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Separable bilayer microfiltration device for viable label-free enrichment of circulating tumour cells.

Zhou MD, Hao S, Williams AJ, Harouaka RA, Schrand B, Rawal S, Ao Z, Brenneman R, Gilboa E, Lu B, Wang S, Zhu J, Datar R, Cote R, Tai YC, Zheng SY - Sci Rep (2014)

Bottom Line: Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

View Article: PubMed Central - PubMed

Affiliation: Micro &Nano Integrated Biosystem (MINIBio) Laboratory, Department of Biomedical Engineering and Materials Research Institute, Pennsylvania State University, University Park, PA 16802, U.S.A.

ABSTRACT
The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

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Related in: MedlinePlus

Characterization of device performance.(A): Capture efficiency is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (B): Enrichment factor from undiluted human blood is measured and plotted. Each column shows the mean value and its standard deviation (n = 4). (C)–(G): LIVE/DEAD assay with representative areas of the SB microfilter after filtration. Viable MDA-MB-231 cancer cells are highlighted with green (C) while dead tumour cells are in red (D). Fluorescence composite (E) and differential interference contrast (DIC)/fluorescence composite (F) of tumour cells detected in A and B. LIVE/DEAD cell assay consisting of viable cell indicator Calcein-AM green and the exclusion dye Ethidium Homodimer-1 were used. (G): Cell viability after filtration is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (H)–(K): Cell viability detected by virus infection. The mCherry-expressing MCF-7 cells were used. Infected viable cancer cells expressed green fluorescence protein. The captured MCF-7 cancer cells were highlighted with red (H) while infected viable tumour cells were in green (I). Fluorescence composite image (J) and differential interference contrast (DIC) image overlaid with fluorescence composite image (K) of tumour cells detected in (A) and (B). Scale bars are 10 μm.
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f3: Characterization of device performance.(A): Capture efficiency is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (B): Enrichment factor from undiluted human blood is measured and plotted. Each column shows the mean value and its standard deviation (n = 4). (C)–(G): LIVE/DEAD assay with representative areas of the SB microfilter after filtration. Viable MDA-MB-231 cancer cells are highlighted with green (C) while dead tumour cells are in red (D). Fluorescence composite (E) and differential interference contrast (DIC)/fluorescence composite (F) of tumour cells detected in A and B. LIVE/DEAD cell assay consisting of viable cell indicator Calcein-AM green and the exclusion dye Ethidium Homodimer-1 were used. (G): Cell viability after filtration is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (H)–(K): Cell viability detected by virus infection. The mCherry-expressing MCF-7 cells were used. Infected viable cancer cells expressed green fluorescence protein. The captured MCF-7 cancer cells were highlighted with red (H) while infected viable tumour cells were in green (I). Fluorescence composite image (J) and differential interference contrast (DIC) image overlaid with fluorescence composite image (K) of tumour cells detected in (A) and (B). Scale bars are 10 μm.

Mentions: The capture efficiency and enrichment were measured using two breast cancer cell lines: MCF-7, estrogen receptor and progesterone receptor (ER/PR) positive and less invasive; and MDA-MB-231, triple negative and highly invasive. The measured capture efficiencies were 83 ± 3% for MCF-7 and 78 ± 4% for MDA-MB-231 (n = 4 each) when ~130 cells in ~1 μL of DPBS were spiked in 1 mL of DPBS (Figure 3A). Assuming perfect capture efficiency, enrichment can be uncoupled from capture efficiency and measured independently38. The enrichment was thus determined by the retention of non-tumour leukocytes. The ratio of leukocytes inside the original blood sample and those left on the device was used as measured enrichment against leukocytes. The measured enrichment factors were 2.0 ± 0.3 × 103 for MCF-7 and 1.7 ± 0.4 × 103 for MDA-MB-231 (Figure 3B). The measured capture efficiency and enrichment factors are comparable to the previous 3D microfilters with similar double layer membrane structure37.


Separable bilayer microfiltration device for viable label-free enrichment of circulating tumour cells.

