Using the FAST disk enables ultra-fast (< 20 s for 3 mL whole blood) CTC isolation with a reduced pressure drop (~1 kPa). The fluorescence images of the membrane after isolation of CTCs from the blood of a cancer patient.
Liquid biopsy in non-small cell lung cancer
EGFR mutation detection by ctDNA analysis is an exciting application of liquid biopsy as recently approved by the FDA. Moreover, we introduce recent research articles studying molecular diagnosis based on the analysis of CTCs and ctDNA and give future perspectives on liquid biopsy for patients with NSCLC.
Technologies for CTCs analysis and clinical applications in NSCLC
Prognosis using commercially available CTCs isolation devices
Using the FAST disc enables ultra-fast (< 20 s for 3 mL whole blood) CTC isolation with a reduced pressure drop (~1 kPa). The overall process of isolating CTCs from 3 mL of whole blood could be completed within 1 minute (Figure 3.1c).
Molecular diagnosis using isolated CTCs by microfluidic chips
Research motivations
This study emphasizes the necessity of standardization based on the development of a robust system and the importance of preliminary validation of each technique with large-scale multicenter studies for the real application of liquid biopsy in clinical routine. We expect that this thesis may suggest expanding the ability to use CTC-based liquid biopsy in routine clinical and longitudinal surveillance of cancer therapy.
Research aims
In addition, the selection of blood collection tubes, blood volume, and type of downstream analysis may affect data quality and may cause inaccuracies and statistical errors. Furthermore, the high durability of the disc was confirmed by the repeatability test over 30 days.
Centrifugal microfluidics for size-based CTC isolation
Centrifugal microfluidics
Membrane filtration
Fluid assisted separation technology (FAST) disc for CTC enrichment
Introduction
Design and function of the FAST drive. a) Schematic image showing the Fluid-Assisted Separation Technology (FAST) disc mechanism. Filtration occurs primarily at the outer edge of the filter with the disc rotating in the non-FAST mode, while more uniform liquid transport occurs with the FAST disc, as shown in the images showing 10 μm red fluorescent beads filtered with conventional (above ) and FAST (below). Scale bar: 100 μm) (b) Images of the lab-on-disk and portable operating system. As we demonstrate, the FAST disk approach enables a self-contained, efficient, user-friendly, robust, and cost-effective lab-on-disk system for CTC isolation at the point of care.
Experimental details
All pumping necessary to generate the flow during the staining process was induced by the rotation of the disk for 6 seconds at 600 RPM. After permeabilization of the cells, the blocking step with 20 μg/ml IgG was followed by staining with different antibodies. The convergence time depends on the inductance (solving) of the exhaust channel, while the characteristic time scale in this case is ͷǤൈ ͳͲିସs, which is much shorter than for the non-FAST case.
The stability of the non-FAST case is highly dependent on the output resistance and the membrane resistance, as shown in Figure 2.9c and Figure 2.9d. To test the CTC isolation efficiency of the FAST disk, blood samples were divided into two aliquots (~3 ml each) and assayed with the FAST disk and ScreenCell (Sarcelles, France), a commercially available size-based isolation kit. .
Result and discussion
The main advantages of the FAST disk, i.e. faster filtration and reduced pressure drop, are further confirmed by whole blood separation. After the validation of the FAST disk platform, we analyzed blood samples from patients with different types of cancer (n = 142) and from healthy individuals (n = 50) as controls (Figure 2.13d, Figure 2.15 and Table 2.2). Performance comparison with the commercially available size-based filter system and clinical test of the FAST disk. a) Collection efficiency and purity were compared for the results of the filtration experiments with MCF-7 cells spiked with blood samples.
The circles in the left figures show the entire membrane area with a diameter of 10 mm and 6 mm for the FAST disc and ScreenCell, respectively. PC9 lung cancer cells added to whole blood were treated with a FAST disc and the lysate of the membrane-captured cells was used as a subject for DNA preparation followed by real-time PCR.
