Chemical and physical properties of carbonated hydroxyapatite affect breast cancer cell behavior

Abstract

Breast microcalcifications are routinely explored for mammographic detection of breast cancer and are primarily composed of non-stoichiometric hydroxyapatite (Ca10-x(PO4)6-x(CO3)x(OH)2-x) (HA). Interestingly, HA morphology and carbonate substitution vary in malignant vs. benign lesions. However, whether or not these changes (i) are functionally linked and (ii) impact malignancy remains unclear due in part to lack of model systems that permit evaluating these possibilities. Here, we have adapted a 96 well-based mineralized culture platform to investigate breast cancer cell behavior in response to systematic changes in the chemical and physical properties of HA. By adjusting the carbonate content of the simulated body fluid (SBF) solutions used during growth, we can control the morphology and carbonate substitution of the deposited HA. Our results suggest that both the combined and individual effects of these differences alter breast cancer cell growth and secretion of tumorigenic interleukin-8 (IL-8). Consequently, changes in both HA carbonate incorporation and morphology impact the behavior of breast cancer cells. Collectively, our data underline the importance of biomineralized culture platforms to evaluate the functional contribution of HA material properties to the pathogenesis of breast cancer.

Statement of significance: Breast microcalcifications are small mineral deposits primarily composed of hydroxyapatite (HA). HA physicochemical properties have been of considerable interest, as these are often altered during breast cancer progression and linked to malignancy. However, the functional relationship between these changes and malignancy remains unclear due in part to lack of model systems. Here, we have adapted a previously developed a 96 well-based culture platform to evaluate breast cancer cell behavior in response to systematic changes in HA properties. Our results demonstrate that changes in HA morphology and carbonate content influence breast cancer cell growth and interleukin-8 secretion, and suggest that characterizing the effect of HA properties on breast cancer cells may improve our understanding of breast cancer development and progression.

Keywords: Breast cancer malignancy; Carbonate contents; Culture platform; Hydroxyapatite; Microcalcifications.

Figure 1

Schematic of HA mineral formation procedure on 96-well polypropylene (PP) plates. According to previous work (Ref. 6) HA mineral growth was induced by coating 96-well PP plates with PLG. Following partial hydrolysis of PLG, a precursor layer was formed by incubation in 4.2 mM [CO₃²⁻] mSBF for 4 days. Finally, mineral coatings of varied carbonate content were formed by incubation in 0–27 mM [CO₃²⁻] mSBF for 4 days. Scale bar = 5 mm.

Figure 2

Adjusting mSBF carbonate content alters the composition of HA mineral coatings. (a) XRD and (b) FT-IR spectra of commercially available HA (Sigma) and HA mineral coatings that were formed by incubation in 0–27 mM carbonate mSBF. (c) Relative carbonate composition of HA mineral coatings as determined by calculating the ratio of the CO₃²⁻ and PO₄³⁻ FT-IR peak areas at 874 cm⁻¹ and 900–1300 cm⁻¹, respectively. (d) Crystallinity index was calculated from FT-IR peaks by dividing the sum of the peak maxima at 565 cm⁻¹ and 603 cm⁻¹ by the height of the valleys between them. * and ^# indicate significant differences as compared to HA and 0 mM conditions, respectively (P < 0.05).

Figure 3

Adjusting mSBF carbonate content alters the morphology of HA mineral coatings. Low (A–H) and high magnification (a–h) SEM micrographs captured after 8 days of mineral formation in 0–27 mM carbonate mSBF. Scale bar = 10 μm (A–H) and 0.5 μm (a–h). Non-hydrolyzed PLG control films are shown for comparison.

Figure 4

Adjusting mSBF carbonate content modulates the surface properties of HA mineral coatings. (a) Representative AFM micrographs of HA mineral coatings and non-hydrolyzed PLG control surfaces as collected in tapping mode. (b) Surface roughness and (c) Surface area of HA mineral coatings as determined by image analysis of representative 5 μm x 5 μm regions. Upper case letter (A) indicates significant difference relative to PLG. Lower case letters (a, b and c) indicate significant differences relative to 0 mM, 2 mM and 4.2 mM conditions, respectively (P < 0.05). Scale bar = 1 μm.

Figure 5

Adjusting mSBF carbonate content during mineral formation regulates subsequent serum protein adsorption and MDA-MB231 cell adhesion. (a) Serum protein adsorption on HA mineral coatings after 1 hour of incubation in serum-containing media as determined by colorimetric analysis. (b) DNA concentration of lysates prepared from adherent MDA-MB231 1 hour after seeding on mineral coatings as measured fluorimetrically. (c) Representative SEM images of MDA-MB231 cells 3 hours after seeding on mineral coatings. Arrows indicate elongated cells. Upper case letter (A) indicates significant difference relative to PLG. Lower case letters (a, b and c) indicate significant differences relative to 0 mM, 2 mM and 4.2 mM conditions, respectively (P < 0.05). Scale bar = 20 μm.

Figure 6

α_v and β₁ integrins regulate breast cancer cell adhesion on PLG surfaces, but not HA mineral coatings. DNA concentration of lysates prepared from adherent MDA-MB231 1 hour after seeding on mineral coatings in the presence and absence of function-blocking (a) α_v or (b) β₁-integrin antibodies as measured fluorimetrically. * indicates significant differences between antibody (α_v, β₁) and control (No ab) conditions (P < 0.05).

Figure 7

HA mineral properties regulate MDA-MB231 growth and IL-8 secretion. MDA-MB231 (a) growth and (b) IL-8 secretion 3 days after seeding onto the different HA and PLG control surfaces as analyzed by fluorimetric quantification of DNA content and ELISA of conditioned media, respectively. Dashed lines indicate a biphasic regulation of cell behavior that is likely due to topographical (0–2 mM) vs. chemical (4.2–27 mM) differences of the resulting mineral coatings. Upper case letter (A) indicates significant difference relative to PLG. Lower case letters (a, b and c) indicate significant differences relative to 0 mM, 2 mM and 4.2 mM conditions, respectively (P < 0.05).

Figure 8

HA mineral properties differentially affect IL-8 secretion of various breast cancer cell lines. IL-8 secretion of (a) MCF7 vs. MDA-MB231 as well as of (b) normal MCF10A vs. pre-malignant MCF10AT1 vs. malignant MCF10DCIS.com as determined by ELISA followed by normalization to DNA content after 3 days of culture on the different mineral coatings. Upper case letter (A) indicates significant difference relative to PLG. Lower case letters (a, b and c) indicate significant differences relative to 0 mM, 4.2 mM and 15 mM conditions, respectively (P < 0.05).