Differentiation as a hallmark Flashcards
Hanahan and Weinberg 2000
6 original hallmarks
Sustained proliferative signalling, evading growth suppressors, evading apoptosis, sustained angiogenesis, enabling replicative immortality and tissue invasion and metastasis
Hanahan and Weinberg 2011
Added two new hallmarks and two enabling characteristics
Hallmarks - Deregulating cellular energetics and avoiding immune destruction
Enabling characteristics - Genome instability and mutation, tumour promoting inflammation
Dominique Bonnet and Jack Dick in 1997
Cells were taken directly from an acute myeloid leukeamia. These live cells were the sorted by preparative flow cytometry after staining for two cell surface markers (CD34 and CD38) which are markers of differentiation in myeloid cells. On the basis of this staining, they were able to identify 4 subpopulations of these cells. They then asked if any particular subpopulation had the capacity to behave like a leukaemia. They then implanted cells from each quadrant into an immunodeficient NOD/SCID mouse. Found that only the CD34hiCD38lo population was able to grow as a leukaemia. This was interesting as when they looked at the cells in these leukaemias - they found included all 4 cell types. Therefore, these cells were behaving as stem cells and gave rise to the other cell types
Al-Hajj et al 2003
Huge paper - almost 12,000 citations. Looked at primary breast cancer or metastases - did flow cytometry using four cell surface markers (CD24, CD44, B38.1, ESA) to sort into different categories. They then injected these into NOD/SCID mice. They found that the ESA+CD44+CD24-/lowLineage- population was >50-fold enriched for the ability to form tumours relative to unfractionated tumour cells and that this subset represented only a fraction (~2%) of the tumour cell population. They then did a second experiment - and showed that transplanting these stem cells gave rise to phenotypically heterogeneous tumours. They analysed the phenotypical diversity arising from CD44+CD24-/lowLineage- cells - and found that these tumours remembled the phenotypic complexity of the tumours from which they were derived. Suggests they can differentiate into normal cells
Quintana et al 2008
Looked specifically at malignant melanoma. Showed that maybe the CSC model does not apply is tumorigenic potential is a common attribute of melanoma cells. They used NOD/SCID mice lacking the IL-2 gamma receptor. These mice were even more immunocompromised. Xenograted melanoma cells (human melanomas grown in NOD/SCID mice) were dissociated, then live human cells were isolated by flow cytometry (getting rid of mouse cells). Then these were deposited in one cell per well in Terasaki plates and then injected into NOD/SCID/IL2rg-/- mice. Depending on the patient (tumours from 4 patients total), 12-65% of single cells formed tumours. Overall, 27% of injections lead to tumour formation. This confirms that single cells with tumorigenic potential are common within human melanomas.
Schepers et al 2012
- Tumours were induced by the conditional deletion of the TSG Apc, mutated in most colon cancers.
- We already knew Lgr5 was a marker of normal tissue stem cells.
- Cross two different strains of mice. Lgr5EGFP-Ires-CreERT2/Apcfl/fl mice. Second strain = R26R-Confetti strain which had the multicolour Cre reporter R26R-Confetti.
- In this cross, Apc deletion was targetted in intestinal stem cells by exploiting the specific expression of the intestinal stem cell-marker genes Lgr5 while concomitantly labelling individual Apc-mutated stem cells in red.
- After the resulting single-coloured tumours had grown to a substantial size, cells expressing Lgr5 in these tumours were induced to switch their colour from red to blue. This was done by injecting the mice with tamoxifen (for the second time).
- They found that blue cells generated large clonal patches in the red tumour, providing evidence for a hierarchical organisation of adenoma growth in vivo - they were able to show all cells were derived from a single adenoma stem cell.
Cortina et al 2017
Used CRC patient derived organioids with CRISPR/Cas9 technology to label defined tumour populations and perform fate-mapping experiments in vitro and in vivo. (1) used CRIPSR/Cas9 to integrate the EGFP reported and lineage-tracing casettes into the LGR5 locus of human CRC organioids. LGR5-EGFP+ cells could then be isolated from organioid derived xenografts (into NOD/SCID mice) using flow cytometry and were shown to express gene programs similar to those of normal intestinal stem cells. Moreover, LGR5-EGFR+ cells innoculated in secondary recipient mice - tumours generated by LGR5-EGFP+ cells were populated by both stem cell-like and differenitated-like tumour cells in similar proportions as the primary xenografts from which they were initially purified from - indicating they undergo self-renewal and differentiation. Lineage tracing (similar procedure to what was previously described) of LGR5-EGFP+ CRC cells in organioid derived xenografts demonstrated their self-renewal and ability to differentiate into mucosecreting and absorptive-like phenotypes in vivo. Interestingly, a population of mitotically inactive LGR5+ cells remained quiescent for prolonged times - could this population fuel tumour growth/recurrence following ablation of actively dividing cells? They only looked at one main PDO (and supported this w findings from a second PDO which was poorly tumorigenic in mice) - looking at different classes of PDOs representative of different classes of CRC would have greatly strengthened the manuscript. Did they look at the fact that there may be more plasticity than the authors gave credit for - coudl Lgr5- cells give rise to Lgr5+ cells?
