The Metastasis & Growth Of Tumours: A Study Of The ‘Path Of Least Resistance’ Using Differential Membrane Lipid Compositions

Jordon Sandland, Charlotte Rowland, Aimie Rendle, Sudeshna Rajbongshi & Birsen Somuncuglu

A Group Project Research Proposal

Presented To The Department Of Chemistry

University Of Hull

2015

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Contents

Chapter 1: Case For Support

Rationale………………………………………………………………………….………….3

Background & Context………………………………………………………………………4

Vision & Strategy……………………………….……………………………………………6

Key Challenges……………..……………………………………………………………...12

References………………………………………………………………………………….16

Lay Summery…………………………………….…………………………………………18

Chapter 2: Justification Of Resources

Research Team & Resources…………………………………………………………….19

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Chapter 1: Case For Support

Rationale

Our vision is to assemble a research team to take part in a new study of cancers and how

they move and grow through tissue in the body. Starting from a primary tumour site, our

studies will show whether cancer grows through a ‘path of least resistance’ by using a

specifically designed microfluidic lab-on-a-chip to mimic the body. This research will give

insight to whether each type of cancer: lung, breast and prostate, grows through similar or

dissimilar metastatic paths. This proposal is a promising new study into the metastasis of

tumours. This research is of high importance as so many people are affected by cancer in

some way. The understanding and treatment of cancer is a global issue, any efforts to

further understand the growth of tumours should be looked at in high regard due to its global

importance.

In the long term this research will be of vital importance to the treatment of metastatic cancer

as understanding the way the tumour spreads would lead to more successful treatment,

leading to better after-patient welfare and the lesser cost on healthcare institutions as cancer

is currently is a huge global expense both socially and economically.

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Background & Context

During 2011 Cancer Research UK recorded a total of 330,000 people had developed some

kind of cancer with over half of them having either breast, prostate or bowel cancer.1, 2, 3 As

of the current date (24/02/2015) there are finally the same number of people who survive

cancer as those who tragically die from it. The scope of this research project is the

development of a model of the understanding of tumour growth and metastasis and has

global socioeconomic influences due to the likelihood of one developing cancer.

Our research group intends to further the research into cancer by understanding a

fundamental level in a poorly understood area, understanding how tumours grow in the body

by a series of simulated lab-on-a-chip experiments. This research builds on that of M. Klahn

et al. where a series of computational molecular dynamic calculations were performed to

understand how lipid concentrations and CHOL levels in membranes alter the fluidity of a

membrane.4 In increased concentration of CHOL in in cancer cells has been found in the

phospholipid bilayer.4 This has the effect of increasing the membrane fluidity and decrease

the order of the system.4 The implications of this is that cancer cells may dislodge from the

primary tumour site and migrate in the body through the blood which is a key parameter we

will be studying. Similarly the work by M. Jobin et al. explained how membrane area is

increased in leukemic cells, and how the cancer has caused a physical change to the

morphology of the cell.5 This may be an explanation for how cancer cells migrate and cause

metastasis through what we believe is the ‘path of least resistance’. It is believed that this is

an important area in not only cancer treatment but also cancer prevention and is at the

forefront of any cancer research as this is an original approach.

The implications of this study is the formation of a model for cancer tumour growth and

metastasis unlike any other. This project has scientific basis in the work laid out by M. Jobin

et al. who found that tumour cells have been shown to possess heightened levels of anionic

lipids in membranes. There have been many studies in the effect of dietary fatty acids on the

body and the possibility of increased cancer risk. For example T. Brasky et al. explained how

high concentration of serum phospholipid long-chain ω-3 fatty acids, which is a biomarker of

usual ω-3 fatty acid intake, was associated with a large increase in the risk of high-grade

prostate cancer.6 suggesting that dietary intake has possibility of having an impact on lipid

membrane composition thus giving scientific credence to our hypothesis.

