lecture 26: breast and cancer stem cells Flashcards Preview

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What defines stem cells?

  • defined by their ability to self-renew and differentiate along multiple lineages 
  • stem cell → common progenitor → committed progenitor → mature cells 
  • stem cell → stem cell 


What is development of the breast?

  • newborn → puberty → pregnancy → lactation → involution (→ pregnancy → etc) 
  • proliferation at puberty
  • proliferation and differentition during pregnancy 
  • differentiation during lactation
  • apoptosis during involution 


What are the three distinct epitheli cell types seen in breast?

  • 18 day pregnant
  • luminal epithelium
    • alveolar
    • ductal
  • myoepithelium 


For what are mammary stem cells required?

  • homeostasis in the mammary gland and growth during pregnancy 
  • remarkable generative capacity of breast tissue 
  • more than 25-fold expansion of breast 'epithelium'


How have in vivo strategies been used to define the mammary stem cell (MaSC)?

  • identification of cell surface markers to allow fractionation of mammary cells by flow cytometry 
  • transplantation studies:
    • perform limiting dilution assays to allow comparison of the relative repopulating frequencies of different subpopulations 
    • demonstrate the multilineage differentiation capacity of SCs. Serial transplantation is the 'gold standard' to prove the self-renewing capability 
  • lineage tracing studies in vivo


What is in vivo characterization using mammary fat pad transplantation?

  • mammary gland not an essential organ
  • cauterize in a young animal (3 weeks) 
  • remove rudimentary tree attached to nippple
  • leave behind an intact fat pad 
  • inject FRESH cells into this 
  • harvest 8 weeks post-transplantation 
  • ask whether we see a ductal tree that has emerged in this area 


What are the multiple cell types of the mammary gland?

  • luminal epithelial cells
  • myoepithelial cells
  • basement membrane (separates epithelial cells from surrounding stroma)
  • fibroblasts 
  • adipocytes 
  • blood vessel
  • lymph node 
  • macrophages 
  • complex microenvironment 


How many epithelial subpopulations were defined by cell surface markers?

  • CD24 + or -
    • heat stable antigen
  • CD29 + or -
    • beta1-integrin
  • DP - double positive - smallest population, 4.8% 
  • CD24+/CD29- 23.9% 
  • double negative = 55.4% 
  • CD29+/CD24- = 5/3% 
  • transplant cells in numbers proportional to their frequency in the overall population 


What was transplantation of subsets of lineage-cells?

  • cells double-sorted
  • rosa 26 donors (lacZ gene in Rosa-26 locus)
  • only one population had repopulating capacity when put into cleared fat pad 
    • CD29hi
    • CD24+ 


Were they able to get generation of a functional mammary gland from a single stem cell?

  • yes
  • beta-galactosidase makes blue
  • capable of multilineage differentiation 


Can the Lin-CD24+CD29hi cell self-renew?

  • serial transplantation studies
  • MaSC 
  • primary transplantation
  • primary outgrowth 
  • secondary transplantation ( first generation self-renewed MaSCs) 
  • secondary outgrowth 
  • tertiary transplantation (second gen self-renewed MaSCs)
  • tertiary outgrowth
  • most could be passaged ~8 times


Do bipotent cells exist and function in vivo?

  • big controversial issue in the field 
  • they had done research with a team in canada → recapitulated findings independently → found a stem cell that could give rise to all of these cells 
  • in 2011 a paper appeared and said that bipotent stem cells do not exist - only unipotent cells exists 
    • there is a myo-SC → myoepithelial cells
    • luminal SC → ductal and alveolar cells 
    • claimed using lineage tracing
  • at the same time they were also carrying out lineage tracing experiments 


What is 3D imaging of the mammary gland?

  • 3 week-old gland prior to puberty
  • this is about a 4mm section of an intact mammary gland 
  • can see elongated myoepithelial cells 


What is the strategy for in vivo lineage tracing?

  • Rosa26 is a strong, ubiquitous promoter 
  • dtTomato is a red fluorescence protein 
  • all daughter cells of labeled parental cell are permanently marked
  • single colour for quantification  
  • toxicyclin inducible system 
  • tet operon that contains cre 
  • third cross where cre induces expression of a promoter gene 
  • triple transgenic mice
  • if the promoter is expressed in a specific cell of interest, in the presence of toxicyclin, it will activate the tet operon → cre mediating recombination → expression of that particular promoter or reporter gene in that particular cell type 
  • indelible marking of all daughter cells of that parental cell 


What is confetti?

