Vimentin, FOXC1, keratin-8, keratin-18, VWF, PECAM-1, CD34, PSMA, CD105, CD309, CD144, and CD202B; and (ii) removing unbound antibody from the sample. 
     
     
         15 . The method of  claim 14 , wherein the detectable label is selected from the group consisting of a fluorescent group, a radioisotope, a stable isotope, an enzymatic group, a chemiluminescent group, and a biotinyl group. 
     
     
         16 . The method of  claim 13 , wherein the cancer is:
 (a) selected from the group consisting of carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, colon cancer, rectal cancer, breast cancer, prostate cancer, renal cell cancer, transitional cell carcinoma, lung cancer, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary, ovarian carcinoma, endometrial carcinoma, glioblastoma, neuroblastoma, Hodgkin lymphoma, and non-Hodgkin lymphomas;   (b) an invasive and/or metastatic cancer;   (c) a stage I cancer, stage II cancer, stage III cancer, or stage IV cancer; and/or   (d) early stage cancer.   
     
     
         17 . A method for analyzing blood vessel characteristics of a tumor in a subject, the method comprising
 (a) obtaining a blood sample from the subject;   (b) filtering the sample by passing the sample at a flow-rate selected from the group consisting of at least about 0.05 mL/min, at least about 0.10 mL/min, at least about 0.15 mL/min, at least about 0.20 mL/min, at least about 0.25 mL/min, at least about 0.30 mL/min, at least about 0.40 mL/min, and at least about 0.50 mL/min through a sieve that comprises a plurality of pores having a diameter selected from the group consisting of at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 12 μm, at least about 14 μm, at least about 16 μm, at least about 18 μm, and at least about 20 μm;   and   (c) detecting and analyzing the sample to determine the number of TECCs present in the sample based on one or more morphological characteristics and/or expression of one or more target biomarkers expressed by TECCs,   wherein an increased number of TECCs compared to the number of TECCs in a baseline sample indicates an increase in tumor blood vessel size in a subject, and   wherein a reduced number of TECCs compared to the number of TECCs in a baseline sample indicates a decrease in tumor blood vessel size in a subject.   
     
     
         18 . The method of  claim 17 , wherein detecting and analyzing a sample for the presence of TECCs further comprises (i) contacting the sample retained on a surface of the sieve with at least one antibody that is coupled to a detectable label to allow binding of the antibody to one or more target biomarkers expressed on the cells, wherein the target biomarkers is selected from the group consisting of PAI-1, Vimentin, FOXC1, keratin-8, keratin-18, VWF, PECAM-1, CD34, PSMA, CD105, CD309, CD144, and CD202B; and (ii) removing unbound antibody from the sample. 
     
     
         19 . The method of  claim 18 , wherein the detectable label is selected from the group consisting of a fluorescent group, a radioisotope, a stable isotope, an enzymatic group, a chemiluminescent group, and a biotinyl group. 
     
     
         20 . The method of  claim 17 , wherein the subject is susceptible to or suffering from cancer. 
     
     
         21 . The method of  claim 20 , wherein the cancer is:
 (a) a group consisting of carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, colon cancer, rectal cancer, breast cancer, prostate cancer, renal cell cancer, transitional cell carcinoma, lung cancer, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary, ovarian carcinoma, endometrial carcinoma, glioblastoma, neuroblastoma, Hodgkin lymphoma and non-Hodgkin lymphomas;   (b) an invasive and/or metastatic cancer;   (c) a stage I cancer, stage II cancer, stage III cancer, or stage IV cancer; and/or   (d) early stage cancer.