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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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Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E2), particularly in the C-H stretching region (3000 – 2750 cm−1) and in the peak associated with nucleic acids (1080 cm−1). 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+ cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm−1 peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.
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pone-0096878-g006: Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E2), particularly in the C-H stretching region (3000 – 2750 cm−1) and in the peak associated with nucleic acids (1080 cm−1). 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+ cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm−1 peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.

Mentions: Our data show that ER level and activity can be dynamically altered and that ER-positive cells may not respond to endocrine therapy if they have undergone EMT. Given the spatially-heterogeneous nature of tumors, an imaging technique to record the individual cellular states within a tumor is needed. While IHC can provide information about protein expression, it is time-consuming and requires antibodies [35]. We hypothesized that using FT-IR microscopy could be used to measure the chemical response of breast cancer cells to hormone treatment or to endocrine therapy without loss of spatial information. We used the same 3D culture samples and conditions so that mRNA expression and FT-IR signatures could be directly compared. After E2 treatment, we observed a large increase in the C-H vibrational region of the spectrum (Fig. 6A), commonly associated with the CH2 bending of fatty acids and lipids [36]. These three peaks (2960 cm−1, 2930 cm−1, and 2850 cm−1) are also correlated with fatty acyl chain peroxidation43. Similarly, the peak at 1080 cm−1, associated with the phosphate backbone of nucleic acids and altered metabolic activity, is increased upon E2 treatment. Thus, changes in absorption pattern can be correlated with biological changes occurring in ER+ breast cancer cells in response to treatment with E2.


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)

Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E2), particularly in the C-H stretching region (3000 – 2750 cm−1) and in the peak associated with nucleic acids (1080 cm−1). 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+ cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm−1 peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096878-g006: Fourier transform-infrared (FT-IR) spectroscopic imaging can be used to monitor hormone response in cells in culture.(A) A change in FT-IR spectroscopic imaging is seen in MCF-7 cells treated with estradiol (E2), particularly in the C-H stretching region (3000 – 2750 cm−1) and in the peak associated with nucleic acids (1080 cm−1). 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+ cells. In samples that have been co-cultured with HMF, this change is not seen at the 1080 cm−1 peak, corresponding to the endocrine-resistant growth that was seen using proliferation assays.
Mentions: Our data show that ER level and activity can be dynamically altered and that ER-positive cells may not respond to endocrine therapy if they have undergone EMT. Given the spatially-heterogeneous nature of tumors, an imaging technique to record the individual cellular states within a tumor is needed. While IHC can provide information about protein expression, it is time-consuming and requires antibodies [35]. We hypothesized that using FT-IR microscopy could be used to measure the chemical response of breast cancer cells to hormone treatment or to endocrine therapy without loss of spatial information. We used the same 3D culture samples and conditions so that mRNA expression and FT-IR signatures could be directly compared. After E2 treatment, we observed a large increase in the C-H vibrational region of the spectrum (Fig. 6A), commonly associated with the CH2 bending of fatty acids and lipids [36]. These three peaks (2960 cm−1, 2930 cm−1, and 2850 cm−1) are also correlated with fatty acyl chain peroxidation43. Similarly, the peak at 1080 cm−1, associated with the phosphate backbone of nucleic acids and altered metabolic activity, is increased upon E2 treatment. Thus, changes in absorption pattern can be correlated with biological changes occurring in ER+ breast cancer cells in response to treatment with E2.

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