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learning objectives?

- to recognise the different levels of tumour heterogeneity
- to understand the models that have been proposed to explain phenotypic heterogeneity within tumours
- to understand the characteristics, limitations and challenges of the cancer stem cell model
- to recognise the underlying therapeutic implications of a cancer stem cell model


What is the history of cancer?

- uncontrolled division of cells that leads to the formation of an abnormal cell mass (tumour)

- first recorded mention: the Ebers papyrus, Egypt, 3500 ya
- hippocrates (460 - 370 BC): first clear definition
- 1906, first international conference on cancer, Heidelberg/Frankfurt, Germany
- 1975/76, discovery of proto-oncogenes (Varmus, Bishop, UCSF)
- 1995/97 Characterisation of cancer stem cells (AML, Bonnect and Dick, Toronto, Canada)


What organs are affected by cancer?

- cancers affect a large number of organs and tissues


What is the molecular basis of cancer?

1. tumour initiation stems from spontaneous/stochastic or environmentally induced genetic alteration
2. initial genetic damage must be non-lethal (bypassing of cell checkpoints)
3. damage must happen to a cell that will proliferate
4. clonal expansion from cell that incurred initial genetic change
5. tumour development frequently requires alteration of at least 2 essential (= driver) genes
6. driver genes usually involve 4 main classes of genes: proto-oncogenes, tumour suppressor genes, programmed cell death genes, DNA repair genes
7. alterations usually need to affect both alleles of a driver gene for maximal impact
8. mutation in other genes have lower to no impact on cancer progression: passenger mutations
9. carcinogenesis is a multi-step process with progressive emergence of intra-tumour heterogeneity


What is inter- and intra-tumour heterogeneity?

- heterogeneity between cancer types
- heterogeinity between tumours of the same organ in different patients (tumour subtypes)
→ different genetic alterations, different cell-of-origins, different micro-environment context etc
- heterogeneity between primary tumour and metastases within the same patient
- phenotypic heterogeneity within a single tumour:
→ presence of different cell types in different proportions
→ genetic or epigenetic level
→ environment/context-driven heterogeneity

means that is difficult to treat the tumour as a single entity

genetic and not genetic reasons for heterogeneity

quite clear genetic differences within the same tumour or at different stages of the same tumour


What are two general models for cancer heterogeneity?

stochastic model/clonal evolution
- self renewal and differentiation are random
- various clones may co-exist (may have different sizes
- all cells have equal but low probability of initiating tumour growth
- strong influence of the microenvironment
extrinsic (mostly) and intrinsic factors

cancer stem cell model
- distinct calsses of cells exist within a tumour
- only a small definable subset has intrinisic ability to initiate tumour growth
- hierarchical organisation with CSC as the source of other cells
- implies a strong instrinsic capability of CSCs to initiate tumorgenesis


How has the presence of stem-like cells in haematopoietic cancer been determined?

self-renewal assay in immunodeficient mice
NOD/SCID: non-obese diabetic/severe combined immunodeficiency
- cancer cells (ex: leukaemia cells) → FACS → take one subset

sublethally irradiated NOD/SCIF mice → injected with the specified CD34+ tumour cells → long-term bone marrow reconstitution

must mean they have stem cell characteristics


Presence of stem-like cells in solid tumours?

- self-renewal assay in NOD/SCID mice
- serial orthotopic implantation
- CD24 expression: marker on non stem cell like
- CD24 + did not form tumour after mammary gland injection
- repeated several times when taking non-CD24+ cells --> generated CD24+ cells


What are cancer stem cells?

cells that have the ability to generate heterogenous tumours with higher efficieny once injected at high dilution in immunocompromused mice
- self renewal
- differentiation into progeny that can't self renew


What properties do cancer stem cells share with normal stem cells?

1. expression of "specific" markers that enrich cells with tumourigeneic potential
2. self renewal
→ tissue specific normal stem cells must self-renew throughout the lifetime of the animal
→ cancer stem cells undergo self-renewal to maintain tumour growth indefinitely
3. potential for differentiation into phenotypically diverse mature cell types
→ give rise to a heterogenous population of cells that compose the organ or the tumour but lack the ability for unlimited proliferation (hierarchical organisation of cells)
4. regulated by similar signalling pathways
→ pathways that regulate self-renewal in normal stem cells are dysregulated in cancer stem cells


What are some experimental approaches to study Cancer stem cells?

- markers
→ very hard to find markers that are extremely exclusive of one cell type and not another
→ often more subtle: gylcosylation, proportions, e.g. CD133 in brain tumours
- enrichment of tumourigenicity
→ force them into suspension, stem cells start forming spheres (clonogenic tumour sphere assay), other cells won't form the spherres
- self renewal
→ seropassaging
→ sphere assay many times
→ in vivo , logistically more difficult
- differentiation potential
→ look in tumours in vivo
→ adherence cell growth in vitro, subtypes only arise from stem cells


What are markers for tissue stem cells and cancer stem cells?

- healthy intestinal epithelium vs colon cancer
- both have LGR5 and ALDH1
- using more than one marker allows you to extract a purer colony of stem cells

breast cancer
- CD44+CD24+ in high numbers does not generate tumour (even when injecting up to 200,000 cells)
- in low numbers it does (low as 200 cells)

- 100 CD133+ cells yes
- 100,000 CD133- cells, no

melanoma: no marker enriches tumourigenic potential vs equal tumorigenic potential of all cells
- therefore melanoma does not respond to cancer stem cell model at all


What are the self-renewal properties of cancer stem cells?