  • a stochastic multicolour cre reporter for clonality studies 
  • 4 fluorescent proteins 
  • if you deliver just a small single pulse of an agent (toxicyclin) → random activation of only one of four colours in that cell 
  • because only one pulse is delivered not flipping backwards and forwards


What was population dynamics of K5-expressing cells in puberty?

  • equi-expression of all four fluorescent proteins 
  • K5 marks long myoepithelial cells 
  • shows that many progenitor cells are involved in morphogenesis of the gland during puberty 


What were they able to show in the end?

  • K5 marks both luminal and myoepithelial cells after induction in the adult 
  • able to show that after an 8 week chase 
  • unicoloured clonal regions 
  • both myoepithelial and cuboidal luminal
  • proved that bipotent stem cells exist 
  • capable of giving rise to all the epithelial cells of the mammary gland
  • also luminal progenitor cell exists 


What is the search for normal human breast stem cells?

  • not able to do lineage tracing but did do cell surface marker analysis 
  • found that there were four distinct populations 
  • only one of these, if you transplant back into the mouse fat pad, has the ability to give rise to ductal outgrowths 
  • same pattern for human and mouse mammary tissue  


What are functional similarities between mammary epithelial subpopulations in mouse and human?

  • can identify bipotent stem cells in both 
  • two types of luminal progenitors 
  • can prospectively isolate all the mature cells as well
  • don't know about all the precursors leading up to myoepithelial cells 
  • both the mouse and human MaSCs lack receptors for the ovarian hormones oestrogen and progesterone 
  • important because increased progesterone and oestrogen are linked with increased breast cancer risk 
  • could they still be influenced?


Are MaSCs sensitive to ovarian hormone deprivation?

  • yes - highly sensitive 
  • stem cells appear to retain a 'memory' of prior steroid hormone deprivation 
  • repopulating frequency:
    • control: 1/58
    • ovariectomy: 1/247
  • also when they could generate tissue, it was only very little 


What happens to numbers of MaSCs during pregnancy?

  • pregnancy is accompanied by an 11-fold increase in the number or activity of MaSCs 
  • the augmented MaSC pool drives secretory cell expansion 
  • seen in mid-pregnancy 
  • transplantation assay measures function 
  • stem cell highly receptive to hormonal signalling 


What is evidence that ovarian hormones (oestrogen and progesterone) profoundly influence stem cell activity?

  • hormone deprivation and anti-oestrogens decrease MaSC pool/activity 
  • excess hormones increase MaSC function
  • ageing is associated with increased MaSC function 
  • pregnancy dramatically increases MaSC pool 


  • ductal luminal cells express ER and PR - remain the most important prognostic markers for breast cancer to date 
  • 70% of breast cancers are ER positive and if they are there is a much better prognosis 
  • how is this signalling to the stem cell when the stem cell doesn't have receptors for the hormones?


What pathway mediates steroid hormone signalling to stem cells?

  • The RANKL/RANK pathway 
  • signals to NFkB 
  • steroid hormones like progesterone stimulate ductal cell to make RANKL 
  • signals to the stem cell pool
  • drives proliferation 
  • probably other molecules involved as well


What is a strategy for assessing gene function in mammary stem and progenitor cells?

  • retrovirus-mediated manipulation of gene expression in discrete populations 
  • harvest virgin mammary gland 
  • single cell suspension
  • cell sorting into subpopulations 
  • plate subpopulation on feeder cells
  • transduce with retrovirus 
  • sort GFP+ cells after single cell suspension 
    • colony forming assay on feeder cells
    • colony forming assay in matrigel
    • transplant into cleared fat pad 
      • puberty or pregnancy 
      • mammary outgrowth


What are pathways often deregulated in cancer?

  • self-renewal pathways in stem cells 
  • decided to look at notch - associated with the most aggressive subtype of cancer (triple negative) 
  • wanted to understand the normal role of notch 
  • wnt, notch and hedgehog are all very important pathways → embryonic and normal development
    • always see them reemerging in cancer 


Can short-term cultured MaSCs reconstitute a functional mammary gland?

  • yes
  • 5 or 6 days of culture did not abrogate this ability 


What does constitutive Notch signalling in MaSCs promote?

  • luminal cell commitment and induces hyperplasia in vivo
  • deregulated notch1 can go on to produce luminal tumours 


What are dual functions for Notch?

  • restricts MaSC proliferation and directs luminal cell fate determination 
  • complex pathway
  • normally it represses proliferation of the mammary stem cell 
  • but when activated it promotes formation of luminal progenitors 


What are GATA transcription factors?

  • six family members (GATA-1-GATA-6)
  • bind DNA at the consensus sequence (A/T)GATA(A/G) 
  • play critical roles in development, cell-fate specification and differentiation