Cytoskeleton analysis of A549 and H2228 cells before and after live cell enrichment by ISET^® and <i>in vitro</i> culture.

<p>Representative images of A549 and H2228 cells showing merged Hoechst (blue), actin (red) and tubulin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref041" target="_blank">41</a>] fluorescence were taken after 72h of <i>in vitro</i> 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.</p

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

<div><p>Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET^® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET^® <i>in vitro</i> performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.</p></div

Overview of ISET^® filtration workflows.

<p>(A) ISET^® workflow for isolation and downstream analysis of fixed Circulating Rare Cells (CRC) from 10 mL of whole blood (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a> section 3 for details). The filter can subsequently be sent by post or stored in biobank for years [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref021" target="_blank">21</a>] for further CRC staining, cyto-morphological analysis and counting, immuno-labeling, <i>in situ</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>, section 4 for details). (C) ISET^® workflow for enrichment and downstream analysis of live CRC from whole blood (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>, 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.</p

<i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.

<p><i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.</p

<i>In vitro</i> assay of ISET^® sensitivity threshold for isolation of fixed A549 cells.

<p><i>In vitro</i> assay of ISET^® sensitivity threshold for isolation of fixed A549 cells.</p

<i>In vitro</i> assay of ISET^® linearity.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>). Error bars (when visible) indicate standard error.</p

Comparison of EpCAM fluorescence on labeled cells before and after live cell enrichment by ISET^®.

<p>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.</p

Assessment of ISET^® intra-assay accuracy and precision.

<p>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.</p

CTC characterization possibilities after CTC isolation or enrichment by ISET^®.

<p>(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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref016" target="_blank">16</a>] and precisely counted. CTC can also be characterized by simple or multiple immuno-fluorescence-labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref011" target="_blank">11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref018" target="_blank">18</a>], simple or multiple immuno-cytochemistry labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref019" target="_blank">19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref020" target="_blank">20</a>], or FISH [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref021" target="_blank">21</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref023" target="_blank">23</a>]. (A2) CTC can be characterized by molecular analysis (PCR, next generation sequencing …) after laser microdissection of the filter ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref024" target="_blank">24</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref026" target="_blank">26</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref100" target="_blank">100</a>]). (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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref027" target="_blank">27</a>]. (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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref101" target="_blank">101</a>] and proteomic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref102" target="_blank">102</a>] can be targeted to CTC after single cell isolation by micromanipulation (manual or by robot such as CellCelector^TM) or dielectrophoresis (DEPArray^TM). 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, <i>in vivo</i> or <i>in vitro</i> expansion and functional assays.</p

Cell sizes and viability before and after live cell enrichment.

<p>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.</p