Characterization of the cell-free DNA released by tumor organoids derived from colorectal cancer patients

Abstract

Introduction: Patient-derived tumor organoids (PDTOs) have histological, molecular and clinical (drug sensitivity) characteristics comparable to those of their originating tumors. However, little is known about their ability to replicate the release of tumor-derived DNA.

Methods: Supernatants from 21 colorectal cancer PDTO cultures, established from 13 patients, were prospectively collected. The presence, structure, and mutational landscape of nuclear (cf-nDNA) and mitochondrial (cf-mtDNA) cell-free DNA as well as extracellular mitochondria (ex-Mito) were analyzed using qPCR, fragmentomics and shallow whole-genome sequencing. Mutation profiling was performed via IntPlex qPCR and whole-exome sequencing (WES).

Results: Cf-nDNA was detected in 95 % of PDTO supernatants with concentrations ranging from 0.009 to 209 ng/mL. Cf-nDNA fragment size analysis revealed patterns consistent with circulating DNA, including mononucleosome-associated profile. Cf-mtDNA was present in all samples (0.27-89.2 pg/mL) and extracellular mitochondria was also detected (0.009-17.4 pg/mL). A strong concordance (>85 %) was observed between oncogenic mutations in cfDNA and the molecular alterations detected in PDTOs and patient tumors.

Conclusion: PDTOs release both nuclear and mitochondrial cfDNA into their culture medium displaying high similarity with patient-derived circulating DNA (cirDNA), including fragmentation patterns and oncogenic mutations. This study strengthens the relevance of the PDTOs as patient tumors models and highlights the potential of analyzing PDTO-derived cfDNA as a non-invasive approach to investigate tumor evolution and as a valuable tool to support functional precision oncology.

Keywords: Cell-free DNA (cfDNA); Circulating DNA; Fragmentomics; Patient-derived tumor organoids (PDTOs).