Label-free flow cytometry of rare circulating tumor cell clusters in whole blood

Abstract

Circulating tumor cell clusters (CTCCs) are rare cellular events found in the blood stream of metastatic tumor patients. Despite their scarcity, they represent an increased risk for metastasis. Label-free detection methods of these events remain primarily limited to in vitro microfluidic platforms. Here, we expand on the use of confocal backscatter and fluorescence flow cytometry (BSFC) for label-free detection of CTCCs in whole blood using machine learning for peak detection/classification. BSFC uses a custom-built flow cytometer with three excitation wavelengths (405 nm, 488 nm, and 633 nm) and five detectors to detect CTCCs in whole blood based on corresponding scattering and fluorescence signals. In this study, detection of CTCC-associated GFP fluorescence is used as the ground truth to assess the accuracy of endogenous back-scattered light-based CTCC detection in whole blood. Using a machine learning model for peak detection/classification, we demonstrated that the combined use of backscattered signals at the three wavelengths enable detection of ~ 93% of all CTCCs larger than two cells with a purity of > 82% and an overall accuracy of > 95%. The high level of performance established through BSFC and machine learning demonstrates the potential for label-free detection and monitoring of CTCCs in whole blood. Further developments of label-free BSFC to enhance throughput could lead to important applications in the isolation of CTCCs in whole blood with minimal disruption and ultimately their detection in vivo.

Deep learning-enabled detection of rare circulating tumor cell clusters in whole blood using label-free, flow cytometry

“…CTCCs were generated using a previously established protocol. 12,18 Briefly, GFP-associated MDA-MB-231 cells were grown on a 10 cm culture plate to 90% confluency. Following a wash step with phosphate buffer saline (Invitrogen), 1.5 mL of 0.25% trypsin (Gibco) was added to cleave the bonds between the cells and the plastic culture plate.…”

“…CTCC formation provides certain advantages to cancer cells, including increased survival rates in the bloodstream and enhanced ability to colonize distant tissues. 1,12 The collective presence of multiple cancer cells within a cluster can provide protection against immune system attacks, promote resistance to therapies, and facilitate the formation of secondary tumors. 1,9,10 While interest in CTC and CTCC detection and isolation has grown, the only FDA-approved technique to date is CellSearch.…”

“…BSFC monitors intrinsic light scattering and fluorescence to detect CTCCs. 12,36 We have previously demonstrated using in vitro BSFC that CTCCs have unique light scattering signatures, 36 which can be used to detect and classify CTCCs in whole blood using machine-learning (ML) based algorithms. 12 However, exogenous fluorescence was used in these studies to identify CTCCs from non-CTCCs (NCs) events.…”

“…12,36 We have previously demonstrated using in vitro BSFC that CTCCs have unique light scattering signatures, 36 which can be used to detect and classify CTCCs in whole blood using machine-learning (ML) based algorithms. 12 However, exogenous fluorescence was used in these studies to identify CTCCs from non-CTCCs (NCs) events. 12 In this study, we aim to improve our ML model for fully label-free detection of CTCCs in whole blood and assess the clinical utility of BSFC for CTCC detection.…”

Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo

“…Currently, there are several common approaches to the construction of in vivo cytometry systems, including photoacoustic flow cytometry (PAFC) [8][9][10][11], ultrafast scanning photoacoustic system [10,12,13], fluorescence [14][15][16][17][18], fluorescence imaging [19][20][21][22][23], scattering [24][25][26][27], diffuse fluorescence [7,[28][29][30][31], stimulated Raman scattering [32] among other cytometry methods.…”