We identified several mutations that were clonally selected through progression for which the allele fraction differed in the different cell lines. focal amplifications acquired during progression. Normalised coverage (y\axis) is plotted according CMK to genomic CMK position (x\axis) PATH-240-315-s005.tif (3.0M) GUID:?677F74D3-85DF-410F-93E1-0DB12ECBE3F9 Figure S5: RNAi modulation of the isogenic MCF10 model in cancer cell line spheroids (A) Micrograph images of MCF10DCIS.com cells subsequent to CMK transfection of siRNA. Images were taken at day 7. (B) Heatmap of spheroid growth after siRNA mediated silencing and gene expression on plastic (2D). Relative spheroid growth is measured by the survival fraction of treated cells relative to siControl. (C) Micrograph images of spheroids at day 1, 4 and 7 of the MCF10 progression series, demonstrating good growth kinetics. (D) Images of spheroids stained with H&E and antibodies against Ki67, phosphor\AKT, and TP53. PATH-240-315-s009.tif (5.9M) GUID:?61271A41-6561-4A35-A614-6E89AF1AF49E Figure S6: Oligo deconvolution in MCF10Ca1a cells in spheroids and on plastic. (A) Barplots showing relative mRNA levels after gene silencing. Statistically significant alterations in mRNA expression were calculated using Student’s test (*p 0.05) (B) CMK Barplots of relative cell growth in 2D and as 3D spheroids with individual oligos. Relative spheroid growth is measured by the survival fraction of treated cells relative to siControl. Statistical comparisons were performed using Student’s and invasive disease is a multifaceted process that results in the acquisition of multiple genomic alterations, including changes in genomic copy number, structural rearrangements, acquisition of mutations, altered gene expression, and Rabbit Polyclonal to ATG4A pathway dysregulation 1, 2, 3, 4. The transition through these states, i.e. non\invasive to invasive disease, is a well\defined and staged process, through which breast cancers progress to acquire the capacity to grow, persist, and eventually spread to secondary sites. High\throughput molecular profiling of breast cancers and their precursor lesions has revealed that they have distinct genomic and transcriptomic alterations 3, 5, 6, 7, 8; however, matched preinvasive lesions and invasive counterparts from the same patient are remarkably similar 6, 7, 8, 9, 10, suggesting that the extent of genomic heterogeneity is determined early in breast cancer development. There is evidence suggesting that the progression from to invasive disease is not exclusively driven by specific genomic aberrations in the preinvasive cells, but is a result of paracrine interactions of tumour cells with the surrounding stromal CMK environment 3, 11, 12, 13. The MCF10 progression series is a product of the normal mammary epithelial cell line MCF10A that is spontaneously immortalised from the MCF10 mortal cell line (MCF10M), which originated from benign fibrocystic disease 14. As MCF10A cells are non\tumorigenic, cells were HRAS\transformed to produce MCF10neoT and MCF10AT1 cells 15, 16 (Figure ?(Figure1A).1A). MCF10AT1 cells were subsequently serially passaged to produce carcinoma clones derived from the same tumour, whereas MCF10Ca1h is derived from a separate tumour (Figure ?(Figure1A).1A). This series of cell lines therefore represents an isogenic model of disease progression, and provides a useful tool for the investigation of molecular changes during the progression of human breast neoplasia and the generation of tumour heterogeneity on a common genetic background 19. Open in a separate window Figure 1 Spectrum of acquired alterations in the MCF10 progression series. (A) Diagrammatic representation of the generation of the MCF10 progression series. Non\invasive cell lines are highlighted in grey, DCIS.com cell are highlighted in green, and invasive cell lines are highlighted in red. Circled numbers represent the number of days for which cell lines were grown in vivo before replantation. (B) Matrix of identified somatic mutations acquired from.