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• W2927656553 abstract "•A single-step, non-toxic clearing agent for 3D imaging of whole organs and tumors•Derivation of an integrative platform to interrogate intratumoral heterogeneity•Profound clonal restriction occurs during neoplastic progression•The epithelial-mesenchymal transition occurs clonally as a frequent event Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors. Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors. To interrogate breast tumor biology and heterogeneity in greater depth, we have developed a 3D imaging protocol based on a rapid clearing agent termed FUnGI that enables large-scale imaging of intact solid tumors. A profound reduction in the number of tumor clones was observed during oncogenesis in distinct mouse models that targeted either basal or luminal progenitor cells, and revealed that the luminal progenitor is a key cell-of-origin. Furthermore, the application of a pipeline involving multicolor lineage tracing, 3D imaging, cell sorting, and RNA sequencing of tumor clones revealed that the epithelial-to-mesenchymal transition (EMT) frequently occurs at a clonal level. This study highlights the inherent plasticity of mammary tumors and suggests that the EMT is not a rare event in vivo. Breast cancer comprises multiple intrinsic subtypes that are characterized by phenotypic and functional heterogeneity (Ellis and Perou, 2013Ellis M.J. Perou C.M. The genomic landscape of breast cancer as a therapeutic roadmap.Cancer Discov. 2013; 3: 27-34Crossref PubMed Scopus (174) Google Scholar). Both the mutational profile and “cell-of-origin” are thought to impact on intertumoral heterogeneity. Similar to other solid tumors, breast cancer exhibits intratumoral heterogeneity, which poses a significant obstacle to effective patient treatment. A breast tumor typically displays marked subclonal diversity that is driven by somatic evolution (Curtis et al., 2012Curtis C. Shah S.P. Chin S.F. Turashvili G. Rueda O.M. Dunning M.J. Speed D. Lynch A.G. Samarajiwa S. Yuan Y. et al.The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.Nature. 2012; 486: 346-352Crossref PubMed Scopus (3696) Google Scholar, Nik-Zainal et al., 2012Nik-Zainal S. Alexandrov L.B. Wedge D.C. Van Loo P. Greenman C.D. Raine K. Jones D. Hinton J. Marshall J. Stebbings L.A. et al.Mutational processes molding the genomes of 21 breast cancers.Cell. 2012; 149: 979-993Abstract Full Text Full Text PDF PubMed Scopus (1282) Google Scholar, Stratton et al., 2009Stratton M.R. Campbell P.J. Futreal P.A. The cancer genome.Nature. 2009; 458: 719-724Crossref PubMed Scopus (2385) Google Scholar). Moreover, intratumoral heterogeneity can be maintained by clonal evolution, stochastic mechanisms, and/or a hierarchical model underpinned by cancer stem cells (Batlle and Clevers, 2017Batlle E. Clevers H. Cancer stem cells revisited.Nat. Med. 2017; 23: 1124-1134Crossref PubMed Scopus (1351) Google Scholar, Kreso and Dick, 2014Kreso A. Dick J.E. Evolution of the cancer stem cell model.Cell Stem Cell. 2014; 14: 275-291Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar, Marusyk et al., 2012Marusyk A. Almendro V. Polyak K. Intra-tumour heterogeneity: a looking glass for cancer?.Nat. Rev. Cancer. 