- if the cells are able to self-renew, it means that they are able to from a stem cell, generate at least one new stem cell again and again
- long term maintenance of tumours
- maintained or increased over several passages
- repeated isolation of CD133+ cells from serial xenografts always generates identical tumours


What is the multi-lineage differentiation potential of cancer stem cells?

colon cancer cells
- isolation with CSC markers (CD133+/CD24+)
- assay of CSC or differentiation markers immediately or after several days in conditions that promote differentiation (Matrigel, +SVF)
- loss of CSC markers (CD24, CD44, CD133)
- acquisition of differentiated markers (CK20)

- expression of stem cell markers starts decreasing and expression of differentiated markers starts increasing

- injection of single CSCs in immunodeficient mice
- generation of terminally differentiated cells following single CSC grafts

- enrichment of CSCs from patient tumour samples
- injection in immunodeficient mice: xenografts look like their tumours of origin (histological appearabce, marker expression...)
- e.g. colon cancer


What pathways that are involved in self-renewals are deregulated in cancer cells?

- Wnt → critical for survival of haematopoietic, epidermal and gut stem cells → the prominent pathway in colon carcinoma, epidermal tumours
- Hedgehog → haematopoieitic, neural, germ line → medullablastoma, basal cell carcinoma
- Notch → leukaemia, mammary, colon

- very often pathways that are important for development
- also adult stem cells


What is the role of CSCs in metastasis development?

- essential for metastasis development
- key step in cancer andfor therapy
- colorectal tumour sample:
→ CD133+CD44+CD26+/CD26- → tumourgenesis
- cell purification and orthotopic implantation

triple positive subpopulation only
- detected as circulating tumour cells in the blood
- give rise to liver metastases
- display high invasive properties in vitro
double positive was quite able to generate primary tumours but could not metastasise

different subtypes of CSCs?


What is the role of CSCs in therapeutic resistance?

- CSCs are often enriched following chemotherapy
- CSCs are involved in post-treatment tumour relapse


What are the mechanisms of CSC resistance to anti-cancer therapy?

- slow cycling (quiescent?) cells: less sensitive to proliferation-targeting treatments
- poor sensitivity to pro-apoptotic signals
- high capacity for drug metabolism (detoxification)
- less prone and less sensitive to DNA damage:
→ low levels of reactive oxygen species
→ very active DNA repair machinery


What are therapeutic implications of a pure cancer stem cell model?

- drugs that kill tumour cells but not cancer stem cells → tumour shrinks but grows back
- drugs that kill tumour stem cells → tumour loses its ability to generate new cells → tumour degenerates

not that simple


Is there a relationship between cancer stem cells and the cell of origin of cancer?

- not the same thing
- cell of origin probably happened years before investigation of the cancer stem cells

- the cancer stem cell and the cell of origin are different concepts
- the cell of origin is not always known at present and may not be a stem cell
- the cell of origin varies depending on cancer types and subtypes, which may play a role in inter-tumour heterogeneity


What is the link between EMT and CSCs?

- EMT: loss of differentiated characteristics by epithelial cells (cell-cell adhesion, planar and apical-basal polarity, lack of motility...) and acqusition of mesenchymal features (motility, invasiveness, increased resistance to apoptosis)
- MET is the reversal of that programme
- important roles in multiple developmental processes (gastrulation, formation of placental, somimtes, heart valves, neural crest, urogeneitcal tract, branching morphogenesis of multiple organs)
- in cancer, EMT is involved in invasion, metastatic dissemination and acquisition of therapeutic resistance
- EMT process (during development and cancer) is highly sensitive to signals that cells receive from their stromal microenvironment
- induction of EMT promotes the CSC phenotype


What is the phenotypic plasticity of cancer cells?

- homeostatic equilibrium of tumourigenic cells
- non tumourigenic cells can generate CSCs (no hierarchy?)
- when placed in non-challenging culture conditions, even non-CSCs can survive and can actually regenerate cells with a CSC-phenotype (eg expressing CSC markers), which is what we call plasticity in this case

consequence for therapy means you probably need combination therapy e.g. chemotherapy and CSC-targeted, elsewise you could still have tumour relapse


What are challenges for therapeutic targeting of CSCs?

- how best to combine CSC targeting with targeting of other tumour cells?
- minimise targeting of adult tissue stem cells
- possible genetic variability of CSCs: do they still share sensitivity to common therapeutic compounds?
- what are appropriate biological responses to measure in preclinical and clinical trials? (what time-course, which CSC markers...?)


Take home messages?

- tumours are phenotypically heterogenous
- two main models have been proposed to explain this heterogeneity:
→ stochastic clonal evolution model
→ cancer stem cell model

- the CSC model implies that:
→ heterogeneity is almost uniquely driven by intrinsic factors
→ a rare subpopulation of tumour cells (CSCs) has self-renewal and tumourigenic properties
→ tumour cells are organised along a determined hierarchy with CSCs as the source
→ specific markers allow for enrichment of the tumourigenic fraction

- the CSC model is compatibile with clonal evolution, but in a determined and NOT stochastic (random) manner

- some cancers do not seem to follow the CSC model (e.g. melanoma)

- the CSC and the Cell of origin are not interchangeable concepts

- relation with porcesses controlled by environmental signals (e.g. EMT) and demonstration of tumour cell plasticity currently challenge of the CSC model

- is there a fixed CSC entity or rather a tumour-initiating (cancer stem-like) cell state?