Yu-Cheng et al 2013
Did a meta-analysis of literature evaluating CD133 and epithelial cell adhesion molecule (stem cell markers) as prognostic factors in HCC. Total looked at 1344 patients. The presence of CSCs was significantly associated with poor survival, including OR and disease free survival.
Wang et al 2009
- Looked for the cell of origin of malignant astrocytic glioma.
- The model they used - used Cre Lox system to generate mutant p53 under GFAP promoter
- Found that accumulation of a detectable level of mutant p53 proteins occurs first in neural SCs in the SVZ
- The p53-positive glioma precursors are remarkably homogeneous and exhibit a lineage marker expression pattern reminiscent of Olig2+ SVZ-C* transit-amplifying progenitor cells.
- Moreover, despite that the p53ΔE5-6 mutation was targeted to diverse CNS cell populations throughout the brain, the p53ΔE5-6-positive glioma precursors were exclusively identified in the two brain areas – corpus callosum and olfactory bulb, which are the migratory destinations of the two differentiated progeny of the multipotent SVZ stem cells.
- Therefore, the lineage marker expression pattern and anatomical location of glioma precursors suggest that the p53ΔE5-6-positive malignant glioma cells may arise from multipotent SVZ stem cells and/or transit-amplifying progenitors.
Poli et al 2018
Showed that MYC-driven epigenetic reprogramming favours the onset of tumorigenesis by inducing a stem cell-like state (1) Transduced human mammary epithelial cells with retroviral vector expressing exogenous c-Myc. Using gene expression profiling, showed that overexpression of MYC induces transcriptional downregulation of mature luminal-specific TFs while luminal progenitor cell-specific regulators were unaltered. (2) they also found sustained myc overexpression confers stem cell-like traits in these cells (measured the difference between the ability of WT and myc-transduced cells to grow for subsequent passages in low adherence conditions as mammospheres).Myc-cell mammospheres showed upregulated stem cell markers. (3) The downregulation of GATA3 and ESR1 (both of which are master regulators of mammary gland morphogenesis and luminal cell differentiation) was shown to be regulated by c-mYC which is able to bind to their cis-regulatory elements.
Wang et al 2008
Tumour cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133. They characterised c-Myc expression in these different tumour population susing RT-PCR, immunoblotting, immunofluorescence and flow cytometry. Found that c-Myc is highly expressed in glioma CSCs relative to non-CSC glioma cells. They used an shRNA to knockdown c-Myc in glioma CSCs - reduced proliferation with induced cell cycle arrest in G0 phase and increased apoptosis. Finally, glioma CSCs were infected with non-targetting control lentivirus or lentivirus expressing c-Myc shRNA. Cells were injected into nude mouse brain. 100% of control mice developed large tumours. No mice injected with cells that were c-Myc depleted developed tumours and after 100 days demonstrated no evidence of neoplastic cells. Therefore, c-Myc appears to be essential for cancer stem cells to form tumours
Ball et al 2017
Focussed on pancreatic ductal carcinoma Analysis of clonal dynamics during serial xenotransplantation of pancreatic cancer samples indicates that long-term growth is not driven by CSCs, but rather by the successive activation of transiently active tumor-initiating cells. This was in contrast with the continuous activity of limited numbers of self-renewing TICs within a fixed cellular hierarchy that has been observed in some of the cancers previously described by the same group (CRC and AML)
Taulli et al 2009
miR-1 and miR-206 (miRNAs associated with muscle differentiation) reduced expression in rhabodomyosarcoma in mice
- In vitro induction of miR-1/206 reduced abiliy of RMS cells to grow, and caused a change to a more myogenic phenotype
- Use of a lentiviral vector to induce miR-206 expression in vivo - blocked the growth of rhabdomyosarcoma xenografts in mice
- Finally, we showed that the product of the MET proto-oncogene, the Met tyrosine-kinase receptor, which is overexpressed in RMS and has been implicated in RMS pathogenesis, was downregulated in murine satellite cells by miR-206 at the onset of normal myogenesis.
- Potential huge benefit for miRNAs as they work on multiple targets, and can be designed to be specic, reducing side effects.
- HOWEVER: Problems with delivery, instability and potential odd target effect (both through immunogenic effect and off target gene expression changes)
Graham et al 2002
- Looked at a specific TKI used for CML
- Stem cells (Lin−CD34+) from the peripheral blood of patients with CML in chronic phase and from healthy donors were labeled with dye to enable high-resolution tracking of cell division.
- Then they were cultured for 3 days with and without growth factors ± TKI.
- After culture, the cells were separated by FACS into populations of viable quiescent versus cycling cells for genotyping.
- For healthy controls, in the presence of growth factors, TKI affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide.
- For CML samples, in the presence or absence of growth factors, the response to TKI was variable.
- In the most sensitive cases, TKI killed almost all dividing cells; however, a significant population of viable CD34+ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone
- These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and SKI.