2012; 12: 323-334Crossref PubMed Scopus (1370) Google Scholar, Visvader and Lindeman, 2012Visvader J.E. Lindeman G.J. Cancer stem cells: current status and evolving complexities.Cell Stem Cell. 2012; 10: 717-728Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar). High-resolution imaging is required to visualize clonal diversity and understand the cellular landscape within entire solid tumors, but this has been hampered by the inability to optically clarify tissue. A comprehensive view of tissue composition, cell shape, cell-cell interactions, and cell-fate decisions in complex biological specimens requires sophisticated 3D imaging and longitudinal imaging technologies. Over the last decade, the development of fluorescent reporter mice, together with evolving light microscopy technologies, has opened avenues for deep imaging. Sample preparation for whole-mount tissue and clearing methodologies to render samples optically transparent and reduce light scattering during image acquisition have become areas of intense focus. This has led to the derivation of several clearing reagents and techniques, including solvent-based 3DISCO (Erturk et al., 2012Erturk A. Becker K. Jahrling N. Mauch C.P. Hojer C.D. Egen J.G. Hellal F. Bradke F. Sheng M. Dodt H.U. Three-dimensional imaging of solvent-cleared organs using 3DISCO.Nat. Protoc. 2012; 7: 1983-1995Crossref PubMed Scopus (654) Google Scholar) and its derivatives (Pan et al., 2016Pan C. Cai R. Quacquarelli F.P. Ghasemigharagoz A. Lourbopoulos A. Matryba P. Plesnila N. Dichgans M. Hellal F. Erturk A. Shrinkage-mediated imaging of entire organs and organisms using uDISCO.Nat. Methods. 2016; 13: 859-867Crossref PubMed Scopus (381) Google Scholar, Renier et al., 2014Renier N. Wu Z. Simon D.J. Yang J. Ariel P. Tessier-Lavigne M. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.Cell. 2014; 159: 896-910Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, Renier et al., 2016Renier N. Adams E.L. Kirst C. Wu Z. Azevedo R. Kohl J. Autry A.E. Kadiri L. Umadevi Venkataraju K. Zhou Y. et al.Mapping of brain activity by automated volume analysis of immediate early genes.Cell. 2016; 165: 1789-1802Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), and aqueous-based clearing agents such as Scale solutions (Hama et al., 2011Hama H. Kurokawa H. Kawano H. Ando R. Shimogori T. Noda H. Fukami K. Sakaue-Sawano A. Miyawaki A. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.Nat. Neurosci. 2011; 14: 1481-1488Crossref PubMed Scopus (865) Google Scholar, Hama et al., 2015Hama H. Hioki H. Namiki K. Hoshida T. Kurokawa H. Ishidate F. Kaneko T. Akagi T. Saito T. Saido T. Miyawaki A. ScaleS: an optical clearing palette for biological imaging.Nat. Neurosci. 2015; 18: 1518-1529Crossref PubMed Scopus (392) Google Scholar), ClearT2 (Kuwajima et al., 2013Kuwajima T. Sitko A.A. Bhansali P. Jurgens C. Guido W. Mason C. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.Development. 2013; 140: 1364-1368Crossref PubMed Scopus (331) Google Scholar), SeeDB/FRUIT/SeeDB2 (Hou et al., 2015Hou B. Zhang D. Zhao S. Wei M. Yang Z. Wang S. Wang J. Zhang X. Liu B. Fan L. et al.Scalable and DiI-compatible optical clearance of the mammalian brain.Front. Neuroanat. 2015; 9: 19Crossref PubMed Scopus (133) Google Scholar, Ke et al., 2013Ke M.T. Fujimoto S. Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.Nat. Neurosci. 2013; 16: 1154-1161Crossref PubMed Scopus (630) Google Scholar, Ke et al., 2016Ke M.T. Nakai Y. Fujimoto S. Takayama R. Yoshida S. Kitajima T.S. Sato M. Imai T. Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent.Cell Rep. 2016; 14: 2718-2732Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), CUBIC (Murakami et al., 2018Murakami T.C. Mano T. Saikawa S. Horiguchi S.A. Shigeta D. Baba K. Sekiya H. Shimizu Y. Tanaka K.F. Kiyonari H. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat. Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (165) Google Scholar, Susaki et al., 2015Susaki E.A. Tainaka K. Perrin D. Yukinaga H. Kuno A. Ueda H.R. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.Nat. Protoc. 