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Reconstruction of dynamic mammary mini gland in vitro for normal physiology and oncogenesis

Nature Methods volume 20, pages 2021–2033 (2023)Cite this article

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Abstract

Organoid culture has been extensively exploited for normal tissue reconstruction and disease modeling. However, it is still challenging to establish organoids that mimic in vivo-like architecture, size and function under homeostatic conditions. Here we describe the development of a long-term adult stem cell-derived mammary mini gland culture system that supports robust three-dimensional outgrowths recapitulating the morphology, scale, cellular context and transcriptional heterogeneity of the normal mammary gland. The self-organization ability of stem cells and the stability of the outgrowths were determined by a coordinated combination of extracellular matrix, environmental signals and dynamic physiological cycles. We show that these mini glands were hormone responsive and could recapitulate the entire postnatal mammary development including puberty, estrus cycle, lactation and involution. We also observed that these mini glands maintained the presence of mammary stem cells and could also recapitulate the fate transition from embryonic bipotency to postnatal unipotency in lineage tracing assays. In addition, upon induction of oncogene expression in the mini glands, we observed tumor initiation in vitro and in vivo in a mouse model. Together, this study provides an experimental system that can support a dynamic miniature mammary gland for the study of physiologically relevant, complex biological processes.

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Fig. 1: Development of mammary mini gland with augmented branching patterns and stabilized structures.
Fig. 2: Mammary mini gland recapitulates the molecular identity and cellular heterogeneity of mammary gland.
Fig. 3: The mammary mini gland recapitulates the reproductive functions of mammary gland.
Fig. 4: The mammary mini gland maintains mammary stem cells.
Fig. 5: The mini gland is able to recapitulate stem cell fate transition.
Fig. 6: The mammary mini gland can be utilized to model cancer initiation.

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Data availability

Sequencing raw data and counts matrices are available under accession number GEO: GSE211527. Computer code for single-cell transcriptome analysis is available at Supplementary Code. Source data are provided with this paper.

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Acknowledgements

We thank the Imaging Facility of Westlake University for assistance in mini gland cleared tissue imaging. We thank the Flow Cytometry Facility of Westlake University for assistance in FACS analysis and sorting. We thank the Westlake Animal Facility for mouse husbandry. This work was supported by National Natural Science Foundation NSF grant 32170803. This work was supported by Westlake Education Foundation.

Author information

Authors and Affiliations

  1. Fudan University, Shanghai, China

    Lei Yuan, Shaofang Xie, Pei Cai & Zuobao Lin

  2. Westlake Laboratory of Life Sciences and Biomedicine, School of Life Sciences, Westlake University, Hangzhou, China

    Lei Yuan, Shaofang Xie, Huiru Bai, Xiaoqin Liu, Pei Cai, Jing Lu, Chunhui Wang, Zuobao Lin, Shuying Li, Yajing Guo & Shang Cai

  3. Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Lei Yuan, Huiru Bai, Xiaoqin Liu, Pei Cai, Chunhui Wang, Zuobao Lin, Shuying Li, Yajing Guo & Shang Cai

  4. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Shaofang Xie & Jing Lu

  5. College of Life Sciences, Zhejiang University, Hangzhou, China

    Xiaoqin Liu

  6. Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China

    Jing Lu

  7. Westlake Disease Modeling Lab, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Chunhui Wang, Shuying Li & Shang Cai

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Contributions

L.Y. and S.C. conceived the study and experimental design, performed and analyzed experiments, and wrote the manuscript. S.X. contributed to qRT–PCR, plasmid design and lentivirus production. H.B. and X.L. contributed to single-cell RNA-seq and single-cell RNA-seq analysis. J.L. participated in microscopy analysis. P.C., C.W., S.L., Z.L. and Y.G. assisted with animal experiment.

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Correspondence to Shang Cai.

