A80-2-36 Red Blood Cells Enable Tumor DNA Uptake From Lung Cancer Cells-Implications for Liquid Biopsy

Rationale

Lung cancer remains the leading cause of cancer death worldwide. Although circulating tumor DNA liquid biopsies are used in advanced disease, reliable methods for early-stage detection are lacking. Improving early cancer detection through liquid biopsy requires understanding how tumor DNA is captured and preserved in biological reservoirs. Our group and others have demonstrated that red blood cells (RBCs) serve as an abundant, easily accessible reservoir of extracellular DNA that may offer potential advantages over conventional circulating tumor DNA assays for the detection of early-stage cancers. However, questions remain about RBC-mediated DNA acquisition and recovery, inter-individual heterogeneity in tumor DNA uptake across RBCs, and the stability of RBC-bound tumor DNA (rtDNA).

Methods

To address these gaps, we characterized RBC uptake of tumor DNA using three human lung cancer cell lines, H1975, A549, and H358 expressing EGFR L858R, KRAS G12S and KRAS G12C mutations respectively. RBCs were immunopurified from healthy donor blood and co-cultured with cancer cells. DNA was extracted and quantified by qPCR to confirm that RBCs acquire tumor DNA. To test whether direct contact is required, transwells were used to separate RBCs from tumor cells during co-culture. We also assessed how exposure duration, RBC number, and storage conditions influenced detectable rtDNA to inform practical handling and clinical workflows.

Results

RBCs acquired tumor DNA across all models, with mean detected copies of 2.79 × 10⁷ ± 5.95 × 106 EGFR L858R, 6.39 × 104 ± 7.53 × 104 KRAS G12S, and 2.30 × 10⁵ ± 1.24 × 10⁵ KRAS G12C following co-culture, underscoring a significant expression but also a pronounced substantial heterogeneity in tumor DNA acquisition by RBCs. Transwell assays confirmed that direct interaction is necessary for efficient tumor DNA uptake by RBCs. Time-course experiments showed detectable rtDNA after exposures as brief as five minutes and at RBC counts as low as 1,000 cells. Importantly, rtDNA remained stable at -80 °C when stored as extracted DNA or within RBC pellets for over six weeks under clinically relevant conditions.

Conclusion

Our findings define key biological and operational parameters of RBC-mediated tumor DNA acquisition, including rapid uptake, contact dependence, inter-individual variability, and robust stability. Ongoing work in our laboratory is focused on identifying host determinants of DNA uptake and optimizing methodologies for tumor DNA recovery. Collectively, this work lays essential groundwork for advancing RBC-based liquid biopsy approaches toward clinical translation in early-stage lung cancer, where sensitive and robust liquid biopsy approaches are lacking.

This abstract is funded by: Senticell,LLC