Category: NCRC Research Project

#JournalClubwithFederica:How small RNAs contribute to neuroblastoma biology

We’ve recently started a new journal club series focusing on papers published by our research group over the past few years. The paper I chose is titled “A Context-Dependent Role for MiR-124-3p on Cell Phenotype, Viability and Chemosensitivity in Neuroblastoma in vitro“. It explores the anti-cancer potential of miR-124-3p in neuroblastoma.

Neuroblastoma is particularly challenging to treat, especially when tumours become resistant to chemotherapy. This resistance is compounded by tumour heterogeneity—these cancers comprise different cell types, specifically adrenergic and mesenchymal cells. This variability affects treatment responses and plays a role in metastasis and how aggressively the cancer can spread.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression, and miR-124-3p has emerged as a promising player in cancer research. A Kaplan–Meier plot in the study (Figure 1) shows a strong association between low miR-124-3p levels and poorer survival rates in neuroblastoma patients, underscoring its potential impact on patient outcomes.

Our group’s study specifically examined how miR-124-3p might help reverse chemotherapy resistance and inhibit tumour cell growth in neuroblastoma. Excitingly, it has the potential to reduce cancer cell survival and increase their sensitivity to chemotherapy—an important breakthrough for treating resistant neuroblastomas.

The study found that miR-124-3p directly targets genes involved in the epithelial-to-mesenchymal transition (EMT), a process that makes cancer cells more invasive and treatment-resistant. By suppressing these genes, miR-124-3p can reverse EMT, shifting cells to a less aggressive, more treatment-sensitive state. Our group observed that increased miR-124-3p significantly reduced neuroblastoma cell invasion (Figure 2). In SK-N-AS cells and their drug-resistant form, invasion dropped by 50% and 70%. In Kelly cells and their resistant form, invasion decreased by 10% and 30%. The most invasive of all, the drug-resistant SK-N-ASCis24 cells, showed the most substantial decrease in invasion after miR-124-3p treatment. This suggests that miR-124-3p could help limit neuroblastoma spread, highlighting its therapeutic potential.

While miR-124-3p isn’t part of my project, seeing how different molecular mechanisms can be harnessed to develop cancer therapies is always inspiring. Using miRNAs to sensitize resistant cancer cells to treatment could complement approaches like immunotherapies or vaccines, like the one I’m working on. Understanding these molecular pathways brings fresh perspectives on weakening cancer cells and making treatments more effective.

Written by Federica Cottone

Congratulations Dr Frawley!

June 9th 2022 – A Big Day for Tom and me. This is the end of the 4th year PhD marathon. A long journey through scattered showers and sunny spells, gale winds and stormy snow with sunshine developing elsewhere, turning chilly under clear skies on some days with temperatures below/above zero. The full spectrum of emotions and hard work spiced up with the COVID19 restrictions’ uncertainty. All these together have moulded into a new high skilled researcher – Dr Thomas Frawley.

The PhD Viva and celebration, June 9th 2022

My greatest thanks to Tom’s examiners Profs Elena Aikawa (Harvard Medical School, USA) and Marc Devocelle (RCSI, Ireland)!!

This work would not be possible without the generous support from the Irish Research Council and National Children’s Research Centre.

Remote Research Projects

Regardless COVID19 pandemic, we continue to host undergraduate students from various Universities for their research projects. Two students, Carla and Chris, from the Technical University of Dublin, carried out BSc projects remotely. Having in-house datasets and many more published in open access, their projects were focused on bioinformatics, re-analysing them and giving a second look. Both Carla’s and Chris’ research received the highest score in their classes. Many congratulations – well deserved!! We wish to thank both for their kind words and willingness to share their story.

Chris Sheridan, the final year student in Biomolecular Science at the Technical University of Dublin, 2021

“My project concerned analysing the exosomal miRNA expression of neuroblastoma cells in response to chemotherapy. Though the project was not too large, it certainly was the largest project I have ever taken part in. The work Dr. Piskareva and her team are conducting is so interesting and novel that I felt very fortunate to be participating in such an exciting field. Despite the novel and complex nature of the topic, the project was extremely engaging, allowing for an opportunity to learn new valuable research and data analysis skills. I was able to get very useful and helpful feedback regularly from everyone on the research team, where there was a very welcoming and positive attitude. This made the topic seem less daunting and my goals more achievable. I was really happy with my results, and I am excited to see where they may lead in the future. Some of the miRNAs identified in the analysis may represent potential biomarkers or therapeutic targets for high-risk neuroblastoma patients. As I have yet to experience any lab-based research, it was cool to see the team’s approaches and applications of lab techniques and analysis strategies to see how research is conducted in the “Real World” after seeing these topics before only in lecture notes. Overall, the project was challenging but very rewarding and enjoyable. Throughout the project, the overall experience, the excitement of the results coming together, and the realisation that I may have something to contribute to this field of research cemented the idea in me that this is certainly the path I wish to pursue in science and for that, I would like to thank Dr. Piskareva and her team for such a positive and educational experience during my time with them.“

