Lecture 21 - Application of experimental models and imaging in cancer

What in-vitro models can be used to analyse cancer cells?

• Cancer cell lines

• Tumour-derived material

• Co-cultures

• 3D-cultures

What are the types of cancer cell lines?

• Spontaneous

  • Derived from naturally arising tumours

• Immortalised 

  • Intentionally induced immortalisation

What are the types of tumour-derived materials?

• Primary cultures 

• Tissue slices 

• Transformed cells 

What are co-cultures?

Stromal and epithelial cell populations mixed in with cancer cell line

What are 3D cultures?

Single or mixed populations embedded in matrix to mimic stromal microenvironment  

What are the three types of 3D culture models?

• Hanging drop  

  • Cells will stick as a spherical structure as they grow 

• Cells are growth within a hydrogel which helps form organoids or spheroids 

• Cells seeded into a spinning bioreactor 

What mouse models can be used in cancer studies?

Injection of cell lines/human tumour tissues 

Over-expression of transgene 

• Powerful promoters 

Depletion of gene 

• Knockout 

• Short inhibitory RNAs 

Site-specific expression  

• Gene expression driven by tissue specific promoter 

Regulated expression 

• Temporal expression  

  • Expression can be turned on at a specific time 

What are the advantages of 2D human cancer cell lines?

• Easily perturbed on many levels (genetics, environment, drugs, etc) so causality is easy to establish

• Can be subjected to highly controlled TME treatments but in vivo complexity is not recapitulated

What are the disadvantages of 2D human cancer cell lines?

• Can be subjected to highly controlled TME treatments but in vivo complexity is not recapitulated

• Immortalized cell lines often poorly representative of in vivo traits

What are the advantages of microfluidics-based in vitro models?

• Can generate a highly controllable, dynamic environment to reconstruct TME conditions

• Can recapitulate major TME features (e.g. ECM, sheer stress, gradients, etc)

• Conventional devices are commercially available but more complex devices require special expertise and equipment

What are the disadvantages of microfluidics-based in vitro models?

• Conventional devices are commercially available but more complex devices require special expertise and equipment

• Only partially recapitulates TME complexity and heterogeneity

What are the advantages of organoid- or spheroid-based in vitro models?

• Can be established from cells originating from patient tissue

• Retains many in vivo cell biology features

• Possible to genetically manipulate

• Amenable to multi-passage culture

• Some phenotyping assays developed on cell lines are applicable

What are the disadvantages of organoid- or spheroid-based in vitro models?

• Amenable to multi-passage culture

• Some phenotyping assays developed on cell lines are applicable

• Stem cell-based organoids lack stromal cells and vasculature

What are the advantages of animal models?

• In vivo model

• Genetic diversity and life history are controllable

• Genetic knockouts can be made

• Highly complex cell and biophysical environment

What are the disadvantages of animal models?

• Highly complex cell and biophysical environment

• Differences between animal models and humans limit relevance

• Physiochemical TME conditions are not controllable

• Difficult to ensure comparable sampling between individual replicates

How can Induced Pluripotent Stem Cells (iPSCs) be used for cancer research?

Need to generate a prostate organoids suitable for disease modelling  

• Can create iPSCs and then induce them to develop into prostate cells 

  • These prostate organoids will self-organise and recapitulate key features of prostate development 

• These iPSCs can also be used to develop prostate cancer models 

  • Can introduce genes into the iPSCs to try and make them look like cancer 

Why are microfluidic models useful in mimicking the physiochemical TME?

These microfluidic devices are designed to mimic the native transport unit of solid tissues including blood/lymphatic vessels 

• Can control blood, interstitial and lymphatic pressure through fluidic ports and central reservoir 

  • Sheer stress 

  • Compressive stress 

  • Hydrostatic pressure 

  • Remove accumulated waste products  

• Can establish chemical gradients

How do Vascular Organoid on a Chip (VOoC) work?

From stem cells an organoid can be made as well as endothelial cells + pericytes can be made

• EC + pericytes can be placed on a chip along with hydrogel

• Microvascular sprouting will then begin occurring in the hydrogel

• The organoid can then be seeded onto the hydrogel

• Vascularisation of the organoid will occur

Why are all the different cancer models useful in their own respect?

Suitability of cancer models will depend on what you want to investigate 

• E.g. molecular models are great at investigating mutations and epigenetics however would be less suited to investigate the TME as a whole 

What is bulk level analysis?

• Looks at the average expression across a cluster of cells 

What is single cell level analysis?

• Looks at the expression of certain genes within a single cell 

What is spatial level analysis?

• Looks at the expression of certain genes within a spatial cut of a tumour 

• Allows particular cells within particular regions of the tumour to be investigated  

What is spatial proteomics?

• Three fluorescently conjugated antibodies applied at each cycle 

• Image for protein expression and distribution 

• Repeat process until all biomarkers of interest are imaged 

• Computational spatial profiling analysis enables cell proportions, clustering, phenotyping and neighbourhoods  

What is Mass Spectrometry Imaging (MSI) used for?

• Visualising in situ molecular heterogeneity in tumour tissue

  • Can generate images by mapping thousands of molecules simultaneously

How is Mass Spectrometry Imaging (MSI) performed?

• Section of tumour added to slide 

• Apply matrix 

• Create virtual raster over the tissue section 

• Acquire mass spectra in each pixel 

• Raw data and imagine processing 

  • Spatial distribution and abundance of biomolecules

• Allows visualisation of heterogeneity of tumour 

What are the roles of imaging in cancer studies?

Identification of:

• Tumour size

• Tumour location

• Tumour spread/confinement

• Involvement of critical anatomical structures

What is a disadvantage of conventional imaging techniques?

• Conventional imagine techniques can only detect quite large tumours (>2mm) 

What is a disadvantage of cancer biopsies?

• Many biopsies fail to recover tumour cells due to the location of the tumour

What are examples of tracers which can be used during a PET scan?

• Glucose metabolism 

• Hypoxia 

• Amino acid metabolism 

• Fatty acid metabolism 

Why are PET scans useful for cancer imaging?

• Allows classification of cancers metabolism  

• Allows early stage tumour detection and response evaluation in therapy

What are the ideal future directions of PET scans?

• Biologically targeted tracers

  • Integration with genomic/proteomic profiling 

• Accelerate precision medicine and direct therapy