Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices
Abstract
:1. Introduction
2. Results
2.1. Recovery Rate and Linearity of Cell RevealTM System
2.2. Identification and Isolation of CTCs
2.3. Average Sequencing Depth and On-Target Percentage
2.4. Mutations in Isolated CTCs
3. Discussion
4. Materials and Methods
4.1. Cell Line and Spiking Test
4.2. Patient Sample Preparation
4.3. CTC Enrichment and Identification
4.4. CTCs Isolation
4.5. Whole Genome Amplification (WGA)
4.6. PCR-Based Targeted Sequencing
4.7. Next-Generation Sequencing Analysis (For Panel_File)
4.8. Next-Generation Sequencing Analysis (For Panel_with_deDup_File)
4.9. Strategies of Prioritizing Variants
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Case No. | 1 | 2 | 3 |
---|---|---|---|
Sex | F | F | M |
Age | 62 | 49 | 67 |
Diagnosis/Staging | Lung AdCa/I | Lung AdCa/IV | Double cancer (lung AdCa/I & thyroid papillary carcinoma/IV) |
Status | Post-operation | Post-treatment | Post-operation |
CTCs | |||
TTF-1+ CTCs | 14/4 mL (2 mL × 2) | 8/2 mL | 16#/8 mL (4 mL × 2) |
TTF-1− CTCs | 18/4 mL (2 mL × 2) | 1/2 mL | 12/8 mL (4 mL × 2) |
Total CTC count | 32/4 mL (2 mL × 2) | 9/2 mL | 28#/8 mL (4 mL × 2) |
Isolated CTCs | |||
TTF-1+ CTC | 6 | 8 | 10 |
TTF-1− CTC | 6 | 0 | 0 |
WBCs | 10 | 2 | 17 |
Purity (%) | 55 | 80 | 37 |
Case No | Average Depth | On-Target Percentage (10×) | On-Target Percentage (100×) | On-Target Percentage (250×) |
---|---|---|---|---|
1 | 7560 | 89% | 84% | 79% |
2 | 8014 | 62% | 57% | 54% |
3 | 5852 | 90% | 79% | 71% |
Gene | Variation | Patient | CTCs AF | Pathway |
---|---|---|---|---|
EGFR | p.T725M | STE504366 | 0.235 | 1. MAPK_SIGNALING_PATHWAY 2. ERBB_SIGNALING_PATHWAY 3. PATHWAYS_IN_CANCER |
ERBB2 | p.N812fs | RCE162277 | 0.087 | 1. ERBB_SIGNALING_PATHWAY 2. PATHWAYS_IN_CANCER |
ERBB2 | RCE385327 | 0.127 | ||
KRAS | p.R97I | RCE162277 | 0.253 | 1. ERBB_SIGNALING_PATHWAY 2. PATHWAYS_IN_CANCER |
STK11 | p.L9fs | STE504366 | 0.2 | 1. MTOR_SIGNALING_PATHWAY 2. ADIPOCYTOKINE_SIGNALING_PATHWAY |
STK11 | p.M18T | STE504366 | 0.267 | |
STK11 | p.V34I | STE504366 | 0.275 |
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Jou, H.-J.; Ho, H.-C.; Huang, K.-Y.; Chen, C.-Y.; Chen, S.-W.; Lo, P.-H.; Huang, P.-W.; Huang, C.-E.; Chen, M. Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices. Int. J. Mol. Sci. 2022, 23, 15139. https://doi.org/10.3390/ijms232315139
Jou H-J, Ho H-C, Huang K-Y, Chen C-Y, Chen S-W, Lo P-H, Huang P-W, Huang C-E, Chen M. Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices. International Journal of Molecular Sciences. 2022; 23(23):15139. https://doi.org/10.3390/ijms232315139
Chicago/Turabian StyleJou, Hei-Jen, Hsin-Cheng Ho, Kuan-Yeh Huang, Chen-Yang Chen, Sheng-Wen Chen, Pei-Hsuan Lo, Pin-Wen Huang, Chung-Er Huang, and Ming Chen. 2022. "Isolation of TTF-1 Positive Circulating Tumor Cells for Single-Cell Sequencing by Using an Automatic Platform Based on Microfluidic Devices" International Journal of Molecular Sciences 23, no. 23: 15139. https://doi.org/10.3390/ijms232315139