Towards 3D neuroblastoma cell models

It seems I have got a conference season. Three conferences within 2 months – no complains though. This time I went to the Matrix Biology Ireland Meeting in Galway. It was fantastic mix of topics and speakers ranging from new approaches in bone and heart repair to new matrixes in reconstruction of body tissues and diseases in the lab to minimise use of animals in pre-clinical studies.

My talk was focused on neuroblastoma microenvironment and cell-to-cell communication through exosomes. I wrote about it in October post. I talked about things that did work and did not as well as new directions. One of the new directions is reconstructing neuroblastoma by growing neuroblastoma cells on collagen based scaffolds in 3D. Collagen constitutes most of our tissues to keep it shape and strength. These scaffolds are sponge-like matrixes built from collagen and other components. Of course cells grow differently on these matrixes. They have a different shape and growing properties in 3D. Neuroblatoma cells look like water drops on the cotton wool-like collagen scaffolds. In contrast, when they grow on plastic in 2D, they are flat. Studies show that cells in 3D respond to cytotoxic stress in a similar pattern as if being within a body (details in recent reviews 1-4).  It would be a great breakthrough once these models are optimised for neuroblstoma research  field. It will help to test all new and known drugs in the environment close to clinical settings. It could be a step forward to personilised therapies for children with neuroblastoma by isolating cancer cells, growing them in 3D and testing how they respond to all therapies available. It will facilitate more efficient design of treatment for relapsed or poorly responding tumours, sparing patients unnecessary rounds of chemotherapy and ultimately increasing survival.

 

Neuroblatoma cells look like water drops on the cotton wool like collagen scaffolds. In contrast, when they grow on plastic in 2D, they are flat. Arrows point towards cells.
This is microscopic images of neuroblatoma cells growing on the collagen scaffolds and plastic. Arrows point towards cells.

 

I’ve always felt that a selection of abstracts for an oral presentation is biased. The overall background and views of conference organisers would affect works selected for an oral presentation.  The same abstract was not selected for an oral presentation by one committee, but was supported by the other.  Never give up!

Readings:

  1. Schweiger PJ, Jensen KB.Modeling human disease using organotypic cultures. Curr Opin Cell Biol. 2016 43:22-29.
  2. Salamanna F, Contartese D, Maglio M, Fini M. A systematic review on in vitro 3D bone metastases models: A new horizon to recapitulate the native clinical scenario? Oncotarget. 2016 7(28):44803-44820.
  3. Picollet-D’hahan N, Dolega ME, Liguori L, Marquette C, Le Gac S, Gidrol X, Martin DK. A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models. Trends Biotechnol. 2016 34(9):757-69.
  4. Nyga A, Neves J, Stamati K, Loizidou M, Emberton M, Cheema U. The next level of 3D tumour models: immunocompetence. Drug Discov Today. 2016 21(9):1421-8.

Quality of life for childhood cancer survivors

For children who do survive cancer, the battle is rarely over.  Over 60% of long‐term childhood cancer survivors have a chronic illness as a consequence of the treatment they received; over 25% have a severe or life‐ threatening illness. How much do we know about quality of life of childhood cancer survivors?

Researchers in health- and illness-related social sciences understand that the there is a life after the treatment completed. The life is full if diverse levels and issues from health related to social adaptation in different shapes and forms. Children and teenagers may experience fear when returning to school due to temporary or permanent changes to their physical appearance (1,2). They worry about their ability to socialise with their friends due to lengthy absences (3–5). Treatment can result in the development of learning disabilities in children and thus marking school as a major source of frustration (1,2). These learning difficulties can affect a child’s confidence and self-esteem, if left without attention and care (1,3). All studies come to the same conclusion. Challenges in education of children with cancer are complex, however most can be tackled efficiently through planning and good communication (1–5).

