L18- Preclinical models

overview of contribution of preclinical models to precision medicine

what material can be studied for in vitro analysis of cancer cells

1. Cell lines

• Spontaneous

• Immortalised (oncogene)

2. Tumour-derived material

• Primary cultures

• Tissue slices

• Transformed cells

3. Co-cultures

• Stromal and epithelial cell populations

4. 3D-cultures

• Single or mixed populations embedded in matrix to mimic stromal microenvironment (more representative of tumour microenvironment) – spheroids, organoids

what are primary cultures

Tumour-derived material

Take samples from biopsy and try to grow in lab 

- tend to die out after few passages  

what is a tissue slice

tumour derived material

biopsy from patient and cut into 200 micron think slices, put in culture and grown- die out after about a week

what are transformed cells

tumor derived material

takes a lot of work and time, need to be validated and characterised which delays models

what is co culture

stomal and epithelial populations mixed to see what effect stroma has  

where are 3D cultures derived from

Derived from stem cells- forms 3D structure containing cancer cells and other cell types from TME, hard to produce, complex 

overview of different \3\d models

1. Primary grown as a spheroid , scaffold free 

2. Mix with other cell types 

   • Is a scaffold 

   • Decellularized organ 

3. Grown in liquid culture, can change pH etc 

   Cancer cells can be hard to grow as haven't got types of growth media right  

how did we go from in vitro platforms to ex vivo models

what are 2 types of proliferation assay

1. direct cell count

2. indirect cell count (growth)

what is direct cell count- proliferation assay

• Haemocytometer (graticule on glass slide)

• Coulter counter (cell numbers passing through light source)

• Flow cytometer –including cell cycle analysis (cell morphometric properties detected as pass through light source)

what is indirect cell count (growth)- proliferation assay

• MTT (mitochondrial turnover)

• Sulforhodamine B (SRB) (cell protein content)

• Tritiated thymidine (DNA replication)

• Scanning microscope (IncuCyte) (cell confluency)

what is an adhesion assay

example- measure integrin-mediated cell-cell interactions

1. coat plates with ECM components

2. add radiolabelled or fluorescently labelles cells

3. allow to adhere, wash wells

4. measure radioactivity/fluorescence

what is cell motility

Essential for multiple cellular processes - requires spatial and temporal regulation of contractile and protrusive forces induced in cell body and adhesion strength

Actin cytoskeletal network (dynamic and versatile intracellular scaffold) – critical for driving projection of cell membrane and orchestrating directed movement

what 3 actions allow for cell motility

Protrusion of cell membrane

– Formation of leading edge

Initiation of new cell-substratum attachment s ites

– Adhesion molecules stabilise structure at leading edge

– Structural and signalling molecule complex form on cytoplasmic side – focal contacts

Contractile forces

– Derived from activity of actin-myosin motors

– Forward movement of cell

Disassembly of adhesions at rear of cell

– Cell retraction

what is the scratch wound migration assay

a motility assay

Cancer cells growing in culture on well 

• Allow to cover whole dish 

• Scratch a wound into cell layer- gap between cells 

• Use microscopes, measure how cells will migrate to fill in the gap  

• Over time, gap fills as cells migrate  

• Put conditions/drugs into environment to see how can modulate cells to be more or less motile  

what is quantitative fluorescent speckle microsopy

a more sophisticated motility assay

Monitor how actin forms macromolecular structure  

Can put drugs in to modulate and see effects  

Fluorescent labelled protein - Rhodamine-G-actin (monomer) incorporated into macromolecular structures (F-actin)

•Visualised by real-time imaging technique

•Fluorescent speckles act as markers of actin dynamics

describe the invASION ASSAY- BOYDEN CHAMBER

Boyden chamber- well of tissue culture disc, insert a chamber that fits into well, bottom part has small holes (smaller than a cell), cells squeeze themselves through to enter well below 

• Chemoattractant in bottom of chamber attracts cancer cells 

• Measure motility to some extent 

• Can put collagen matrix at bottom of chamber- a barrier for cancer cells to cross, in order for them to cross they need to digest using proteases  

• Take bottom part if chamber and stain it- darker areas in image are cancer cells, smaller than pores, but can squeeze through, can change shape and size to be motile and invasive  

what is an issue with using cancer cell lines

Issue with cancer cell lines- come from patients with advanced cancer with lots of mutations 

Useful for looking at late stage of cancer  

• Not many models for early stages  

describe the applications of cell lines to stratify cancer types based on activating mutations

NSCLC 139 cell line panel as models for therapeutic intervention – mutational profile defined by DNA sequencing

• Characteristic cell line mutations reflect mutations observed in NSCLC patient sub-groups

• Investigate how patients with specific mutation will respond to different therapeutic agents

How are cell lines used in non-small cell lung cancer research?

