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Identification of genetic loci that control mammary tumor susceptibility through the host microenvironment.

Zhang P, Lo A, Huang Y, Huang G, Liang G, Mott J, Karpen GH, Blakely EA, Bissell MJ, Barcellos-Hoff MH, Snijders AM, Mao JH - Sci Rep (2015)

Bottom Line: Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls.We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency.Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

ABSTRACT
The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genes involved in cytokines, including TGFβ1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.

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TGFβ1 regulates growth factor-induced branching.(A) Representative phase-contrast images of BALB/c or SPRET/EiJ organoids induced to branch with TGFα in growth factor-reduced Matrigel. Organoids were grown for 5 days after treatment. (B) Quantification of the number of BALB/c and SPRET/EiJ organoids in each condition that had three or more branches (n = 6 experiments, >200 organoids/condition). (C) TGFβ1 levels after treatment with TGFα. ELISA analysis of culture media harvested from organoid cultures (n = 6 independent experimental sets). (D) Merged channel images BALB/c and SPRET/EiJ mammary gland sections stained with DAPI, latent-TGFβ1 and active-TGFβ1 (n = 6 experiments). (E) Representative phase-contrast images of BALB/c organoids induced to branch with TGFα. After 24 hours, organoids were treated with SPRET/EiJ culture media either alone or with TGFβ1 blocking antibody and grown for 5 days. Quantification of the number of BALB/c organoids in each condition that had three or more branches (n = 6 experiments, >100 organoids/condition). The p-values were obtained by t-test.
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f5: TGFβ1 regulates growth factor-induced branching.(A) Representative phase-contrast images of BALB/c or SPRET/EiJ organoids induced to branch with TGFα in growth factor-reduced Matrigel. Organoids were grown for 5 days after treatment. (B) Quantification of the number of BALB/c and SPRET/EiJ organoids in each condition that had three or more branches (n = 6 experiments, >200 organoids/condition). (C) TGFβ1 levels after treatment with TGFα. ELISA analysis of culture media harvested from organoid cultures (n = 6 independent experimental sets). (D) Merged channel images BALB/c and SPRET/EiJ mammary gland sections stained with DAPI, latent-TGFβ1 and active-TGFβ1 (n = 6 experiments). (E) Representative phase-contrast images of BALB/c organoids induced to branch with TGFα. After 24 hours, organoids were treated with SPRET/EiJ culture media either alone or with TGFβ1 blocking antibody and grown for 5 days. Quantification of the number of BALB/c organoids in each condition that had three or more branches (n = 6 experiments, >100 organoids/condition). The p-values were obtained by t-test.

Mentions: Parental strain differences in branching and invasion into the fat pad during mammary development led us to investigate which regulators of these physiological processes differ between BALB/c and SPRET/EiJ. Our SNP mapping and bioinformatic analysis identified the TGFβ pathway, a known regulator of mammary branching and development24, as a potential mediator of mammary tumor susceptibility. We further examined the involvement of TGFβ1 signaling on breast cancer risk using an ex vivo organoid culture model to assess stromal invasion25. Ductal fragments from BALB/c, SPRET/EiJ and their F1 hybrid mammary glands were cultured in growth factor-reduced Matrigel (Figure 5A), and the number of branching organoids were quantified (Figure 5B). We observed that SPRET/EiJ and F1 hybrid organoids were unable to form any branched structure, whereas BALB/c organoids branched as reported previously; similar results were obtained using standard 3D culture conditions in 3 mg/ml collagen I (Figure S6). These results suggest that SPRET/EiJ and BALB/c epithelial cells sense and respond to their microenvironments differently.


Identification of genetic loci that control mammary tumor susceptibility through the host microenvironment.

