Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Abstract

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

Figure 1:. Workflow for the epithelial organoid generation with example organoids.

(A) Schema of workflow for organoid generation from mouse or human tissue without passaging. (B) Representative images of isolated epithelial organoids from mouse WT mammary glands, mouse mammary tumors, and human breast tumors in media following isolation. Each image was adjusted individually for brightness and contrast for enhanced visualization. Images were taken in brightfield on an inverted epi-fluorescent microscope at 10x magnification. Scale bar represents 20 μm. Mammary tumors were isolated from MMTV-PyMT mice; normal mammary tissue was isolated from FVB mice. Mice ranged from 8–14 weeks of age.

Figure 2:. Imaging organoids in the extracellular matrix.

Each image was adjusted individually for brightness and contrast for enhanced visualization for this collection. Prior to imaging, samples were fixed with 4% paraformaldehyde. Wild-type samples were stained with phalloidin 568 to visualize the cell membrane and all samples were stained with Hoechst to visualize nuclei, showing organoid morphology and density. Images were taken at 10x magnification on a confocal microscope and further enlarged with a scanning zoom of 3.003. The laser wavelength used to detect DAPI was 405 nm with a power of 5 and the laser wavelength used to detect phalloidin was 561.0 nm for channel 3 with a power of 0.5. Confocal pinhole size was maintained at 19.16 for all images. (A) Representative brightfield image of mouse mammary WT organoid embedded in BECM at Day 0. (A’) 1:250 Hoechst labeling nuclei and (A’’) 1:250 phalloidin labeling actin immunofluorescent images of A. Scale bar represents 20 μm. Normal mammary tissue was isolated from FVB mice. Mice ranged from 8–12 weeks of age. (B) Representative brightfield image of mouse mammary tumor organoid embedded in BECM at Day 0. (B’) 1:250 Hoechst labeling nuclei and (B’’) mTomato-labeled immunofluorescent image of B. Scale bar represents 20 μm. Mammary tumors were isolated from MMTV-PyMT mice. Mice ranged from 12–14 weeks of age. (C) Representative brightfield image of mouse mammary tumor organoid embedded in Collagen I at Day 3. The image represents an organoid demonstrating “invasive” property. (C’) 1:250 Hoechst labeling nuclei and (C’) mTomato-labeled immunofluorescent image of C. Scale bar represents 20 μm. Mammary tumors were isolated from MMTV-PyMT mice. Mice ranged from 12–14 weeks of age. (D) Representative brightfield image of human breast tumor organoid embedded in BECM at Day 0. (D’) Hoechst labeling nuclei and (D’’) 1:250 Actin-targeting phalloidin-labeled immunofluorescent image of D. Scale bar represents 20 μm. (E) Sectioned image of an organoid embedded in BECM and stained with H&E taken at 20x magnification. Scale bar represents 20 μm. H&E staining was performed by the University of Texas Southwestern Tissue Management Shared Resource.