TY - JOUR
T1 - Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer
AU - Cackowski, Frank C.
AU - Wang, Yugang
AU - Decker, Joseph T.
AU - Sifuentes, Christopher
AU - Weindorf, Steven
AU - Jung, Younghun
AU - Wang, Yu
AU - Decker, Ann M.
AU - Yumoto, Kenji
AU - Szerlip, Nicholas
AU - Buttitta, Laura
AU - Pienta, Kenneth J.
AU - Morgan, Todd M.
AU - Taichman, Russell S.
N1 - Funding Information:
The authors graciously thank Emily Caruso, Megan Kuczler, and Amy Kasputis for management of sample collection. We also thank Dr Arul Chinnaiyan, Dr David Smith, and Dr Zachary Reichert for helpful discussions. We thank Melissa Coon and Dr Robert Lyons of the University of Michigan DNA Sequencing Core for sample preparation and generation of raw RNA-Seq data. Most importantly, we thank all the patients who donated samples to make this study possible. Direct funding was provided by the NIH/NCI P01-CA093900, Department of Defense W81XWH-14-1-0403 and The Prostate Cancer Foundation Challenge award 16CHAL05. RT received support as the Major McKinley Ash Colligate Professor. FC receives support from a Career Enhancement Award from the NIH/NCI Prostate Cancer Specialized Program in Research Excellence (SPORE) at the University of Michigan F048931, sub-award F036250, and Prostate Cancer Foundation Young Investigator Award 18YOUN04.
Publisher Copyright:
© 2019 Wiley Periodicals, Inc.
PY - 2019/10
Y1 - 2019/10
N2 - Background: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. Methods: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. Results: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients’ plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). Conclusion: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.
AB - Background: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. Methods: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. Results: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients’ plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). Conclusion: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.
KW - disseminated cancer cell
KW - dormancy
KW - flow cytometry
KW - metastasis
UR - http://www.scopus.com/inward/record.url?scp=85071486338&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85071486338&partnerID=8YFLogxK
U2 - 10.1002/pros.23896
DO - 10.1002/pros.23896
M3 - Article
C2 - 31449673
AN - SCOPUS:85071486338
SN - 0270-4137
VL - 79
SP - 1715
EP - 1727
JO - Prostate
JF - Prostate
IS - 14
ER -