(P-BT-9) Detection of  Replicative Potential in Cancer Cells After Photoinactivation

Background/Case Studies: Puck and Marcus (1956) described a cell culture technique using cell lines to assess the colony-forming ability of single mammalian cells plated in culture. The clonogenic assay detects cells that conserved their replicative capacity after a treatment affecting their viability. Cells that survive a given treatment typically form colonies within 21 days. Our research focuses on an autologous cell-based immunotherapy product designed to act as a vaccine against cancer. The vaccine production includes photoinactivation, which involves the photosensitizer riboflavin (Rb) and UV light to render the cells unable to replicate. We compared the UV dose response in cell lines and evaluated our product by measuring the cell's capacity to form colonies after photoinactivation.

Study

Design/Methods: HeLa and Caov-3 cell lines were treated with increasing doses of UV light in the presence of Rb and cultured in optimal conditions to measure their ability to replicate after the treatment. Our study design comprised three parts: 1) a dose-response analysis using UV doses of 0, 100, 300, and 600 Joules (J) to determine the effective killing dose. 2) evaluation of the sensitization effect of Rb in HeLa cells by exposing them to UV irradiation without Rb. 3) determination of photoinactivation efficiency in patient-derived tumor cells at the growth-restrictive dose using the clonogenic assay.

Results/Findings: Our colony counting results showed reduced viability with increasing doses of radiation. At doses of 50 and 100J, the colony count decreased from 1,788 to 401 in HeLa and 2,354 to 636 in Caov-3 cells. Moreover, doses ≥300J eliminated the cells' ability to form colonies. Consequently, we found no survivors at doses of 300 and 600J, resulting in no colony formation. The absence of Rb during irradiation drastically increased cell survival. At doses of 50 and 100J, our colony count went from 2,354 (Rb) to 5,425 (No Rb) and from 401 to 1,506, respectively. Also, HeLa cells treated with 300J and no Rb showed survivors with an average of 11 colonies; however, there were no survivors at 600J. Finally, the patient-derived tumor cells treated with Rb and 300J showed no growth after photoinactivation.

Conclusions: All cell lines showed a dose-response dependency and different sensitivity to the treatment. Doses < 300J were insufficient to disable replication, leading to treatment resistance and proliferation. All cell lines tested showed cell killing at doses ≥300J, and no colonies formed after the photoinactivation. The Rb effect is evident in HeLa cells, which produce an average of 3.5x more survivors when treated without Rb. Even 300J without Rb showed incomplete inactivation. Finally, patient-derived tumor cells treated with 300J and Rb demonstrated efficient and complete cell killing.