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:


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.

We are now a Supporter Group of The Brain Tumour Charity

We’re delighted to be able to announce that Make William Well has become a Supporter Group of The Brain Tumour Charity

Becoming a Supporter Group means that we can raise money for a cause we believe in without having to register as a charity – The Brain Tumour Charity has agreed to ringfence 100% of any money we raise though our donations page for use on research we want them to spend it on

Things have come a long way since we first proposed carrying research into cannabinoids over two years ago and we are extremely grateful to the Children’s Brain Tumour Research Centre for agreeing to write the cannabidiol research proposal which we pushed so hard for and helped raise funding and awareness to make happen

Our hope now is that this research can be built upon such that parents aren’t having to spend thousands of pounds and in some cases break the law on something that, provided it is found to be effective, should be available on NHS prescription

Please visit our Brain Tumour Charity webpage (which contains links to other useful pages on their website) and consider making at donation by clicking here

Also, watch this space for a couple of exciting developments we’ve got in the pipeline…


British Medical Journal – Article on History of Medicinal Cannabis

When many people hear the words “medicinal cannabis” they automatically associate the term with phrases like “backdoor legalization for recreational use” and assume it has no actual medicinal value

Having been thrust into the world of medicinal cannabis I came across not only anecdotal stories of people being effectively treated with various cannabinoids (compounds found within the cannabis plant), but numerous academic studies on how they can be effective on various conditions, as well as cannabis based products made by large pharmaceutical companies

I also came across suggestions as to why the US criminalised cannabis in the early 1900’s and used scare stories to vilify its use in order to save the jobs of those working for what is now called the Drug Enforcement Agency (DEA).  I read these stories with a high degree of scepticism, whilst at the same time believing things like ‘there is no doubt that cannabis causes schizophrenia’…

The more I read, though, the more the “conspiracy stories” about the covering up of the value of medicinal cannabis started to make sense.  I learned more about cannabis and how different cannabinoids can have different effects on different disorders – how some, for example CBD can actually counteract the psychoactive effects of THC, another cannabinoid.

Given this I read with great interest an article by a professor of neuropsychopharmacology and psychiatrist at Imperial College London, David Nutt, particularly since it was published in such a prestigious medical journal – the British Medical Journal (BMJ), which in just three pages gives validity to many of the conclusions I’d come to.

My reason for writing this blog post is to persuade people to see through the illegalities to the medical possibilities of cannabis in the hope that misinformed biases can be overcome and much needed research into cannabinoids and childhood brain tumours can be carried out.


Hadrian’s Wall Walk

It might not come as a surprise to those who keep up to date with our blog but we’ve decided to put the Hadrian’s Wall Challenge back to the second bank holiday in August.

Is going to be an interesting period for me personally since I also managed to secure a place in the Great North Run which is a couple of weeks later!

The timing does, however, mean that we’ve got more time to find something worthwhile to raise money for – less than three months left ’till the end of the CBD Research Project… fingers crossed there’ll be something off the back of that we can support.

If you’re interested in taking part, please visit our Facebook Event Page and/or message me and I’ll send you a link to the WhatsApp group we’ve started.

Hadrian’s Wall Challenge 2019

Every year since William’s diagnosis we’ve organised some kind of sponsored event to raise money for charities we believe can directly help William and other children with brain tumours.

To mark him making it to five years since diagnosis (nope, we can’t believe it either!), we have decided to organise our most ambitious event yet – walking the entire length of Hadrian’s Wall – all 84 miles from the East Coast of England to the West Coast.

It’s still early days organising the event but at the moment we’re looking to do it during the first May Bank Holiday of 2019 so we’ll be starting at Bowness-on-Soloway on the West Coast on Saturday 4th May with a view to finishing at Wallsend on the East Coast on Monday 6th. At the moment we’re planning on camping along the way. At this stage, though, we’re open to suggestion.

It’s a very exciting but uncertain time for research into the area we believe will be of most help to William and kids like him so we haven’t decided exactly where any money raised will go but participants can rest assured that we’ll make sure, by the time it comes to raising money, everything raised will go directly towards the research we believe will help William and kids like him.

If you’re interested in taking part either as an individual or as part of a group, please get in touch. For updates you can follow our Blog, Facebook Page or dedicated Web Page. There’s also a Facebook Event Page.

CBD Research Project – 12 Month Update

For anyone who hasn’t followed William’s story closely… after his terminal brain tumour shrank by two thirds having started him on CBD and a Ketogenic Diet, we proposed and helped fund a £100,000 research project at the Children’s Brain Tumour Research Centre into CBD and his tumour type – the project started in early 2018…

Almost 12 months on and the project “show(s) that we can kill tumour cells with CBD whilst the astrocyte population is unchanged”. However, Prof Grundy, who has been leading the research, has cautioned that “these results, though promising are very preliminary and there are a range of increasingly complex studies that need to be conducted to assess whether cannabinoids may have a role in treating children’s brain tumours”.

Despite the cautionary tone, it’s hard to contain our excitement at this latest development, not least because it means that our efforts to date have not been in vain. The next six months of this project will be critical but given these results and current events we are increasingly hopeful that clinical trials into cannabinoids and childhood brain tumours are increasingly imminent.

There’s much more to be done and we’ll be continuing to do all we can to make sure that childhood brain tumours aren’t forgotten as cannabinoids are increasingly being shown to be effective against cancer. Please watch this space for how you can help – in the meantime, please like and share our page and posts so as we can continue to spread awareness.

Make William Well Registered as Official NICE Stakeholder

The National Institute for Health and Care Excellence (NICE) has been asked to carry out a review of cannabis-based products for medicinal use and is in the process of setting up a committee to discuss prescription guidelines.