2015; 10: 1709-1727Crossref PubMed Scopus (403) Google Scholar, Tainaka et al., 2014Tainaka K. Kubota S.I. Suyama T.Q. Susaki E.A. Perrin D. Ukai-Tadenuma M. Ukai H. Ueda H.R. Whole-body imaging with single-cell resolution by tissue decolorization.Cell. 2014; 159: 911-924Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), “active” and “passive” CLARITY (Chung et al., 2013Chung K. Wallace J. Kim S.Y. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (1386) Google Scholar, Tomer et al., 2014Tomer R. Ye L. Hsueh B. Deisseroth K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues.Nat. Protoc. 2014; 9: 1682-1697Crossref PubMed Scopus (576) Google Scholar, Yang et al., 2014Yang B. Treweek J.B. Kulkarni R.P. Deverman B.E. Chen C.K. Lubeck E. Shah S. Cai L. Gradinaru V. Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell. 2014; 158: 945-958Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar), UbasM (Chen et al., 2017Chen L. Li G. Li Y. Li Y. Zhu H. Tang L. French P. McGinty J. Ruan S. UbasM: an effective balanced optical clearing method for intact biomedical imaging.Sci. Rep. 2017; 7: 12218Crossref PubMed Scopus (53) Google Scholar), and Ce3D (Li et al., 2017Li W. Germain R.N. Gerner M.Y. Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D).Proc. Natl. Acad. Sci. U S A. 2017; 114: E7321-E7330Crossref PubMed Scopus (173) Google Scholar). These protocols often require complex multi-step protocols and/or long incubation times to obtain optically cleared tissues (Richardson and Lichtman, 2015Richardson D.S. Lichtman J.W. Clarifying tissue clearing.Cell. 2015; 162: 246-257Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Moreover, samples typically cannot be stored and require imaging within a short time frame. Although solvent-based reagents are very effective in rendering tissues transparent, they have been reported to induce significant tissue shrinkage and, in the case of the mammary gland, marked structural deformation (Lloyd-Lewis et al., 2016Lloyd-Lewis B. Davis F.M. Harris O.B. Hitchcock J.R. Lourenco F.C. Pasche M. Watson C.J. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.Breast Cancer Res. 2016; 18: 127Crossref PubMed Scopus (64) Google Scholar). Immunolabeling, a key technique to reveal morphology and the molecular composition of biological samples, has been mostly restricted to conventional thin-section preparations or small samples owing to the difficulty of labeling and performing deep imaging on complex, intact specimens. Protocols for immunolabeling deep within tissues have been pioneered (such as with iDISCO) but require weeks of incubation for deep antibody penetrance and rapidly quench endogenous fluorescence (Erturk et al., 2012Erturk A. Becker K. Jahrling N. Mauch C.P. Hojer C.D. Egen J.G. Hellal F. Bradke F. Sheng M. Dodt H.U. Three-dimensional imaging of solvent-cleared organs using 3DISCO.Nat. Protoc. 2012; 7: 1983-1995Crossref PubMed Scopus (654) Google Scholar, Pan et al., 2016Pan C. Cai R. Quacquarelli F.P. Ghasemigharagoz A. Lourbopoulos A. Matryba P. Plesnila N. Dichgans M. Hellal F. Erturk A. Shrinkage-mediated imaging of entire organs and organisms using uDISCO.Nat. Methods. 2016; 13: 859-867Crossref PubMed Scopus (381) Google Scholar, Renier et al., 2014Renier N. Wu Z. Simon D.J. Yang J. Ariel P. Tessier-Lavigne M. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.Cell. 2014; 159: 896-910Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar), thus negating the combined use of a fluorescent reporter strategy with immunolabeling. For the mammary gland, imaging of expansive ductal regions in their native stroma has been demonstrated (Davis et al., 2016Davis F.M. Lloyd-Lewis B. Harris O.B. Kozar S. Winton D.J. Muresan L. Watson C.J. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny.Nat. Commun. 