Conclusions
The estimated purity is on the order of 0.1% and 1.0% for 10 and 100 cells added with 3 ml of whole blood, respectively, as the log depletion of WBC with FAST disk has been shown to be approximately 3.0. The FAST disc uses the entire membrane surface for filtration and significantly reduces clogging problems. Although there are many reports on the importance of CTCs as a novel biomarker for cancer metastasis,83 few studies have been conducted to date to determine whether CTCs are also useful for the early detection of solid tumors.84, 85 With highly sensitive, cost-effective With the effective and robust FAST disk technology, we were able to detect a significant number of CTCs not only in cancer patients with metastases, but also in patients in relatively early stages of cancer without distant metastases, raising the potential to use CTCs as early diagnosis. Highlighter.
Furthermore, CTCs isolated from the FAST disk are not fixed, but rather live, allowing them to be easily used for standard assays such as immunostaining, high-resolution imaging, and mutational analysis. which are particularly important for personalized therapy. Because the FAST disk mechanism can be applied to a variety of size-based filtering techniques, we expect it to be useful in a wide range of applications.
Introduction
Although most current CTC enumeration technologies rely on signal averaging across individual heterogeneous CTCs, facile analysis of CTCs at single-cell resolution is highly desirable to assess cell heterogeneity and reveal its clinical consequences. Although much progress has been made and the clinical significance of CTCs has been demonstrated, remaining challenges include the high cost, low throughput and complexity of the process, as well as false positive/false negative results, which will hinder wider adoption of CTCs hinder. CTC-based liquid biopsy as routine practice in clinical settings. In this report, we present the preclinical validation of a liquid-assisted separation technology (FAST) disk 108, which provides rapid (>3 ml/min), reproducible, and label-free isolation of CTCs directly from unprocessed whole blood of patients with NSCLC.
We performed serial monitoring of CTC counts, mutation detection, and single-cell multiplex gene expression of CTCs from a prospective cohort of patients with NSCLC receiving treatment with EGFR-TKIs. The study highlights the potential of a CTC-based liquid biopsy for assessing therapy efficacy and emerging drug resistance.
Prognostic values of CTCs through single cell analysis
Experimental details
CTC enumeration, immunofluorescence staining, and detection by the CellSearch epithelial cell assay (Menarini Silicon Biosystems, Inc.) were performed using the CellSearch™ protocol (www.cellsearchctc.com) at Brigham and Women's Hospital (BWH), Boston, MA BWH Number IRB is 2016P001708). To detect mutations in isolated CTCs, genomic DNA was extracted using the QIAamp DNA blood mini-kit (Qiagen). Survival time was assessed using the Kaplan-Meier method, and the difference in survival between groups was assessed using a log-rank test.
Single-cell cDNA was prepared using the Single Cell-to-Ct kit (Life Technologies) and the specific target was pre-amplified for gene expression analysis. mRNA expression values of target markers normalized to GAPDH values using the 2−ΔCt method were summed for each E/M group, and the relative percentage was used to classify CTCs according to their epithelial-to-mesenchymal transition (EMT features).
Results and discussion
In addition, the t-SNE analysis, shown in Figure 3.6b, clearly represents the inter-cell lineage and inter-patient heterogeneity of CTCs. Individual cells from four different NSCLC cell lines and three different NSCLC patients showed different E/M hybridization signatures (Figure 3.6c and Figure 3.10a). Despite their heterogeneity, single cells from the same cell lines clustered together when we plotted the gene expression of the mesenchymal markers, vimentin and CD44, against that of the epithelial markers, EpCAM, KRT7, KRT18 and KRT19 (Figure 3.11).