As someone who has had a very keen interest in cannabis and cancer for two years since William’s tumor reduction (having given him Cannabidiol), I applied for a lay-member position on the NICE committee although, since the draft scope didn’t include cancer treatment as something cannabis-based products could be prescribe for, my application was declined.

This prompted us to apply to have Make William Well registered as a stakeholder in the committee and we’re delighted to be able to announce that, just before the deadline for submitting comments on the draft scope passed, we received confirmation that our application was successful.

The comments we submitted emphasized the importance of having cancer treatment considered by the committee… watch this space… if you have any comments you would like to be put forward to the Committee, please let us know here.

NICE Stakeholder

First Ever UK Paediatric Brain Tumour Symposium


I attended this event on Tuesday organised by the charity Brains Trust and found it really interesting.  It was aimed at children and families living with a brain tumour diagnosis, researchers, clinicians and people with a professional interest in brain tumour care.

It was a long day with some really interesting topics covered, from how drugs can be better delivered to brain tumours to promising new therapies to late effects of brain tumours and their impact on education. (click here for a copy of the programme and here for details of the speakers).

Luckily all of the presentations were filmed and posted on YouTube and can be viewed by clicking here so I don’t feel as though I need to cover everything in this blog as I have done for events that I have attended in the past

Instead I’d like to focus in on some of the things I found particularly exciting in some of the presentations.

Drug Delivery

Professor David Walker (Professor of Paediatric Oncology) presented on the challenges of drug delivery and how difficult it is to get the correct amount of a drug to a brain tumour, particularly since it isn’t easy for many drugs to pass through what is know as the blood brain barrier.

One of the approaches David spoke about (there were more in subsequent presentations) was the use of micro-catheters and their use in delivering drugs directly to the tumour although the most exciting thing for me was the prospect of being able to re-visit drugs which had previously been written off due to their inability to cross the blood brain barrier.

Delivering Drugs During Surgery

This presentation was delivered in two sections by Dr Ruman Rahman (an academic assistant professor) and Ms Catherine Vasey (a PhD student).  They both spoke about the exciting procedure of placing drugs within a “container” near to where a tumour was removed during surgery so that the drug could be gradually released at the exact point it’s needed.

Catherine went on to discuss nano-particles and how they could be used to contain drugs and only release them when they enter a cancerous atmosphere.

Electrical Field Therapy

Dr Stuart Smith, a Clinical Associate Professor in Neurosurgery, gave a presentation on electrical fields and how they could be used to disrupt the growth of fast growing cells.  Very exciting but very expensive.

This, in addition to the allure of other promising research that had been presented throughout the morning, prompted me to ask about the availability of such approaches and why our children can’t have access to them now – unfortunately though it was time for lunch and the debate was cut short…

The afternoon session covered some of the practical consequences of having a brain tumour for children from cerebellar mutism syndrome to late effects to the impact on education.

Impact on Education

Louise Robinson, a Neuro Oncology Specialist, working for the charity CLIC Sargent, who supported William whilst we were living in the area gave a really useful presentation on education. She shared some useful links:

A Teachers Guide for Pupils with Brain Tumours

The Brain Tumour Charity – Education Resource

Practical Strategies for the Classroom

Parents Perspectives

A fellow parent of a child with a brain tumour, Pam Wouters, gave a very touching account of her son’s brain tumour journey.  What was really encouraging was hearing another parent speak about the importance of a holistic approach, in particular diet.

It was also humbling to speak to parents who had lost their child but still found the courage to attended the event, many of whom were using their experience to help other children.  I’m not sure I’d have the courage…

The MANY Advantages of Legalising “Last Resort” Medicinal Cannabis for Cancer Treatment

Next Tuesday the deadline for commenting on what conditions medicinal cannabis can be legally prescribed for in the UK will pass (for some reason this fact appears to have gone largely unnoticed).

At the moment the list of conditions includes chronic pain, intractable nausea and vomiting, spasticity and epilepsy but not cancer treatment.  This is because, despite a significant amount of research proving that cannabis compounds can kill cancer cells in the laboratory, there is evidently not as much as for the other conditions to warrant its inclusion (even for use when all else has failed as a last option).

Traditionally, the process of getting to the level of evidence required for the inclusion of a condition typically involves gold standard randomized controlled trials, which cost £millions.

With cancer rates in general continuing to rise and oncologists having to rely on the decades old treatment options of surgery, chemotherapy and radiotherapy for many conditions (only one drug has EVER been developed specifically for a childhood brain tumour), this “gold standard” approach is clearly failing.

Luckily there are those in the scientific community who recognize the limitations of expensive controlled trials and the importance of other approaches such as adaptive clinical trials which evaluate treatments by observing their effects on patients and making changes based on these observations.

I would like to see people being given the option of having medicinal cannabis prescribed to treat their cancer in the first instance although I think at this stage there is something more achievable we should be fighting for – I believe what we currently have is a golden opportunity to advance research into cannabis and cancer

I’m by no means a researcher or scientist but would it be so hard to imagine the benefits of allowing the prescription of medicinal cannabis to patients for whom all other treatments have failed and for this to be done as part of an adaptive clinical trial?

As well as generating much needed clinical research, this would also mean that patients and the parents of children with cancer would have access to medicinal cannabis under the supervision of a doctor as opposed to having to self fund and medicate using drugs of often questionable quality and content.

Finally, who knows, it may end up saving lives…

If you haven’t already, please consider signing our petition to Have Cancer Treatment included in the Medicinal Cannabis Review but hurry, we only have until 4th December.

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