3D in vitro modelling using material from the Breast Cancer Now Tissue Bank
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1 @QMBCI bartscancerinstitute 3D in vitro modelling using material from the Breast Cancer Now Tissue Bank Richard Grose Barts Cancer Institute Ed Carter
2 Normal Breast Architecture Calponin / CK8 Myoepithelial Cell Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer Normal breast duct consists of a ring of polarised luminal epithelial cells surrounded by myoepithelial cells Myoepithelial cells: Provide polarisation cues to luminal cells Contract during lactation to propel milk through the ductal tree
3 Myoepithelial and s: A Close Relationship Myoepithelial Cell Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer Relationship begins to break down in ductal carcinoma in situ and falls apart in invasive breast cancer Approximately 50% of DCIS cases will progress to invasive breast cancer
4 Myoepithelial Cells may Promote Tumourigenesis Normal Tumour Suppressing z DCIS Tumour Promoting Structural z MMP2 BM Synthesis MMP Activity TIMP-1 z z VEGF CD10 Maspin VEGF z SMA z Understanding relationship will lead to novel prognostic markers and new therapeutic targets Need a model to study this...
5 Primary Breast Epithelial Cells Breast tissue samples can be reduced to their individual cellular components for tissue culture applications Readily accessible through Breast Cancer Now tissue bank Collagenase/ Hyaluronidase Breast Tissue Organoids Trypsin Single MECs Sedimentation CD10 EPCAM Fibroblasts Myoepithelial Luminal Contact details:
6 Modelling the Breast Bilayer Aim - Generate a model of the breast duct bilayer to interrogate myoepithelial / luminal interactions in the progression of breast cancer Breast ducts from reduction mammoplasty Isolation of luminal and myoepithelial cells 2D culture of individual cell populations infection with viral particles Combine cells in 3D matrix and culture analyse by confocal microscopy
7 Myoepithelial and s can be Isolated from Reduction Mammoplasty Specimens Myoepithelial 40% CD10 + APC 40% EpCAM + Luminal PE
8 P63 CK14 Calponin Primary Myoepithelial and s Retain their Characteristics in vitro Myoepithelial Luminal Calponin CK14 P63 CK8 CK19 HSC70 10 days in 2D culture CK19 CK8
9 Myoepithelial and s Coalesce in Collagen Gels to form Spherical and Ductal Structures Collagen Gel Breast Section Day 0 Day 21 Day 3 Myoepithelial / Luminal Day 21 CK8 / P-Cadherin / Nuclei
10 Matrigel Does Not Promote Reformation of Bilayer Myoepithelial / Luminal CK8 P-Cadherin Merge Matrigel promotes bipotency of luminal and myoepithelial cells
11 Modelling DCIS in a Physiologically Reflective Setting Myoepithelial Cell HER2 Expression in s Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer Physiological bilayer can be recreated through recombination of myoepithelial and luminal cells in 3D gels Does HER2 overexpression recapitulate DCIS?
12 Modelling DCIS in a Physiologically Reflective Setting HER2 Virus Dox (µg/ml) HER2 HSC70 D0: Embed Cells D14: Ducts Form -Induce Overexpression D21: Analyse
13 Overexpression of HER2 Destabilises Bilayer Control +DOX +DOX +Trastuzumab HER2
14 Quantifying Luminal Filling Collaboration with James Gopsill, University of Bristol Cell volume / body volume ratio Control ** ** Doxycycline Doxycycline + Trastuzumab
15 Luminal HER2 expression recapitulates DCIS HER2-Expressing Spheroid DCIS Breast Section HER2/Vimentin Doxycycline Myoepithelial Cell HER2 Expression in s Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer
16 ITGB6 αsma DCIS to Invasive Cancer - Integrin α V β 6 Integrin α V β 6 is upregulated in the myoepithelial cells of high grade DCIS breast cancer Expression promotes MMP9 production and matrix remodelling Normal Low Grade DCIS High Grade DCIS Allen, M.D. et al. (2014) Clin. Can. Res. 20(2):344-57
17 Progressing DCIS to Invasive Cancer Myoepithelial Cell HER2 Expression in s β6 Expression in Myoepithelial Cells Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer Luminal HER2 expression recapitulates DCIS Does co-overexpression of ITGB6 in myoepithelial cells push the model to invasive cancer?
18 Progressing DCIS to Invasive Cancer Myoepithelial Cell HER2 Expression in s β6 Expression in Myoepithelial Cells Cancerous Normal Breast Duct Ductal Carcinoma in-situ Invasive Breast Cancer ç ç
19 Luminal / ITGB6 Myoepithelial Luminal / Myoepithelial ITGB6 Induction in Myoepithelial Cells Promotes Invasion Vimentin / Her2 / Nuclei
20 Summary Normal breast cells can be isolated, manipulated and recombined in 3D cultures These models allow us an objective view on cell-cell and cell-matrix interactions in the development of the breast duct and in the progression of breast cancer These insights will allow increased understanding of cell signalling in the breast and help in identifying novel biomarkers of disease progression Meet the expert.. Ed Carter Poster Block 13 Carter, E.P. et al. (2017) Breast Cancer Research. 19(1):50.
21 Acknowledgements Ed Carter Louise Jones Jenny Gomm James Gopsill Lucía Fernández Adrienne Morgan Mike Allen Iain Goulding Linda Haywood John Marshall Breast Cancer Now tissue bank donors
University of Bristol - Explore Bristol Research
Carter, E. P., Gopsill, J. A., Gomm, J. J., Jones, J. L., & Grose, R. P. (2017). A 3D in vitro model of the human breast duct: A method to unravel myoepithelial-luminal interactions in the progression
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