Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy.

Circulating tumor cells (CTCs) are considered as surrogate markers for prognosticating and evaluating patient treatment responses. Here, 226 blood samples from 92 patients with breast cancer, including patients with newly diagnosed or metastatic refractory cancer, and 16 blood samples from healthy subjects were cultured in laser-ablated microwells. Clusters containing an increasing number of cytokeratin-positive (CK+) cells appeared after 2 weeks, while most blood cells disappeared with time. Cultures were heterogeneous and exhibited two distinct sub-populations of cells: 'Small' (≤ 25 μm; high nuclear/cytoplasmic ratio; CD45-) cells, comprising CTCs, and 'Large' (> 25 μm; low nuclear/cytoplasmic ratio; CD68+ or CD56+) cells, corresponding to macrophage and natural killer-like cells. The Small cell fraction also showed copy number increases in six target genes (FGFR1, Myc, CCND1, HER2, TOP2A and ZNF217) associated with breast cancer. These expanded CTCs exhibited different proportions of epithelial-mesenchymal phenotypes and were transferable for further expansion as spheroids in serum-free suspension or 3D cultures. Cluster formation was affected by the presence and duration of systemic therapy, and its persistence may reflect therapeutic resistance. This novel and advanced method estimates CTC clonal heterogeneity and can predict, within a relatively short time frame, patient responses to therapy.


Immunophenotyping of cells in culture
Media was removed without disturbing the clusters, and cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, St Louis, MO) at room temperature for 10 min. Fixed cells were then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, San José, CA) in PBS on ice for 1 min. Primary antibodies (Supplementary Table 4) were diluted in 200 μl of PBS supplemented with 2% bovine serum albumin (BSA) and added to the patterned dishes. Dishes were incubated for at least 1 h on ice. For secondary labeling, dishes were washed gently and incubated for ~1 h on ice with respective Dylight 488 or 594 secondary antibodies (Abcam, Cambridge, United Kingdom) and counterstained with Hoechst dye (Invitrogen).

Immunophenotyping of cells via cytospots
CTC cultures or control cell lines (embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), macrophages, endothelial cells, MCF-7 and MDA-MB-231) were trypsinized and concentrated in PBS. Cytospots were generated with 100 μl of cell suspension at 600 rpm for 5 min on frosted slides (Thermo Fisher Scientific) using a Cytospin 4 cytocentrifuge (Thermo Fisher Scientific). Slides were fixed and permeabilized as previously described for cells and incubated with a range of primary antibodies (Supplementary Table 4) for 30 min at 4°C. These antibodies were validated for their specificity with negative controls using either MCF7, MDA-MB-231 or lysed blood samples at Day 0 before culture. Slides were washed and incubated for another 30 min on ice with respective Dylight 488 or 594 secondary antibodies (Abcam, Cambridge, United Kingdom) and counterstained with Hoechst dye (Invitrogen). Putative CTCs were identified as CK+/CD45-/Hoechst+ cells. Antibody staining was conducted independently of pan-CK antibody staining to prevent cross-reactivity. Beta-galactosidase staining was performed at pH 6 to identify senescent cells.

DNA FISH
Dehydrated slides were treated with 4 mg/ml RNase (Sigma-Aldrich) in PBS for 45 min at 37°C, washed in 1× PBS/0.2% Tween-20 thrice and denatured with a 70% formamide/2×saline sodium citrate (SCC) (Sigma-Aldrich) solution for 10 min at 80°C. The slides were then dehydrated via another round of ice-cold graded ethanol series. Slides maintained at 42°C were then hybridized with probes (Supplementary Table 5), and denatured by incubation at 74°C for 5 min. Slides were sealed with rubber cement and kept at 42°C in dark and humid conditions for 16 h. Hybridized slides were washed with 50% formamide/2×SSC, followed by 2×SSC, both at 42°C with shaking, and then counterstained with Vectashield ® mounting medium containing 4,'6'-diaminido-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) prior to mounting with a glass coverslip (Thermo Fisher Scientific). Slides were imaged with an epifluorescence microscope (Nikon, Japan) and z-stacks for each channel were obtained at 63 ×. ImageJ (NIH, Bethesda, MD) was used to achieve the projected image (maximum intensity) and the fluorescence signal dots were enumerated accordingly.

RNA FISH
Cytospots were prepared as described above. Cells were then permeabilized and incubated with customized probes obtained from iDNA (Affymetrix, Santa Clara, CA) (Supplementary Table 3) using the Quantigene kit (Affymetrix) according to recommended protocols. Slides were sealed with rubber cement and kept at 42°C in dark and humid conditions for 16 h. Hybridized slides were treated with components of the kit as instructed and counterstained with Vectashield ® mounting medium containing DAPI (Vector Laboratories) prior to mounting with a glass coverslip (Thermo Fisher Scientific). Probes were selected using breast cancer expression profiling databases to identify the most differentially expressed genes based on their highest and lowest EMT scores [1][2]. All probes were added to the same treated slide, incubated overnight, washed and amplified according to the recommended protocol. Slides were then counter-stained with DAPI before mounting. Slides were imaged as described for DNA FISH.

Phagocytosis assay for macrophages
Fluorescein-labelled polystyrene microbeads (1 μm) were added at high densities to a culture dish at Day 14 of culture. Beads were incubated with the CTC cultures under optimal conditions as described for the 24-h timepoint. Dishes were then washed with PBS, fixed with 4% PFA as described and imaged with confocal microscope at 20 × magnification.