Fig 1.
Overview of ISET® filtration workflows.
(A) ISET® workflow for isolation and downstream analysis of fixed Circulating Rare Cells (CRC) from 10 mL of whole blood (See Methods section 3 for details). The filter can subsequently be sent by post or stored in biobank for years [21] for further CRC staining, cyto-morphological analysis and counting, immuno-labeling, in situ hybridization and molecular analyses (with or without laser capture microdissection). (B) ISET® workflow for dual collection of plasma and enrichment of fixed CRC from whole blood (See Methods, section 4 for details). (C) ISET® workflow for enrichment and downstream analysis of live CRC from whole blood (See Methods, section 5 for details). Optionally, single-cells can be isolated by micromanipulation for further analyses or CRC can be purified by immune-magnetic depletion of CD45+ cells before further molecular or cell growth assays.
Fig 2.
ISET® sensitivity in vitro assays using individually micropipetted tumor cells.
(A) In vitro sensitivity assay using the ISET® workflow described in Fig 1A (See Methods sections 3 and 6 for details). Image: example of recovered fixed cell stained with Cell TrackerTM Orange (larger panel: TRITC filter only, lower magnification, or smaller panels (higher magnification, TRITC filter only or merge of TRITC filter and brightfield)). (B) In vitro sensitivity assay using the ISET® workflow described in Fig 1C (See Methods, sections 5 and 6 for details). Image: example of recovered live cell stained with Cell TrackerTM Orange (TRITC filter). (C) In vitro sensitivity assay using the ISET® workflow described in Fig 1A (See Methods sections 3 and 6 for details) with precise counting of cells by dilution. Image: example of recovered fixed cell stained with Cell TrackerTM Orange (TRITC filter only or merge of TRITC filter and brightfield)
Fig 3.
Cell size assessment of tumor cells from human and mouse tumor cell lines.
(A) Box plot of cell sizes (diameters) according to each cancer cell line tested. Each box has its ends at the quartiles, and the median of distribution is marked by a line within the box. Error bars indicates maximum and minimum diameters. Cells from human and mouse tumor cell lines were incubated 3 min with the Rarecells® Buffer without blood and recovered on standard (8 micron-pore) ISET® filters. MMTV-PyMT*: values measured for MMTV-PyMT cells isolated using 5 micron-pores ISET® filters. Cells were stained on filters using May-Grünwald Giemsa before analysis by microscopic observation. Ns: Not significant. (B) Size distribution of two series of 50 MMTV-PyMT cells on ISET® filters isolated using 8 micron-pore filters (8a and 8b) and 5 micron-pores filters (5a and 5b).
Table 1.
In vitro assay of the repeatability and reproducibility of ISET® sensitivity tests for isolation of fixed cells.
Table 2.
In vitro assay of ISET® sensitivity threshold for isolation of fixed A549 cells.
Table 3.
In vitro assay of ISET® sensitivity using various types of cancer cells.
Fig 4.
In vitro assay of ISET® linearity.
(A) Number of tumor cells (A549) detected on ISET® filters plotted against expected number of tumor cells. Cells were spiked into whole blood after counting by dilution (30 to 300 cells) or counting by individual cells micromanipulation (2 cells). 0 cells (n = 4 replicates), 2 cells (n = 30), 30 cells (n = 9), 100 cells (n = 13) or 300 A549 cells (n = 2) were added to 1 mL of blood. Error bars (when visible) indicate standard error. (B) Mean % of tumor cells (A549) detected on ISET® filters plotted against expected number of tumor cells spiked into whole blood. (C) Number of tumor cells (A549) observed on ISET® filters plotted against expected number of tumor cells after extrapolation to 10 mL of blood (log scale). In addition to the test with 2 cells spiked in 10 mL (n = 6 replicates), to facilitate comparison with different volumes of blood, tests in 1 mL or 5 mL were plotted as their equivalent in 10 mL (See Methods). Error bars (when visible) indicate standard error. (D) Number of tumor cells (HeLa) detected on ISET® filters plotted against expected number of tumor cells. Cells were added into the whole blood after their counting by dilution (50 to 100 cells) or after their counting by individual cells micromanipulation (1 or 3 cells). 1 cell (n = 3 replicates), 3 cells (n = 3), 50 cells (n = 14) or 100 cells (n = 9) were added to 1 mL of blood (See Methods). Error bars (when visible) indicate standard error.