Recently researchers working in FRED HUTCH Cancer Research Center asked adult childhood cancer survivors a number of health related questions about the quality of lives (6,7). The results are far from optimistic: “chance of surviving childhood cancer has improved — but survivors’ overall health has not”. You can find more by following the link.

It is important not only to recognise the problems but to start changing the situation. Apparently much more could be done more efficiently if patients are involved in setting up future research agenda.

Reading

  1. Gurney JG, Krull KR, Kadan-Lottick N, Nicholson HS, Nathan PC, Zebrack B, et al. Social outcomes in the childhood cancer survivor study cohort. J Clin Oncol. 2009;27(14):2390–5.
  2. McDougall J, Tsonis M. Quality of life in survivors of childhood cancer: A systematic review of the literature (2001-2008). Supportive Care in Cancer. 2009. p. 1231–46.
  3. Barrera M, Shaw AK, Speechley KN, Maunsell E, Pogany L. Educational and social late effects of childhood cancer and related clinical, personal and familial characteristics. Cancer. 2005;104(8):1751–60.
  4. Langeveld NE, Stam H, Grootenhuis MA, Last BF. Quality of life in young adult survivors of childhood cancer. Support Care Cancer. 2002;10(8):579–600.
  5. Klassen AF, Anthony SJ, Khan A, Sung L, Klaassen R. Identifying determinants of quality of life of children with cancer and childhood cancer survivors: A systematic review. Support Care Cancer. 2011;19(9):1275–87.
  6. Yeh JM, Hanmer J, Ward ZJ, Leisenring WM, Armstrong GT, Hudson MM, et al. Chronic Conditions and Utility-Based Health-Related Quality of Life in Adult Childhood Cancer Survivors. J Natl Cancer Inst [Internet]. 2016;108(9):4–7.
  7. Armstrong GT, Chen Y, Yasui Y, Leisenring W, Gibson TM, Mertens AC, et al. Reduction in Late Mortality among 5-Year Survivors of Childhood Cancer. N Engl J Med. 2016;374(9):833–42.

E-poster at SIOP2016

Ok. Now, when the stress of the presentation is over, I am happy to share new technologies used during the SIOP2016. As I mentioned yesterday, my work was selected for e-poster presentation. It looked this way:

This is e-poster station, where anyone can look up all posters displayed during the meeting.
This is e-poster station, where anyone can look up all posters displayed during the meeting.

 

It is definitely a step forward. Anyone can look up any poster, listen to a commentary recorded by the author, zoom in and out and send a request/comment to the author. It looks cool and trendy. Though, you can feel invisible as no physical copy displayed in a designated area. No crowds of poster presenters and judges. No waiting faces desperate to share their study…

The actual Poster Discussion session was a traditional presentation when my poster was up on the big screen, I had 8 minutes to convince the audience navigating through figures. This session was late and no many attendees survived to come and challenge your statements. Nevertheless, it was enjoyable experience. 🙂

siop-2016-banner-728-x-90

 

 

 

 

Last minute discovery with SIOP2016

 

SIOP is the International Society of Paediatric Oncology. It is a global multidisciplinary society representing doctors, nurses, other health care professionals, scientists and patients or their relatives. The Society’s motto is ‘no child should die of cancer’. The meeting 2017 is being held in Dublin, the city where I live and work.

Indeed, it was appealing to attend the key meeting in childhood oncology field. As any participant, I had an opportunity to submit an abstract about my research.  To no surprise at all, I received email notifying me on my work being selected for e-Poster presentation.  Common stuff. The email also said that it would be displayed at designated stations, like big screens throughout the meeting. Very unusual format, but we are living in the digital technology era; things are changing all the time. So, I would not need to stay by the poster this time. Great – more time for networking and talks.

Then I received another email informing about a Poster Discussion session, which I assumed to be a standard procedure when a group of selected piers stand by your poster and ask Qs. None comes in majority cases. A participant stands and waits and waits till the session is over. So, of course I took it easy.

