Arginine, Glioblastoma and the Immune System

An interesting piece of work came to my attention earlier this week that I think is worth sharing for a number of reasons. Firstly, I think it illustrates a way of working that really should be encouraged - it has significance beyond the specific interventions mentioned. Secondly, I think there are lessons here for those who are interested in anti-cancer agents not patented by the drug companies (such as curcumin, quercetin, resveratrol etc). Finally, it has interest for those suffering from glioblastoma (GBM) - a form of brain cancer that is particularly aggressive and hard to treat.

The work in question looked specifically at the phenomenon of immune suppression in GBM patients, and let's note at the outset that immune suppression is a problem for all cancer patients, not just those with GBM. Brain cancer patients - not just those with GBM - at the University of Colorado Hospital had blood taken and analysed, along with blood from non-cancer patients. Analysis revealed that the blood from GBM patients in particular showed lower levels of T-cell proliferation and released lower levels of interferon-gamma compared to normal blood samples - in other words they had lower levels of immune function. The specific forms of this immune suppression were worked out in a series of lab experiments that ultimately showed that one of the factors associated with this suppression was a high level of the enzyme Arginase I (ArgI). This enzyme consumes the amino acid Arginine, which is essential for T-cell activation.

So far so good - it's interesting, in an academic way, but so what? The experimenters then took the next step and looked at chemically blocking ArgI with a specific drug. Doing this improved the immune response in the test tube. Levels of interferon-gamma rose to normal levels. The next step, however, is where it gets really interesting from a patient point of view. Rather than blocking ArgI with a drug, the team looked at increasing levels of Arginine (which is what ArgI consumes). By adding more Arginine the immune response improved. In simple terms the ArgI was basically using up the circulating Arginine and thus starving the T-cells of it, but by increasing the amount of Arginine the researchers showed that they could 'feed' the ArgI and have enough left over to be used by the T-cells, which could become activated as normal.

Q&A With Dr Anthony Howell - The Reverse Warburg Effect

One of the fundamental ideas in cancer has been that cancer and normal cells differ in their metabolism. First proposed by Nobel prize-winner Otto Warburg in 1924, the 'Warburg Effect' is the hypothesis that cancer cells generate energy by the non-oxidative (without oxygen) breakdown of glucose (a process called glycolysis). This is in contrast to normal cells, which generate energy through an oxygen-dependent pathway. Tumours use the non-oxidative pathway, which is less efficient than normal metabolism, even in conditions where there is plenty of oxygen available. We can see this greediness for glucose in PET scans, for example, where radioactively labelled sugar is sucked up by tumours and not by normal tissue.

For many years the Warburg effect has been investigated as a possible Achilles heel for tumours, and there is still lots of work being done on ways to attack tumours by interrupting this glycolytic process. Such work ranges from dietary interventions (see for example the article on diets and cancer, and the interview with Dr Gerald Krystal), to looking for drugs which disrupt the process at different points.

However, in recent years Dr Michael Lisanti and a group of colleagues have proposed a fundamental re-appraisal of the theory. They have proposed the cancer cells in advanced solid tumours are able to induce the Warburg Effect in the cells that surround them (the tumour stroma), and that they then consume the by-products of glycolysis produced by the stromal cells. In other words they cannibalise the tissues around themselves, inducing the Warburg Effect so that they can feed off the nutrients produced by the stromal cells. They have termed this new theory, which is a radical departure from what is currently accepted, the 'Reverse Warburg Effect'.

For those who wish to learn more there are a number of freely available papers by Lisanti and co available via PubMed.

Dr Anthony Howell is one of Lisanti's co-authors and a researcher and clinician based in Manchester. He has kindly agreed to an interview so that we can learn more about this new theory and what it may mean in terms of clinical practice. Although the language in this interview is more technical than much of what is published on this site, I feel it's important to bring these new theories and results to as wide an audience as possible.

PP: A recent editorial in the journal Cell Cycle described the work of Michael Lisanti, yourself and others as ‘iconoclastic’. Just how revolutionary is the theory of the ‘reverse Warburg’ effect and the idea of ‘cancer as a parasite’?

AH: Warburg was a great scientist as reflected in the award of the Nobel Prize for his pioneering work on tumour metabolism. His major contribution was to show that tumour cells showed enhanced glycolysis and he thus assumed that mitochondrial respiration was impaired in tumour cells. It certainly looks like this when you put tumour cell lines such as the breast MCF-7 cell line into culture by themselves. These cells have few mitochondria and there is increase in the sugar degradation glycolytic pathway. What Michael Lisanti and his colleagues have shown is that this may be an artefact of culturing the cells alone. When co-cultured with human fibroblasts, the MCF-7 cells synthesise mitochondria and thus use oxidative phosphorylation as their main source of energy. In the body tumour cells are always associated with what is called a supportive stroma derived from the host, so that the co-culture system is a better mimic of what might be happening in-vivo, and indeed we find up-regulation of mitochondria in most breast cancers compared with normal breast tissue. Cultured alone the fibroblasts have a large number of mitochondria. However, when co-cultured with tumour cells they degrade the mitochondria and depend upon gylcolysis. So all these observation are opposite to Warburg’s observations. It is as if the Warburg effect occurs in tumour associated fibroblasts rather than the tumour cells themselves which has lead Lisanti to call what is happening in co-culture ‘The Reverse Warburg Effect’. I think this is where the implication that these observations are iconoclastic comes from.

