Abstract
The effects of the stiffness of the microenvironment on the molecular response of 3D colony organization, at the maximum level of mammographic density (MD), are investigated. Phenotypic profiling reveals that 3D colony formation is heterogeneous and increased stiffness of the microenvironment, within the range of the MD, correlates with the increased frequency of aberrant 3D colony formation. Further integrative analysis of the genome-wide transcriptome and phenotypic profiling hypothesizes overexpression of ERBB2 in the premalignant MCF10A cell lines at a stiffness value that corresponds to the collagen component at high mammographic density. Subsequently, ERBB2 overexpression has been validated in the same cell line. Similar experiments with a more genetically stable cell line of 184A1 also revealed an increased frequency of aberrant colony formation with the increased stiffness; however, 184A1 did not demonstrate overexpression of ERBB2 at the same stiffness value of the high MD. These results suggest that stiffness exacerbates premalignant cell line of MCF10A.
Highlights
This paper has two overarching goals. It advances the current understanding of the role of increased stiffness on colony organization and its molecular endpoints, within the range of mammographic density (MD), through detailed confocal microscopy and molecular profiling
Measuring the stiffness of the MD provides the basis for engineering matrices for 3D cultures and profiling colony formation with embedded protocol[24]
Colony organization of the 3D cell culture models is an important endpoint for phenotypic profiling, which can be mapped to the genomic aberrations
Summary
Within the range of mammographic density, is quantified in histology sections. This observation is consistent with the MCF10A cell lines. Having reduced the technical variations, 3D colony profiling revealed that aberrant colony formation is gradually increased as a function of the stiffness of the microenvironment These observations are consistent for MCF10A and 184A1. ERBB2 was not overexpressed in (i) a genomically stable cell line of 184A1, and (ii) in 2D cultures of MCF10A at the high stiffness value of the microenvironment. A second experiment with a genomically stable 184A1 HMEC revealed that ERBB2 is not overexpressed with the increased stiffness Both MCF10A and 184A1 are immortalized premalignant cell lines, MCF10A has probably lost more barriers toward transformation, and uniquely expresses ERRB2 under high stiffness of the microenvironment.
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