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Nature Methods thanks Cedric Blanpain, Silvia Fre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Optimization of culture conditions for murine mammary gland Organoid.

a, Confocal images of immunostaining of mammary gland at E13.5, E15.5, E17.5 and P1 showing the cellular heterogeneity, polarity and architecture during mammary gland development (top). Model representation of embryonic mammary rudiment formation from E13.5 to birth (bottom). b, Immunofluorescence staining for the indicated markers of organoid. c, Mammary organoid culture for extended time. d, Representative images of organoids cultured for 20d in sphere formation media. e, Bright-field (top and middle rows) and immunofluorescence (bottom row) images of organoids cultured for 3d in the indicated medium, showing the essential factors for symmetry breaking. f, Representative images of organoids cultured for 3d in the indicated inhibitors. g, Representative images (top and middle rows) of organoids cultured for 3d in basic media with/without addition of FGF2 or the indicated inhibitors. Bottom row: Immunofluorescence staining of the frozen section of the organoids. h, Immunofluorescence staining for the indicated markers of the frozen section of the organoids. i, Quantification of Ki67+ cells in mammary organoids under various conditions. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. j, Motility trajectory plots of individual cells after treatment of indicated factors. X and Y represent spatial coordinates. The unit for X and Y axis is μm. k, Quantification of the speed of movement for the individual cells under indicated treatments. Each panel displays superimposed trajectories of 20 individual cells. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. l, Time-lapse movie of mammary organoid formation and initiation of symmetry breaking. The red dashed line indicates the formation and healing of the cavity. m, Representative images of organoids cultured in collagen I. n, Representative images of organoid outgrowths in ECM of different ratio of Matrigel and Collagen I. The scale bar represents 500 μm (black) in b, c, d, e, f, g, m, n, 50 μm in a, b, e, g, h (white), l.

Source data

Extended Data Fig. 2 Establishment of mini gland culture system.

a, Brightfield images of organoids without feeder cells for 24 days in branching media. b, Immunofluorescence staining of organoid in branching media for indicated markers at d24. c, Representative FACS plots showing mammary cell population from digested organoids without feeder cells. d, Immunofluorescence staining of organoids for indicated markers without feeder cells after pseudo estrus cycles at d20. e, Representative FACS plots showing basal and luminal cell populations from digested organoids without feeder cells after pseudo estrus cycles. f, qRT-PCR analysis of gene expression of mammary gland markers in mammary organoids, compare with primary mammary glands. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. g, Tracking of the size of the mini gland during one month culture. n = 10, biological replicates. Data are presented as mean ± SEM. h, Immunofluorescence staining for indicated markers from the adult primary mammary gland (left) and mammary mini gland (right). i, Comparison of the TEB widths of the mammary mini-glands and the primary mammary glands. n = 10, biological replicates. Data are presented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. j, Comparison of the duct widths of the mammary mini glands and the primary mammary glands. n = 4, biological replicates. Data are presented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. k, Bright-field images of organoids cultured for 6d in the indicated medium. l, Bright-field images of mini glands cultured in the indicated medium, showing the effect of Fgf10 and Fgf2 in mini gland culture. The scale bar represents 500 μm in a, k, l, 50 μm in b, d, h.

Source data

Extended Data Fig. 3 Growth factor requirement of mini gland culture.

a, Representative images of mini gland outgrowths in medium of indicated composition with feeder cells and pseudo estrus cycle. b, Sizes of mini glands tracked from d0 to d14 after seeding in different culture conditions with feeder cells and pseudo estrus cycle. n = 3 biological replicates. c, Representative images of mini gland outgrowths in medium without serum of indicated composition with feeder cells and pseudo estrus cycle. Data are presented as mean ± SEM. The scale bar represents 500 μm in a, c.

