Engineered Breast Cancer Cell Spheroids Reproduce Biologic Properties of Solid Tumors

Abstract

Solid tumors develop as 3D tissue constructs. As tumors grow larger, spatial gradients of nutrients and oxygen and inadequate diffusive supply to cells distant from vasculature develops. Hypoxia initiates signaling and transcriptional alterations to promote survival of cancer cells and generation of cancer stem cells (CSCs) that have self-renewal and tumor-initiation capabilities. Both hypoxia and CSCs are associated with resistance to therapies and tumor relapse. This study demonstrates that 3D cancer cell models, known as tumor spheroids, generated with a polymeric aqueous two-phase system (ATPS) technology capture these important biological processes. Similar to solid tumors, spheroids of triple negative breast cancer cells deposit major extracellular matrix proteins. The molecular analysis establishes presence of hypoxic cells in the core region and expression of CSC gene and protein markers including CD24, CD133, and Nanog. Importantly, these spheroids resist treatment with chemotherapy drugs. A combination treatment approach using a hypoxia-activated prodrug, TH-302, and a chemotherapy drug, doxorubicin, successfully targets drug resistant spheroids. This study demonstrates that ATPS spheroids recapitulate important biological and functional properties of solid tumors and provide a unique model for studies in cancer research.

Keywords: aqueous two-phase systems; cancer stem cells; drug resistance; hypoxia; spheroids.

Figure 1

(a) Spontaneous spheroid formation using aqueous two phase system (ATPS) microtechnology; a compact MDA-MB-157 spheroid forms within 24 hrs of incubation (right). (b) This robotic approach produces spheroids of highly consistent diameter (b) with a normal distribution (inset).

Figure 2

Optimization of PrestoBlue assay to determine viability of cells in spheroid cultures. PrestoBlue is directly added to wells containing spheroids and metabolized by live cells. Based on the cellular metabolic activity, the solution emits a fluorescence signal detectable with standard plate readers. Incubation of spheroids with PrestoBlue for 3–4 hours optimally resolves cell viability.

Figure 3

(a) H&E staining of a 1.5×10⁴ cell density MDA-MB-157 spheroid shows a compact intercellular network. Blue and purple represent nuclei and cytoplasm, respectively. (b–d) Immunohistochemical analysis for ECM proteins (shown in red) show deposition of (b) collagen I, (c) fibronectin, and (d) laminin in ATPS spheroids. Blue represents nuclei staining with Hoechst.

Figure 4

Immunostained cryosections of (a) 1.5×10⁴ and (b) 1.0×10⁵ cell density spheroids show the distribution of Ki-67 positive (pink) proliferative cells. Analysis of stained sections is used to compare the distribution of nuclei (blue) and proliferative cells throughout each section. The larger spheroid contains a non-uniform distribution of proliferative cells compared to the smaller spheroid.

Figure 5

Pimonidazole staining (pink) of cryosections of (a) 1.0×10⁵ and (b) 1.5×10⁴ cell density spheroids of MDA-MB-157 shows presence of a hypoxic core only in the larger spheroid. (c) q-PCR analysis of expression of the hypoxic marker CA IX in cells of both spheroids validates the results of immunostaining. mRNA levels are normalized with respect to a monolayer of MDA-MB-157 cells. Error bars represent the standard error of mean for three trials. Blue represents nuclei staining with Hoechst. (*p < 0.01)

Figure 6

(a) Dose-response of 1.5×10⁴ and 1.0×10⁵ cell density MDA-MB-157 spheroids to doxorubicin treatment shows drug resistance of larger spheroids. Error bars represent the standard error of mean. (b), (c) Gray values of fluorescence intensity measurements along a sample line crossing spheroids of both densities show complete penetration of doxorubicin into spheroids after 48 hours of incubation. (d) Doxorubicin localization in the nuclei of 1.0×10⁵ cell density spheroids after 48 hrs of treatment. Blue represents nuclei staining with Hoechst.

Figure 7

Surface plot of viability of 1.0×10⁵ cell density spheroid of MDA-MB-157 cells co-treated with varying concentrations of doxorubicin and TH-302 shows synergistic enhancement in toxicity due to combination treatment. Color bar represents the cell viability range. Green and yellow squares represent cell viability of spheroids from treatment with doxorubicin only and TH-302 only, respectively.

Figure 8

Dose-response of MDA-MB-157 spheroids made with 1.0×10⁵ and 1.5×10⁴ cells to cisplatin treatment. Error bars represent the standard error of mean.

Figure 9

q-PCR analysis of expression of (a) CD24, (b) CD133, and (c) Nanog in 1.5×10⁴ and 1.0×10⁵ cell density spheroids of MDA-MB-157 cells normalized against mRNA levels of a monolayer of cells. Expression levels are relative to -actin and hypoxanthine phosphoribosyltransferase (HPRT) and calculated using the ΔΔC_t method. Fold change in mRNA expression represent 2^−ΔΔCt. Error bars represent the standard error of mean for three trials. Largest cryosections of 1.5×10⁴ and 1.0×10⁵ cell density spheroids immunostained for cancer stem cell markers (d) CD24 (green) and (e) CD133 (red). Blue represents nuclei staining with Hoechst. (*p < 0.05)