3D HETEROGENEOUS BREAST TISSUE MICROENVIRONMENT USING POLYLACTIDE BEADS.
Breast cancer remains the most common cancer type in women and the second leading cause of death in the US . Normal breast tissue is rather heterogeneous as are the breast cancer types mainly originating from the ducts . Additionally, the microenvironment plays a critical role in the cancer progression through both the chemical and cell-cell interactions [3, 4]. Indeed, while two-dimensional (2D) cultures have provided key information, 3D cultures more closely recapitulate the breast tissue and the associated cancer progression [5, 6].
3D models of breast tissues and of breast cancer progression should allow the tuning of both breast structure and functions mimicking the natural physiology of the breast. Should three dimensional models would be invaluable in the understanding of breast cancer development, progression and in the evaluation of potential treatment regimens. Specifically, such 3D models should deepen our understanding of the complex cell-cell and cell-matrix interactions that are involved in the development of breast tissue as well as cancer initiation and progress. Three dimensional models of breast tissues also can serve as monitoring system for cellular processes that lead to tumor growth and invasion and as an alternative method for investigation new drugs or drug regimen. Importantly, 3D model may also be used as potential in vivo implants personalized to each patient's needs.
The challenges in the generation of 3D breast tissues remain to control specific features of normal breast tissue, the generation of functional acinus- and duct-like structures and the modulation of the complex cell-cell and cell-ECM interactions. In particular, current 3D models of breast tissue do not account for heterogeneous density observed within breast tissue. In prior investigations , we demonstrated the key importance of the composition and density of the extracellular matrix (ECM) in the generation of functional acini and ducts.
Here, we further our inquiry of the role of the density on the matrix using embedded poly-lactide beads to mimic the breast tissue heterogeneity. Our results indicate that the nature of the beads and more significantly of their coating affected the size/complexity of the 3D breast tissue model generated.
Breast cells. MCF10A were purchased from ATCC (Manassas, VA) and used within passages 4-12. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Cellgro, Manassas, VA, USA) supplemented with 5% horse serum (Lonza, Allendale, NJ, USA), insulin (10 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), epidermal growth factor (20 ng/ml; Sigma-Aldrich), hydrocortisone (0.5 mg/ml; Sigma), amphotericin B (Cellgro), and gentamycin (Cellgro). Cell viability and counts were assessed using trypan blue dye (Cellgro). Cells were expended in 2D cultures conditions in the media described above, detached using trypsin, washed, counted and used in the 3D model.
Polylactide Beads. Polylactide beads were a gift from Dr. Burg lab (Clemson University). Polylactide beads were prepared and stored under vacuum until use as described elsewhere .
3D breast heterogeneity models. After sterilization, beads were immersed in either PBS, F12/DMEM media or collagen I solutions overnight and rinsed in sterile PBS prior to use in 3D models. Bead diameters were determined post-coating. Diameter of the beads were derived from microphotographs obtained using a IX70 microscope displaying a calibrated scale using Image J software.
After layering and gelling a mix collagen I/Matrigel[R] (Biosciences, Bedford, MA, USA; 1:1 mixture) [7, 9], MCF10A cells alone or admixed with coated beads (17ug) were added to each well and the growth of breast like structures assessed overtime (up to 14 days) through microscopy (IX70 Olympus with camera DP70). For monitoring purposes, MCF10A cells were stained with the nuclear vital dye Hoechst 33342 (Promega, Madison, WI, USA; excitation 350 nm, emission 461 nm). Multiple overlapping microphotographs were stitched together to capture the entire structures. 3D model parameters measured using Image J software (NIH) included the total area of the structure generated and the length of the branching.
Extracellular protein and collagen remodeling. After a 14-day incubations, 3D structures were assessed for collagen and non-collagenous protein deposit following stain with Sirius Red and Fast Green (Chondrex, Redmond, WA), respectively with the MCF10A cells labeled with nuclear vital dye.
Statistical Analyses. All experiments were independently repeated (n=2-3) and data are presented as mean [+ or -] SEM. One-way and two-way ANOVAs along with post-hoc tests were used to assess differences in coated bead sizes, 3D area, branching overtime and matrix reorganization. A priori, p<0.05 was considered significant.
Coating poly-lactide beads with media or collagen I solution affected the diameter of the poly-lactide beads (133 [+ or -] 3 [micro]m, 117 [+ or -] 4 [micro]m vs 124 [+ or -] 2 um for PBS, Media and Collagen I coating, respectively, p<0.05).
