Research Article: Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration

Date Published: January 27, 2017

Publisher: Public Library of Science

Author(s): Sreeraj G. Pillai, Peixuan Zhu, Chidananda M. Siddappa, Daniel L. Adams, Shuhong Li, Olga V. Makarova, Pete Amstutz, Ryan Nunley, Cha-Mei Tang, Mark A. Watson, Rebecca L. Aft, Harriet Wikman.

http://doi.org/10.1371/journal.pone.0170761

Abstract

Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.

BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.

Filtering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.

We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.

Partial Text

Disseminated tumor cells (DTCs) are detected in the bone marrow (BM) of up to 40% of early stage breast cancer patients at the time of diagnosis and are an independent prognostic factor for recurrent disease development[1]. DTCs found in the BM are shed from primary breast cancers and are thought to be intermediaries in the metastatic process[2]. DTCs are rare cells, found with a frequency of about 1 cancer cell per million nucleated BM cells[1], are molecularly heterogeneous, and are often molecularly distinct from their primary tumor of origin[3–5]. Not all patients with BM DTCs, detectable by conventional epithelial markers such as cytokeratin, will develop metastatic disease [6], indicating that DTCs themselves likely differ with regard to further metastatic potential. Molecular analysis of DTCs offers the possibility of identifying specific DTCs with metastatic potential, designing targeted therapies to eliminate these cells, monitoring alterations in tumor cell phenotype and genotype, and predicting therapeutic response[2]. Analysis of DTCs offers information that may not be obtainable in early stage breast cancer patients by studying circulating tumor cells (CTCs), which are found with greater rarity in the peripheral circulation[7,8].

In this study, we have demonstrated the effective use of a sized based filtration-method for DTC enrichment from BC patient BM aspirate specimens. While eliminating biases associated with antigen-capture based approaches, we demonstrate that this method allows for efficient recovery and enrichment of DTCs, which may then be readily utilized for sensitive downstream detection methodologies such as immunocytochemical, in situ hybridization, or qPCR-based gene expression assays. Though many technologies have been used to enrich for CTCs, few of these have been tested using BM for DTCs (summarized in[33]) and we believe that this is the first report of using size filtration for DTC enrichment.

 

Source:

http://doi.org/10.1371/journal.pone.0170761