Fig 5.
Assessment of ISET® intra-assay accuracy and precision.
50 A549 cells were spiked into 5 mL of blood (n = 3 experiments, 43 to 49 cells per 5 mL). The number of tumor cells found on each spot after ISET® filtration (each corresponding to the filtration of 1 mL of blood) was recorded. Experiments were done on 5 spots but for intra-assay precision and accuracy only the assessment of the number of cells found on single spots or randomly grouped spots (any 1, any 2, any 3, any 4 spots) is relevant. The cell counting on the combination of all the 5 spots was used as reference. Results show that cell counting on four spots exhibited a representative mean tumor cells value per mL of blood. (A) Bar chart with the mean tumor cell number per spot and corresponding standard error of the mean (error bars) depending on the number of spots analyzed. Error bars are calculated using the standard deviation in different combinations of any 4 spots, any 3 spots, any 2 spots or any 1 spot, respectively. If only one spot is considered, standard deviation is higher than when counting 4 spots, showing that counting on four spots gives a reliable mean tumor cells' value per mL of blood. (B) Table indicating the number of tumor cells found on each spot for each of the five experiments, the 95% confidence interval (CI), the precision (%CV) and the accuracy (%error) depending on the number of spots analyzed (1 to 4) as compared to the analysis on five spots.
Table 4.
In vitro assay of ISET® sensitivity with dual collection of tumor cells and plasma.
Table 5.
In vitro assay of ISET® sensitivity for enrichment of live tumor cells.
Fig 6.
Cell sizes and viability before and after live cell enrichment.
Cell sizes (median diameter) (A) or viability (B) according to each cancer cell line tested before or after live cell enrichment (n = 3 experiments). Error bars indicate standard error. Cells from human and mouse tumor cell lines were incubated 5 min with the Rarecells® Live Cells Buffer with blood and recovered on standard (8 micron-pore) ISET® filters. MMTV = MMTV-PyMT. Cell size and viability were analyzed using the TC20™ Automated Cell Counter (Bio Rad) and Trypan Blue stain.
Fig 7.
Comparison of EpCAM fluorescence on labeled cells before and after live cell enrichment by ISET®.
Representative images of MCF-7 cells before live cell enrichment showing EpCAM fluorescence alone (A1) and merged Hoechst and EpCAM fluorescence with bright field image (A2) are each presented with a scale bar of 50 microns. Representative images of MCF-7 cells after live cell enrichment showing EpCAM fluorescence alone (B1) and merged Hoechst and EpCAM fluorescence with bright field image (B2) are each presented with a scale bar of 50 microns. (C) Comparison of cell distribution across three levels of EpCAM expression for 50 MCF-7 cells before and 50 MCF-7 cells after live cell enrichment. The low EpCAM group comprises cells with corrected total cell fluorescence (CTCF) below 91000 units, the medium EpCAM category regroups cells with CTFC between 91000 and 200000 units and the high EpCAM group contains cells with CTCF above 200000 units. (D) Comparison of median corrected total cell fluorescence (CTCF) calculated on 50 MCF-7 cells before and 50 MCF-7 cells after live cell enrichment. Error bars indicate standard error.
Fig 8.
In vitro culture of A549 tumor cells after enrichment from blood using ISET®.
Representative bright field images of A549 cells after live cell enrichment from blood and in vitro culture were obtained at 3 distinct time points. Scale bar of 50 microns. (A1, A2) Images at day zero (D0 = 2h of culture) showing A549 cells after live cell enrichment from blood obtained using the 10X (A1) and the 40X objective (A2). At 40X (A2) a single normal leucocyte is pointed by a single black arrow and a representative tumor cell is pointed by two arrows. (B1, B2) Images at day 2 (D2 = 2 days of culture) showing A549 tumor cells growing after live cell enrichment from blood obtained using the 10X (B1) and the 40X objective (B2). (C1, C2) Images at day 5 (D5 = 5 days of culture) showing A549 tumor cells growing after live cell enrichment from blood obtained using the 10X (C1) and the 40X objective (C2).
Fig 9.
Cytoskeleton analysis of A549 and H2228 cells before and after live cell enrichment by ISET® and in vitro culture.
