An Ex vivo cultivation model for circulating tumor cells: The success rate and correlations with cancer response to therapy

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Cancer mortality is closely linked to recurrence and distant metastasis, posing challenges in real-time tracking due to the invasiveness of current methods. Circulating tumor cells (CTCs) show promise as potential tools; however, their scarcity remains a significant obstacle. In this prospective study, we validated a simple culture protocol and investigated the correlation between clinical response and CTC growth status. Following negative selection, the isolated cells were subjected to ex vivo cultivation in a two-dimensional environment supplemented with cytokines for up to 21 days, followed by immunofluorescence staining for analysis. Among 37 participants with solid tumors and distant metastasis (34.8% head and neck cancer), 47 samples were collected, from which CTCs were detected. The percentages of CTCs, atypical CTCs, and white blood cells during cultivation from days 7–21 were significantly different ( p < 0.001, p < 0.001, and p = 0.330, respectively). Patients were further categorized into progressive disease (PD) and non-PD groups based on disease status, revealing significant differences in CTC growth rates, which increases from Days 7–21 between groups (5.5x vs. 2.8x growth, respectively; p < 0.001). With the proposed protocols, we cultured CTCs from patients with various cancers for 21 days and identified a tool for predicting cancer response. The actual cancer status (PD or non-PD) at CTC isolation correlates to CTC growth rate, guiding the required observation time and parameters for culture. • Demonstrated increased CTC count and percentage using stage IV cancer patient culture samples. • Achieved 100% CTC capture rate and 97.9% culture success, unique to our study. • Validated success across eight cancer types with distinct therapy responses. • High 2D success linked to common cytokine regimens and negative selection. • Simplified protocol advances CTC culture in standard labs

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