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Miniaturized protein profiling permits targeted signaling pathway analysis in individual circulating tumor cells to improve personalized treatment - PubMed

  • ️Mon Jan 01 2024

Miniaturized protein profiling permits targeted signaling pathway analysis in individual circulating tumor cells to improve personalized treatment

Mahdi Rivandi et al. J Transl Med. 2024.

Abstract

Background: Traditional genomic profiling and mutation analysis of single cells like Circulating Tumor Cells (CTCs) fails to capture post-translational and functional alterations of proteins, often leading to limited treatment efficacy. To overcome this gap, we developed a miniaturized 'protein analysis on the single cell level' workflow-baptized ZeptoCTC. It integrates established technologies for single-cell isolation with sensitive Reverse Phase Protein Array (RPPA) analysis, thus enabling the comprehensive assessment of multiple protein expression and activation in individual CTCs.

Methods: The ZeptoCTC workflow involves several critical steps. Firstly, individual cells are labeled and isolated. This is followed by cell lysis and the printing of true single cell lysate preparations onto a ZeptoChip using a modified micromanipulator, CellCelector™. The printed lysates then undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader. Finally, signal quantification is carried out with Image J software, ensuring precise measurement of multiple protein levels.

Results: The efficacy of ZeptoCTC was demonstrated through various applications. Initially, it was used for measuring EpCAM protein expression, a standard marker for CTC detection, revealing higher levels in single MCF-7 over MDA-MB-231 tumor cells. Furthermore, in Capivasertib (Akt-inhibitor)-treated MCF-7 single cells, ZeptoCTC detected a 2-fold increase in the pAkt/Akt ratio compared to control cells, and confirmed co-performed bulk-cell western blot analysis results. Notably, when applied to individual CTCs from metastasized breast cancer patients, ZeptoCTC revealed significant differences in protein activation levels, particularly in measured pAkt and pErk levels, compared to patient-matched WBCs. Moreover, it successfully differentiated between CTCs from patients with different Akt1 genotypes, highlighting its potential to determine the activation status of druggable cancer driving proteins for individual and targeted treatment decision making.

Conclusions: The ZeptoCTC workflow represents a valuable tool in single cell cancer research, crucial for personalized medicine. It permits detailed analysis of key proteins and their activation status of targeted, cancer-driven signaling pathways in single cell samples, aiding in understanding tumor response, progression, and treatment efficacy beyond bulk analysis. The method significantly advances clinical investigations in cancer, improving treatment precision and effectiveness. The workflow will be applicable to protein analysis on other types of single cells like relevant in stem cell, neuropathology and hemopoietic cell research.

Keywords: Breast cancer; Circulating tumor cell; Personalized medicine; Protein analysis; Single cell proteomics.

© 2024. The Author(s).

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1

Overview of the established workflow. Depicted is the established workflow for isolating and analyzing single CTCs with Reverse Phase Protein Arrays (RPPA). The workflow consists of three primary steps: (A) Capture and verification of CTCs using Diagnostic Leukapheresis (DLA) combined with the CellSearch® system. (B) Single cell/CTC picking with a glass capillary, followed by in situ lysing and mixing on a Micropick 48 slide™ and subsequent printing onto ZeptoChip using a modified CellCelector™ instrument. (C) RPPA analysis of the chip using the ZeptoReader

Fig. 2
Fig. 2

Optimization of conditions and hardware for single cell lysis and manipulation. (A) Monitoring of single cell lysis efficiency upon variation of lysis buffer composition. For better visualization, MCF-7 cells were prelabeled with Celltracker Green and NucBlue™ Live ReadyProbes™, and fluorescence images under the microscope were taken at indicated time points during lysis; Scale bar: 25 μm, Magnification:10x; DAPI (nucleus) and FITC (cytoplasm); (B) Lysis efficiency: comparison of complete lysis durations (min) for different CLB1:CSBL1 ratios; (C) Design of the holder for the ZeptoChip (left) and the adapter for the Micropick 48® slide, the Magnetpick® slide, and for the ZeptoChip (plus its holder) to position them precisely onto the CellCelector’s microscope stage (right)

Fig. 3
Fig. 3

EpCAM protein expression signals measured from single cell preparations using ZeptoChip. Quantification of fluorescence signals depicted from two MCF-7 and two MDA-MB-231 single cell lysate samples, respectively. The mean fluorescence signals (MFI) of both MCF-7 single cells were significantly higher than those of both MDA-MB-231 cells (p-value = 0.0435). Error bars indicate the standard deviations of the mean technical replicate spot signals (n = 3)

Fig. 4
Fig. 4

Expression signals of Akt and pAkt (Ser473) in treated single MCF-7 cells (description see main text)

Fig. 5
Fig. 5

Evaluation of phosphorylated Akt and Erk1/2 proteins in single CTCs derived from a MBC patient. (A) False color clippings of RPPA Zeptosens fluorescence array images (exposure 4s) for analysis of pAkt and pErk1/2 protein levels in CTC and WBC single cells from a MBC patient, and comparison to bulk cell QC (MCF) controls (3 replicate spots printed per sample). (B) Mean fluorescence signals (MFI) as quantified by ImageJ: elevated pAkt and pErk1/2 levels in CTCs compared to WBCs in line with QC lysates; pErk1/2 abundancy signal 3–11 times higher than pAkt as evident from different MFI scales

Fig. 6
Fig. 6

Measuring pAkt to Akt protein in single CTCs derived from two index MBC patients. (A) (left) RPPA Zeptosens analysis of pAkt and Akt protein signals (MFI) in single CTC and WBC samples derived from index patient 1 harboring an Akt1(E17K) mutation, in addition to co-printed quality control samples (QC treated, QC control); (right) pAkt/Akt mean signal ratios of respective patient 1 single CTC and WBC samples. (B) (left) RPPA Zeptosens analysis of pAkt and Akt protein signals (MFI) in single CTCs and WBCs from index patient 2 (wild type); (right) pAkt/Akt mean signal ratios of respective patient 2 single CTC and WBC samples

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