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L. Nicholson. et al. rationalized that phospholipid replacement therapy can result in a

reorganisation of the lipid membranes and there were toxic, mutagenic or carcinogenic

effects for doses of up to 3.75 g/Kg of body mass.7 Thus it has been shown that lipid

membranes can be altered in the living body without any significant ill effects. We predict

that the outcome of our experiments will result in a new lipid therapy that can be used to

strengthen lipid membranes and reduce the growth of tumours and mitigate against

metastasis. However altering the protein chemistry or lipid membrane thickness could have

drastic unknown biological effects which does not make for a good cancer therapy, thus the

proteins of the cancer cells and healthy cells will not be investigated due to their delicate and

specific nature in cellular biology. Nor will we be investigating the depth of lipid membranes

as altering the thickness could similarly have unknown biological effects and cause the cell

to no longer function properly.

Our key goals are to build on the research done before using computational molecular

dynamic calculations and taking them into a laboratory environment for experimentation.4

Our research group intends to take tissue samples from biopsies of prostate, breast and lung

cancer tissue samples and layer them into a lab-on-a-chip matrix designed as a ‘cellular

assault course’. Our theory is that cancer is spreads through taking the path of least

resistance. Therefore we will be able to clearly measure where the cancer spreads on the

web structure of the lab-on-a-chip and detected using fluorescence spectroscopy. The

surrounding cells and infected cells are harvested, the cells separated via centrifugation. The

lipids are extracted into DCM and separated by chromatography to separate out the mixtures

and coupled to a mass spectrometer to collect qualitative and quantitative data on the

elemental composition, thus giving information on the lipid structure and on the number of

such lipids. Collectively this data allows for elucidation of lipid composition on the

concentration of lipids in the cells. We believe there will be a significant relationship in the

composition of the lipid membranes and the relative direction of cancers spread from

tumours. Thus allowing for the creation of a reproducible metastasis model. This is a cost

effective model and can be replicated with ease, the use of human tumour tissue samples

increases the similarity to in vivo experimentation.

As further study it may be possible to develop a lipid supplement which selectively alters the

lipid membranes of cells in tissues most likely to be afflicted with cancer. It is well known that

dietary fatty acids directly affect the composition of lipid membranes so the development of a

lipid supplement is not beyond the scope of this research as a further study. This mitigating

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initial tumour growth and minimizing possibility of metastasis. Therefore the cancer tumour

can be removed by operation rather than expensive and debilitating chemo or radiotherapy

limiting the side effects of such therapies by reducing the need for them. Thus reducing the

strain on medical institutions such as the NHS which will inevitably improve quality of care

and have the economic effect of reducing overheads.

Vision & Strategy

The initial aim of the proposed research group is to pioneer a new lab on a chip model

incorporating model techniques seen in “workshop meeting report organs on chips: human

disease models” to create a ‘cellular obstacle course’ in order to follow the metastasis of

cancer cells from a primary tumour site in what we call the ‘path of least resistance’.8 The long

term aim is to deduce the path of movement of cancer cells to allow researchers to integrate

the simplistic model designed here and apply it to real in vitro models in human cancer

patients. By creating a better understanding of the metastasis mechanism, this approach could

propose unprecedented opportunities for the production and design of novel drugs.

To achieve these aims, the proposed project will deliver:

An in vivo study, where cancer cells are held in a compartment of a microfluidic chip.

This will enable a real time study that will mimic the complex cellular interactions and

processes in the human body and will also provide a proposal for the mechanism of

disease development from primary tumour tissue cells through to metastasis.

A plastic chip containing compartments with a varying thickness of endothelial

membrane networks in order to provide the cancer cells with optional pathways to

travel. This will relate accordingly to the cells composition and structure.

A nutrient media vital for cancer cell survival. The production of an environment in

which the parameters and conditions are closely controlled for maximum relation to

real biological systems.

A determination of how a variety of cancer tissues have similar or dissimilar metastasis

paths.

Fluorescent labelling techniques that can be used to trace movement of cancer cells

to their desired destination.

Quantification of lipid composition of cancer cell membranes, along with a reference

comparison to healthy cells of the corresponding cell type.