Source data

Extended Data Fig. 4 Effect of Wnt signaling in mini gland morphogenesis.

a, Schematic diagram showing the introduction of pseudo-estrus cycles into the mammary culture system. b, Representative brightfield images and FACS plots showing mammary cell population from digested organoids without feeder cells. c, Schematic diagram showing the introduction of exogenous Wnt3a (200 ng/ml) and pseudo-estrus cycles into the mammary culture system. d, Representative brightfield images and FACS plots showing mammary cell population from digested organoids without feeder cells but with exogenous Wnt3a. e, Schematic diagram showing the introduction of stromal cells and pseudo-estrus cycles into the mammary culture system. f, Representative brightfield images and FACS plots showing basal and luminal cell populations from digested mini gland. g, Schematic diagram showing the introduction of exogenous Dkk1 (200 ng/ml), stromal cells and pseudo-estrus cycles into the mammary culture system. h, Representative brightfield images and FACS plots showing basal and luminal cell populations from digested mini gland with exogenous Dkk1. i, Comparison of the size of the mammary mini glands under indicated treatments. n = 4, biological replicates. Data are presented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. j, Quantification of the percentage of Sca1−/+ and Cd61-/+ cells in luminal cells. n = 4, biological replicates. Data are represented as mean ± SEM. The scale bar represents 500 μm in b, d, f, h.

Source data

Extended Data Fig. 5 Molecular identity of mammary cells in mini gland in vitro.

a, Expression of Cd61 and Sca1 in luminal cells of the mini gland. b, Immunofluorescence staining of mini gland for indicated markers at d26. c, Brightfield images and representative FACS plots showing basal and luminal cell populations from dissociated mini gland. d, qRT-PCR analysis of gene expression of mammary gland markers in the basal-like and luminal-like cells of the mini gland. n = 3 biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. e, qRT-PCR analysis of gene expression of Sca1- and Sca1+ cell populations from luminal cells in mini gland, compared with primary mammary gland. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. The scale bar represents 500 μm in c, 50 μm in b.

Source data

Extended Data Fig. 6 Characterization of transcriptome of mini gland.

a, UMAP plots comparing the expression of cell identity markers in primary mammary gland epithelial cells and the mini gland. b, UMAP plot comparing the expression of cell identity markers in two basal subpopulations.

Extended Data Fig. 7 Capacity of mini gland undergoing estrus cycle.

a, Multiple pseudo-estrus cycles for mini gland culture. Dashed lines indicate that side branches forming at the primary ducts after estrus cycle. b, Representative images of mini gland outgrowth with or without FGF2 during pseudo estrus cycle. c, Growth curve of mini gland tracked from d0 to d14 with or without FGF2 during pseudo estrus cycle. n = 4 biological replicates. Data are presented as mean ± SEM. d, Transcriptional characterization of the pseudo estrus cycle. n = 3 biological replicates. Data are represented as mean ± SEM. The scale bar represents 500 μm in a, b.

Source data

Extended Data Fig. 8 Capacity of mini gland undergoing lactation.

a, Comparison between different lactation strategies. b, Immunofluorescence staining of E-cadherin in mini gland of lactation. White dashed lines indicate the binucleated cells. c, Lipid metabolomics of mini gland and mini gland in lactation. d, Bright-field images of mini gland morphogenesis under lactation induction and involution induction. e, H&E staining of mini gland morphogenesis under lactation induction and involution induction. f, Nile red staining of mini gland morphogenesis under lactation induction and involution induction. g, Immunofluorescence for indicated markers in mini gland under lactation induction and involution induction. h, Bright-field images of mini gland morphogenesis under two round of lactation induction and involution induction. The scale bar represents 500 μm in a, d, h, 50 μm in e, f, g, 10 μm in b.

Source data

Extended Data Fig. 9 Exploration of the dynamics of the stem cell fate transition using mini gland.