A day before the meeting, I downloaded the meeting app and started to browse along the content and features. Out of curiosity, I checked details of the Poster Discussion session. This was the moment of mental breakdown – I discovered being selected for an oral poster presentation! My chances were 1 in 1475 (the number of submitted abstracts). I should probably also buy a lottery ticket tonight. Could lucky things come together?

I will reflect on the new e-poster presentation experience later today…

 

siop-2016-ppt

 

http://www.siop2016.kenes.com/

 

 

 

How we work with cancer cell lines?

How we work with cancer cell lines?

The very first human cancer cell line was developed from a patient with an aggressive cervical cancer in 1951. This cell line was called HeLa after the patient name – Henrietta Lacks. This is the most popular and robust cancer cell line in biomedical research. Since then, other cancer cell lines were developed including neuroblastoma.

The first successful neuroblastoma ‘cell lines’ were cell populations from tumours that were adapted to grow for a short period in the lab environment in 1947. These tumour cell populations were used as a tool for diagnosis. This success inspired other researchers to develop long-term or immortal neuroblastoma cell lines. To date different neuroblastoma cell lines exist.

Cancer cell lines are sensitive and delicate in handling. They can only grow in the safe environment. Researchers have to protect them against bacteria, low temperatures, and too acidic/alkaline conditions. We protect cancer cells from bacteria contamination by handling them in cabinets where all plastic and media are sterile.

Handling neuroblastoma cells in the cell culture cabinet.
Handling neuroblastoma cells in the cell culture cabinet.

Cancer cells like to grow in conditions similar to conditions in human body. They like temperature of 36.6 – 37C. To achieve it special ‘green cell houses’ – CO2 incubators are built, which maintain the constant temperature, humidity and CO2 concentration.

We place cells in plastic dishes or containers called flasks and keep flasks in the ‘green cell houses’.
We place cells in plastic dishes or containers called flasks and keep flasks in the ‘green cell houses’.

The cell growth and well being are checked regularly using microscopes. Healthy cells are to have similar shape, even distribution and grow attached to the plastic surface. Most microscopes have a camera attached to the top and linked to a computer. It helps to take picture of growing cells and record changes in cell behaviour.

Microscopic examination of drug resistant neuroblastoma cells KellyCis83. Cells look healthy and can be kept for another 2-3 days to form a more dense population.
Microscopic examination of drug resistant neuroblastoma cells KellyCis83. Cells look healthy and can be kept for another 2-3 days to form a more dense population.

 

Recommended reading

  1. Skloot, R. The Immortal Life of Henrietta Lacks 2011
  2. Thiele CJ. Neuroblastoma Cell Lines. Human Cell. 1998. 1-35 p.
  3. Murray M, Stout A. Distinctive Characteristics of the Sympathicoblastoma Cultivated in Vitro: A Method for Prompt Diagnosis. Am J Pathol. 1947;23(3):429–41.

 

Drug resistant neuroblastoma cells

Children with neuroblastoma undergo several cycles of intensive chemotherapy to stop disease progression with the final aim to eliminate the tumour. Chemotherapy includes carboplatin or cisplatin in various combinations with drugs such as cyclophosphamide, ifosfamide, doxorubicin, etoposide, topotecan and vincristine (1). Nevertheless, in average 1 in 5 children with stage 4 disease do not respond to therapy. Up to 50% of children that do respond experience disease recurrence with tumour resistant to multiple drugs and more aggressive behaviour that all too frequently results in death.

The development of drug resistance is the major obstacle in treatment of neuroblastoma. To tackle this problem, researchers need to study different models of disease using cell lines, 3D tumour cell models, mice models and have access to clinical samples.

