Abstract LB260: Assessing the suitability of our exosomal small ncRNAs to serve as biomarkers for breast cancer & elucidating the relationship of exosomal ncRNAs and cancer cells from t-47d and hc1143 cell lines
Authors: Kobayashi Ken D. , Jijiwa Mayumi , Nasu Masaki , Deng Youping 
Published: April 2023

Abstract
Background: Exosomes are known to play the role of cargo, carrying biologically important materials such as non-coding RNAs to their recipient cells. Small non-coding RNAs such as miRNA have been shown to exert strong effects on tumorigenesis. The liquid biopsy, which utilizes small RNAs from plasma exosomes, is therefore a promising tool for precision oncology. The purpose of this study is to discover exosomal miRNAs that serve as detection markers of breast cancer. Our hypothesis are 1) if exosomes play the role of cargo, then there should be an abundance of upregulated ncRNAs in exosomes that are also upregulated in the cancer cells themselves. 2) It is likely that the miRNAs in exosomes that are highly expressed have critical roles in cancer cell activity. We performed small RNA-seq and examined the ncRNAs inside cancer cells and exosomal ncRNAs secreted by cancer cells into the culture media.
Methods: Two cancer cell lines T-47D and HCC1143, and primary Human Mammary Epithelial Cells (HMEC) were used. We prepared 5 samples of cellular RNA extracted from T-47D and HCC1143 and HMEC. We also extracted 8 samples of exosomal RNA from the media; which included those used for each cell culture and those never used for culture with or without FBS. Exosomes in FBS were depleted using Exosome Depletion Kits. The FASTQ files were uploaded onto QIAGEN GeneGlobe for analysis. The data was primarily normalized using geNorm.
Results: Exosomal ncRNAs from both cancer cells were compared to exosomal ncRNAs from normal cells. A total of 879 ncRNAs were detected, 108 of which were significantly upregulated and 104 of which were downregulated in cancer cells. hsa-miR-196a-5p is a ncRNA that had a fold-change of 84.08. Prior studies suggest that this miRNA is associated with MIR196A2 methylation and affects the malignancy of many types of cancers. Comparing cancer exosomal ncRNAs vs. ncRNAs inside cancer cells, a total of 879 ncRNAs were found with a FDR q-value &lt;0.05 and cut-off of 2 fold change. 251 ncRNAs were significantly upregulated in the exosomes. hsa-miR-1-3p had a fold change value of 4025.68 and an associated p-value of 0.0002. High levels of hsa-miR-1-3p are known to be associated with large tumors and is strongly correlated to a lower breast cancer survival rate.
Conclusions: The preliminary results indicate that there were significant differences between exosomal ncRNAs from normal cells and exosomal ncRNAs from cancer cells. The data suggests that exosomal ncRNAs are a promising source of prognostic biomarkers for breast cancer as ncRNAs with a high fold change are repeatedly validated to have a significant impact on breast cancer progression. The data supports our hypothesis that the upregulation of ncRNAs in cancer cells & exosomes is linked to cancer activity. The results will be scrutinized further, as other avenues for data analysis will be explored. The next step in our experiment will be to check the cancer survival rates with the TCGA database and attempt to validate our hypothesis through further cell culture experiments.
Citation Format: Ken D. Kobayashi, Mayumi Jijiwa, Masaki Nasu, Youping Deng. Assessing the suitability of our exosomal small ncRNAs to serve as biomarkers for breast cancer & elucidating the relationship of exosomal ncRNAs and cancer cells from t-47d and hc1143 cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB260.