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Experimental analysis of metastasis and fluorescent probing: In this study we will

examine cancer tissues of lung, breast and prostate from cancer patient biopsies. Each type

will have their own individual chip to measure their metastasis rates and progress. Healthy

tissue of the same type (eg. Human heart tissue) will be placed in compartments A, B and C.

The experiment will be monitored over a time frame that is representative of cancer metastasis

in humans. Every week over a six month period fluorescent images will be taken of the chip.

Cancer cell movement will be visually tracked by fluorescence probing of the primary tumour

tissue cells. Fluorescent imaging will provide a qualitative representation of the concentrations

of cancer cells in compartments A, B and C. The fluorescence dye used to label the lipid

membranes will be a stock solution of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-

indacene-3- propionic acid (BODIPY) in ethanol, which should be incubated with the cells for

5-10 mins before washing away any excess. Once added to the tissue samples the cells will

be highlighted with a high concentration of probe around the membrane of cell. Therefore

conclusions can be drawn of the preferred pathway and movement of the cancer cells will

take.9

Fabrication of the microfluidic chip: The successful delivery of this project hinges on the

engineering of the chip, which needs to be designed to replicate the biological systems in

humans. Firstly, the chips micro-channels and compartments will be made by creating two

epoxy based photoresist moulds made from a photomask on a silicon wafer by UV

photolithography using a UV-lamp. This will allow the intricate geometric patterns of the circuits

to be drawn out. A cast using Polydimethylsiloxane (PDMS) will be created of each mould by

heating the PDMS to 75°C for 1 hour in an oven. The compartments can then be characterized

with a thin PDMS membrane heated in the same way. The endothelial cell membranes can

then be bound to the PDMS layers of A, B and C and then the two casts can be sealed together

leaving the inlets and outlets open to pump through the micro-fluid.10

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The chip design can be seen in Figure 1 below.

Figure 1. The microfluidic chip is formed in two layers. The lower layer consists of the

compartments where the healthy cells and cancer cells are held. The blood circulatory system

is the higher layer of the chip. It has an inlet and outlet, to stop any backflow of blood. The

cancer cell compartment is equidistant from all the other compartments, allowing equal

probability of metastasising to secondary sites. The three different blue compartments A, B,

and C contain healthy cells, where A, B, and C correspond to the different thickness of the

endothelial membranes.

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Figure 2. The chip shows the division between the potential metastatic sites A, B and C and

the blood circulating channel. Each will have a 10 μm-thick microporous polyester membrane

of polydimethylsiloxane (PDMS) for biological barrier modeling. The independent micro-circuit

blood circulation channel is of 100 m x 500 m dimension. Each tissue culture compartment

is connected over 6 mm length, by this micro-circuit. Compartment A, B and C has respectively

a one, two and three cell thick endothelial cells mounted on each PDMS porous layer.

The enclosed environments inside the microcircuits of the chip will be performed under

conditions that optimally mimic the human body functions.

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Composition of media: The nutrient media in which the cells will be immersed in will be made

up of; essential amino acids, vitamins, carbohydrates and inorganic salts. Some serum or

further components can be added to the media to provide the complete mix necessary for

growth.11

Physical environment conditions: The serum must be buffered to allow a stable pH, ideally

around 7.2-7.4. This will be visually monitored by adding a pH indicator (phenol red) to the

media and it will be easily seen as phenol red has very clear colour changes at various pH’s.11

The temperature of the whole system will be maintained at 36.11°C and this will be preserved

using a water bath. The cultured cells will require a CO2 atmosphere of around 5% and a

monitored O2 atmosphere required for cell respiration. Circulation of the nutrient media will be

done via a pump in through the inlets to fill the compartments and back out through the outlet.

Here parameters such as; flow rate, pressure and humidity levels will be monitored.11 Blood

plasma will be taken from human samples in which the pressure and flow rate maintained by

a pump will mimic the natural flow of blood through the human circulatory system.

