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Integration of molecular profiling and chemical imaging to elucidate fibroblast-microenvironment impact on cancer cell phenotype and endocrine resistance in breast cancer.

Holton SE, Bergamaschi A, Katzenellenbogen BS, Bhargava R - PLoS ONE (2014)

Bottom Line: Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth.Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies.Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.

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Three-dimensional co-culture models and analytical approaches in this study.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-7M) or in a separate collagen layer in a sandwich culture (MCF-7S). 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.
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pone-0096878-g001: Three-dimensional co-culture models and analytical approaches in this study.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-7M) or in a separate collagen layer in a sandwich culture (MCF-7S). 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.

Mentions: In order to study the interactions between ER+ breast cancer cells and mammary fibroblasts, we utilized four 3D culture models, shown schematically in Figure 1A and 1B. We used a number of approaches to translate these findings to human disease states (Fig. 1C and 1D). The breast cancer model was comprised of ER+ MCF-7 breast cancer cells grown on Matrigel to form 3D spheroid structures, denoted hereon simply as MCF-7. Primary human mammary fibroblasts (HMF), isolated from normal breast tissue, were commercially obtained. Several co-culture geometries were used in order to systematically characterize the mechanism by which tumor-adjacent stromal fibroblasts exert influence over cancer cell growth. An indirect co-culture, the ‘sandwich’ (MCF-7S), represents the compartmentalization of cancer cells and fibroblast-rich stroma, as may be observed in locally confined tumors. Because the fibroblasts are grown in a separate collagen layer, the two cell types are able to communicate continuously via molecular diffusion of soluble factors while remaining in their respective layers [16]. After a prescribed co-culture time, the layers are easily separated, and molecular analyses can be performed without any cell crossover. The direct, or ‘mixed’, co-culture (MCF-7M), consists of both MCF-7 and fibroblasts grown together on Matrigel, allowing for cell-cell interactions arising from direct contact. To understand the transient influence of soluble factors on cancer cell phenotypes, a conditioned medium (CM) culture was employed which consists of medium from MCF-7M added to the MCF-7 cells. This medium was subsequently analyzed to identify the secreted factors that characterize cancer cell-fibroblast interactions in this model system. These models were used to comprehensively interrogate the differential fibroblast-cancer cell interactions that influence cancer cell behavior and resistance to therapy.


Integration of molecular profiling and chemical imaging to elucidate fibroblast-microenvironment impact on cancer cell phenotype and endocrine resistance in breast cancer.

Holton SE, Bergamaschi A, Katzenellenbogen BS, Bhargava R - PLoS ONE (2014)

Three-dimensional co-culture models and analytical approaches in this study.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-7M) or in a separate collagen layer in a sandwich culture (MCF-7S). 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016150&req=5

pone-0096878-g001: Three-dimensional co-culture models and analytical approaches in this study.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-7M) or in a separate collagen layer in a sandwich culture (MCF-7S). 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.
Mentions: In order to study the interactions between ER+ breast cancer cells and mammary fibroblasts, we utilized four 3D culture models, shown schematically in Figure 1A and 1B. We used a number of approaches to translate these findings to human disease states (Fig. 1C and 1D). The breast cancer model was comprised of ER+ MCF-7 breast cancer cells grown on Matrigel to form 3D spheroid structures, denoted hereon simply as MCF-7. Primary human mammary fibroblasts (HMF), isolated from normal breast tissue, were commercially obtained. Several co-culture geometries were used in order to systematically characterize the mechanism by which tumor-adjacent stromal fibroblasts exert influence over cancer cell growth. An indirect co-culture, the ‘sandwich’ (MCF-7S), represents the compartmentalization of cancer cells and fibroblast-rich stroma, as may be observed in locally confined tumors. Because the fibroblasts are grown in a separate collagen layer, the two cell types are able to communicate continuously via molecular diffusion of soluble factors while remaining in their respective layers [16]. After a prescribed co-culture time, the layers are easily separated, and molecular analyses can be performed without any cell crossover. The direct, or ‘mixed’, co-culture (MCF-7M), consists of both MCF-7 and fibroblasts grown together on Matrigel, allowing for cell-cell interactions arising from direct contact. To understand the transient influence of soluble factors on cancer cell phenotypes, a conditioned medium (CM) culture was employed which consists of medium from MCF-7M added to the MCF-7 cells. This medium was subsequently analyzed to identify the secreted factors that characterize cancer cell-fibroblast interactions in this model system. These models were used to comprehensively interrogate the differential fibroblast-cancer cell interactions that influence cancer cell behavior and resistance to therapy.

Bottom Line: Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth.Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies.Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.

Show MeSH
Related in: MedlinePlus