Profiling protein expression in circulating tumour cells using microfluidic western blotting

Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.


Supplementary Figure 2. Sensitivity of EpCAM antigen to enzymatic detachment. (A)
Fluorescence intensity histograms of MCF7 cells labeled with isotype control (AlexaFluor488mouse IgG) or anti-EpCAM-Alexa Fluor 488 antibody. Cells were detached either by 5mM EDTA (top) or trypsin-EDTA (bottom). In both cases, >98% of labeled cells were positive for EpCAM, using the isotype controls for EpCAM-negative gating (99±0.06% for trypsin/EDTA and 98±0.05% for EDTA, n=4 for both groups). (B) Forward scatter vs. side scatter plot of the cells detached by Trypsin-EDTA and stained with EpCAM, showing the gating strategy for live MCF7 cells. Figure 3. Inter-and intra-assay technical variation of protein expression in scWB. (A) Comparison of GAPDH protein expression in scWB technical replicates using the indicated cell lines (scWB performed as described in the main text). Mann-Whitney U-test p-values were 0.1257, 0.7578 and 0.7815 for BT20 (n=59 and 65), SKBR3 (n=34 and n=30) and MCF7 (n=42 and 40) respectively, confirming the null hypothesis that the distributions of the technical replicates are equal. The red horizontal line represents the mean, and the upper and lower whiskers represent the 75 th and 25 th percentile respectively. (B) Brightfield and fluorescence images of MCF7-GFP cells in microwells, representative fluorescence micrograph and intensity profiles of GFP in the well and anti-GFP antibody probe signal, and table containing GFP and antibody probe signal values used to estimate intra-assay technical variation threshold. GFP-expressing cells are imaged by fluorescence microscopy in scWB wells prior to cell lysis and fluorescence of the cell is quantified. The scWB is performed (as described in the main text) and GFP is detected with anti-GFP antibodies. Antibody probe signal (area-under-the curve, AUC) and coefficient of variation (CV) is quantified for cells that had <5% variation in intact GFP cell fluorescence. The technical variation threshold is calculated as the mean CV (11.0) plus three standard deviations (7.1, for a 99.7% confidence interval) yielding a CV threshold of 32.4%. Notably, all protein expression CVs from Fig. 2C (12 proteins, all three cell lines) are above the technical variation threshold, and thus we measure biological variation in the cell lines. Figure 4. Patient-derived CTCs present a more lysis-hardy biophysical phenotype than breast cancer cell lines as determined by rare-cell scWB. (A) Lysis conditions of the CTCs processed in the scWB. Differences in cell phenotype between breast cancer cell lines and CTCs required additional lysis optimization to assay proteins in CTCs. The lysis conditions tested were mild (10s lysis, 0.5% SDS, 0.1% Triton X-100, 0.25% Na-DOC at 45 o C), moderate (15s lysis, 0.5% SDS, 0.1% Triton X-100, 0.25% Na-DOC at 55 o C and 15s lysis, 1.0% SDS, 0.1% Triton X-100, 0.25% Na-DOC at 55 o C) and harsh (20s lysis, 1.0% SDS, 1.0% Triton X-100, 0.5% Na-DOC at 60-65 o C). Lysis effectiveness, the ability to resolve 1 or more proteins in a CTC, was rated for the CTCs assayed (e.g. No proteins resolved (poor/-), GAPDH only resolved (moderate/+), GAPDH and other proteins resolved (thorough/++)). (B) scWB molecular mass calibration shows expected relationship between logarithm of molecular mass (M r x 10 -3 ) and relative mobility (migration distance normalized to distance migrated for smallest protein target) for the 8-target protein sub-panel (eIF4e, EpCAM, GAPDH, ERK, βTUB, panCK, ER and mTOR, with EpCAM considered a tetramer). Comparison of calibration to patient-derived CTC proteins show as-expected electromigration by all targets, except ER and panCK (as discussed in the main text).

Supplementary Figure 5. scWBs of patient-derived cells positive for the tumor protein profile.
Large, nucleated cells isolated via the Vortex chip were individually deposited into microwells for subsequent scWB analysis. The 8-plex protein profile was applied to each isolate cell. Scale bar is 100 μm.

Supplementary Figure 6. Expression level distributions of the 8-protein panel for patientderived CTCs and WBCs as determined by rare-cell scWB. While both WBCs and CTCs
show detectable CD45 and GAPDH levels, the WBCs show no other detectable protein from the panel assayed. For all the plots, the box represents the 25 th /75 th percentile, and the small square box represents the mean. Here, the whiskers are determined by the outermost data point the falls within the upper and lower inner fence (75 th percentile ± interquartile range).

Supplementary Table 1. Rare-cell scWB protein panel includes both surface and intracellular proteins.
Proteins and corresponding molecular masses are interrogated by the rare-cell scWB assay for both the cell lines (SK-BR-3, MCF7, BT20) and the patient-derived CTCs from ER+ metastatic breast cancer blood samples. Note EpCAM is thought to also exist in both dimer and tetramer forms, with electromigration measured by scWB on cell line lysates consistent with the molecular mass of a tetramer.

Supplementary Table 2. Protein expression variance among breast cancer cell lines as determined by rare-cell scWB.
Supplementary Table 3. Protein expression correlations among the spiked cell lines as determined by rare-cell scWB. The Spearman correlation and p-values were calculated for all protein pairs as described in the main text. Items highlighted in green represent significant correlations with p-values < 0.01.