A Cure Can’t Wait

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Ok, so donations are starting to come in at our JustGiving Team Page (thank you so much to everyone who has donated so far) and in just over two weeks time we’ll be setting off on our Hadrian’s Wall Challenge (fingers crossed for better weather than today!)

This year we’ve teamed up with The Brain Tumour Charity and just like in previous years we’re looking to raise money and awareness for a specific purpose.

Two years ago we wanted research into cannabidiol to be carried out on childhood brain tumours so that one day it could lead to clinical trials – that 18 month research project has now been completed!

Back then the prospect of a clinical trial seemed like a pipe dream although with:

  1. recent developments in the press and in law, and
  2. the increasing acceptance of adaptive clinical trials

we now believe that trials into cannabinoids could be imminent, especially given who we’ve decided to team up with…

I recently received a personal email from Sarah Lindsell – CEO of The Brain Tumour Charity and this one sentence, which still makes me very emotional, I believe, says all you need to know about why I am so confident that teaming up with them is the best way forward for William and kids like him:

We are working in a system that is decades old and not fit for a different world. We have to change this, no matter how hard, for the sake of all those with this disease today and diagnosed tomorrow. I can promise you, we will not give up until we have

Discussions with research institutions are happening now in relation to the setting up of adaptive clinincal trials into cannabinoids and gliomas although as usual there are numerous bureaucratic hurdles that need to be overcome and no doubt the intention will be to look into adult brain tumours first…

As I’ve mentioned in a previous post only three drugs have EVER been developed for childhood cancers, only one of which was for a childhood brain tumour compared to the dozens there have been for adults – this is because children are second thoughts in the drug development process and paediatric tumours are usually very different from their adult equivalents

Please help us change this and for once make children a priority by donating to one of our fundraising pages

I’d like to sign off in the same way Sarah does in her emails to me…

A Cure Can’t Wait

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Wax Forfeit…

Ok so… someone suggested that a good way to increase our sponsorship money for The Brain Tumour Charity would be to carry out forfeits!

The total on our Facebook Team Page stands shy of £400 and the more money raised, the more of me I’m going to be having waxed on 10th August

If you want to get a bit more for your money, I’ll also be waxing more as the number of tickets sold for our Fundraiser increases

And to top it off, if I reach the goal of £3,000 and 100 tickets it will be filmed live on Facebook…

So, here’s me signing off safe in the knowledge that given how many donations we’ve had so far and how many tickets we have sold, there won’t be a whiff of wax a week on Saturday!!!

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All Party Parliamentary Group on Brain Tumours – the Ketogenic Diet

I attended this event last week – it was held at Portcullis House just opposite Big Ben and the Houses of Parliament.

I was invited to The All Party Parliamentary Group on Brain Tumours, basically a meeting held to discuss brain tumours attended by various members of parliament, by the charity Brain Tumour Research.

During the last meeting, which I was unable to attend, Dr Wai Liu, who I went to see present at St Georges University last year, gave a presentation on Cannabis (minutes here). This time the main topic was the Ketogenic Diet.

The main speaker was Sue Wood from Matthews Friends (who was there with Matthews mum and founder of the charity Emma Williams) and she gave an excellent presentation on the likely reasons for why the diet might influence the development of brain tumours.

There were also first person perspectives from a medical professional and an extremely knowledgable researcher whom I had met previously, Andrew Scarborough. They both recounted compelling accounts of how they had used the Diet to manage their own brain tumours.

It was clear from the event that much more research needs to be done before the diet can be recommended clinically although there seemed to be a general consensus in the room that those diagnosed with a brain tumour should at least be given information on the potential benefits.

Through speaking to those involved with the diet as well as parents who have asked for my advice further to Williams progress, I would say that at best the situation is confused and at worst there are medical professionals who (extremely ignorantly in my opinion) vilify the very suggestion that such a diet should even be considered.

As mentioned above, further research is clearly needed although in my opinion patients should be given information on the potential benefits of such a diet on brain tumours and at the very least those who so vehemently oppose the diet should be properly educated.

CBD Project – 18 Month Update

Well, 18 months has elapsed since the start of the 18 month CBD Research Project we proposed and helped fund into childhood brain tumours and it certainly looks to have been worthwhile with more research continuing as a result – full report below:

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In vitro evaluaton of the effect of cannabidiol as an adjuvant therapy for paediatric brain tumours: Update June 2019

The purpose of this research project is to conduct a range of experiments to explore how paediatric brain tumour cells react with CBD. Brain Tumour Action, the Astro Brain Tumour Fund, Amelia’s Appeal, Make William Well and the Jessica Hope Foundation are jointly funding a research technician and experiment costs.

