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In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone

View Article: PubMed Central - PubMed

ABSTRACT

Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps.

No MeSH data available.


Related in: MedlinePlus

Upper panel: in vitro modeling of cancer cell extravasation and early invasion. (a) Microfluidic model of breast cancer cell (BCC) extravasation towards a bone-mimicking microenvironment containing mesenchymal stem cells (BMSCs) and osteo-differentiated BMSCs (OBs); (b) perfusable microvascular networks (green) allowing cancer cell flow, adhesion and trapping within capillary-like structures; (c–g) Representative figures showing expression of bone and vascular specific markers (red), namely osteocalcin (OCN, c), bone alkaline phosphatase (ALP, d), vascular endothelial (VE)-cadherin (e), zonula occludens (ZO)-1 (f) and α smooth muscle actin (g). Endothelial cells (ECs): green; nuclei: blue. Reproduced by permission from [47] Jeon J.S.; et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA2015; Lower panel: engineered model for the study of the effect of microvasculature on BCC dormancy. Left: BMSCs were seeded alone (stroma) or in co-culture with ECs (microvasculature niche). BCCs were seeded onto stroma or microvasculature niche and laminin rich ECM (LrECM) was deposited to create a 3D environment for cancer cell study. YFP: yellow fluorescent protein; Right: T4-2 BCCs seeded on bone marrow (BoMa)-like stroma (scale bar: 100 μm) or BoMa-like stroma + ECs (scale bar: 50 μm) showing how the presence of microvasculature significantly reduced the presence of Ki-67 positive cells and induced a dormant state. T4-2 cells: white; Ki67: green; CD31: red; nuclei: blue. Reprinted by permission from [48] Ghajar, C.M.; et al. The perivascular niche regulates breast tumour dormancy. Nat. Cell Biol.2013.
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ijms-17-01405-f003: Upper panel: in vitro modeling of cancer cell extravasation and early invasion. (a) Microfluidic model of breast cancer cell (BCC) extravasation towards a bone-mimicking microenvironment containing mesenchymal stem cells (BMSCs) and osteo-differentiated BMSCs (OBs); (b) perfusable microvascular networks (green) allowing cancer cell flow, adhesion and trapping within capillary-like structures; (c–g) Representative figures showing expression of bone and vascular specific markers (red), namely osteocalcin (OCN, c), bone alkaline phosphatase (ALP, d), vascular endothelial (VE)-cadherin (e), zonula occludens (ZO)-1 (f) and α smooth muscle actin (g). Endothelial cells (ECs): green; nuclei: blue. Reproduced by permission from [47] Jeon J.S.; et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA2015; Lower panel: engineered model for the study of the effect of microvasculature on BCC dormancy. Left: BMSCs were seeded alone (stroma) or in co-culture with ECs (microvasculature niche). BCCs were seeded onto stroma or microvasculature niche and laminin rich ECM (LrECM) was deposited to create a 3D environment for cancer cell study. YFP: yellow fluorescent protein; Right: T4-2 BCCs seeded on bone marrow (BoMa)-like stroma (scale bar: 100 μm) or BoMa-like stroma + ECs (scale bar: 50 μm) showing how the presence of microvasculature significantly reduced the presence of Ki-67 positive cells and induced a dormant state. T4-2 cells: white; Ki67: green; CD31: red; nuclei: blue. Reprinted by permission from [48] Ghajar, C.M.; et al. The perivascular niche regulates breast tumour dormancy. Nat. Cell Biol.2013.

Mentions: Microfluidic models allow one to overcome these limitations, thus representing promising tools to analyze the molecular mechanisms driving BCC extravasation. Recently, our group has developed two microfluidic models to study BCC transmigration/colonization of a bone-mimicking microenvironment generated with hydrogel-laden osteo-differentiated BMSCs. In the first simplified model, microfluidic channels containing BMSCs embedded in a collagen matrix were endothelialized, and MDA-231 cells were flowed through these biomimetic microvessels, showing a preferential transmigration towards the bone-like matrix compared to empty collagen matrix. More in detail, it was demonstrated that the CXCL-5-CXCR-2 signaling axis was involved in the extravasation process, since gradients of CXCL-5 generated through the microfluidic device were able to attract BCCs to control acellular matrices, while antibodies blocking CXCR-2 significantly reduced BCC transmigration towards bone-mimicking microenvironments. Interestingly, the addition of CXCL-5 not only affected BCCs transmigration, but also their migration distance once extravasated [45]. Despite this system representing the first microfluidic model mimicking the organ-specificity of BCCs metastases to bone, it did not fully recapitulate the transmigration of cancer cells through capillary-like vessels nor the effects of physiological flows. Microvascular networks were then developed within fibrin matrices containing osteo-differentiated BMSCs and mural-like BMSCs, which wrapped around microvessels (Figure 3, upper panel). BCCs (BOKL, bone seeking clone of MDA-231) were then infused into the perfusable microvessels and extravasated towards the bone-mimicking microenvironment, while a control muscle-mimicking matrix was not able to attract cancer cells. Several molecules can be involved in the anti-metastatic features characterizing the skeletal muscle. In particular, the muscle-secreted adenosine was demonstrated to reduce bone metastases in vivo through interaction with the A3 adenosine receptor, which is expressed by BCCs [46], despite its role in extravasation not being previously clarified. Surprisingly, the addition of adenosine was able to reduce BCC extravasation to the bone-mimicking microenvironment, despite increasing vascular permeability. Conversely, blocking the A3 adenosine receptor (PSB-10 antagonist) on BCCs injected in the muscle-mimicking microenvironment increased BCC transmigration. These data clearly demonstrated that muscle-secreted adenosine was able to impair BCCs extravasation, but also showed how endothelial permeability was not the key factor driving extravasation [47].