The first stage in testing drugs is to understand their killing ability of cancer cells. At this stage, researchers test drugs using cell lines. Cell lines are derived from tumours which were surgically removed from children with neuroblastoma. Researchers usually take a small piece of tumour straight after surgery and bring it into the laboratory.  Here, they place this piece into special solution that has enzymes to separate cells from each other. Then the suspension of all kind of tumour cells is placed into plastic dishes or flasks in a highly nutrient media to let cells grow. Cells that can adapt to these conditions start to grow, divide and produce a new generation of cancer cells. Researchers look after their growth, inspect their shape and behaviour; and test them on the presence of tumour markers. Once identity of these cells is confirmed they become a cell line and obtain a name. These cells keep majority of characteristics of the parental tumour and represent very useful tools in cancer research.

In our lab we use such cell lines to study neuroblastoma resistance to drugs. To understand changes in neuroblastoma biology during the development of drug resistance, we created drug resistant neuroblastoma cell lines (2). We treated three neuroblastoma cell lines CHP212, SK-N-AS and Kelly with cisplatin – a common drug in anticancer therapy. SK-N-AS and Kelly cells are sensitive to this drug, while CHP212 cells responded to this drug at much higher levels that the other two. Cells were grown in media containing cisplatin for several weeks. During this period most of the cells responded to cisplatin and died. Then we let cell survivors to recover in media without drug. This cycle was repeated several times until we got a population of cell survivors that can stand doses of cisplatin that can kill 50% of parental cells.  It took us more than 6 months to generate cisplatin resistant neuroblastoma cell lines CHP212Cis100, SK-N-ASCis24 and KellyCis83.

At the next step, we studied differences between these cell lines. We first compared their behaviour and cell shapes. Two resistant cell lines KellyCis83 and CHP212Cis100 started to grow faster, but SK-N-ASCis24 – slower than their parental cell lines. Interestingly, these cells also became more resistant to other drugs such as doxorubicin, etoposide, temozolomide, irinotecan and carmustin. These results are very important as they demonstrate that one drug can activate the cell defense systems that allow to escape toxicity of other drugs. These cell lines can be used to test new drugs and find those that can overcome developed resistance.

Cisplatin resistant cells also changed their appearance. Most dramatic changes occurred in SK-N-ASCis24 cells (see Figure 1).

nbl-cells

Figure 1. Microscopic images sensitive and drug resistant neuroblastoma cells (adapted from (2)) 

Two drug resistant cell lines SK-N-ASCis24 and CHP212Cis100 cells developed additional mobility skills – they became more invasive than their parental counterparts.

 

resistant-cells

 

Then we asked a question: what type of changes allowed cells to adapt to cytotoxic environment?  We examined changes in their genomic DNA first. We found that some genes increased their copy number, other went missing.

We identified changes in protein expression. More intriguingly, some proteins with the increased presence in the cells did not increase their presence in genomic DNA. We sorted these proteins on their role in cell processes such as migration, growth, cell cycle, etc. We found that each cisplatin resistant cell line developed a unique set of features that help them to escape cytotoxic stress (2). The similar patterns are found in clinic. Each patient responds to treatment differently.

What did we learn from this study?

  • One drug, in our study cisplatin, can activate the cell defense systems that allow to escape toxicity of other drugs.
  • The development of drug resistance gives cells new advantages and changes their behaviour and appearance, e.g. mobility skills, different cell shape, response to drugs, etc.
  • Each cisplatin resistant cell line developed a unique set of features that help them to escape cytotoxic stress.
  • These cell lines can be used to test new drugs and find those that can overcome developed resistance.

References

  1. Davidoff AM. Neuroblastoma. Semin Pediatr Surg. 2012; 21(1):2–14.
  2. Piskareva O, Harvey H, Nolan J, Conlon R, Alcock L, Buckley P, et al. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett. 2015;364(2):142–55. 

