Ex Vivo Expansion and Drug Sensitivity Profiling of Circulating Tumor Cells from Patients with Small Cell Lung Cancer
Cancers2020,12, 3394. doi:10.3390/cancers12113394
Abstract
Small cell lung cancer (SCLC) represents one of the most aggressive malignancies among cancer types. Not only tumor sample availability is limited, but also the ability for tumor cells to rapidly acquire drug resistance are the rate-limiting bottlenecks for overall survival in current clinical settings. A liquid biopsy capable of capturing and enriching circulating tumor cells (CTCs), together with the possibility of drug screening, is a promising solution. Here, we illustrate the development of a highly efficient ex vivo CTC expansion system based on binary colloidal crystals substrate. Clinical samples were enrolled from 22 patients with SCLC in the study. The CTCs were enriched and expanded from the collected peripheral blood samples. Expanded cells were analyzed for protein expression and observed for drug sensitivity with the use of immunofluorescence and ATP titer evaluation, respectively. Successful CTC spheroid proliferation was established after 4 weeks within 82% of all the collected peripheral blood samples from enrolled patients. Upon immunofluorescence analysis, the enriched cells showed positive markers for EpCAM, TTF-1, synaptophysin and negative for CD45. Additionally, the expanded CTCs demonstrated marked heterogeneity in the expression of E-cadherin and N-cadherin. In a preliminary case series, the drug sensitivity of patient-derived CTC to cisplatin and etoposide was studied to see the correlation with the corresponding therapeutic outcome. In conclusion, our study demonstrates that it is possible to efficiently expand CTCs from SCLC within a clinically relevant time frame; the biomarker information generated from enriched CTCs can assist the selection of effective drugs and improve disease outcome.
Ex vivo expansion of CTCs from small cell lung cancer (SCLC) patients. (a) Light microscopic images (upper panel) and quantitative analysis of spheroid numbers (lower panel) of the expanded CTCs 14 (left) and 40 (right) days. (b) The viability of expanded CTCs. On day 14 and day 40, the expanded CTCs were stained with a LIVE/DEAD staining kit. Green and red colors, respectively, indicate live and dead cells. The bar graph presents the percentage of live and dead cells in expanded CTCs.
Bright-field images of CTC-derived spheroid phenotypes. Representative examples of expanded CTCs morphology are shown, (a) large cohesive CTC spheroids (left), (b) small regular and irregular cohesive CTC spheroids (middle) and © discohesive CTC spheroids (right). Scale bar indicates 100 μm.
Molecular characterization of expanded CTCs from SCLC patients. Immunofluorescence staining revealed (a) EpCAM+ (green)/CD45- (red)/Hoechst+ (blue) cultured CTCs. Prevalent SCLC biomarkers of (b) TTF-1 and © synaptophysin were used to confirm that expanded CTCs derived from lung tissues. (d) PD-L1 expression in SCLC. Nuclei of cells were stained with Hoechst 33342 (blue). Scale bar indicates 20 μm. Images are representative of two independent SCLC patient samples.
The heterogeneous composition of expanded CTC. Co-staining for E-cadherin (green) and N-cadherin (red) in expanded CTC spheroids reveals the presence of marked heterogeneity for expressions of epithelial and mesenchymal markers. Arrows indicate high levels of N-cadherin.
Expanded CTCs recapitulate the clinical response. SCLC patients’ response to standard of care treatment (cisplatin/etoposide) was evaluated by contrast-enhanced CT images before (a–c) and after (g–i) treatment. Arrows indicate tumor lesions. Before treatment, blood samples were collected to amplify CTCs and expanded CTCs were plated in 96-well plates for drug sensitivity assay. (d–f) Cell viability was detected by CellTiter Glo, and results were normalized to the untreated control group. Patients (№14 and №20) were sensitive to cisplatin/etoposide or (№15) resistant to cisplatin/etoposide as evaluated by a CTC drug sensitivity assay and clinical CT images.
