Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

Abstract

The optimization of a purely negative depletion, enrichment process for circulating tumor cells, CTC's, in the peripheral blood of Head and Neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 109 to 8.04 × 103 cells/ml and still recover, on average, 2.32 CTC per ml of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per ml of blood, with a range of 282 to 0.53 CTC per ml of blood. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.76 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTC's or other cell type relevant to the specific question (i.e. does the CTC have predominately epithelia or mesenchymal characteristics?).

Characterizing deformability and surface friction of cancer cells

“…5 cells per h (∼10 4 cells detected for 6 min), which is sufficient for interrogating patient samples provided upstream enrichment strategies are used (e.g., CD45 depletion) (39,40). Therefore, in the future it may be feasible to use the SMR with constriction to identify CTCs in patient blood samples based on biophysical properties, which may reveal populations of CTCs that have gone undetected by common methods involving specific molecular probes.…”

Non-destructive and Selective Imaging of the Functionally Active, Pro-invasive Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Enzyme in Cancer Cells

“…However, in addition to the TIMP-free active MT1-MMP enzyme, there is an excess of the latent proenzyme and the enzyme⅐TIMP inactive complexes on cell surfaces. Current detection methodologies, including immunocytochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, do not discriminate among these MT1-MMP species and do not allow tracing of the cellular MT1-MMP activity (53). To specifically image the active MT1-MMP alone, we have previously developed genetically encoded FRET biosensors and showed that these biosensors were capable of visualizing MT1-MMP activity in live cells (54).…”

Circulating tumor-cell detection and capture using microfluidic devices

“…An interesting approach with a negative CTC-enrichment technique was also developed utilizing the combination of viscous flow and magnetic force to facilitate the recovery of unlabeled cells (CTCs) from whole-blood samples obtained from cancer patients [76,77]. The method was based on removal of erythrocytes by lysis followed by the magnetic separation of immunomagnetically-labeled (with CD45 antibody) leukocytes.…”