Representative images of A549 and H2228 cells showing merged Hoechst (blue), actin (red) and tubulin [41] fluorescence were taken after 72h of in vitro culture using the 63X objective and are each presented with a scale bar of 20 microns. Images of A549 cells before filtration show examples of cells in G1/S phase (A1) and during mitosis (A2) for comparison with images showing A549 cells after filtration in G1/S phase (B1) and during mitosis (B2). Images of H2228 cells before filtration show examples of cells in G1/S phase (C1) and during mitosis (C2) for comparison with images showing H2228 cells after filtration in G1/S phase (D1) and during mitosis (D2). Comparisons of median corrected total cell fluorescence (CTCF) of actin (E) and of tubulin (F) calculated on 30 A549 and 30 H2228 cells before and 30 A549 and 30 H2228 cells after live cell enrichment. Error bars indicate standard error.
Table 6.
In vitro assay of ISET® sensitivity for enrichment of live tumor cells followed by CD45-immunomagnetic mediated leukocytes depletion.
Fig 10.
Ion TorrentTM molecular characterization of A549 and HCT116 cells before and after live cell enrichment from blood using ISET®.
Comparison of variants' mutant allele frequencies in bulk extracted DNA of approximately 104 A549 (A) and 104 HCT116 (B) cells at 72h of in vitro culture, before and after live cell enrichment. Correlation of variant allele frequencies in bulk extracted DNA of approximately 104 A549 (C) and 104 HCT116 (D) cells at 72h of in vitro culture, before and after live cell enrichment.
Fig 11.
Ion TorrentTM molecular characterization of single HCT116 and leukocytes enriched from blood using ISET®.
(A) Non-sense variants mutant allele frequency from Catalogue Of Somatic Mutation In Cancer (COSMIC) database in whole genome amplified DNA from single HCT116 tumor cells (H1 to H3), whole genome amplified DNA single leukocytes (L1 to L3), whole genome amplified bulk HCT116 DNA (WH), unamplified bulk HCT116 DNA (BH) and unamplified bulk extracted DNA from the blood donor (BL). (B) Venn diagram showing the concordance of COSMIC non-sense variants determination in whole genome amplified DNA from single HCT116 tumor cells (H1 to H3) as compared to unamplified bulk HCT116 DNA (bulk HCT116) and whole genome amplified bulk HCT116 DNA (WGA). # indicates SMARCB1 R201* and ## indicate CTNNB1 S45P, NOTCH1 L1574P and RB1 E137*. (C) Venn diagram showing the concordance of COSMIC non-sense variants determination in whole genome amplified DNA from single leukocytes (L1 to L3) as compared to unamplified bulk extracted DNA from the blood donor (bulk).
Table 7.
In vitro sensitivity and recovery of various CTC methods.
Fig 12.
CTC characterization possibilities after CTC isolation or enrichment by ISET®.
(A) CTC characterization possibilities after fixed CTC isolation by ISET®. (A1)-Enriched cells are stained on the filter and CCC can be identified by cytopathology [16] and precisely counted. CTC can also be characterized by simple or multiple immuno-fluorescence-labeling [11, 17, 18], simple or multiple immuno-cytochemistry labeling [10, 12, 19, 20], or FISH [10, 21–23]. (A2) CTC can be characterized by molecular analysis (PCR, next generation sequencing …) after laser microdissection of the filter ([10, 17, 24–26, 100]). (A3) CTC can be characterized by molecular DNA and RNA analyses without microdissection using sensitive methods for detection of mutation such as Competitive Allele-Specific TaqMan® (CAST)-PCR, co-amplification at lower denaturation temperature (COLD)-PCR, Digital PCR, next generation sequencing, or RT-PCR [27]. (B) CTC characterization possibilities after live CTC enrichment by ISET®. (B1) Enriched CTC are collected in suspension and can be optionally immuno-stained or further enriched by CD45 depletion. CTC can be precisely counted after immune-labeling. (B2) Molecular analysis such as PCR and sanger sequencing, next generation sequencing (this study), RNA analysis, DNA methylation analysis [101] and proteomic [102] can be targeted to CTC after single cell isolation by micromanipulation (manual or by robot such as CellCelectorTM) or dielectrophoresis (DEPArrayTM). Additionally, mutation detection can be performed without single cell isolation on samples in which CTC have been identified using sensitive mutation-detection methods such as CAST-PCR, COLD-PCR, Digital PCR or next generation sequencing. (B3) Samples can be used for short-term culture, in vivo or in vitro expansion and functional assays.