Carla Tejeda Monné, the final year Technological University of Dublin Biomolecular Science Student, specialising in Biotechnology, Therapeutics, and Drug Development, 2021

“During my final year project, I had the unique and amazing opportunity to work under the supervision of Dr. Olga Piskareva. The purpose of my thesis was to assess the clinical significance of Tumour Necrosis Factor Receptor Superfamily Member 1B and Member 4 (TNFRSF1B and TNFRSF4) in neuroblastoma patients. I accomplished this by analysing the gene profiles of several tumours using bioinformatic tools. In addition, I investigated the potential of microRNAs as therapeutic agents for neuroblastoma treatment. I thoroughly enjoyed carrying out this research project, and I hope the findings from my thesis can aid future research into the pathogenesis of neuroblastoma and the development of effective treatments for these children.“

Best of luck to Chris and Carla in their next endeavour!

Sparkles of A Researcher Day

Once I mentioned the importance of the publication track record for a career in science. My team has been productive despite the COVID pandemic. Two review articles were published.

The first was a review written by Tom and published in Cancers focusing on the small extracellular vesicles produced by cancer cells that can transfer various growth signals to the tumour microenvironment aka neighbourhood and promote tumour expansion. The signal in focus was a protein called epidermal growth factor receptor (EGFR). It contributes to the healthy fitness of many different cells in the body. However, many cancer cells produce an excess of this protein giving them an advantage of growth over normal cells. Increased EGFR can be seen in breast, lung, glioblastoma and head and neck cancers.

Cancers 2020, 12(11), 3200; https://doi.org/10.3390/cancers12113200

The second review has been published in Journal of Personalized Medicine on March, 16th. Originally, it was a small review project for Nadiya, a medicine student, last summer. However, it became a big one with all data systematically collected, analysed and condensed. The focus of this review was on Retinoic Acid (RA), widely known as Vitamin A and its role in neuroblastoma. RA plays a vital role in human development. The main feature of RA is to push neuroblastoma cells to become neuron-like cell stopping their aggressiveness and cancer fate. So, we wanted to know more about the ongoing research both in the labs and the clinic. We reviewed primary research articles reporting basic and translational findings as well as clinical trials. Hopefully, it would help other researchers to get a full picture of this topic and a structured resource of experimental models and drugs tested.

J. Pers. Med. 2021, 11(3), 211; https://doi.org/10.3390/jpm11030211

Annual NCRC Symposia 2020

As the year comes to an end, you are looking back and seeing all achievements in a different light, a light of the COVID glaze. Lab research was at bay for a while, challenges to return and re-start experiments, no scientific meetings in the traditional format where you build your new collaborative net at coffee breaks. Despite all, the team has expanded and we welcomed Ellen and Erin in October.

The NCRC Winter Symposia is a lovely way to wrap the year putting together all hard work and look at the progress done so far. We have an exciting project that has two arms: a blue-sky science and a translational. Working together John and Tom were able to generate promising results on understanding how small membrane-bound vesicles or exosomes can send signals from neuroblastoma cells to cells responsible for new blood vessels formation. They developed a protocol to scale up the production of exosomes, isolate them and characterise. We have a dataset on what these exosomes carry on and now can test how they promote new blood vessels formation. Indeed, more left to do but knowing the direction makes this journey meaningful.

Hallmarks of Research

Research is a fascinating journey no doubt. Inquisitive minds try to solve burning puzzles. It takes time. Some puzzles are more complected than the others. One of the hallmarks is the conversion of the resolved puzzle into a scientific story to tell to your peers.

We write and publish these stories. The publishing is another caveat that often makes your story sharper and neater. However, while you are in the process you feel that the mission is impossible.

Delighted to see that one of the missions is completed – a great hallmark for John which coincided with his new research adventure starting in a few days. This is his first first author paper! It is not tautology! It is his first original research paper where he is the first author. This position is a success measure in a research career. His teamwork skills secured him another few original papers. Well done John! Well deserved!