Zhang P, Lo A, Huang Y, Huang G, Liang G, Mott J, Karpen GH, Blakely EA, Bissell MJ, Barcellos-Hoff MH, Snijders AM, Mao JH - Sci Rep (2015)

TGFβ1 regulates growth factor-induced branching.(A) Representative phase-contrast images of BALB/c or SPRET/EiJ organoids induced to branch with TGFα in growth factor-reduced Matrigel. Organoids were grown for 5 days after treatment. (B) Quantification of the number of BALB/c and SPRET/EiJ organoids in each condition that had three or more branches (n = 6 experiments, >200 organoids/condition). (C) TGFβ1 levels after treatment with TGFα. ELISA analysis of culture media harvested from organoid cultures (n = 6 independent experimental sets). (D) Merged channel images BALB/c and SPRET/EiJ mammary gland sections stained with DAPI, latent-TGFβ1 and active-TGFβ1 (n = 6 experiments). (E) Representative phase-contrast images of BALB/c organoids induced to branch with TGFα. After 24 hours, organoids were treated with SPRET/EiJ culture media either alone or with TGFβ1 blocking antibody and grown for 5 days. Quantification of the number of BALB/c organoids in each condition that had three or more branches (n = 6 experiments, >100 organoids/condition). The p-values were obtained by t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352890&req=5

f5: TGFβ1 regulates growth factor-induced branching.(A) Representative phase-contrast images of BALB/c or SPRET/EiJ organoids induced to branch with TGFα in growth factor-reduced Matrigel. Organoids were grown for 5 days after treatment. (B) Quantification of the number of BALB/c and SPRET/EiJ organoids in each condition that had three or more branches (n = 6 experiments, >200 organoids/condition). (C) TGFβ1 levels after treatment with TGFα. ELISA analysis of culture media harvested from organoid cultures (n = 6 independent experimental sets). (D) Merged channel images BALB/c and SPRET/EiJ mammary gland sections stained with DAPI, latent-TGFβ1 and active-TGFβ1 (n = 6 experiments). (E) Representative phase-contrast images of BALB/c organoids induced to branch with TGFα. After 24 hours, organoids were treated with SPRET/EiJ culture media either alone or with TGFβ1 blocking antibody and grown for 5 days. Quantification of the number of BALB/c organoids in each condition that had three or more branches (n = 6 experiments, >100 organoids/condition). The p-values were obtained by t-test.
Mentions: Parental strain differences in branching and invasion into the fat pad during mammary development led us to investigate which regulators of these physiological processes differ between BALB/c and SPRET/EiJ. Our SNP mapping and bioinformatic analysis identified the TGFβ pathway, a known regulator of mammary branching and development24, as a potential mediator of mammary tumor susceptibility. We further examined the involvement of TGFβ1 signaling on breast cancer risk using an ex vivo organoid culture model to assess stromal invasion25. Ductal fragments from BALB/c, SPRET/EiJ and their F1 hybrid mammary glands were cultured in growth factor-reduced Matrigel (Figure 5A), and the number of branching organoids were quantified (Figure 5B). We observed that SPRET/EiJ and F1 hybrid organoids were unable to form any branched structure, whereas BALB/c organoids branched as reported previously; similar results were obtained using standard 3D culture conditions in 3 mg/ml collagen I (Figure S6). These results suggest that SPRET/EiJ and BALB/c epithelial cells sense and respond to their microenvironments differently.

Bottom Line: Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls.We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency.Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

ABSTRACT
The interplay between host genetics, tumor microenvironment and environmental exposure in cancer susceptibility remains poorly understood. Here we assessed the genetic control of stromal mediation of mammary tumor susceptibility to low dose ionizing radiation (LDIR) using backcrossed F1 into BALB/c (F1Bx) between cancer susceptible (BALB/c) and resistant (SPRET/EiJ) mouse strains. Tumor formation was evaluated after transplantation of non-irradiated Trp53-/- BALB/c mammary gland fragments into cleared fat pads of F1Bx hosts. Genome-wide linkage analysis revealed 2 genetic loci that constitute the baseline susceptibility via host microenvironment. However, once challenged with LDIR, we discovered 13 additional loci that were enriched for genes involved in cytokines, including TGFβ1 signaling. Surprisingly, LDIR-treated F1Bx cohort significantly reduced incidence of mammary tumors from Trp53-/- fragments as well as prolonged tumor latency, compared to sham-treated controls. We demonstrated further that plasma levels of specific cytokines were significantly correlated with tumor latency. Using an ex vivo 3-D assay, we confirmed TGFβ1 as a strong candidate for reduced mammary invasion in SPRET/EiJ, which could explain resistance of this strain to mammary cancer risk following LDIR. Our results open possible new avenues to understand mechanisms of genes operating via the stroma that affect cancer risk from external environmental exposures.

Show MeSH
Related in: MedlinePlus