a, Bright field image of d6 organoids. b, The workflow to analyze the fate of labeled cells after 1d tracing. c, FACS dot plots of luminal cells and basal cells gated within the tdTomato positive and GFP positive populations in Krt14rtTA/TetO-Cre/mTmG organoids with 1day doxycycline induction. Data were collected 1 days after the induction. d, Confocal images of immunostaining of Krt14, Krt8 and GFP in 7d organoids with 1day doxycycline induction. Images showed clonal induction in mammary ducts (arrow). e, qRT-PCR analysis indicating that TNFa level is increased from d2 to d14. mRNA expression was normalized to β-actin; n = 3, Data are presented as mean ± SEM. f, Schematic diagram showing the workflow to study the fate transition with the treatment of TNFa. g, Bright-field image of d14 mini gland with or without the treatment of TNFa. h, Representative FACS dot plots of luminal cells and basal cells gated within the tdTomato positive and GFP positive population in Krt14rtTA/TetO-Cre/mTmG mini gland with the treatment of TNF after doxycycline induction at d6. i, FACS quantification of tdTomato positive and GFP positive basal cell (gray) and luminal cell (black) populations in Krt14rtTA/TetO-Cre/mTmG mini gland after doxycycline induction with TNF treatment; n = 3, Data are presented as mean ± SEM. j, Confocal images of immunostaining of Krt14, Krt8 and GFP in mini gland at d14 with the induction at d6 by 100 ng doxycycline per ml with or without TNF treatment. The staining showed clonal induction in mammary ducts (arrow). k, Schematic diagram showing the workflow to analyze the fate of cells in mini glands labeled at d14 with the treatment of anti-TNF blocking antibody adalimumab. l, Representative FACS dot plots of luminal cells and basal cells gated within the tdTomato positive and GFP positive populations in Krt14rtTA/TetO-Cre/mTmG mini gland after doxycycline induction at d14 and adalimumab treatment for 12 days. m, FACS quantification of tdTomato positive and GFP positive basal cells (gray) and luminal cells (black) after doxycycline induction at d14 with or without the treatment of adalimumab; n = 3, Data are presented as mean ± SEM. n, Confocal images of immunostaining of Krt14, Krt8 and GFP in 26d mini gland induced by 100 ng/ml Doxycycline at d14 with anti-TNF blocking antibody adalimumab treatment. Images showed clonal induction in mammary ducts (arrow). The scale bar represents 500 μm in a, g, 50 μm in d, j, n.

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Extended Data Fig. 10 Long-term cultures and in vitro modelling breast cancer using mini gland.

a, Brightfield images of long-term culture of organoid without feeder cells. Organoid collapsed and formed many vacuolar structures (arrow). b, Percentage of well-maintained organoids over time, cultured without feeder cells. c, Representative FACS plots showing basal and luminal cell populations from 117d mini gland. d, Sphere formation from flow cytometry-sorted basal and luminal cells of the 117d mini gland. e, Brightfield images of the development of a mini gland into a branching structure. f, 293T cell lines were infected with mCherry-LSL-PyMT-GFP virus, and then with or without transient transfected Cre plasmid. g, qRT-PCR analysis of PyMT expression with or without transient transfection of Cre plasmid. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. h, Confocal images of immunostaining of Krt8 and GFP in d38 mini gland with a tumor-like structure and control cells with mock vector (without PyMT). i, Representative FACS dot plots, H&E staining, and confocal images of immunostaining of Krt14, Krt5, Krt8 and Ki67 in spontaneous mouse model of MMTV-PyMT. j, k, Representative H&E staining, and confocal images of immunostaining of Krt14, Krt5, Krt8 and Ki67 in d38 mini gland with a tumor-like structure. The scale bar represents 500 μm in a, d, e, 200um in f, 50 μm in h, i, j, k.

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Supplementary information

Supplementary Information

The supplementary information for an example of the FACS gating strategy.

Reporting Summary

Supplementary Table 1

Summary of the experimental procedures.

Supplementary Table 2

Comparison with previous research of generation of mammary gland organoids versus our work.

Supplementary Table 3

Primers for qPCR.

Supplementary Video 1

Time-lapse movie of mammary organoid formation and initiation of symmetry breaking.

Supplementary Code

Supplementary code.

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Yuan, L., Xie, S., Bai, H. et al. Reconstruction of dynamic mammary mini gland in vitro for normal physiology and oncogenesis. Nat Methods 20, 2021–2033 (2023). https://doi.org/10.1038/s41592-023-02039-y

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