Abstract 3757: Establishment of 3D culture system of circulating tumor cells with immune cell evasion strategy

Abstract Introduction: Circulating tumor cells (CTCs), are cancer cells shed from primary tumors, travel through the vasculature and give rise to metastatic lesions. CTCs have more advantages as a diagnostic tool compared with other liquid biopsy components such as cell-free DNA, because they contain all cellular components could provide more information of cancer cells. However, the number of CTC in the blood is very small, there are limitations in applying various kinds of analyses. Therefore, we have developed CTC 3D culture system for expanding number of CTCs after the enrichment of CTC with automated Smart BiopsyTM Cell Isolator. In this study, a CTC-mimicking model with the cancer cell lines to establish a 3D culture environment suitable for CTC growth and a pilot test of CTC 3D culture with clinical samples were performed. Method: To establish the 3D culture system with cancer cell lines, we spiked H358 (lung cancer), HCT116 (colon cancer) with peripheral blood mononuclear cells (PBMCs) (cell:PBMC ratio= 1:10 ∼ 1:250) and initiated culture under 3D conditions. To retain only the minimal number of cancer cells required for growth we tested various approaches including dynamic culture and static culture. Then mixed cell suspension was embedded into the matrigel and incubated until growth. A pilot test for CTC 3D culture was performed with clinical samples of prostate cancer after the approval of institutional review board of Ewha Womans University Mokdong Hospital. Result: We observed that immune cell movement increased both in frequency and speed when higher numbers of PBMCs were present within the gel. As a result, following significant PBMC migration out of the dome, cancer cells progressively expanded and formed small clusters. Starting with 100 cells per dome, cancer cell clusters reached approximately 100-150 μm in diameter by week 2. By recapitulating in vitro the key challenges encountered in clinical samples, such as extremely low number of CTCs and their susceptibility to immune-mediated elimination, our model enables systematic evaluation of these limitations and the identification of strategies to overcome them. Furthermore, pilot application of this system using clinical blood samples yielded promising preliminary results. Conclusions: We have established the 3D CTC culture system with various kinds of in vitro tests for defining culture conditions that allow CTC-like cells to survive immune pressure and our culture system was validated with successful growth of CTC with ex vivo culture of clinical samples of prostate cancer. Citation Format: Ji Eun Moon, Jihyun Lee, Soee Kim, Minseon Hwang, Jung Won Kim. Establishment of 3D culture system of circulating tumor cells with immune cell evasion strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3757.