Since our last update, we have again made progress with the research project and have been fortunate to have three MSc students who wished to be part of the project in an extended role. These students are all very committed and have tackled additional questions not part of the original application, but which have provided further insight.

We have continued to experience problems with the DIPG cell lines which have become infected when they are grown in antibiotic free media. Despite discussing with colleagues working with DIPG cell lines no solution has been found and we have been unable to move forwards with these cells at this time. However, we still have five cell lines which we are able to perform experiments on: SF188 and KNS42 (paediatric high grade gliomas), BxD-1425EPN and DKFZ (ependymomas) and normal astrocytes which are used as control cells.

Recently high impact journals have increased the number of experimental repeats required for publication from 3 to 6. We have therefore increased our experiments accordingly. The data we have obtained to date has been presented in poster format at a recent Nottingham Paediatric Roadshow Conference which allows research being performed locally to be celebrated. This poster will also be presented at the 2019 International Cannabinoid Research Symposium in the USA at the end of June. The research technician employed on the project presented his data to the Children’s Brain Tumour Research Centre Spring meeting in May 2019 which sparked lots of questions into the potential role of this therapy. The data has also been discussed with the NCRI Paediatric England trials group.

In our last report, we reported that after our experiments with CBD the cells die and we are now trying to elucidate the mode of cell death. There 2 main modes of cell death are so-called Apoptosis (programmed cell death) and Autophagy (self-eating/ self-destruction). In addition we wish to study the effect of CBD on the cells both in normal oxygen conditions and also when oxygen levels are limited. This  is thought to better represent the 3-dimensional structures of tumours where the centre of the tumour is deprived of adequate oxygen supply and in the brain. To do this, we have used the hypoxic chamber for experiments. One of the successes over the last few months is that we have been able to maximise our use of the hypoxic chamber and perform lots of experiments in it. Below is a summary as to the status of all of the different experiments being performed in this study. For the purposes of this report, data from SF188 cells only are shown in the figures.

1. Metabolism Assay

Cells used: SF188 and KNS42 (paediatric high grade gliomas), BxD-1425EPN and DKFZ (ependymomas) and normal astrocytes which are used as control cells.

Metabolism assays are used to quantitatively measure the effect of a drug on the proliferation of cells i.e. how quickly the cells are growing and dividing. We have conducted a range of experiments so that we can extrapolate the concentration of the drug needed to achieve 50% cell death. The concentration of CBD which causes 50% cell death (EC50) had previously been confirmed in the SF188, BxD-1425EPN and astrocyte cells in both normal and hypoxic conditions as well as the 24 hour and 5 days’ time points. It has now also been confirmed in the KNS42 and HSJD07 cells with experiments continuing on the DKFZ and T8/18 cell lines. The figure below shows an example of the effect of increased CBD concentration on SF188 cell viability.

 

Graph 1Graph 4Graph 3Graph 2

Figure 1

Figure 1 – Shows the results of metabolism assays on SF188 cells in both short term culture (24 hours) and long term culture (5 days) in both normoxic and hypoxic conditions. The EC50 decreased from 17.6µM at 24 hours to 14.8 µM at 5 days. The graphs in the lower panels show that the level of cell death is decreased under hypoxic conditions when compared to the cells under normoxic conditions (upper graphs). In fact an IC50 (50% cell death) is not achieved with 24 hours incubation in hypoxia. This implies that higher concentrations of drug may be necessary.

2. Western Blotting

Western blots are used to detect specific proteins within cells. The cells are cultured either alone, in the vehicle the drug is diluted in, in CBD or in the presence of two different control drugs, staurosporine (initiates apoptosis) or chloroquine (initiates autophagy). The cells are snap frozen at the end of the experimental time and then protein is extracted from the cell pellets. Western blot analysis is then used to identify which proteins have been released to allow the mode of cell death to be established.

