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Transient activation of the WNT pathway after disruption/remodeling of colorectal cancer cell clusters promotes a malignant phenotype

ABSTRACT: Cancer progression and wound healing share some characteristics. Most colorectal cancers are differentiated adenocarcinomas that maintain three-dimensional structures to some extent. Hence, disruption of the architecture can provoke remodeling similar to the remodeling of normal intestinal epithelium. We used our recently developed three-dimensional culture system to investigate the response of cancer cell spheroids to mechanical disruption. Specifically, we developed a protocol for homogenous disruption of the spheroids that maintained the cell-cell contacts. After disruption, 9 spheroids from 9 patient samples reformed within a few hours, and 2 showed accelerated spheroid growth. Stemness increased after spheroid disruption, as assessed by marker expression, spheroid forming capacity, radiation sensitivity, and tumorigenesis. The spheroid-forming capacity increased in 6 of 11 spheroids. The disruption signature, as determined by gene expression profiling, supported the incidence of remodeling and predicted the prognosis of the colorectal cancer patients. WNT and HER3 signaling was increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increases in growth and stemness after disruption. Thus, disorganized architecture in patient tumors might reflect the processes of disruption and subsequent remodeling and represent a cause rather than simply a consequence of malignancy progression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE75867 | GEO | 2015/12/10

SECONDARY ACCESSION(S): PRJNA305551

REPOSITORIES: GEO

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Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

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Transient activation of the WNT pathway after disruption/remodeling of colorectal cancer cell clusters promotes a malignant phenotype

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