In both the presence and absence of polylactide beads regardless of the coating, MCF10A formed breast like structures including duct-like structures (Fig 1A). When cultured in the presence of beads, the area and the length of the duct-like formation differed (Fig 1 A,B).
When compared the areas generated in the presence of media-coated or collagen I coated beads was significantly higher than the area generated without bead or in the presence of PBS coated beads (2-way ANOVA, overtime, p<0.001, Fig 2).
The number and length of the extensions outward was highly variable and tended to decrease especially in the presence of beads coated with media or collagens compared to no beads controls (n.s.)
To better ascertain the matrix changes associated with the presence of the coated beads, the presence of collagen I was determined. While overall collagen concentrations were not significantly different, the location of non-collagenous proteins overlapped the location of cells, whereas in highly rich collagen I areas, no MCF10A cells were present (Fig 3).
In the present study, we assessed the potential of polylactide beads to mimic the heterogeneous breast tissue environment. Indeed, the density of the microenvironment, in particular of the ECM, defines, to a large extent, both the normal breast tissue and the breast progression [3, 9]. The results indicate that in the presence of beads coated with media or collagen I, the area of the structure generated by MCF10A cells alone is significantly increased. Moreover, the differentiation of outward tubule-like structures appeared to be limited while the complexity of the branching tended to increase.
These results confirmed the observations made using various concentrations of collagen/Matrigel[R] leading to increased matrix density associated with drastic changes in the formation and function of the generated 3D tissue [7, 9]. Others have successfully developed matrices and cell combinations that closely mimic breast tissue [5, 6, 10]. However, the heterogeneity inherent to breast tissue remains a key challenge in developing 3D culture test system mimicking breast and breast cancer progression. Our data represent a first step in the generation of such models.
Key to the optimization of 3D modeling of breast tissue are the generation of standard tools including software and assays to confirm the structure and functionality of the generated 3D breast tissues. Recent works will facilitate those analyses including the development of new software allowing the estimation of volumes of 3D cell-based structures .
Moreover, the results presented here focus on a monoculture (i.e., one cell type), current and future 3D models aim to mimic not only the heterogeneity of the matrix, of the chemical milieu but also of cell types present [3, 4, 9]. Clinically, the generation of 3D in vitro systems derived from cells isolated from individual patients may be used to further personalize the data gathered on the cancer type, progression, prognostic and especially best treatment approach [12, 13].
As the categorization of breast cancers and cancers in general become more refined and thus more amendable to specific targeted therapy, such 3D breast tissue and/or breast cancer progression models hold great promises both as predictive diagnostic tools and as approach to assess multiple therapeutic options and select the most beneficial.
Long-term, the data presented here will participate to the definition of reliable tools for the evaluation of breast cancer progression and the testing of specific treatments. Many steps remain before breast tissue 3D models can reliably be used as diagnostic and drug predictive tools in the clinical environment. Nevertheless, 3D models are closer to fulfill the potential of bioengineered tissues as viable options to further our understanding of breast cancer progression and as personalized test tissue systems to assess drug combinations relevant to the treatment of breast cancer.
The authors would like to thanks Dr. Karen Burg (Georgia State University, Athens, GA) and her students at Clemson University for the preparation of the polylactide beads. Dr. Dreau also would like to acknowledge the participation of numerous undergraduate and graduate students that spent time in his laboratory collecting the data. This research was supported in part through NSF funding (EFRI program).
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Bryanna Sierra and Didier Dreau
Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte NC, USA
Caption: Fig 1. 3D structures generated by MCF10A in the absence (A) or presence (B) of coated polylactide beads. Microphotographs taken on day 11 and stitched together highlighting the presence of 3D structures including duct-like and the complexity of the network formed.
Caption: Fig 2. Media (B) or collagen I (C) coated beads significantly (ANOVA, p<0.001) increased the areas of the structures generated by MCF10A in contrast with PBS coated poly-lactide beads (A) or the absence of beads. On microphotographs taken overtime and stitched together, the total area was determined and compared to 3D MCF10A cultures without beads. Areas are expressed in % of the entire well.
Caption: Fig 3. In 3D cultures, cells clustered in area rich in non-collageous proteins rather than in area rich in collagen. Representative microphotographs of MCF10A cells in 3D cultures with media-coated beads stained on day 14 with Hoechst (nuclear dye, A), non-collagenous proteins (green, B) and collagen I (red, B). The overlapping microphotograph (C) further highlights the co-localization of non-collagenous proteins and MCF10A cells.
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|Author:||Sierra, Bryanna; Dreau, Didier|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Apr 1, 2018|
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