2016; 7: 13053Crossref PubMed Scopus (82) Google Scholar, Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (378) Google Scholar) but 3D imaging of the whole organ and associated breast tumors for concurrent immunolabeling and native fluorescence has not yet been feasible. In this study, through the development of a rapid 3D imaging protocol that enables the imaging of tumors on a large-scale at single-cell resolution, we have explored the cellular organization of mammary tumors and the inherent behavior of clones during oncogenesis. To establish a facile and reproducible platform for the imaging of mammary tissue, a soft tissue that comprises a high fat content, we first evaluated a range of clearing techniques shown to be effective for other tissues. The mammary ductal tree is composed of two epithelial layers, an inner layer of luminal cells and an outer layer of basal or myoepithelial cells, which are embedded in a fat pad predominantly composed of adipocytes and fibroblasts. Several agents proved to be inefficient in clearing this organ, even after a prolonged period (Figure S1A). Although 3DISCO (Erturk et al., 2012Erturk A. Becker K. Jahrling N. Mauch C.P. Hojer C.D. Egen J.G. Hellal F. Bradke F. Sheng M. Dodt H.U. Three-dimensional imaging of solvent-cleared organs using 3DISCO.Nat. Protoc. 2012; 7: 1983-1995Crossref PubMed Scopus (654) Google Scholar) provided the highest degree of optical transparency, this technique alters lipid structure, which is relevant to fatty tissues such as the mammary gland (Lloyd-Lewis et al., 2016Lloyd-Lewis B. Davis F.M. Harris O.B. Hitchcock J.R. Lourenco F.C. Pasche M. Watson C.J. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.Breast Cancer Res. 2016; 18: 127Crossref PubMed Scopus (64) Google Scholar). Another limitation is that organic solvents rapidly quench endogenous fluorescence and necessitate prompt imaging. We therefore sought to develop a clearing agent that could be used in a rapid one-step procedure for both immunolabeled mammary tissue and native fluorescent proteins, and one that reduced light scattering without removing cellular components and leading to loss of fluorescence. Here we describe a protocol spanning only 3 days that utilizes a clearing agent comprising fructose, urea, and glycerol for imaging (FUnGI), termed large-scale single-cell resolution 3D (LSR-3D) imaging. FUnGI was effective in clearing mammary tissue in 2 h at room temperature, and tissue transparency was maintained (Figure 1A). Comparison of FUnGI with the clearing agent Ce3D and glycerol (60% or 80%) revealed that both FUnGI and Ce3D, but not glycerol, could efficiently clear the mammary gland, as verified through quantification using the bulk tissue clarity index (BTCi) (Magliaro et al., 2016Magliaro C. Callara A.L. Mattei G. Morcinelli M. Viaggi C. Vaglini F. Ahluwalia A. Clarifying CLARITY: quantitative optimization of the diffusion based delipidation protocol for genetically labeled tissue.Front. Neurosci. 2016; 10: 179Crossref PubMed Scopus (23) Google Scholar) (Figures S1B and S1C). In addition, FUnGI appeared to have a faster and higher clearing capacity than Ce3D. To immunolabel the mouse mammary gland in its entirety, we incorporated additional components in the protocol: mild detergents to enhance the antibody penetration, an antioxidant reagent (ascorbic acid) to preserve fluorescence over a prolonged period, and purified BSA to reduce background. Immunofluorescence labeling was performed on intact mammary glands using antibodies that recognize lineage-specific proteins, Keratin 5 (K5) (myoepithelial-specific) and E-cadherin (luminal-specific). After a one-step 2-h incubation in FUnGI, we could visualize the entire mammary ductal tree at low resolution by stereomicroscopy or optical projection tomography (Figure 1B; Video S1). The uniformity of staining indicated effective penetration of antibodies