At present we have optimised two of the four antibodies we intend to use to identify the mode of cell death (LC3B for autophagy and PARP for apoptosis). We have an MSc student who is running the Western blots on SF188, BxD-1425EPN and astrocytes using these antibodies. This work is due to be completed over the next month. We are currently optimising the other antibodies (P62 for autophagy and Caspase-3 for apoptosis). From the work completed to date there is strong evidence that there is increased expression of the autophagy marker, LC3B, 24 hours after exposure to CBD however, there was no increase in this marker after 5 days, indicating autophagy is the immediate mode of cell death. In contrast, the apoptosis marker, PARP, was expressed after in those culture left in the CBD media for 5 days but not after 24 hour exposure, indicating that both modes of cell death occur but at different time points. We have also observed stronger band staining as the concentration of CBD increases indicating there is an increase in the expression of both apoptosis and autophagy markers as the concentration of CBD is increased. Importantly, there was no evidence of either mode of cell death in the astrocyte cells exposed to CBD for 25 hours.

Figure 2

Figure 2

Figure 2 – Western blot showing SF188 cells grown in various drugs (C= cells alone, V = vehicle, 10 = 10µM CBD, EC = 17.6µM CBD at 24 hours and 14.8µM CBD at 5 days or 20µM, for 24 hours and 5 days. Antibody control cells were grown in sta = staurosporine for 4 hours or Chl = chloroquine overnight. Antibodies used were LC3B for autophagy (A), PARP for apoptosis (B) and GAPH as control (C). (D) Astrocytes were also evaluated with LC3B to ensure CBD was not causing cell death to a control cell line death in astrocyte cells exposed to CBD for 24 hours (D).

3. 3D Spheroids

3D spheroid culture is thought to better represent the growth of brain tumours in vivo. As previously described, in the CBTRC we have developed a model for culturing cells in 3D which allows end point evaluation by immunohistochemistry (IHC) a technique usually reserved for pieces of tissue. To date 3D spheroids of SF188, BXD-1425EPN and astrocytes have been cultured, processed and embedded in wax ready for a number of antibodies to be screened by IHC. We are using the same antibodies across our 3D spheroid culture and Western blot experiments, which means we are using different methods to validate the data. We will also investigate whether there is a change in the proliferation rate of the spheroids cultured in CBD using the antibody Ki67.

Figure 3

Figure 3

Figure 3 – SF188 spheroids cultured in the presence of the vehicle (Veh) or the EC50 concentration (14.8µM) of CBD for 5 days show a clear decrease in spheroid size. The box and whisker plot shows the decrease in spheroid size from cells cultured in the absence of CBD (1) to those cultured in CBD (14.8 µM) (2). The portion of the box coloured blue represents  spheroids with a volume 25% above the median, and the portion of the box coloured green represents those spheroids with a volume below the up to 25%.

4. Lactate Dehydrogenase Assay

Lactate dehydrogenase (LDH) is a soluble enzyme present in most cells which is released into the culture medium upon cell death due to damage of the plasma membrane. The breakdown of the membrane may be due to either necrosis, or in smaller amounts, apoptosis or autophagy. To date we have this assay on the SF188, BXD-1425EPN and astrocyte cells in both normoxia and hypoxia after 24 hours and 5 days

Figure 4

Figure 4

Figure 4 – LDH assays on SF188 cells cultured in normoxia for 24 hour or 5 days with 15µM CBD (EC50) indicates that after 24 hours this concentration causes approximately 30% cell death not the 50% seen in the metabolism assays, whereas after 5 days in culture the same concentration of CBD led to approximately 58% cell death.

5. Immunofluorescence

Immunofluorescence is the detection of proteins using fluorescent markers. In the project we intend to use the same antibodies used in the Western blot analysis and in the IHC thus confirming the mode of cell death by a further method. Currently, three of the four antibodies have been optimised. Because of the methodology, using cells cultured in chamber slides, the immunofluorescence for the optimised antibodies, LC3B, P62 and Caspase 3, will begin once the Ki67 antibody has been optimised.

6. RNA Analysis

RNA analysis to look at potential gene expression changes caused by exposure to CBD is still to be performed. Any changes in expression can be quantified and may provide multiple pathways which are being effected by the CBD. The changes in RNA expression, either by switching on genes, or switching off genes, could provide key evidence to potential resistance, mode of action and cause of cell death.

RNA microarrays will be used to compare SF188, BXD-1425EPN and astrocyte cells cultured in the presence and absence of CBD for 24 hours and 5 days in normoxia and for the SF188 and astrocyte cells in hypoxia. The cell pellets to be used in this analysis have been collected and will be sent to our collaborating group in the coming weeks.

