title: Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells.
abstract: Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.
authors: Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, Australia. wanchen.huo@gmail.com.
authors: Cecilia W
authors: CW
authors: Huo
authors: St. Vincent's Institute of Medical Research, Melbourne, Australia.
authors: Dexing
authors: D
authors: Huang
authors: Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, Australia.
authors: Grace L
authors: GL
authors: Chew
authors: Department of Pathology, St. Vincent's Hospital, Melbourne, Australia.
authors: Prue
authors: P
authors: Hill
authors: Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, Australia.
authors: Ambika
authors: A
authors: Vohora
authors: School of Medicine at the Queen Elizabeth Hospital, University of Adelaide, Adelaide, Australia.|Robinson Research Institute, University of Adelaide, Adelaide, Australia.
authors: Wendy V
authors: WV
authors: Ingman
authors: School of Medicine at the Queen Elizabeth Hospital, University of Adelaide, Adelaide, Australia.|Robinson Research Institute, University of Adelaide, Adelaide, Australia.
authors: Danielle J
authors: DJ
authors: Glynn
authors: Cell Cycle and Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia.|The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia.
authors: Nathan
authors: N
authors: Godde
authors: Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, Australia.|Division of Surgery, Peter MacCallum Cancer Centre, Melbourne, Australia.
authors: Michael A
authors: MA
authors: Henderson
authors: Department of Surgery, St. Vincent's Hospital, University of Melbourne, Melbourne, Australia.|Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology and Translational Research Institute, Brisbane, Australia.
authors: Erik W
authors: EW
authors: Thompson
authors: The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia.|Metastasis Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia.
authors: Kara L
authors: KL
authors: Britt
mesh_terms: D000328:Adult; D000818:Animals; D001940:Breast / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D000071060:Breast Density; D001943:Breast Neoplasms / Q000473:pathology; D049109:Cell Proliferation / Q000502:physiology; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000254:growth & development; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D009940:Organoids / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D061251:Primary Cell Culture; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D017364:Collagenases
keywords: FACS; biochambers; breast cancer; collagenase digestion; mammary stem cell; xenograft