• Cell lines have been DNA sequenced, so all mutations are known

• Large bank of ~139 cell lines representing different patient-like phenotypes

• Used to study drug effects by:

  • Applying a drug to a specific cell line

  • Observing the resulting cellular response

How are in vitro assays used to study angiogenesis?

• Use endothelial cells from capillaries

• Grow in:

  • 2D (monolayer on plastic)

  • 3D → form endothelial vessel-like structures

• Tubule formation assays:

  • On Matrigel → capillary-like networks

  • In 3D culture on beads → vessel structures

• Models angiogenesis stages:

  • Angioblasts → vascular remodelling → vessel wall maturation (with pericyte

How is the ex vivo angiogenesis assay performed and what is it used for?

• Rat aortic ring explant assay:

  • Take blood vessel (rat aorta) and culture it

  • Observe endothelial cell outgrowth and capillary sprouting

• Used to:

  • Study angiogenesis in a more tissue-like context

  • Test drugs by adding them to see if they block angiogenesis

How is the chick chorioallantoic membrane (CAM) assay used to study angiogenesis in vivo?

• Uses 7-day-old chick embryos (lack a mature immune system)

• Remove eggshell to access the CAM (highly angiogenic membrane)

• Place test substances, tumour cells, or drug pellets onto the membrane

• Observe capillary/angiogenic response after several days

• Readout: visible changes in blood vessel growth

• Useful for drug screening to assess pro- or anti-angiogenic effects

How are chamber assays used to study angiogenesis in vivo?

• Transparent chamber in rabbit ear, dorsal skinfold (mice), cranial window (mice)

• Allows continuous measurement of angiogenesis, blood flow, pH, tumour growth and wound healing

• Invasive and technically difficult

Lose skin in back of mouse, attach chamber that holds skin and can visualise under microscope- look at blood flow in response to being treated with drug- look at vessels and what's happening inside them  

How are tumour models used in in vivo assays of angiogenesis?

• Tumours grown in mice subcutaneously or orthotopically

• Implant tumour cells in skin of mouse (skin at back is very loose)

• Can see tumour grow under skin and follow its size

• Effect of test substance on tumour size and animal survival established at regular intervals

• Tumour can be taken then looked at under microscope

• Histological analysis determines effects of drug on necrosis, thrombosis and microvessel density

what different things can be done using murine models in cancer studies

Injection of cell lines/ human tumour tissue

– Subcutaneous, tail vein, cardiac, orthotopic,

metastatic site (bone)

• Over-expression of transgene

– Powerful promoters eg. CMV, SV40

• Depletion of gene

– Knockout

– short inhibitory RNAs

• Site-specific expression

– Gene expression driven by tissue specific promoter

• Regulated expression

– Temporal gene induction

describe the application of microfluidic device to study the metastasis cascade

Mimic intermediate steps of metastasis in platform , put different cell types into ECM, introduce channels and cell types, changer interstitial pressure, introduce drugs  

• Look at cell-cell and cell -matrix interactions  

give an example of using a 3D microfluidic model to investigate invasion

Tumour cells implanted and endothelial capillary cels growing  

• Tumour cells labelled with red colour so can see how invade towards capillary vessels  

• Use as a model and put different things in to see if you can modulate growth  

how can 3D bioprinted models be used for applications in cancer biology

3D printer- reconstruct TME introducing cell types and cancer cells, cells that form capillaries, use it to study interaction with immune cells etc 

• Much more complex 

what are the limitations of murine models in cancer studioes

Might not be representative of human- e.g. drug works in mouse but not when put into human  

HOWEVER

• EGFR knocked out in mouse- skin inflammation and hair follicle damage  

• Human patient treated with small molecule inhibitor (mimics the knockout )- many features seen in the human are the same as in the mouse- very similar pathology as in mouse  

What is the biological role of c-Myc and how does it affect cell behaviour?

• c-Myc expression correlates with high proliferative activity

• c-Myc is a transcription factor rapidly induced by mitogenic stimuli

  • Drives G1–S progression

• Inhibits terminal differentiation and sensitizes cells to apoptosis

• In vitro manipulation:

  • Overexpression → increases proliferation and promotes apoptosis

  • Knockdown → reduces proliferation

• Around 50% of cancers have affected Myc (usually upregulated)

  • Helps drive cancer cells

How is c-Myc deregulated in cancers and studied in murine models?

• Elevated/deregulated c-Myc in ~50% of cancers

  • Breast, colon, cervical, small-cell lung carcinoma, osteosarcomas, glioblastomas, melanomas, myeloid leukaemias

  • Associated with aggressive, poorly differentiated tumours

• Mouse models:

  • Knockout Myc → lethal (embryos don’t survive) → shows importance of Myc

  • Transgene overexpression of Myc → induces many cancer types

What happens when c-Myc is disrupted or constitutively expressed in in vivo murine models?

