Tag: #research

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)

Welcome!

Hi readers!

This blog is about neuroblastoma biology, its research challenges, and people and media perception of this disease. I am researcher and active supporter of science communication.  I hope you will find interesting to read my blog and ask questions. Your questions would help me to cover topics which I have not heard of or may not plan to cover yet.

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