Hemocytometry: The heterogeneous samples of cells A, B and C in the respective

compartments will be measured separately in order to count the number of cancer cells that

have moved from the primary tissue, to that particular compartment. Two protocols can be

implemented here; a hemocytometer (Improved-Neubauer) or an electronic counter such as

the Z2 from Beckman Coulter. The hemocytometer is appropriate when studying a small

number of samples and is the least expensive approach. However the Z2 allows rapid and

accurate counting of large numbers of cultured cells.11 This will allow qualitative and

quantitative determination of which pathway the cancer metastatic cells prefer to travel; via

the thin (1 cell), medium (2 cell) or thick (3 cell) membranes.

Cancer cell isolation: The metastatic cancer cells will be separated from the healthy cells

they combined with in each compartment by cancer-affinity micro-chromatography can be

used. This method selectively captures suspended cancer cells from a heterogeneous cell

population through binding of substrate-immobilized high-affinity ligands. An antibody-based

cell-affinity technique can be used, where a compatible integrin for each tumour type is bound

to a PDMS microchannel surface. Therefore, this technique will separate the cancer cells from

the healthy cells with a high rate of recovery. Another technique will be the use of CTC

(circulating tumour cells) chip, which can give greater separation efficiency. This method flows

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the cells through a dense array of 78,000 silicon micro-pillars (100 l height, 100l diameter)

functionalized with anti-epithelial-adhesion-molecule antibodies (Anti-EpCAM), to enhance

the likelihood of cell-antibody interactions. The flow velocity and shear force around the micro-

posts is capable of sorting metastasis cells from other cells in the sample with 50% purity.12

Lipid extraction by Folch’s method: Once isolated the fluorescent labels can be detached

from the cancer cells, by centrifuging the cells at 3000 rpm for 1 min using an Eppendorf

centrifuge 5415D. A homogenizer is used to break up the tissue from the compartment A,B or

C. The supernatant containing the cell membranes can be drawn off in layers. The cell

membranes can be broken down into their individual lipid components using homogenization.

Optimum lipid extraction should result when homogenizing the separated cancer cells with a

mixture of chloroform and methanol (2:1). This will mix the matrix water in the cells with the

membrane components creating a monophasic solution. The resulting homogenate is then

diluted with water/chloroform producing a biphasic system. The chloroform layer (lower) will

contain the lipids and the methanol-water layer (upper) will contain the non-lipid cell

components. The chloroform can be isolated and filtered leaving the lipid residue behind. The

residue is then purified with hydrochloric acid and aqueous wash.13

Lipid separation: Non-aqueous capillary electrophoresis (NACE) can be used to separate

the unsaturated & saturated fatty acids based on their differences in charge-to-mass ratios.

Ammonium acetate and acetic acid will be used to buffer the solution and methanol and

acetonitrile used as additives to increase solubility and selectivity. This will yield the

information on molecular structure to show what lipids are in the membrane.14

Mass Spectrometry- composition of membranes: Coupling the NACE with electrospray

ionization-mass spectrometry allows the phospholipids to be quantified. The mass spectrum

of each lipid will indicate its molecular ion peak; fragmentation patterns and integration will

give the relative concentration of that lipid in the sample. Therefore the determination of the

molecular mass of each lipid and the amount of that particular lipid in the sample is possible.

The percentage composition of each lipid in one cell can be determined by averaging the

concentration over the amount of cancer cells in the sample. This will allow a conclusion to be

drawn on how the level of metastasis of cancer cells differs with increasing/decreasing path

length. Hence, determining whether they prefer the path of ‘least resistance’.

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Conclusions: This process can be repeated with samples A, B and C. To gain further

knowledge, the lipid quantification parts of the experiment can be done for a healthy cell

selection of the corresponding type to the cancer cell used in the experiment. This will create

a reference for comparison of lipid composition of the infected, cancer cells and normal

functioning healthy cells, to deliberate whether the increased movement of cancer cells is due

to increased/decreased fluidity/rigidity (entropy) of the membrane.

