Profiling protein expression in circulating tumour cells using microfluidic western blotting

Abstract

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.

Figure 1. Microfluidic rare-cell workflow for multiplexed western blotting of single patient-derived CTCs.

Patient-derived CTCs are: Step i: enriched from 2 to 4 ml of blood using a size- and deformability-selective microfluidic tool (Vortex HT chip) followed by Step ii: cell-enriched effluent (300 μl) is deposited directly in a mesofluidic chamber on planar scWB device. Putative CTCs are visually identified using Hoescht 33342 nuclear stain, and each identified CTC is micropipetted (under microscopy) into a 50 μm diameter microwell (micrographs in inset). Step iii: after the seating of one CTC into one microwell, single-cell western blotting proceeds as in-microwell chemical CTC lysis, single-CTC protein PAGE, covalent immobilization of proteins to the gel (photo-blotting) and in-gel immunoprobing. Step iv: single-CTC lysate is analysed by western blotting and rounds of immunoprobing support the multiplexing of 12 proteins, with expression is compared among patient-derived CTCs and to spiked cell line validation studies. Scale bars, 25 μm (for the cell micrographs) and 250 μm (the separation micrographs).

Figure 2. The rare-cell scWB measures 12 unique protein targets in single cells from three cancer subtypes.

(a) Fluorescence micrographs and intensity plots from rare-cell scWB handling and analysis of healthy blood samples, each spiked with a cancer subtype: EGFR+(BT-20), ER+(MCF7) and HER2+(SK-BR-3). Negative controls include analysis of WBCs only and blank microwells (that is, devoid of a cancer cell). Protein panel comprises the following: control and housekeeping proteins (GAPDH and β-tubulin), oncoproteins (HER2, ER and EGFR), signalling proteins (ERK, eIF4E and mTOR), common CTC classifiers (EpCAM, panCK and CK8) and a WBC indicator (CD45). Scale bar, 50 μm. (b) Comparative protein expression for each cancer cell (BT-20: n=27; SK-BR-3: n=27; MCF7: n=35), with mean and CV noted for each marker. Protein expression is graphed using a log-scale. Ranked oncoprotein expression for each cell line agrees with cancer subtype. Less than 1% of total protein signal is attributable to CD45.

Figure 3. Optimization of the rare-cell scWB for direct protein measurement in patient-derived CTCs.

(a) CTC counts normalized to the blood volume processed by the isolation tool. CTC count for metastatic ER+ breast cancer patients (n=12): 0.33–23.25 CTCs per ml; CTC count for age-matched healthy donors (n=6): 0.33–1.00 CTCs per ml. CTC threshold was set by mean+2 s.d. from healthy donor data at 1.06 CTCs per ml, with 81.8% of the breast cancer patients classified as positive for CTCs. Enumeration for Patient 5 was not possible, as the sample was consumed by the scWB. (b) Fluorescence micrographs and intensity plots from rare-cell scWB analysis of GAPDH in CTCs from Patients 2, 5, 6 and 10 of CTC lysis conditions. Asterisks mark GAPDH peaks. (c) Fluorescence micrographs and intensity plots from rare-cell scWB handling and analysis of representative patient-derived CTCs using nomenclature from Fig. 2a. Micrographs of rare-cell scWB of patient-derived CTCs in representative cases where CD45 was not detected (CD45−). Scale bars, 50 μm.

Figure 4. scWB profiles for eight proteins in each individual CTC derived from three ER positive metastatic breast cancer patients.

(a) Expression for each protein marker and each patient-derived CTC, with comparison with CD45 levels from scWB analyses of pure WBC controls. (b) CVs for protein expression (AUC) from the patient-derived CTCs. Dashed line indicates the threshold in protein expression variation established using GFP-expressing MCF7 cells (see Supplementary Fig. 3). (c) Biaxial plots report protein expression for all markers for each patient-derived CTC from Patients 5 (n=6), 6 (n=2) and 10 (n=12).