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Delayed processing of CPT-collected blood preserves transcriptomic and genomic features of propagated circulating tumor cells

ABSTRACT: Circulating tumor cells (CTCs) provide a minimally invasive window into metastatic disease but are constrained by the need for rapid specimen processing after blood collection. We evaluated whether a workflow utilizing BD Vacutainer CPT-based blood collection could support delayed processing while preserving molecular features of propagated CTC-derived material. In a pilot cohort of four patients, paired CPT tubes were collected; one tube was processed within 2 hours of phlebotomy, whereas the second was stored at 4°C and processed 24 hours later. Both tubes were then harvested for CTCs, which were propagated ex vivo and analyzed by bulk RNA sequencing and whole-exome sequencing. Transcriptomic analyses showed that paired CPT-derived samples demonstrated phenotypes consistent with CTCs, including reduced immune-associated signatures and enrichment of epithelial-mesenchymal transition and KRAS signaling pathways. Direct comparison of paired early- and delayed-processed CPT samples demonstrated strong within-patient gene expression concordance. Whole-exome sequencing further revealed preservation of patient-specific oncogenic alterations and high overlap of detected variants across paired processing conditions. These findings altogether support the feasibility of a CPT-based delayed processing workflow for propagating and studying CTCs, thereby reducing a key logistical barrier to broader CTC research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE333160 | GEO | 2026/05/27

REPOSITORIES: GEO

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Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma. We adapted a microfluidic platform, the HbCTC-Chip (Stott et al., 2010, Pubmed ID: 20930119), to capture melanoma CTCs derived from mouse tumors, using panels of antibodies against melanoma-specific cell surface markers, followed by staining for melanoma antigens and optimized on-chip fluorescent imaging. We used a tamoxifen inducible BRAF(CA/+)/PTEN(flox/flox) melanoma mouse model (Dankort et al., 2009, Pubmed ID: 19282848) derived from a C57BL/6 background. Such mice received focal subcutaneous injection of tamoxifen (Sigma) (50ul at 5mg/ml in 50% EtOH suspension) at the left flank at 6-7 weeks after birth. Blood samples were collected from five mice with high tumor burden following tamoxifen injection. Blood from each mouse was split and processed through the CTC-chips functionalized with anti-CSPG4/MCAM antibody and control IgGs, respectively. Matched primary (from the tamoxifen injection site) and metastatic (from upper or lower back) tumors were harvested from the same mouse and immediately flash-frozen in liquid nitrogen. RNA extraction, single molecular sequencing and determination of Digital Gene Expression was as in Yu et al., 2012 (Pubmed ID: 22763454). One of the five mice yielded no CTC or IgG data. In addition, skin was taken from a sixth mouse, which was a complete wild type C57BL/6 mouse (without the BRAF/PTEN transgenes).The skin was taken after euthanasia of the animal and was processed as the tissue from the matched primary and metastatic tumors.

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Delayed processing of CPT-collected blood preserves transcriptomic and genomic features of propagated circulating tumor cells

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