This study is an excellent example of the many roles that small RNA molecules such as miR-124-3p can play in neuroblastoma pathogenesis. The ability of this miRNA to work together with standard chemo drugs can be exploited further in the development of new anticancer therapeutics targeting relapse and drug-resistant tumours.

Preclinical models for neuroblastoma: Advances and challenges

What a great start for 2020! Our long-lasting and productive collaboration with our colleagues from Tissue-Engineering Research Group Brough to live an important overview of the preclinical models for neuroblastoma. We particularly focused on the 3D in vitro models available.

During this exercise of searching and reading research papers, we found that researchers in neuroblastoma are looking for alternatives of traditional 2D culture. It is may be slow at the moment but the interest is there.

Nolan et al, Cancer Letters 2020

3D neuroblastoma models worked well in both validating known chemotherapies and screening new. The concepts and materials that were initially developed for bone or tissue regeneration can be used to a miniature model of neuroblastoma.

3D tissue-engineered models can accelerate drug discovery and development, reducing the use of animals in preclinical studies.

Full version is available at https://www.sciencedirect.com/science/article/pii/S0304383520300239?via%3Dihub

Scientific part of my journey

Reading my posts, it looks like I am more enjoying the cultural part and almost forgot the main reason I crossed the Atlantic with the Fulbright wings.

The first month in the lab was more a warming up. Where is my desk? Where is the cell culture rooms? How do they run it? How different is it? So, many microscopes – am I capable of imaging? And so on and so forth…

My typical day starts at 8-8.30 am and finishes once all is done. It may be 6pm or 10pm. Once the experiment is set up, I have to monitor cells every 24 hours for 5-7 days with no weekends or days off. The monitoring includes imaging. Lots of imaging. Every condition has 20-30 single cells to follow up. Each cell gets its own GPS tag manually to be able to image exactly the same cell as it grows and becomes a group of hundreds by multiplication. For example, I am running 8 different cell lines in 3 experimental conditions. So, 20-30 cells per all 24 combinations give us 480-720 individual cells to follow up. The imaging takes ~5 hours every day. After 5 days, I will have 2400 – 3600 pics of cells to analyse. It will be fun! I may need lots of Guinness to fly through that numbers.

Tagging cells. The left arrow points to a group of neuroblastoma cells. The arrow in the middle point to the same cells, but this image allows you to see the actual number of the cells. This group has 8 cells. The right arrow points to individual GPS tags for each cell

At the next step, I will select some of the conditions for video recording to trace cell fate from a single neuroblastoma cell to a metastatic niche consisting of hundreds of them. This video will show me how it all happens minute after minute.

Is not it exciting? I am thrilled!

 

8th OLCHC RESEARCH & AUDIT CONFERENCE

This was our 2nd time attending the OLCHC Research & Audit Day on May 25th, 2018. The conference provides a great forum for paediatric clinicians to share and update knowledge across different specialties through talks and poster presentations. It is insightful for basic biomedical researchers like us to see other perspectives.

I was delighted to know that two our studies were shortlisted. It is a rewarding feeling to see your Dream Team doing very well. One was the project of the Erasmus+ student Hanne Pappaert and the other was the project of NCRC funded Postdoc John Nolan. Hanne explored our 3D tissue-engineered model of neuroblastoma using collagen-based scaffolds with distinct mechanical properties. These new scaffolds were designed and manufactured by our collaborator Dr Cian O’Leary from Pharmacy Department and Tissue Engineering and Research Group (TERG) headed by Prof Fergal O’Brien. Hanne grew 5 neuroblastoma cell lines on the 3 scaffolds: hard like a rock, soft and fluffy like a cotton wool and a jelly-like. All cells liked the jelly-like environment. This environment is similar to bone marrow – the most common site of neuroblastoma metastasis. We were excited to see the difference as it means we are one step closer to reconstruct this type of tumour spread.

John has expanded our exploration of our 3D neuroblastoma model by examining the content of exosomes – little parcels sent by cancer cells in 3D and as tumours grown in mice.  We were thrilled to see a high similarity in the exosomal content. This finding additionally proved the great applicability of our 3D model as a tool to study neuroblastoma.

 

How current IT advances help in research?

Here is the perfect example of the teamwork troubleshooting protein extractions. My Dream Team 2018 in action. The current information and communication technologies allow to stay connected and respond quickly.

Five minutes later in the lab: troubleshooting is the exchange of experiences!