Engineering Collagen Scaffolds for Advanced Ex Vivo Culture of - - PowerPoint PPT Presentation

Engineering Collagen Scaffolds for Advanced Ex Vivo Culture of Breast Tumour

David S. Monahan1, Jean McBryan 2 , Fergal J. O’Brien3,4,5, Tanya J.Levingstone 3,4,5

1 School of Biological Sciences, Dublin Institute of Technology. 2 Department of Molecular Medicine, Royal College of Surgeons in Ireland (RCSI). 3 Tissue Engineering Research Group (TERG), Dept. of Anatomy, RCSI. 4 Trinity Centre for Bioengineering, TCD. 5 Advanced Materials and Bioengineering Research (AMBER) Centre, RCSI & TCD.

Introduction

1.67 million cases of breast cancer per year with 522,000 deaths1

• 12% of all cancer cases
• Most common cancer in women
• 2nd most common cancer world wide

Average of 2,883 new cases in Ireland annually

• 1/10 women
• 690 deaths (16%)
• 1. Ferlay et al, Int J Cancer. 2015;

136: 359-86.

Introduction

The tumour microenvironment has an important role in cancer development2

• Extracellular matrix (ECM)
• Cells – Immune cells, adipocytes, endothelium,

fibroblasts

ECM altered during cancer progression2

• Promotes adhesion and signalling in cancer cells
• Stiffer matrix due to enzyme release
• 2. Inusa-Rodríguez and Oskarsson, Adv Drug Deliv
• Rev. 2016; 97: 41-55.

Introduction

Traditionally, cells are cultured in plastic flasks in a 2D environment. Limitations of 2D culture include

• Lack of cell-cell interactions
• Lack of cell-matrix interactions
• Lack of mechanical signals

Introduction

Advantages of 3D culture

• Mimics the extracellular matrix
• Provides cell-cell interactions
• Provides cell-matrix interactions
• Reduces drug development costs and late stage

drug failures

• Reduces animal use
• More accurate investigation of tumour biology

Introduction

• Previous success with a 3D gelatin based ex vivo

culture system3

• Gelatin poorly resembles the natural ECM
• Collagen makes up 1/3 of total proteins in humans
• Collagen scaffolds have previously shown success

for investigation of metastatic prostate cancer4

• 3. Dean et al, cell cycle. 2012;11:2756-61.
• 4. Fitzgerald et al, Biomaterials. 2015;66:53-66.

Aims

• Fabricate a range of collagen and

collagen/gelatin scaffolds

• Characterise the scaffolds in terms of their

structural, mechanical and degradation properties

• Determine if fresh or frozen tumour samples

are optimal for use in this ex vivo culture system

• Determine if the addition of fibronectin

increases the proliferation of breast tumours

Gelatin

Type 1 Collagen 0.5% Collagen DHT (180°C for 48 hours) 0.5% Collagen + 0.1% Gelatin 0.5% Collagen + 0.25% Gelatin Spongostan 0.5% Col Col DHT Col + 0.1% Gel Col + 0.25% Gel

Type 1 Collagen Gelatin Collagen only or Collagen + Gelatin

0.05M Acetic Acid

Blend @ 15,000 RPM

Controlled freezing too

• 40°C

Freeze drying

Cut Freeze dried sheet to cubes

• f ~1cm3

Adapted from 5. Ryan and O’Brien,

• Biomaterials. 2015;73:296-307.

Methods

• Scaffolds were then analysed using:
• Porosity
• Scanning electron microscopy
• Degradation measurements
• Mechanical testing
• Frozen patient/ fresh xenograft tumours

cultured for 5 days on selected scaffolds

• H&E and KI67 expression for tumour

analysis

0.5% Col Porosity = 99.41% Col DHT Porosity = 99.15% Col + 0.1% Gel Porosity = 99.05% Col + 0.25% Gel Porosity = 98.85% Spongostan Porosity = 98.83%

Fabricated scaffolds have a greater stiffness

One-way ANOVA revealed a strong statistical significance between the groups (p<0.001). *Post hoc tukey analysis revealed all scaffolds had a significantly higher stiffness than Spongostan (p<0.05).

Fabricated scaffolds survive longer in culture

Degradation rates

Patient 1 Patient 2 Patient 3

0.5% Col Col + 0.1% Gel Original Tumour Spongostan Not Cultured

Frozen patient tumours @ day 5

Original tumour 0.5% Col Col + 0.1% Gel Col + 0.25% Gel Spongostan Col (f) Col + 0.1% Gel (f)

Example

• f Ki67

Fresh xenograft tumours @ day 5

Proliferation maintained across all groups

Conclusions

• Collagen and Collagen gelatin scaffolds

were successfully fabricated and characterised

• The optimal scaffold was determined to be

the 0.5% Col + 0.1% Gel scaffold.

• Fresh tumours are superior for ex vivo

culture

• Fibronectin increases proliferation by

providing a breast cancer specific environment.

• This study presents a novel system for

ex vivo breast cancer culture for tumour biology and drug development.

• Small sample size
• Lack of human tumours

Limitations

• Hydroxyapatite to mimic

bone metastases

• Analysis of culture media –

MMPs secretion

Current research

• Option to test tumour

response to novel drugs

• Expansion into other

tumour types

Future directions

References

• 1. Ferlay J, Soerjomataram I, Dikshit R et al. Cancer incidence and mortality

worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J

• Cancer. 2015; 136: 359-86.
• 2. Inusa-Rodríguez J, Oskarsson T. The extracellular matrix in breast cancer. Adv

Drug Deliv Rev 2016; 97: 41-55.

• 3. Dean JL , McClendon AK , Hickey TE et al . Therapeutic response to CDK4/6

inhibition in breast cancer defined by ex vivo analyses of human tumours . Cell cycle 2012;11:2756-61.

• 4. Fitzgerald KA, Guo J, Tierney EG et al. The use of collagen based scaffolds to

stimulate prostate cancer bone metastases with potential for evaluating delivery

• f nanoparticulate gene therapeutics. Biomaterials. 2015;66:53-66.
• 5. Ryan AJ and O’Brien FJ. Insoluble elastin reduces collagen scaffold stiffness,

improves viscoelastic properties, and induces a contractile phenotype in smooth muscle cells. Biomaterials 2015;73:296-307

Acknowledgments

• My supervisors Dr. Leanne Harris and Dr. Tanya

Levingstone

• Dr. Jean McBryan for advice with tumour

material

• Dr. Tony O’Grady and Mr. Colm Buckley for

allowing me to use their histology equipment.

• Dr. Damir Vareslija, Sinead Cocchiglia and

Professor Leonie Young for provision of fresh xenograft tumour material.