3D Cell Culture-Based Global miRNA Expression Analysis Reveals miR-142-5p as a Theranostic Biomarker of Rectal Cancer Following Neoadjuvant Long-Course Treatment

Abstract

Altered expression of miRNAs in tumor tissue encourages the translation of this specific molecular pattern into clinical practice. However, the establishment of a selective biomarker signature for many tumor types remains an inextricable challenge. For this purpose, a preclinical experimental design, which could maintain a fast and sensitive discovery of potential biomarkers, is in demand. The present study suggests that the approach of 3D cell cultures as a preclinical cancer model that is characterized to mimic a natural tumor environment maintained in solid tumors could successfully be employed for the biomarker discovery and validation. Subsequently, in this study, we investigated an environment-dependent miRNA expression changes in colorectal adenocarcinoma DLD1 and HT29 cell lines using next-generation sequencing (NGS) technology. We detected a subset of 16 miRNAs differentially expressed in both cell lines cultivated in multicellular spheroids compared to expression levels in cells grown in 2D. Furthermore, results of in silico miRNA target analysis showed that miRNAs, which were differentially expressed in both cell lines grown in MCS, are involved in the regulation of molecular mechanisms implicated in cell adhesion, cell-ECM interaction, and gap junction pathways. In addition, integrins and platelet-derived growth factor receptors were determined to be the most significant target genes of deregulated miRNAs, which was concordant with the environment-dependent gene expression changes validated by RT-qPCR. Our results revealed that 3D microenvironment-dependent deregulation of miRNA expression in CRC cells potentially triggers essential molecular mechanisms predominantly including the regulation of cell adhesion, cell–cell, and cell–ECM interactions important in CRC initiation and development. Finally, we demonstrated increased levels of selected miR-142-5p in rectum tumor tissue samples after neoadjuvant long course treatment compared to miR-142-5p expression levels in tumor biopsy samples collected before the therapy. Remarkably, the elevation of miR-142-5p expression remained in tumor samples compared to adjacent normal rectum tissue as well. Therefore, the current study provides valuable insights into the molecular miRNA machinery of CRC and proposes a potential miRNA signature for the assessment of CRC in further clinical research.

The Extracellular Matrix-Derived Biomarkers for Diagnosis, Prognosis, and Personalized Therapy of Malignant Tumors

“…For example, using 3D culture model with decellularized ECM scaffolds (dECM) allowed to identify fulllength Collagen VI as a driver of breast cancer cell invasion in obesity and metastasis (89). In colorectal cancer, the patterns of expression of miRNA dependent on 3D microenvironment have been characterized, and one of them (miR-142-5p) was identified as a theranostic biomarker (90). As applied to clinical scenarios, the 3D cell culture models incorporating TME are referred to as a "patient's avatar" (91) and "patient-on-a-chip" (92) models, and can allow to identify biomarkers of individual response to the therapy.…”

Comprehensive Analysis of miRNA-Mediated Regulatory Network and Identification of Prognosis Biomarkers in Rectal Cancer

“…ROC curve analysis showed that miR-21 and miR-328 could provide valuable information for individualizing treatment in rectal cancer patients ( Campayo et al, 2018 ). 3D cell culture-based global miRNA expression analysis revealed increased levels of miR-142-5p in rectal tumor tissue samples after neoadjuvant long course treatment, which may be a theranostic biomarker of rectal cancer ( Kunigenas et al, 2020 ). Low plasma level of exosomal miR-486-5p was associated with organ-invasive primary tumor, which attributed to adverse prognosis of rectal cancer ( Bjornetro et al, 2019 ).…”

CRISPR-Cas9 Screen Identifies DYRK1A as a Target for Radiotherapy Sensitization in Pancreatic Cancer

“…Our results also simultaneously exemplify the possibility of performing CRISPR-Cas9 screens in an in vitro model such as 3D cell culture. In particular, we demonstrated that the sensitivity of 3D cells to radiation is no different from that of 2D cells, despite their growth in matrigel and special culture media [ 17 , 33 , 34 ]. As a result, we irradiated cells with two distinct growth patterns using a standard radiographic screening approach.…”