C. U. Ihemelandu1, A. Naeem2, O. Rodriguez2, C. Albanese2 1Georgetown University Medical Center, Surgical Oncology, Program In Peritoneal Surface Oncology, Washington, DC, USA 2Georgetown University Medical Center, Department Of Oncology And Pathology, Lombardi Comprehensive Cancer Center, Washington, DC, USA
Introduction: Conventional imaging modalities, such as CT, PET and MRI lack the sensitivity to detect and appraise tumor burden in patients with peritoneal metastasis of a colorectal cancer origin (CRC-PM). We propose utilizing circulating tumor exosome-associated miRNA as diagnostic/screening markers for early detection of CRC-PM recurrence and to guide targeted molecular therapy. Our hypothesis is that identification of specific miRNA associated with circulating CRC-PM tumor-derived exosomes represent early markers for detection of CRC-PM recurrence.
Methods: Exosomes were isolated from the serum of 6 CRC-PM patients, 4 healthy donors, and two CRC cell lines (SW620, HT116). Isolation of exosomes from serum samples was performed using the ExoQuick Plasma Prep kit. The size of the exosomes was verified using transmission electron microscopy. Exosomes were also characterized using markers CD63, HSP70, and CD9. RNA was isolated from exosomes using the miRNeasy Micro Kit. Qiagen miRNA library prep kit was used to prepare the miRNA library. RNAseq was performed on a HiSeq 2500 (Illumina, single-end 50-bp read length), using equimolar amounts for each sample.
Results: RNA sequencing analysis detected a total of 128 differentially expressed (DE) miRNA (p<0.01) in patients vs. healthy controls, and 203 miRNAs (p<0.01) in cell lines vs. healthy controls. Among the 128 DE-miRNA, expression levels of 56 DE-miRNA were downregulated, while those of 72 were upregulated in patients as compared to expression levels in controls (p< 0.05). Only one miRNA was differentially upregulated with FDR q<0.001. PCA analysis and unsupervised hierarchical clustering of the samples indicate a distinction among all groups i.e., patients, healthy, and cell lines. miR-3168 expression was noted to be significantly induced in cancer than in healthy controls with a log2 fold change of 3.8 (p<0.01, q<0.01) and in CRC cell lines as compared to healthy controls (log2 FC 1.2, p-value 0.02). Using MiRge 2.0, miRNA-3168 was annotated to biological functions such as response to stress, catabolic process, regulation of molecular function, and immune system processes. Furthermore, miRNA target filter analysis (Ingenuity Pathway Analysis @ Qiagen) revealed that miRNA-3168 is directly or indirectly involved in the regulation of well-studied CRC biomarkers EGFR and c-Met . Through its interaction with various molecules RPL17 (Ribosomal Protein L17), YBX1 (Y-Box Binding Protein 1), BDKRB2 (Bradykinin Receptor B2), miRNA-3168 is predicted to regulate the expression of EGFR and c-Met.
Conclusion: miRNA-3168 is significantly induced in CRC-PM patients and could serve as an additional biomarker in exosomes using liquid biopsy to detect and appraise peritoneal tumor burden.