 

 

 

 

A mother’s battle with neuroblastoma

A very nice piece of journalist’s story about neuroblastoma through a mother’s view. I met the mother personally at the 4th Neuroblastoma Research Symposium in Newcastle-upon-Tyne, UK in November 2015. Susan Hay and other same minded parents of children with neuroblastoma joint their efforts to raise money not only for current kids battling this nasty cancer, but more importantly for research in the cancer biology, diagnostics and new therapies which are to give a better deal for children with neuroblastoma.

https://www.theguardian.com/society/2015/nov/15/a-mothers-battle-with-neuroblastoma

Clinical trials in children commonly go uncompleted or unpublished

Currently, the only popular trend in science is to publish only those results that look as a breakthrough or display statistically significant data. Obsession with positive outcome leads to discontinuation and non-publishing the all other data which don’t meet the requirements.  As a result researchers and public did not know mistakes, efforts and drilling details of non-positive studies, so they have no opportunity to review this data, refine the research hypothesis and technical performance. This leads to waste of time and funding money.

The very recent study by Natalie Pica, MD, PhD, and Florence Bourgeois MD, MPH from the Department of Pediatrics at Harvard Medical School and Boston Children’s Hospital, both in Massachusetts has again confirmed the problem. The researchers carried out a retrospective, cross-sectional study of childhood randomized controlled trials and published their findings in Pediatrics (DOI: 10.1542/peds.2016-0223). They collected information from all trials that were registered ClinicalTrials.gov from 2008 to 2010, then searched scientific publications based on the trials. They also verified final status of the selected trials (completed or discontinued) by the end of 2012. If researchers found no publication, they contacted investigators and sponsors associated with trials to clarify the issue.

The main findings were:

  1. 19% of 559 trials were discontinued early representing approximately 8369 children. The most common reason for discontinuation – difficulty with patient accrual (37%).
  2. 30 % of the 455 completed trials were not published, representing 69 165 children participants.
  3. Only 42 unpublished trials posted results on ClinicalTrials.gov.
  4. Trials were less likely to be dropped if they were funded by industry.
  5. Trials funded by industry were more than twice as likely to result in nonpublication and a longer mean time to publication when compared with trials sponsored by academia.

Researchers concluded that “withdrawal and nonpublication were common, resulting in thousands of children exposed to interventions that did not lead to informative or published findings. Trial funding source was an important determinant of these outcomes, with both academic and industry sponsors contributing to inefficiencies.” (Pica N & Bourgeois F, 2016, e 20160223)

Pica N & Bourgeois F, Discontinuation and Nonpublication of Randomized Clinical Trials Conducted in Children. PEDIATRICS V 138(3) 2016:e 20160223 Access to the study can be found here:

http://pediatrics.aappublications.org/content/pediatrics/early/2016/08/02/peds.2016-0223.full.pdf

 

 

Neuroblastoma summary

Neuroblastoma is a childhood cancer caused by the abnormal growth and development of non-mature nerve cells, called neuroblasts [1]. The disease commonly affects children age 5 years or younger. Approximately 50% of children have tumours that have spread at diagnosis [1]. The main challenge in treating neuroblastoma is to stop tumour spread and resistance to multiple drugs. Despite major advances in available therapies, children with drug resistant and/or recurrent neuroblastoma have a dismal outlook with 5 year survival rates of less than 20% [2-4]. Therefore, this cancer needs more research and funding as well as people awareness of these needs.

 

  1. Davidoff, A. M. Neuroblastoma. Semin. Pediatr. Surg. 21, 2–14 (2012).
  2. Gatta, G. et al. Childhood cancer survival in Europe 1999-2007: Results of EUROCARE-5-a population-based study. Lancet Oncol. 15, 35–47 (2014)
  3. Peinemann, F., Tushabe, D. A., van Dalen, E. C. & Berthold, F. Rapid COJEC versus standard induction therapies for high-risk neuroblastoma. The Cochrane database of systematic reviews 5, CD010774 (2015).
  4. Peinemann, F., van Dalen, E. C., Tushabe, D. A. & Berthold, F. Retinoic acid post consolidation therapy for high-risk neuroblastoma patients treated with autologous hematopoietic stem cell transplantation. Cochrane database Syst. Rev. 1, CD010685 (2015)