Key Challenges

The study of Metastasis and growth tumours is one that has been extensively researched but

we proposal an original method, but in this study we are focusing on building a microfluidic

chip, and monitoring the growth of the cells using techniques like NACE, hemocytometry,

mass spectrometry to analyse further the lipid composition. The first challenge would be to

isolate the right cell type i.e. healthy cells from a healthy

Microfluidic pumping mechanism: To successfully perform this the excess fluid needs to

exit through the outlet and nutrition needs to entering through the inlet system. David Beebe

proposed a controlled passive pump technology to induce flow.8 This occurs via surface

tension gradients and capillary forces, large droplet is deposited in the entrance of the open

Microfluidic channel causing the droplet/fluid to be sucked into the channel and a liquid droplet

forms at the exit site. This pump mechanism is suitable due to its simple requirements and

cost effectiveness. The main challenges faced include standardization which is difficult when

cells cultured from difference sources have different genetic material. Only 0.01% of circulating

tumour cells will give rise to metastases this means a greater deal of investigation is required

to discover cancer cells with invasive potential. When filtering blood to produce cancerous

cells independently the filtering method can damage the cells.8

Fluorescent probing: Another challenge is to monitoring the path of the cancer cells,

Fluorescence spectroscopy will be used to monitor cell growth and metastasis which is an

inexpensive method. However the challenge of transporting the fluidic cell to the spectrometer

and taking measurements will require integration of the chip to the spectrometer which is a

non-standard fit. Additionally the ‘life support’ of the lab-on-a-chip will need to be transported

to the fluorescence photospectrometer.

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Overdosing the use of fluorophore compounds often has toxic effects. Fluorophore use can

influence cell signalling pathways, induce apoptosis and inhibit the function of ion channels.

To optimize fluorescent probing an ideal fluorophore must be selected with an ideal maximum

excitation/emission wavelength, extinction coefficient, quantum yield, and lifetime as well as

ideal stokes shift. The selection of the fluorophore will depend on the type of tissue under

investigation.15 To maximise the effectiveness of fluorescent probing any compounds that may

supress fluorescence must be removed, this again depends on the sample under

investigation. The type of fluorophore needs to be considered as some have low purity and

are expensive which should be considered. A consideration of the redox properties of the

fluorophore must be considered, redox reactions occur endogenously as well as exogenously

and the products of redox reactions can either supress an excited fluorophore and reduce

intensity and therefore sensitivity of the method or create fluorophore compounds and produce

pseudo signals.15 Foe this research BODIPY in ethanol was suited considering the

disadvantages. Also the chip needs to be protected from light when fluorescent dye is used.16

Non-aqueous Capillary Electrophoresis: The main problems arising with capillary

electrophoresis (CE) are the capillary dimensions and nature of separating electrolyte

including pH, ionic strength, salt nature and additives. Several further problems include the

applied electric field dependency and the capillary temperature. Where possible these

problems will be addressed and potential solutions provided. The viscosity of the sample

subjected to separation must be low as the viscosity has an inverse behaviour to the

electroosmotic and electrophoretic mobilities. The solution is to simply keep the viscosity of

the sample low to encourage separation within a reasonable timeframe. The sample under

investigation must have at least a suitable autoprotolysis constant this makes inert solvents

unsuitable for electrophoresis.17 A further problem is the use of a particular organic solvent,

pKa values of organic solvents significantly different from water improve sensitive through an

improved separation process.17 The bore size for the capillary must be controlled carefully,

ideally for lipid separation using NACE the bore hole should be large, organic electrolyte

solutions have lower thermal conductivities compared to water which has a slightly greater

resistance to heat transfer. Low heat production results in enhanced separation efficiency due

to lowered longitudinal diffusion and an evenly distributed temperature inside the capillary.

Because this method uses a non-aqueous electrolyte the dissipation of Joule heat is less

dramatic and enabling a better loading ability. To reduce the heating power more the use of

longer carbon chain solvent molecules is ideal.18

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Cell Isolation: Cancer-affinity micro-chromatography is used to isolate the cancer cells but

this is not 100% effective as slight change in pH will hinder the process. However two

alternative methods for isolation are available, CTC; positive enrichment and negative

enrichment. Positive enrichment requires isolating target cells using cell size, surface protein

expression, whereas negative enrichment requires eluting non-target cells. The former

produces CTC in high purity whereas the latter produces low purity. It is therefore useful to

use a two-stage microfluidic chip for selective isolation of circulating tumour cells.

