All specimens were collected into vacutainer (Becton-Dickinson) tubes containing the anticoagulant EDTA and were processed through the HB-Chip within 6?h of blood draw
All specimens were collected into vacutainer (Becton-Dickinson) tubes containing the anticoagulant EDTA and were processed through the HB-Chip within 6?h of blood draw. mean?=?386??238?CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological staining, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of malignancy. numbers (20), adapted for isolation of rare cells from whole blood by varying the ratio of height of the grooves to that of the channel, chevron sizes, and periodicity. The herringbone grooves are staggered periodically, with each mixing cycle defined by two sequential regions of ten chevrons shifted asymmetrically (Fig.?1that is relevant in this system (Fig.?S1 chimeric transcript, confirmed by nucleotide sequencing, in a patient whose main tumor harbored this translocation, but not in a patient whose tumor did not have the chromosome fusion (Fig.?3and and Fig.?S5and Fig.?S5and for additional details. Study Subjects and Blood Processing. MUC16 Patients with advanced prostate and lung malignancy were recruited according to a protocol approved by the institutional review table (IRB). Blood specimens from healthy volunteers were collected under a separate IRB-approved protocol. A total of 19 malignancy patients (15 prostate, 4 lung), who were treated at the Massachusetts General Hospital Cancer Center, donated 10C20?mL of blood on one or more occasions for analysis around the HB-Chip. All specimens were collected into vacutainer (Becton-Dickinson) tubes made up of the anticoagulant EDTA IDO-IN-4 and were processed through the HB-Chip within 6?h of blood draw. Samples were run on the previously explained microfluidic processing machine (17). Briefly, a 5?mL aliquot of blood was placed in an air-tight conical tube on a rocker assembly, and 4?mL of blood were pneumatically driven through the chip at a circulation rate of 1 1.5C2.5?mL/hr. Following, the HB-Chip was flushed with 2.5?mL of PBS at 2.5?mL/hr to remove any nonspecifically bound cells. CTC Staining and Enumeration. After processing of blood around the HB-Chip, cells were fixed with 4% paraformaldehyde, and subsequently permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS. Cells were immunostained with main antibodies, either a rabbit polyclonal antibody to PSA (DAKO), monoclonal antibody against cytokeratin 7/8 (CAM 5.2 clone, BD Biosciences), or a monoclonal mouse IgG2b antibody to PSMA (Santa Cruz Biotechnology). Following, the appropriately matched secondary AlexaFluor 488-conjugated antibody (Invitrogen) was used to identify epithelial cells. Nuclei were stained with DAPI. All samples were counterstained with mouse IgG1 antihuman anti-CD45 (Clone H130, BD Biosciences), followed by Alexa Fluor 594 fluorophore (Invitrogen) to identify any bound leukocytes. After completion of staining, all devices were IDO-IN-4 washed PBS and stored at 4?C. All devices were imaged using our previously explained imaging system (19). See for additional details. Statistical Analysis. Data are reported as mean??standard error of the mean as noted. If groups experienced a normal distribution and IDO-IN-4 homogenous variances, the group means were compared by an independent for details on the hematoxylin and eosin staining, on-chip FISH, RT-PCR analysis and sequencing, cell line preparation, circulation visualization, and malignancy cell line experiments. Supplementary Material Supporting Information: Click here to view. Acknowledgments. We express our gratitude to the all patients who participated in this study and the healthy volunteers who contributed blood samples. We are also grateful to Octavio Hurtado, Jochen Lennerz, Douglas Rubinson, Vijay Ambravaneswaran, Salil Desai, Ravi Kapur, John Walsh, Tom Barber, Justin Wong, Zev Nakamura, Matthew Ulman, Suchismita Paul, Brian Brannigan, Alessandra Moore, Maria Kempner, Brooke Nentwig, and Laura Libby for expert technical support. This work was supported by Dream Team Awards from your Prostate Malignancy Foundation and Stand Up To Malignancy (to D.A.H. and M.T.), a Quantum Grant from the National Institute for Biomedical Imaging and Bioengineering (to M.T.), National Institutes of Health/National Malignancy Institute (NIH)/(NCI) Grant CA89138 (to S.M.), and nice donations from your Ellison Foundation, AstraZeneca, the Martell Foundation, Alex and Sonja Spier, the Monell Foundation, and institutional support from Massachusetts General Hospital. S.L.S. is usually supported through an American Malignancy Society New IDO-IN-4 England Division Research Grant, S.N. is usually supported through an NIH Directors New Innovator Award, R.J.L. is usually supported by a Physician Research Training Award (Department of Defense, Prostate Malignancy Research Program) and a Career Development Award (The American Society of Clinical Oncology (ASCO) Malignancy Foundation), and D.A.H. is usually supported by the Howard Hughes Medical Institute..