UK Clinical Trials Gateway

I have previously written about how to search for clinical trials, describing the difference between Phase I, II and III trials, as well as how to use the US National Institutes of Health Clinical Trials database (http://clinicaltrials.gov/). However, the fact it's currently very difficult to find out what trials are going on in the country as a whole. What's missing is a central database of all clinical trials. A step in the right direction is the UK Clinical Trials Gateway (http://www.ukctg.nihr.ac.uk/default.aspx).

While not a central registry itself, the UKCTG does enable the user to search a number of international registries through the site. Users can search on specific diseases (e.g. a specific type of cancer, like lung cancer), specific drugs (e.g. celecoxib) or other forms of therapy (e.g. photodynamic therapy), as well as combinations such as "celecoxib AND cancer". For each record returned by the search you can find out whether the trial is recruiting patients, where the trial is taking place and more details on the actual treatment itself. For patients looking for a trial it's a really useful place to start.

However, there is still room for a central registry of clinical trials. Not only would it provide doctors and patients with a single database to query for all trials in the UK, it would also allow us to track the level of activity for different diseases. For example doing a query on the UKCTG shows that there are no trials for osteosarcoma in the UK, which is sad if true, but I know that there are some trials on-going at the moment (the SUCCEED trial, run by Merck and Ariad Pharmaceuticals).

Just as importantly a central registry will enable us to see what trials are aiming to do, what protocol they use and what outcomes they are tracking. With this information we can see which trials publish their results, and also see whether they succeed in what they set out to do or whether they changed what they say they are looking for in order to present the best possible gloss on their results. And yes, this is something that does happen...

Until then, at least the UKCTG provides us with yet another useful tool to help patients and their doctors find appropriate clinical trial information.

Test Tube Cancer Cells and People

Test tube cancer cells vs. real cancer cells

In a previous article (How To Read A Cancer Paper Part 1 - ), I looked at the pitfalls of in vitro studies. These are studies performed in the lab that look at cancer cells under glass (actually a Petri dish rather than a test tube as such). These studies - and there are tens of thousands of them - are often performed to see how cancer cells respond to specific anti-cancer agents. And, as I said in the article:

...you have to take these test tube studies with a huge pinch of salt. In the test tube substance X kills cancer, but that really doesn’t mean much in the real world. At most it gives you reason to carry on looking at substance X in more detail, but that’s about it.

The obvious alternative is to look at in vivo studies, which are discussed in the second article in the series. These are studies that use animals (normally mice or rats), with real tumours rather than cells grown under glass. While these are better, there are still major pitfalls, which I discuss in the article. One problem that I highlight is that the tumours that are grown are derived from standard cell lines. These are cancer cells that have been grown for year after year after year under lab conditions. The intention is to have some standard cell lines that are indicative of the tumour type or sub-type (e.g. androgen responsive prostate cancer, triple negative breast cancer etc). A key point I make in that article is:

Furthermore, cancer cells mutate and adapt, so cells that have been taken from a patient biopsy twenty or thirty years ago and then kept under glass for generation after generation will have changed in order to survive in the test tube environment.

A new study, published in the Proceedings of the National Academy of Sciences, looks at precisely this issue and comes to a disturbing conclusion. The experimenters were specifically looking at cancer cells that are drug-resistant. This is of major clinical importance as the fact is that we still don't fully understand why some cells can become resistant to anti-cancer drugs. Using state of the art technology, the researchers took a panel of standard cell lines and checked which genes are expressed. They did this both in the test tube and also from tumours grown in animals from these same standard cell lines. They then compared these with a set of cells taken directly from patient biopsies. The results are clear:

No correlation was found between clinical samples and established cancer cell lines....all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model.

In other words, the standard cell lines, originally from different types of tumour, have come to resemble each other genetically, whether used in the test tube or implanted in animals. They resemble each other more than they resemble 'live' samples taken from cancer patients.

A numb lip

The first sign that my son, George, had a problem in the jaw was a funny tingling in the upper lip, on the right hand side. This developed into a feeling of numbness that wouldn’t go away. He had had a basal cell carcinoma surgically removed from the left side of the skull, just under the hair line above the ear, a few weeks previously. Was the numbness related to that? We checked with the surgeon and were told it was unrelated.

Was it a dental problem? We then got into a cycle of being shunted around from one hospital or clinic to the next. We went from dentists to maxillofacial and back again. The numbness was slowly getting worse but we were not getting anywhere. Having had two cancers already, we checked with the Royal Marsden and did the rounds there too. A very senior paediatric oncologist saw us and referred us to a number of people. My son’s jaw was ultrasounded and there, nestling under his chin was an odd looking little lump. A tumour? No, probably not we were told.

The paediatric oncologist was very clear. Whatever the problem was, it wasn’t oncological. Which was a relief. But the problem wasn’t going away. And he was starting to get pain in the jaw too.

Finally we tried another dentist. She x-rayed and saw that his wisdom tooth had been pushed out - it wasn’t right. She referred us back to maxillofacial - immediately.

I can vividly remember the day. My son was seen by a registrar called Amir Ketabchi. He recognised immediately what was going on. We could tell by the flurry of activity around us that this was serious. George was booked into a CT scan the next day. The result was clear – a tumour in the right mandible.

The only question was what kind of tumour? We desperately hoped for something benign and easy to treat. But luck was against us. The ‘not an oncological problem’ was an osteosarcoma.