‘Cell life support’: As the chip is made from PMDS polymer (plastic) previous studies shows

cells are very difficult to remove from plastic due to presence of serum in the medium. To

overcome this careful washing of cells required. There are more common problems which

includes cells are not sticking to the plastic and cells are clumping together, which hinders the

cell movement through plastic. Insufficient amounts of serum, dissociating agent and

mycoplasma contamination causes cells not to stick to the plastic. If there is an absence or

low concentration of certain additives, cell contamination by bacteria or low cell density the

rate of cell growth is decreased. 11

pH is a major factor slight variations can cause cell death by denaturing proteins. Similar

effects can occur with the variations in the concentration of CO2.18 Therefore a serum with pH

7.2-7.4 is used. The culture medium needs to be washed with a buffer solution of phosphate-

buffered saline (PBS) without calcium chloride and magnesium chloride. Then the flask will be

incubated at 370C for 2 mins after trypsin is added to the culture. Before the experiment the

cells are washed with PBS again without calcium chloride and magnesium chloride. Detachin

is used in this step instead of trypsin to the culture flask, and the flask was incubated at 370c

until the cells indicates detachment from the flask surface.19

Since we are mimicking the human body the chip needs to be at 370C, slight change in

temperature will tremendously alter the cell by denaturing the proteins in the cell membranes.

Micro incubators can be used to overcome the challenge, which is built-in the microfluidic

system and can create in vivo microenvironment of the culture. Long term culture over 6

months should be possible, this is achieved via a tight regulation of the cellular environment

and homeostasis.8

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Environmental, economic and ethical challenges: Several factors come into play when

identifying ethical concerns with regards to research biopsies. The first and foremost is the

consent of the participate this is due to the moral and legal aspects. Expanding on the legal

agenda it is also important that the participant is fully informed and that the details of the

experiment should not be ambiguous as this is often one of the main arguments put forth when

a legal case is raised with the participant. To provide a solution to this it would be ideal to

provide a document fully explaining the research, essentially a waiver to legally bind the

participant. It would also be advisable to follow this up with a questionnaire pre-trails and post-

trails so that the participant gains a sense of quality nurture and that further experiments can

be tailored to better accommodate the participants.20

Another main problem with research biopsies is that no direct benefit to the patient is

established, due to the type of experiment this is unavoidable but it may be comforting to

inform the participants that drugs and techniques now in circulation would have once begun

the same way and that this research may indirectly benefit the participant or their relatives.

This in some eyes may be seen as persuasion, manipulation and at an extreme perhaps

coercion but there is no denying that research is the only method of progression and that

details are thoroughly outlined in the waiver. Often participants have an expectation of

personal benefit and therefore follow through with these experiments in the hope of receiving

the next best assessment techniques or general cures rather than out of an altruistic motive,

this is the free will of the participant and cannot be controlled by the research team, the only

way around this problem is the evaluation of the research itself. Is the research novel and will

it actually have a greater impact on future patients or is it just a gimmick that will cost the

taxpayers more when it’s sold to the NHS yet provide no great advantage?! If a case is put

forward on the fundamental nature of lab-on-a-chip and future possibilities this is at least a

step in the right direction.20

The cell growth and fluorescence measurements will be taken over a 6 month period

sequentially. Thus there is a high probability of interruption due to fire alarms and electrical

faults / black outs, thus the ‘cell life support’ systems and pumps will need to be connected to

an emergency generator to mitigate damage from the possibility of a power short, the

experimental parameters do not allow for any variation in the temperature, partial pressures

or the saturation of gasses in blood ect. This has an economic concern as the money could

be used to support other research.

