7 research outputs found
Secreted proteins that were increased 5-fold or higher in co-culture conditioned medium (CM) compared to CM from naïve MCF-7.
<p>Secreted proteins that were increased 5-fold or higher in co-culture conditioned medium (CM) compared to CM from naïve MCF-7.</p
Treatment of cancerous and normal breast cells with conditioned medium (CM) alters gene expression.
<p>(A) Gene expression changes in MCF-7 cells treated with CM reveal a 10-fold increase in SNAIL, a 7-fold increase in SLUG, and elimination of detectable E-Cadherin mRNA. The increase in EMT markers is accompanied by a down-regulation of ERα mRNA. (B) After treatment of HMEC grown in monolayer culture with CM, there were changes in expression of EMT markers and increases in IL-1β and the growth factor amphiregulin (AREG), suggesting an important role for paracrine signaling between the tumor microenvironment and adjacent normal epithelium.</p
Hierarchical clustering of breast cancer patient samples based on the secreted protein signature reveals distinct subgroups.
<p>(A) Hierarchical clustering of primary tumors reveals three distinct classes (B) Kaplan–Meier method and the log-rank test was used to compare the mean survival rates across the three identified classes. Patients in the red class (i.e. with higher gene expression of the signature secreted proteins) are significantly associated with a poorer prognosis. (C) ONCOMINE analysis shows that the protein signature is also overexpressed in IDC compared with DCIS in an independent dataset. The protein signature is also significantly correlated with the stroma of breast tumors</p
Three-dimensional co-culture models and analytical approaches in this study.
<p>Schematic of the several three-dimensional co-culture models we utilized to study the interactions between MCF-7 breast cancer cells and primary mammary fibroblasts. The MCF-7 were grown as spheroids in a Matrigel overlay culture. Fibroblasts were incorporated either in a direct-contact mixed culture (MCF-7<sup>M</sup>) or in a separate collagen layer in a sandwich culture (MCF-7<sup>S</sup>). To study the effects of paracrine signaling, conditioned medium (CM) studies were done in which CM was taken from the mixed culture and used to treat MCF-7 or normal mammary epithelial cells (HMEC) grown alone. The CM was also profiled using protein arrays to obtain the secreted protein interaction signature (iSig). We used gene expression and phenotypic assays to study response to hormone and the expression of markers of EMT. This molecular profiling approach was correlated to label-free FT-IR spectroscopic imaging and also gene expression from patient samples.</p
Spectroscopic signatures determined using 3D co-culture models can be translated to human breast tissue samples.
<p>(A) Tissue microarray (TMA) biopsy cores (1.5 mm core diameter) were IHC stained for ERα and also imaged using FT-IR imaging (N-H/O-H band at 3300 cm<sup>−1</sup> visualized here for clarity). Images classified using Bayesian classifier are displayed as well to highlight the ability of FT-IR to discriminate between cell types in complex samples. Scale bar represents 250 µm. (B) Differences between epithelial pixels in patient samples with high (>80%) and low (<20%) expression of ERα can be seen in peaks at 1080 cm<sup>−1</sup> (phosphate) and 1030 cm<sup>−1</sup> (glycosidic bonds). Interestingly, there are more apparent differences in these peaks when pixels from fibroblasts are analyzed. Full spectrum (3800 – 950 cm<sup>−1</sup>), C-H stretching region (3000 – 2750 cm<sup>−1</sup>), and biochemical fingerprint region (1750 – 950 cm<sup>−1</sup>) are shown.</p
Mammary fibroblasts induce an epithelial-to-mesenchymal transition in ER<sup>+</sup> breast cancer cells in 3D culture.
<p>(A) Over the course of 6 days in the sandwich co-culture, MCF-7<sup>S</sup> display increased mRNA levels of EMT markers SNAIL and SLUG with a decrease in E-cadherin mRNA. (B) Mixed co-cultures were prepared at two fibroblast seeding densities, low- and high-density (relative to MCF-7) and EMT markers were modulated up or down as seen with the sandwich co-culture, and a dose-dependent response to the fibroblast presence was observed. (C) Immunohistochemistry was used to confirm decrease in overall E-cadherin protein level inMCF-7<sup>M</sup> (D) Co-culture with human mammary fibroblasts (HMF) increases the invasiveness of MCF-7 breast cancer cells.</p
Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.
<p>(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E<sub>2</sub>), particularly in the C-H stretching region (3000 – 2750 cm<sup>−1</sup>) and in the peak associated with nucleic acids (1080 cm<sup>−1</sup>). However, when cells are co-cultured with human mammary fibroblasts (HMF), the response to hormone is lost. (B) Spectral changes are also seen in MCF-7 cells treated with tamoxifen (Tam). While there is a slight induction in peaks associated with the C-H stretching region upon treatment with Tam, the peak associated with nucleic acids, and therefore proliferation, is decreased in MCF-7 cells. This is correlated with the anti-proliferative effects of tamoxifen on ER<sup>+</sup> cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm<sup>−1</sup> peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.</p