On the other hand, 18 CTCs from patient LP39 were more heterogeneous, with the intermediate expression of mesenchymal markers (Figure 3.6c and Figure 10a). As shown in the 3D t-SNE plots in Figure 3.12c and Figure 3.13c, each individual CTC showed a different gene expression pattern in hierarchical clustering, but CTCs from the same time point were well clustered. In addition, the gene expression profiles of CTCs isolated at different time points were clearly different, which was also demonstrated by the correlation matrix analysis (Figure 3.16 and Figure 3.17).
When we classified individual CTCs based on epithelial or mesenchymal gene expression signature, the heterogeneity of individual CTCs, as well as the dynamic variation of the M score, was evident, as shown in Figure 3.12d.
Conclusions
A number of studies have suggested that EMT may be associated with EGFR-TKI resistance in NSCLC patients 133, 134. It was also suggested, based on liquid biopsy applications, that CTCs expressing EMT-related genes were associated with progression of disease and poor therapeutic responses in breast 116 , gastric 117 , pancreatic 114 , and lung 135 cancer patients. 118 characterized single CTCs and demonstrated their inter- and intra-patient heterogeneity in four pancreatic cancer patients.
Furthermore, analysis of EMT scores of individual pretreatment CTCs isolated from three patients with NSCLC indicated that the patients who showed relatively poor drug responses had CTCs with high M scores, while the patient who showed a relatively good response on EGFR-TKI treatment, had CTCs. with high E-scores. Although we could frequently observe CTC clusters from patients with other types of cancer, CTC clusters were rarely observed from patients with NSCLC (Figure 3.2b).
General conclusions and future perspectives
General conclusions
Plan for future
Next, downstream analysis of single CTCs and CTC groups from each patient is necessary to observe distinct characteristics associated with clinical outcomes. Therefore, a robust high-efficiency CTC isolation system and a high-throughput molecular analysis of CTCs become increasingly necessary. Our powerful FAST drive enables a comprehensive characterization of CTCs through mRNA profiling by multiplex PCR with 48 RNA markers, which has already been used to report on analyzes of single CTCs and CTC clusters.
We will investigate a strategy for isolation of single CTC and CTC clusters from the FAST drive and phenotypic characterization of CTC clusters based on mRNA profiling and demonstrate the correlation between mRNA expression of CTC clusters and clinical outcomes in patients with pancreatic cancer. unsectable.
Future perspective
3DUN6P :RQJ'- 2RL&& .XUW]'0 9HUPHVK2 $DOLSRXU$ 6XK6 3LDQ./ &KDERQ--/HH6+0ROHFXODUSURILOLQJRIVLQJOHFLUFXODWLQJWXPRUFHOOVIURPOXQJ FDQFHUSDWLHQWV3URFHHGLQJVRIW KH1DWLRQDO$FDGHP\RI6FLHQFHV .. 0XUOLGK DU 9 =HLQDOL 0 *UDEDXVNLHQH 6 *KDQQDG|5H]DLH 0 : LFKD 0 6 6LPHRQH'0 5DPQDWK1 5HGG\501DJUDWK6$5DGLDO)ORZ0LFURIOXLGLF'HYLFHIRU 8OWUD|+LJK|7KURXJKSXW$IILQLW\Ǧ%DVHG,VRODWLRQRI&LUFXODWLQJ7XPRU&HOOV6PDOO. 2JGHQ , 0 &DWDORQD : &KXPVUL 6 7DQJ & 0 &ULVWRIDQLOOL 0 &LUFXODWLQJ JLDQW PDFURSKDJHVDVDSRWHQWLDOELRPDUNHURIVROLGWXPRUV3URF1DWO$FDG6FL86$. LP/6 +X0 +XDQJ0& &KHRQJ:& *DQ$7/ /RRL;/ /HRQJ6 0 .RD\(6&/L0+0LFURVLHYHODEFKLSGHYLFHIRUUDSLGHQXPUDWLRQDQGIOXRUHVFHQFHLQ VLWXK\EULGL]DWLRQRIFLUFXODWLQJWXPRUFHOOV/ DE&KLS.
XDQJ6% :X0+ /LQ<+ +VLHK&+