In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone
Upper panel: in vitro modeling of cancer cell extravasation and early invasion. (a) Microfluidic model of breast cancer cell (BCC) extravasation towards a bone-mimicking microenvironment containing mesenchymal stem cells (BMSCs) and osteo-differentiated BMSCs (OBs); (b) perfusable microvascular networks (green) allowing cancer cell flow, adhesion and trapping within capillary-like structures; (c–g) Representative figures showing expression of bone and vascular specific markers (red), namely osteocalcin (OCN, c), bone alkaline phosphatase (ALP, d), vascular endothelial (VE)-cadherin (e), zonula occludens (ZO)-1 (f) and α smooth muscle actin (g). Endothelial cells (ECs): green; nuclei: blue. Reproduced by permission from [47] Jeon J.S.; et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA2015; Lower panel: engineered model for the study of the effect of microvasculature on BCC dormancy. Left: BMSCs were seeded alone (stroma) or in co-culture with ECs (microvasculature niche). BCCs were seeded onto stroma or microvasculature niche and laminin rich ECM (LrECM) was deposited to create a 3D environment for cancer cell study. YFP: yellow fluorescent protein; Right: T4-2 BCCs seeded on bone marrow (BoMa)-like stroma (scale bar: 100 μm) or BoMa-like stroma + ECs (scale bar: 50 μm) showing how the presence of microvasculature significantly reduced the presence of Ki-67 positive cells and induced a dormant state. T4-2 cells: white; Ki67: green; CD31: red; nuclei: blue. Reprinted by permission from [48] Ghajar, C.M.; et al. The perivascular niche regulates breast tumour dormancy. Nat. Cell Biol.2013.
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ijms-17-01405-f003: Upper panel: in vitro modeling of cancer cell extravasation and early invasion. (a) Microfluidic model of breast cancer cell (BCC) extravasation towards a bone-mimicking microenvironment containing mesenchymal stem cells (BMSCs) and osteo-differentiated BMSCs (OBs); (b) perfusable microvascular networks (green) allowing cancer cell flow, adhesion and trapping within capillary-like structures; (c–g) Representative figures showing expression of bone and vascular specific markers (red), namely osteocalcin (OCN, c), bone alkaline phosphatase (ALP, d), vascular endothelial (VE)-cadherin (e), zonula occludens (ZO)-1 (f) and α smooth muscle actin (g). Endothelial cells (ECs): green; nuclei: blue. Reproduced by permission from [47] Jeon J.S.; et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA2015; Lower panel: engineered model for the study of the effect of microvasculature on BCC dormancy. Left: BMSCs were seeded alone (stroma) or in co-culture with ECs (microvasculature niche). BCCs were seeded onto stroma or microvasculature niche and laminin rich ECM (LrECM) was deposited to create a 3D environment for cancer cell study. YFP: yellow fluorescent protein; Right: T4-2 BCCs seeded on bone marrow (BoMa)-like stroma (scale bar: 100 μm) or BoMa-like stroma + ECs (scale bar: 50 μm) showing how the presence of microvasculature significantly reduced the presence of Ki-67 positive cells and induced a dormant state. T4-2 cells: white; Ki67: green; CD31: red; nuclei: blue. Reprinted by permission from [48] Ghajar, C.M.; et al. The perivascular niche regulates breast tumour dormancy. Nat. Cell Biol.2013.
Mentions: Microfluidic models allow one to overcome these limitations, thus representing promising tools to analyze the molecular mechanisms driving BCC extravasation. Recently, our group has developed two microfluidic models to study BCC transmigration/colonization of a bone-mimicking microenvironment generated with hydrogel-laden osteo-differentiated BMSCs. In the first simplified model, microfluidic channels containing BMSCs embedded in a collagen matrix were endothelialized, and MDA-231 cells were flowed through these biomimetic microvessels, showing a preferential transmigration towards the bone-like matrix compared to empty collagen matrix. More in detail, it was demonstrated that the CXCL-5-CXCR-2 signaling axis was involved in the extravasation process, since gradients of CXCL-5 generated through the microfluidic device were able to attract BCCs to control acellular matrices, while antibodies blocking CXCR-2 significantly reduced BCC transmigration towards bone-mimicking microenvironments. Interestingly, the addition of CXCL-5 not only affected BCCs transmigration, but also their migration distance once extravasated [45]. Despite this system representing the first microfluidic model mimicking the organ-specificity of BCCs metastases to bone, it did not fully recapitulate the transmigration of cancer cells through capillary-like vessels nor the effects of physiological flows. Microvascular networks were then developed within fibrin matrices containing osteo-differentiated BMSCs and mural-like BMSCs, which wrapped around microvessels (Figure 3, upper panel). BCCs (BOKL, bone seeking clone of MDA-231) were then infused into the perfusable microvessels and extravasated towards the bone-mimicking microenvironment, while a control muscle-mimicking matrix was not able to attract cancer cells. Several molecules can be involved in the anti-metastatic features characterizing the skeletal muscle. In particular, the muscle-secreted adenosine was demonstrated to reduce bone metastases in vivo through interaction with the A3 adenosine receptor, which is expressed by BCCs [46], despite its role in extravasation not being previously clarified. Surprisingly, the addition of adenosine was able to reduce BCC extravasation to the bone-mimicking microenvironment, despite increasing vascular permeability. Conversely, blocking the A3 adenosine receptor (PSB-10 antagonist) on BCCs injected in the muscle-mimicking microenvironment increased BCC transmigration. These data clearly demonstrated that muscle-secreted adenosine was able to impair BCCs extravasation, but also showed how endothelial permeability was not the key factor driving extravasation [47].

View Article: PubMed Central - PubMed

ABSTRACT

Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps.

No MeSH data available.


Related in: MedlinePlus