In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment

Abstract

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAF V600E ) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

MYCN Impact on High-Risk Neuroblastoma: From Diagnosis and Prognosis to Targeted Treatment

“…Interestingly, circulating messenger RNA (mRNA), circulating tumor cells (CTCs) and circulating NB exosomes can also be found in biological fluids and used as biomarkers for diagnosis and prognosis assessment [ 44 ]. In particular, CTCs can provide comprehensive tumor profiling involving RNA, protein and/or metabolic information, while cfDNA only contains a genomic statement [ 45 , 46 ]. Of course, this method is more expensive and slower than cfDNA use [ 47 ], but it can be extensively considered as complementary in HR-NB evaluation, as suggested in other cancer studies [ 48 , 49 ].…”

Recent advances in microfluidic devices for single-cell cultivation: methods and applications

“…13,204 Single-cell analysis using microfluidic systems at the downstream has been extensively studied in probing cellular phenotypes and cell-cell interactions. Numerous cells, such as bacteria, yeasts, tumor cells, immune cells, and neurons, have been studied to analyze genes and genomes, 18,49,144,146,205,206 transcriptomes, 20,21,193,[207][208][209][210] proteins, 57,116,149,[211][212][213][214] and metabolites. 19,139,215,216 The types of microfluidics for downstream analysis can be divided based on the location of the analysis, i.e., on-chip and offchip.…”

“…After drug treatment, the average SERS intensities from three surface protein markers decrease over time, indicating the reduction of cell malignancy. 211 In addition to advanced methods, a chemiluminescent (CL) detection-based low-cost 3D printed microfluidic array is used for single-cell proteomics. The device consists of 5 inlets, 5 sample chambers (i.e., sample, biotinylated secondary antibodies (Biotin-Ab 2 ), streptavidin conjugated with the polymer of horseradish peroxidase (St-Poly-HRP), CL reagent, and PBS buffer chamber), 8 detection chambers, programmable micropumps, and an ultrasonic lysis probe.…”

Application of Single-Cell Multi-Omics in Dissecting Cancer Cell Plasticity and Tumor Heterogeneity

“…Recently, single-cell proteomics techniques have also been developed to investigate tumor heterogeneity and uncover the mechanisms of tumor progression (Wagner et al, 2019;Liu L. et al, 2020;Reza et al, 2021). For example, Wagner et al analyzed 144 human breast tumors and 50 non-tumor tissues using cytometry by time-of-flight (CyTOF).…”