into the tissue. Furthermore, native fluorescence in the Elf5-rtTA-IRES-GFP reporter line (Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (378) Google Scholar) was preserved 1 week after clearing (Figure S1D). Notably, the combination of fructose and glycerol circumvented tissue shrinkage and distortion, which are associated with the use of urea (Figures 1A and S1E) (Richardson and Lichtman, 2015Richardson D.S. Lichtman J.W. Clarifying tissue clearing.Cell. 2015; 162: 246-257Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Moreover, the mammary ducts remained open (Figure 1C; Video S2). FUnGI also contrasts with many previously reported clearing agents that are relatively toxic and therefore are not optimal for tissue preparation on a daily basis (Figure S1A). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxYzMxZDRhYjIzZjU5ZDRiODYwMGE1NjYxNzQxYmFjMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4Nzc0NzQ2fQ.g1rpK9Y2_k3Xl2Bqw_wCLVLWPGWA0ISyIWO4qPwhi1uyyVW83685ETap2bVvYwmPY86i4jozyGkF3cIJjzT1KEkq1T0L4lUNfnRHqZQ4KECJ_hnqrG2KTkGLTdGZiJGIhQIgBB0Mjf6zizKDKEKRPRJqqYLAuW3slKgRy2tJFuz8x8ZUWc2jhYHay6RltP6y2jgeFnuUxcKD_1CjP1HhElX9b6xt0t69P8QD6iYcwRZNVQln9yIXQAFCL7HWIVKgrCGYfKGABTg6XVawGLCsllB7I4gNevvMp1Nv2OYAFtezkpPHlgYowZqHed6wqUGAzUOwPXF9SF7EOf2o4RIH7A Download .mp4 (20.13 MB) Help with .mp4 files Video S1. An Entire Mouse Mammary Gland Immunolabeled for Keratin 5 (Red) and E-cadherin (Green), Related to Figure 1 Snapshots Presented in Figure 1B eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjM2ZlMzk3YTZmZDc2NTQ3OWU3NDE4NTE3NjY5ZmYwMCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4Nzc0NzQ2fQ.WnnyDfJ-qnjRHGQZJjERAFeZXVqYHcFlX1hklaa1V-ZSWDItUn8XXoLejv7NSaeNrDUOxCxQw16VByvU_3o3qrhGOHkJQtlDXYA89Z6TO0ML1QitWqT2pZIdaVvF3BT9Yr1xBJhGRZp5sB31tXLAfYLOJKhyaQtSt6GtcCOwwxWE-2WnsAB2HngOfpilPntXVVEcDcs45Bj2LRsONJ-_V7whG9pKcejJgy6fHO0nAuHpDg5XqixY7lwkHSJUr5VQiMEjUJhYjHCXFCnMK4vYAONqY8VtBBIEWek9uZ8jQYo1sGD8T7fWrsgJBLc6NsizsJ5e_Vzzza9-npMEumAAxg Download .mp4 (24.48 MB) Help with .mp4 files Video S2. Video through a Whole-Mounted Mouse Mammary Duct in 3D after Labeling for Keratin 5 (Red) and E-cadherin (Green), Related to Figure 1 Snapshots Presented in Figure 1C The LSR-3D imaging pipeline was next tested on a range of whole-mouse adult organs including the reproductive tract, small intestine, lung, and lymph node (Figure S2), and late embryonic structures (data not shown). FUnGI was effective in clearing these and other organs within 2 h, also reflected in the quantification by BTCi (Figures S3A–S3I). Interestingly, high-resolution 3D imaging of the adult lung revealed that K5+ basal cells were spatially localized to the upper bronchiolar region and exhibited a unique morphology, characterized by an elongated body and protruding dendrites (Figure S2C). Collectively, we describe a rapid immunolabeling protocol and a clearing agent that is non-toxic, efficiently clarifies tissue, and maintains structural integrity to the organ. Another notable advantage of the FUnGI imaging pipeline is that it permits storage of tissues at −20°C before microscopic analysis, with no overt difference in fluorescence between freshly prepared versus frozen-preserved tissue. Mammary glands frozen in FUnGI clearing agent retained fluorescence and integrity for at least 18 months (Figures S3J and S3K). Studying the cellular architecture of intact human breast tissue is an important step toward understanding early cellular changes that lead to oncogenesis. Notably, human breast tissue is considerably more fibrous than its mouse counterpart due to the abundance of fibroblasts. Moreover, the human ductal network is composed of terminal ductal lobular units (TDLUs) that emanate from the primary ducts and represent sequential stages of differentiation (Russo et al., 1992Russo J. Rivera R. Russo I.H. Influence of age and parity on the development of the human breast.Breast Cancer Res. Treat. 