Zhou MD, Hao S, Williams AJ, Harouaka RA, Schrand B, Rawal S, Ao Z, Brenneman R, Gilboa E, Lu B, Wang S, Zhu J, Datar R, Cote R, Tai YC, Zheng SY - Sci Rep (2014)

Characterization of device performance.(A): Capture efficiency is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (B): Enrichment factor from undiluted human blood is measured and plotted. Each column shows the mean value and its standard deviation (n = 4). (C)–(G): LIVE/DEAD assay with representative areas of the SB microfilter after filtration. Viable MDA-MB-231 cancer cells are highlighted with green (C) while dead tumour cells are in red (D). Fluorescence composite (E) and differential interference contrast (DIC)/fluorescence composite (F) of tumour cells detected in A and B. LIVE/DEAD cell assay consisting of viable cell indicator Calcein-AM green and the exclusion dye Ethidium Homodimer-1 were used. (G): Cell viability after filtration is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (H)–(K): Cell viability detected by virus infection. The mCherry-expressing MCF-7 cells were used. Infected viable cancer cells expressed green fluorescence protein. The captured MCF-7 cancer cells were highlighted with red (H) while infected viable tumour cells were in green (I). Fluorescence composite image (J) and differential interference contrast (DIC) image overlaid with fluorescence composite image (K) of tumour cells detected in (A) and (B). Scale bars are 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260227&req=5

f3: Characterization of device performance.(A): Capture efficiency is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (B): Enrichment factor from undiluted human blood is measured and plotted. Each column shows the mean value and its standard deviation (n = 4). (C)–(G): LIVE/DEAD assay with representative areas of the SB microfilter after filtration. Viable MDA-MB-231 cancer cells are highlighted with green (C) while dead tumour cells are in red (D). Fluorescence composite (E) and differential interference contrast (DIC)/fluorescence composite (F) of tumour cells detected in A and B. LIVE/DEAD cell assay consisting of viable cell indicator Calcein-AM green and the exclusion dye Ethidium Homodimer-1 were used. (G): Cell viability after filtration is measured and plotted for two cell lines, MCF-7 and MDA-MB-231 human breast cancer cell lines. Each column shows the mean value and its standard deviation (n = 4). (H)–(K): Cell viability detected by virus infection. The mCherry-expressing MCF-7 cells were used. Infected viable cancer cells expressed green fluorescence protein. The captured MCF-7 cancer cells were highlighted with red (H) while infected viable tumour cells were in green (I). Fluorescence composite image (J) and differential interference contrast (DIC) image overlaid with fluorescence composite image (K) of tumour cells detected in (A) and (B). Scale bars are 10 μm.
Mentions: The capture efficiency and enrichment were measured using two breast cancer cell lines: MCF-7, estrogen receptor and progesterone receptor (ER/PR) positive and less invasive; and MDA-MB-231, triple negative and highly invasive. The measured capture efficiencies were 83 ± 3% for MCF-7 and 78 ± 4% for MDA-MB-231 (n = 4 each) when ~130 cells in ~1 μL of DPBS were spiked in 1 mL of DPBS (Figure 3A). Assuming perfect capture efficiency, enrichment can be uncoupled from capture efficiency and measured independently38. The enrichment was thus determined by the retention of non-tumour leukocytes. The ratio of leukocytes inside the original blood sample and those left on the device was used as measured enrichment against leukocytes. The measured enrichment factors were 2.0 ± 0.3 × 103 for MCF-7 and 1.7 ± 0.4 × 103 for MDA-MB-231 (Figure 3B). The measured capture efficiency and enrichment factors are comparable to the previous 3D microfilters with similar double layer membrane structure37.

Bottom Line: Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

View Article: PubMed Central - PubMed

Affiliation: Micro &Nano Integrated Biosystem (MINIBio) Laboratory, Department of Biomedical Engineering and Materials Research Institute, Pennsylvania State University, University Park, PA 16802, U.S.A.

ABSTRACT
The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Show MeSH
Related in: MedlinePlus