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Fabrication of lab-on-a-chip devices is expensive and requires clean rooms and expensive

equipment. Most research facilities are unable to manufacture in house and will require

expensive out sourcing. Therefore for further research to be conducted by another institution

this will present a considerable challenge thus the reproducibility of the experiment is

dependent on this process.8 Given the tremendous challenges that remain in elucidating

mechanisms of metastasis, this is just the start of studying metastasis using microfluidic

devices, more research may be required to improve on this model.21

References

1. Cancer Research UK- Let`s Beat Cancer Sooner Online,

http://www.cancerresearchuk.org/cancer-info/cancerstats/, (Accessed February 2015).

2. Cancer Research UK, Cancer Incidence in the UK in 2011,

http://publications.cancerresearchuk.org/downloads/product/CS_REPORT_INCIDENCE.pdf

(Accessed February 2015).

3. Cancer Research UK, Cancer Incidence and Mortality in the UK

http://publications.cancerresearchuk.org/downloads/product/CS_REPORT_TOP10INCMOR

T.pdf (Accessed February 2015).

4. M. Klahn and M. Zacharias, Phys. Chem. Chem. Phys., 2013, 18, 14427.

5. M. L. Jobin and I. D. Alves, Biochimie, 2014,107,154.

6. T. M. Brasky, A.K. Darke, X. Song, C. M. Tangen, P.J. Goodman, I. M. Thompson, F. L.

Meyskens Jr, G. E. Goodman, L. M. Minasian, H. L. Parnes, E. A. Klein, A. R. Kristal, J Natl

Cancer Inst, 2013, 105, 1132.

7. G. L. Nicolson and M. E. Ash, Biochim Biophys Acta, 2014, 1838, 1657.

8. A. van de Stolpe and J. deen Toonder, Lab Chip, 2013,13,3449.

9. I. Mikhalyov and A.Samsonov, BBA Biomembranes, 2011, 1808, 1930.

10. Y. Temiz, R.D. Lovchik, G.V. Kaigala and E. Delamarche, Microelectron Eng, 2015, 132,156.

11. Cancer Cell Culture Methods and Protocols, ed. S.P. Langdon, Humana Press, New Jersey,

2010.

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12. J. Chen, J. Libc and Y. Sun, Lab Chip, 2012, 12, 1753.

13. E. G. Bligh and W. J. Dyer, Can J Biochem Phys, 1959, 37, 912.

14. A. C. Otieno and S. M. Mwongela, Anal. Chim. Acta, 2008, 624, 163.

15. C. C. Winterbourn, Biochim. Biophys. Acta., 2014, 1840, 730.

16. M. Hitzbleck and E. Delamarche, Chem. Soc. Rev., 2013, 42, 8494.

17. M. L. Riekkola, M. Jussila, S. P. Porras, I. E. Valko, J. Chromatogr. A., 2000, 892, 155.

18. M. Jussila, S. Palonen, S. P. Porras, M. L. Riekkola, Electrophoresis., 2000, 21, 586.

19. S. Tanzi, M. Matteucci, T. L. Christiansen, S. Friis, M. T. Christensen, J. Garnaes, S. Wilson,

J. Kutchinskyb and R. Taborysk, Lab Chip, 2013, 13, 4784.

20. J. Peppercorn, I. Shapira, D. Collyar, T. Deshields, N. Lin, I. Krop, H. Grunwald, P.

Friedman, A. H. Partridge, R. L. Schilsky, and M. M. Bertagnolli, J Clin Oncol, 2010, 28, 1.

21. E. W. K. Young, Integr. Biol., 2013, 5, 1096.

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Lay Summary

Cancer is the one of the widespread diseases that causes a death - which is characterized as

an uncontrolled growth of cells and ultimately invade surrounding tissues, in other words, leads

to metastasis. According to the Cancer Research UK, 161. Half of all cancer deaths in UK are

mainly lung, bowel, breast and prostate cancer. Hence, we will develop a model to understand

how the tumour grows and how cancer spreads from one organ or tissue to another. In this

research project the cancer cells that are going to investigate includes; lung, breast and

prostate only.