1992; 23: 211-218Crossref PubMed Scopus (186) Google Scholar, Russo and Russo, 2004Russo J. Russo I.H. Development of the human breast.Maturitas. 2004; 49: 2-15Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar): Type I TDLUs are the most undifferentiated and occur in young virgin tissue, while type IV lobules are the most differentiated. Breast tissue from healthy women who had undergone a reduction mammoplasty was immunolabeled for K5, E-cadherin, and K8/18 to distinguish the myoepithelial (K5+) and luminal layers (E-cadherin+ and K8/18+), and then subjected to high-resolution 3D imaging after clearing. Type I and II TDLU structures could be visualized in young adult breast tissue, including a substantial proportion of progesterone receptor-positive (PR+) cells lining the ducts and lobules. By contrast, breast tissue from post-menopausal women largely lacked TDLU structures, as well as PR+ cells, compatible with the lack of ovarian hormones at this stage (Figure 2). FUnGI efficiently cleared the breast and did not result in tissue shrinkage (Figures 2H–2I). We next applied the LSR-3D technology to study whole breast tumors at cellular resolution. Figure 3 shows a patient-derived breast tumor xenograft that was subjected to the rapid immunolabeling protocol (3 days) and then imaged by multiphoton technology for deep imaging. FUnGI efficiently cleared the entire tumor to an approximate volume of 50 mm3 in 4 h and multiphoton imaging provided high resolution at a depth of 0.9 mm (Figures 3A–3D). Labeling penetrance to a depth of 1.9 mm (using a 2 mm working distance lens) could also be demonstrated for solid tumors (Figures S3L and S3M), which appears to be substantially greater than that possible using SeeDB or CUBIC (>200 μm) (Lloyd-Lewis et al., 2016Lloyd-Lewis B. Davis F.M. Harris O.B. Hitchcock J.R. Lourenco F.C. Pasche M. Watson C.J. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.Breast Cancer Res. 2016; 18: 127Crossref PubMed Scopus (64) Google Scholar). To test the LSR-3D imaging pipeline on a multicolored tumor model, MDA-MB-231 breast cancer cells were stably transfected with GFP, BFP or RFP fluorescent reporter genes, and then transplanted into mammary fat pads to generate tumors (Figures 3E–3H). Small tumor masses of approximately 0.5 mm3 were evaluated after clearing in FUnGI by confocal imaging 2 weeks post-transplantation (Figures 3E and 3F). FUnGI preserved native fluorescence and revealed multicolored domains at this time point. At 5 weeks post-transplantation, larger tumors were analyzed in combination with immunolabeling for Pecam/CD31 to visualize the tumor and surrounding vasculature. Established tumors remained multicolored, with some unicolored patches present, indicating clonal expansion. In addition, distinct vasculature patterns were visible throughout the tumor (Figures 3G and 3H). Taken together, this approach provides a rapid and flexible imaging pipeline to analyze whole tumors that harbor fluorescent reporter genes and/or have been immunolabeled. We next addressed the compatibility of an in vivo multicolor fluorescent reporter system with the FUnGI-based protocol in order to gain insight into mechanisms that drive tumor progression. Genetically engineered mice harboring the cre-inducible four-color fluorescent reporter allele Rosa26R-confetti (Snippert et al., 2010Snippert H.J. van der Flier L.G. Sato T. van Es J.H. van den Born M. Kroon-Veenboer C. Barker N. Klein A.M. van Rheenen J. Simons B.D. Clevers H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.Cell. 2010; 143: 134-144Abstract Full Text Full Text PDF PubMed Scopus (1324) Google Scholar) represent a powerful tool for clonal analysis (Kretzschmar and Watt, 2012Kretzschmar K. Watt F.M. Lineage tracing.Cell. 2012; 148: 33-45Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, Malide et al., 2012Malide D. Metais J.Y. Dunbar C.E. Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.Blood. 2012; 120 (e105–116)Crossref PubMed Scopus (34) Google Scholar), which we previously exploited to track stem and progenitor cells in the mouse mammary gland (Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (378) Google Scholar). To evaluate native fluorescence from the confetti locus, mammary glands were analyzed before and 2 h after clearing in FUnGI, using F-actin staining to highlight the entire mammary gland. High-resolution 3D imaging of confetti-labeled tumors was achieved, as illustrated in Figures S4A–S4F. To track changes in clonality during oncogenesis, we first analyzed a hormone-driven (medroxyprogesterone acetate [MPA]) and carcinogen-induced (7, 12 dimethylbenz(a)anthracene [DMBA]) mouse model (Aldaz et al., 1996Aldaz C.M. Liao Q.Y. LaBate M. Johnston D.A. Medroxyprogesterone acetate accelerates the development and increases the incidence of mouse mammary tumors induced by dimethylbenzanthracene.Carcinogenesis. 1996; 17: 2069-2072Crossref PubMed Scopus (78) Google Scholar, Gonzalez-Suarez et al., 2010Gonzalez-Suarez E. Jacob A.P. Jones J. Miller R. Roudier-Meyer M.P. Erwert R. Pinkas J. Branstetter D. Dougall W.C. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis.Nature. 2010; 468: 103-107Crossref PubMed Scopus (434) Google Scholar, Yin et al., 2005Yin Y. Bai R. Russell R.G. Beildeck M.E. Xie Z. Kopelovich L. Glazer R.I. Characterization of medroxyprogesterone and DMBA-induced multilineage mammary tumors by gene expression profiling.Mol. Carcinog. 2005; 44: 42-50Crossref PubMed Scopus (35) Google Scholar). To allow temporal and spatial control of expression, doxycycline (Dox)-inducible basal or luminal-specific promoters were used for lineage tracing, independent of tumor induction (Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (378) Google Scholar). Dox induction of the “confetti” locus in either the Krt5-marked basal compartment or the Elf5-marked luminal progenitor population resulted in labeling efficiencies (∼6%) conducive with clonality studies, also reflected in 3D imaging (Figures S4G and S4H). After Dox induction, mice were treated sequentially with MPA and DMBA. Fluorescent reporter gene expression was then visualized in mammary glands at different stages of preneoplasia and in established tumors. In the Krt5-rtTA/TetO-cre/R26R-confetti model, multicolored ducts with labeled elongated myoepithelial cells were present at the earliest preneoplastic stage (2 weeks post-treatment with DMBA) (Figures 4A–4C), whereas unicolored patches prevailed in the ducts and lobular-like hyperplastic structures from 4 weeks after DMBA treatment (Figures 4D–4F). Morphologic analysis of clone composition at early time-points (2-4 weeks post-treatment) revealed a high proportion of mixed but luminal-enriched clones (Figures 4E and 4F). These findings suggest that luminal cells generated from bipotent stem cells in the K5+ basal compartment (Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (378) Google Scholar) serve as cell" @default.
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• W2927656553 date "2019-04-01" @default.
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• W2927656553 title "Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging" @default.
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• W2927656553 doi "https://doi.org/10.1016/j.ccell.2019.02.010" @default.
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