Establishing a CTC-driven workflow for mRNA-transfected moDC production in personalized cancer immunotherapy

Background: After its success during SARS-CoV-2 pandemic, RNA-based vaccines are now being explored for cancer therapy. One of the strategies is the use of monocyte-derived dendritic cells (moDCs) transfected with tumor mRNA, but access to tumor tissue - especially from metastatic sites - is a limitation. Circulating tumor cells (CTCs) offer an alternative source for tumor material. Here, we developed a workflow integrating CTCs and mRNA technology for personalized cancer therapy. Methods: Our first aim was to compare the recovery rate (RR) of 3 different CTCs isolation platforms (Parsortix [P], RosetteSep [RS], or ScreenCell [SC]) by spiking fluorescently-labeled breast cancer SKBR3 cells (200, 50, 30; triplicate) into healthy donor (HD) blood and counting the number of recovered CTCs using the Incucyte System. In parallel, moDCs were differentiated from HD monocytes and transfected with 500 ng of GFP-mRNA using Lipofectamine™ MessengerMax™ (LMM) to standardize conditions for CTC-mRNA transfection. GFP expression was assessed at 6, 24, 48, 72, and 96h by confocal microscopy, with non-transfected and mock-transfected moDCs as controls. Results: Spiking tests showed that P and SC had similar performance and higher RR compared to RS, which yielded the lowest RR (Table 1). Regarding moDCs transfection, GFP expression was detectable at 6h, persisted through 24-48h and declined after 72h, with no signal in the controls. These conditions are currently being tested on SKBR3 cells recovered with SC, to assess the possibility to transfect moDCs with CTC-derived mRNA. Conclusion: SC and P showed similar RR, but SC's simplicity and lower cost make it more suitable for deployment with our collaborators in Brazil. moDCs transfection was effective and LMM successfully delivered the target sequences for moDC processing and expression, supporting this as an immunotherapeutic approach. Future studies will be performed to validate the workflow also in clinical samples. Recovery rates (RR) of each enrichment platform Parsortix® ScreenCell® RosetteSep™ 30 spiked cells* 30.0% ± 4 33.3% ± 9 10.8% ± 2 50 spiked cells* 44.0% ± 3 53.5% ± 5 5.0% ± 4 200 spiked cells* 59.4% ± 29 60.75% ± 8 10.9% ± 4 *spiking experiments were conducted in triplicate