questions ennit Flashcards
What negative control could be used for competition assay?
Could use MCF-7: it is not usually metastatic. When WT mice are injected with MCF-7, no metastases are found.
Also paul shore’s lecture.
What are 4T1 cells? WHy are they suitable to model human mammary cancer?
4t1 is highly tumorigenic and invasive
suitable model:
1. tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site
2. as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor.
3. progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to human mammary cancer
aim, methods and purpose of the experiments?
Whether dormant BrCaCs mimic quiescent LT-HSCs.
Investigate SNO cells of the endosteal niche that are important in HSC quiescence so maybe in BrCaC dormancy.
How BrCaCs interact with SNOs
Characterise dormant BrCaCs and how they interact with the endosteal niche.
How Notch inhibition and inflammation affects dormancy.
What was your overall aim?
Find the mechanism for dormancy in the bone and what wakes it up. Whether it’s the same as the HSCs use.
Why is your overall aim important?
Understanding how dormant BrCaCs behave in the bone and what stimuli keeps them dormant or wakes them up could be used to treat them.
How did you address your aim experimentally?
kj
What were your key findings and conclusions?
- Subpopulation of BrCaCs migrates towards endosteum.
- SNOs exhibit atypical phenotype and function.
- SNOs inhibit breast cancer proliferation in vitro
- Notch pathway involved in SNO-mediated dormancy. inhibition of general notch stops this SNO-dormancy and sirna of notch1 and notch2 brings them back.
- notch2low subpopulation of brCaCs in mda and pc3 but not in mcf7
- notch2high cells are dormant in vivo, not ki67+
- notch2high BrCacs express hsc stemness markers
- notch inhibition wakes up dormant cells in vivo
- inflammation does not wake it up but chronic inflammation increases metastasis
Why didnt you look at notch1, why just notch2?
notch2 has highest expression in mda on sno and highest increase in proliferation when it’s inhibited. Would ideally also look at notch1.
Why did you focus on notch?
Notch is involved in regulation of HSCs in embryogenesis. HSCs express notch which interact with jagged 1 on osteoblasts. Notch is involvd in regulating stem cells. Overactivation of Notch leads to increased stem cells and repressed differentation.
Why did you look at inflammation?
It’s known to be pro-oncogenic.
Wanted a reason why women can go years and years with nothing happening then suddenly the dormant cancer awakes
Why do you think notch1 is similar in effect as notch2?
Not sure because read that they can have very different effects, but similar in others. Cleavage causes generation of different intracellular notch domains so act differently on the dna. But differences may depend on the strength of the ligand binding and reaction, not their different functions??
In the reading i looked at, notch1 seemed correlated with worse prognosis in breast cancer and notch2 with better prognosis
Notch cause or cure other kinds of cancers?
Read some studies where notch caused increase in cancer. and inhibiting notch reduced the cancer progression- seems mixed. may help active cancers differently from dormant ones.
Breast cancer stem cells- notch made it worse
esophageal cancer- notch made it worse
How did they visualize HSCs in the bone in vivo?
Fed mice with water and brdu. After 70 days the brdu had been diluted out fo most cells but in slow-cycling cells they
How did they visualize HSCs in the bone in vivo?
Fed mice with water and brdu. After 70 days the brdu had been diluted out of most cells but in slow-cycling cells it stays around so can detect them. Then because some of the cells weren’t HSCs, but other slow-cycling cells, they stained with mature lineage markers and most cells in the bone marrow marked positive but most of the brdu+ cells atatched to the endosteum were lineage negative and positive for sca1 and ckit (HSC markers). HSCs were found in spongy areas where the OBs are, not in the long parts. so suggested OBs had role.
Costained with N-cadherin and found the HSCs attached to SNOs, and also positive for n-cadherin.N-cadherin and beta-catenin provide an adhesive interaction between SNO and HSC.
Increased SNO number led to increased HSC number.
Summarise Zhang et al.’s findings?
-Showed that HSCs are enriched in the trabecular bone area.
HSCs attach to SNOs on bone surface.
- BMP signalling controls the number of SNO cells, which in turn controls the HSC number.