To investigate how cancer cells spread to the other parts of the body, we are designing a lab-

on-a-chip which is a device that incorporates one or several laboratory functions on a single

chip. With lab-on-a-chip experiments it will allow real time monitoring of cellular process, by

interacting cancer cells with three different thicknesses of endothelial membrane of healthy

cells under closely controlled conditions. By doing this we can provide possible pathways to

the cancer cells, observe which pathway it chooses and hence verify if it chooses “pathway of

least resistance”.

In order to keep the both cancer cells and healthy cells alive, a nutrient media is provided and

physical environment conditions are used keep conditions as close to human biological

systems. In addition to this, the spreading of the cancer cells are investigated using fluorescent

probe every week over six month period which is time frame that is illustrative of cancer

metastasis in humans. Once the experiment is completed, the concentration of the cancer

cells in three different thickness of endothelial membrane will be measured and this enables

us to determine which pathway the cancer cells choose to spread more.

According prior research, tumour cells cause to increase the level of lipids and this will result

in increase in fluidity of the membrane. In order to find the lipid composition in a membrane,

the lipids were extracted and masses of the each lipid was determined by using non-aqueous

capillary electrophoresis coupled with electrospray ionization-mass spectrometry and the

percentage composition of each lipid in a cell is calculated by averaging the concentration

over the amount of cancer cells in the sample and comparison was made with healthy cells to

see in which membrane does the lipid concentration increases more.

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To conclude the research will give successful and reliable results to validate or disprove the

theory of ‘pathway of least resistance’. However more research should be done to increase

the reliability and to apply it to real in vivo models in human cancer patients. The experiment

reliability increases by; doing more repeats, designing new lab-on-a-chip with different

pathways, increasing the variability of different thickness of membrane and varying the types

of cancer cells.

Chapter 2: Justification Of Resources

Research Team & Resources

Research Team: The proposed project is highly interdisciplinary requiring input from two full

time academic research (£43,000 per annum) staff and three technicians (£25,000 per

annum) in order for the project to succeed. This project has foundations in chemistry,

biochemistry and biology thus a well-reasoned research team is required. The project

requires supervision from academic research staff who will manage the logistics of setting up

the experimental parameters and organising the five post graduate students (£13,000 per

annum pro rata) responsible for the upkeep of the cancer cells in the lab on a chip as they

will need their vitals checked periodically. There will be collaboration with the technicians

who manufacture the lab-on-a-chip devices. Technicians and full time post graduate

students will undertake the measurements from the fluorescence spectroscopy and the

mass spectroscopy in which should take no longer than 1 day to perform analysis. The time

scale of the project requires analysis of cancer cells every week for a 6 month period thus

the PG’s performing the spectroscopic analysis will only be required for 1 day per week for

full analysis of all samples.

Equipment & Consumables: The research group will require funds for the manufacture of

lab-on-a-chip devices, purchase of pumps and nutrient serums which may be produced in

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house by GS’s. The proposed project requires apparatus for hemocytometry (£5,000) which

can be performed in the biology division, cancer-affinity micro-chromatography (£5,000),

non-aqueous capillary electrophoresis (NACE) and NACE mass spectrometry (£250,000)

will require substantial funds due to the requirement of a full time operator (£24,000 per

annum) and staff technical training. The cancer tissue and blood samples will be obtained by

donations to science. A precision syringe pump (£3500) will be required for the blood serum

fluid control in micro-fluidic devices. A milli-Q- water filtration unit will be required (£700 per

item) for the filtration systems and three bench top coolCLAVE (£900) sterilisation units will

be required to sterilise equipment and microfluidic chips. General glassware and laboratory

equipment along with safety equipment (including personal safety) will be provided

(£10,000). Finally a total of 7 personal computers (£4000) is required for the duration of the

project and funds are also allocated for general laboratory upkeep and maintenance

(£15000).

Travel & Outreach: Minimal funds will be required for travel expenses (£10,000), however

funds will be allocated for outreach activities (£25,000) from such organisations as the Royal

Society of Chemistry and Royal Institution science and engineering week as well as

communication and publication in relevant journals to spread awareness of the new

developments. Training may also be given to those able and willing to attain conferences

(£1000).