• Targeted gene disruption of both c-Myc alleles → embryonic lethal (day 9.5–10.5)

• Transgenic mice with c-Myc constitutively expressed develop variety of phenotypes:

  • Lymphomas, skin ulcerations, multiple tumours

What are the limitations of constitutive c-Myc expression models and how can they be overcome?

• c-Myc constitutively expressed in single cell type via tissue-specific promoter

  • Problem: expressed during embryonic development (long latency)

• Issue: tumour in humans doesn’t arise in embryo but later stage

• Overcome:

  • Express Myc using specific promoter

  • Only expressed when activating agent is added

  • Turn on in one cell type and specific organ

What are conditional transgenic c-Myc models and why are they useful?

• Conditional transgenic:

  • c-Myc can be switched on in specific adult tissues

  • Activating agent stabilises and activates MYC signalling

• Models are useful to:

  • Study tumour development in adult context

  • See how different organ backgrounds react

How is conditional transgenic c-Myc expression used in different tissue environments?

• Conditional transgenic (c-Myc) expression:

  • Subrabasal epidermis (involucrin promoter)

  • Pancreatic islet β-cells (insulin promoter)

• Constitutive Myc expression (inactive), tamoxifen acts as activating ligand

• One model: gene only in skin basal cells → skin tumours

• Another model: under insulin promoter → pancreatic tumours

• Need to ensure model reflects what you're trying to study

What are the tumour characteristics of c-Myc activation in different tissues?

• In subrabasal epidermis:

  • c-Myc activation induces proliferation and impaired differentiation

  • No apoptosis

• In pancreatic islet β-cells:

  • Rapid proliferation (1 day)

  • Apoptosis of β-cells and onset of diabetes in 9 days

• Difference:

  • Skin tumours → no apoptosis

  • Pancreatic tumours → high apoptosis levels

why is imaging in cancer important

Accurate positional information of tumour/ metastases are required for modern clinical treatment

what are the important things to learn from imaging in cancer

Tumour location

– Many biopsies fail to recover cancer cells

• Tumour size

– Conventional imaging techniques detect tumours > few mm (>109 cells)

• Tumour confined/spread locally to lymph nodes

– Stage 1 cancers frequently >90% 5 year survival

– Early stage tumours often curative

• Involvement of critical anatomical structures

– Vital organ/ blood supply may alter treatment strategy

describe the features of metastases imaging techniques in mouse models

what do results of different imaging techniques look like

what is a common model for intravasation of breast cancer cells

in vivo multiphoton microscopy of GFP labelled mammary tumour

how can intravascular trafficking be imaged

Cancer cells moving through bloodstream 

• HT1080 cancer cells – Nuclear green, Cytoplasmic red

what is positron emission tomography (PET)

• Widely used in clinical staging and restaging for many malignancies

• [18F]fluorodeoxyglucose (FDG) – glucose analogue used in >90% of cancer scans

• FDG selectively taken up by cells with high rate of glucose metabolism (tumours)

• FDG-PET used for staging of many cancers (eg breast, colorectal, oesophageal, head

and neck, melanoma, lymphoma and non-small cell lung cancers)

How is FDG-PET imaging used to predict tumour response to therapy and survival?

• Sequential FDG-PET imaging in advanced cancer is often a more accurate predictor of treatment response than clinical or histopathological criteria

• Detects metabolically active tumour sites

• Combining PET and CT:

  • Visualises active tumour regions

  • Shows where cancer has metastasised

• Applications:

  • Whole body FDG-PET/CT image fusion

  • Identification and manual segmentation of FDG-avid malignant lesions

  • Example: tumours in lungs and mediastinal lymph nodes

• Can assess treatment response (e.g. lymphoma pre vs post scans)

• Other tracers:

  • 18F PET probe detects brain receptor activity (yellow/orange)

  • Antagonist blocks receptor activity

What are the limitations of current imaging probes and the advantages of ‘smart’ probes in tumour imaging?

• Current imaging probes:

  • Non-specific (small molecules, leak into extracellular space → high background)

  • Targeted (monoclonal antibodies – unbound antibodies give high background)

• Smart probes:

  • Optically silent in native (quenched) state when injected

  • Highly fluorescent following enzyme-mediated release of fluorochrome at target site

  • Signal amplification – several 100 fold

  • Highly specific, enzyme cleavage specific peptide sequences

  • Single enzyme can cleave multiple fluorochromes

What are the components of smart probes in tumour imaging?

• Smart probes comprise 3 biocompatible building blocks:

  1. Near infrared fluorochrome

  2. Enzyme-specific peptide substrate link

  3. Delivery vehicle – dextran backbone

Why are cancer-associated proteases used as targets for smart probes?

• Important in invasion, metastases, angiogenesis

• Frequently overexpressed at tumour site

  • Cathepsins, MMPs, Caspases, PSA, Thrombin

• Smart probe activated → emits strong signal detected by imaging