Cancer Cell Invasion of Mammary Organoids with Basal-In Phenotype

Abstract

This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal-in phenotype is the use of a minimal gel scaffold that the epithelial cells self-assemble around and encapsulate. When MDA-MB-231 breast cancer cells are co-cultured with epithelial cells from day 0 under these conditions, cells self-organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA-MB-231 invasion in a tumor progression-relevant direction relative to epithelial cell-basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.

Keywords: basal-in phenotype; basement membrane; breast cancer; invasion; organoid.

Figure 1.. Cell-assisted minimal scaffolding is critical for organoid formation.

Schematic representations (a, d, f) and brightfield images (b, c, e, g) of 1.5% Matrigel added to warm media, MCF10A cells with no Matrigel, and 1.5% (120 ± 10 μg/mL) Matrigel added to cold media, respectively. Black arrow in a and b shows a pocket of Matrigel. (h) H&E image of a day 16 organoid section. (i) Laminin-1,2 stain of a day 16 organoid section. All scale bars represent 200 μm.

Figure 2.. MCF10A organoids exhibit a basal-in phenotype.

(a) Commonly observed, basal-out structure of MCF10A acini grown in gels. (b) Basal-in phenotype of MCF10A organoids described here. Smaller internal acinar structures maintain basal-out morphology. (c) Laminin-5 staining of a day 16 organoid section. Scale bar represents 200 μm. Inset shows 40x image, with laminin-5 layer on the inner side of the structure. Scale bar represents 50 μm. (d) Integrin alpha-6 staining of a day 16 organoid section. Scale bar represents 200 μm. (e) Laminin-5 staining of a day 12 organoid with a lesser extent of hollowing. Scale bar represents 200 μm. Inset shows 40x image of internal acinar-like structures that do not exhibit a basal-in morphology. Scale bar represents 20 μm.

Figure 3.. Self-organizing behaviors of MCF10A and MDA-MB-231 co-culture.

(a) Time course development of co-culture without Matrigel. (b) Area and (c) roundness of co-culture both with and without Matrigel. The mean values are reported, and error bars represent the standard deviation; n = 20 organoids were used for each condition. (d) Time course development of co-culture with 1.5% (120 ± 10 μg/mL) Matrigel. Black arrow in d indicates the peripheral ball of MDA-MB-231 cells. H&E image (e) and laminin-5 stain (f) of a day 16 co-culture organoid where a peripheral MDA-MB-231 ball remained attached to the main organoid body. H&E image (g) and laminin-5 stain (h) of a day 16 co-culture organoid where a peripheral ball of MDA-MB-231 cells dissociated from the organoid, which represents a majority of the co-culture organoids. All scale bars represent 200 μm, except the inset in (f) which represents 50 μm. * denotes a p-value <0.005 and ** denotes a p-value <0.000001.

Figure 4.. MCF10A organoid model as a cancer invasion platform.

(a) Schematic of cancer invasion experiments. Organoids are grown to partial maturity, and MDA-MB-231 cancer cells are introduced on day 7 or 16. Due to the basal-in nature of the organoids, there is easy apical-surface access, represented by the outside of the organoid. Organoids were maintained in culture for an additional 9 days before fixation. (b) E-cadherin staining of MCF10A organoids after MDA-MB-231 cells invaded inside. Scale bar represents 200 μm. (c) Confocal slice at midplane of a cleared organoid showing MDA-MB-231 cancer invasion introduced at day 7 and maintained for 9 additional days. Scale bar represents 300 μm. (d) Laminin-5 staining of the basement membrane development over time at day 8 and (e) day 16. Scale bars represent 200 μm. (f) Quantification of percent of organoids showing cancer invasion when MDA-MB-231 cells were added on day 7 (n = 8) or day 16 (n = 5) of MCF10A organoid culture.