7. Cell Cycle Analysis

Cell cycle analysis enables the position of a cell through its replication cycle when it undergoes and event to be established. This event may be cell death or it may be that the cell becomes static. We have performed some preliminary experiments on the SF188 and BXD-1425EPN cells to look at this but have not discovered a specific stage at which cell death is more likely to occur. Cell cycle analysis of the astrocyte cells will be performed over the next few months. The more slowly growing cell lines will not be investigated by this means as this would probably not be fruitful.

8. Additional Work and Future Plans

We have begun working with other groups to develop additional experiments such as an ELISA assay to look for immune markers expressed on spheroids which may be released into the cell suspensions as a way to investigate the effects of CBD on immune reactions. We have also held preliminary discussion with another group to look at CBD more specifically with T and B cells (immune cells). In order to pursue this work, additional grant funding would be required.

As previously mentioned we currently have three MSc students working on additional CBD projects.

i. Western blot analysis for apoptosis and autophagy markers. The cells will also be grown in normoxic and hypoxic conditions in the presence of CBD to investigate if this has any effect on levels of apoptosis and autophagy

ii. Currently some patients are taking cannabis oil as an adjuvant therapy for their brain tumour. The treatment is to take the oil for CBD 3 days on, 3 days off. We would like to replicate this in vitro by giving the cells 1µM CBD for 3 consecutive days and then looking at cell viability using viability tests (Resazurin) and Western blotting for apoptosis and autophagy markers. The cells will also be grown in normoxic and hypoxic conditions in the presence of CBD to investigate if this has any effect on levels of apoptosis and autophagy.

iii. Investigate the mechanism of action of CBD in further detail on paediatric brain tumours to see if we can determine the receptors through which CBD is having its action (CB1 or CB2) suing proliferation assays to determine the drug’s effects on the cells.

On behalf of the research team, we would like to thank Brain Tumour Action, Astro Brain Tumour Fund, Amelia’s Appeal, Make William Well and the Jessica Hope Foundation for making this study possible. We are excited about the results and the potential impact we can share with the scientific community later in 2019 and 2020 at the next International Society of Paediatric Oncology Conference.

Stable Scan

Was by far the most nerve racking scan we’ve ever had but the results we got yesterday showed no increase in William’s tumour size

He has been on metronomic chemotherapy so there’s every chance that it could have been that which has had the effect although we suspect his altered cannabinoid regimen and continued diet have had a lot to do with it

All this gives us added incentive to push for clinical trials to be conducted into cannabinoids and childhood brain tumours. If you’d like to help, please visit our JustGiving Page for the Hadrians Wall Challenge – why no consider taking part yourself?

Why not join us on 26th August for last day of Challenge?

Hadrians Wall 2019

We’re so close to making clinical trials into cannabinoids and childhood brain tumours happen soon but, like in 2017, we can’t make progress like we made back then without your support

Needless to say we’re looking for donations which you can do by visiting our JustGiving Page although if you’d like to join the team, you could always take part yourself…

Some of us will be walking the full length of the wall in just three days and we’d be more than happy to have you join us

If, though, you’d just like to join us on the last day or even the last few miles (or even just for the central section on the Sunday), we’d still appreciate your support

Evan's WallEven if you can’t take part in the walk, our first business supporter will be holding one heck of a party at their Evan’s on the Wall venue (which just so happens to be at the end point of Hadrian’s Wall) especially since one of their owners will be taking part in the challenge!
Roundtable

The challenge will also be supported by Whitley Bay Round Table. Our local Round Table at the time was so generous when we were looking for a way to fund our first bottle of CBD and we’re grateful to have Roundtable support again

Hadrians Wall 2019

Exciting Developments

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I’m really exited to be able to announce a number of things in this post:

  • The Brain Tumour Charity have confirmed that research into cannabis and childhood brain tumours will be the focus of our fundraising efforts for them.  There’s a real chance that this will lead to imminent clinical trials – we just need your support to help make this happen sooner
  • Back when we were struggling to buy our first bottle of CBD, our local Round Table took no time agreeing to pay for it – having recently revived our local Whitley Bay Round Table, primarily to raise money to support local causes, it was agreed that the Hadrian’s Wall Challenge will be the first sponsored event we support
  • We have our first business supporter, Evan’s Bistro – one of their owners will be taking part in the challenge and they just happen to own a restaurant at Segedunum – the official end of Hadrian’s Wall.  Needless to say celebrations will be involved – watch this space for further information…

If you would like to support us then please visit our Just Giving Page

There are still places on the challenge if you’d like to help support us – we’ll be setting out on 24th August