#IrishResearchCouncil – Blog about neuroblastoma research

#JournalClubwithRabia: How Can Fish Help Us Study Anticancer Drugs?

Hi all! Rabia here, I came across an intriguing paper highly relevant to my work on the rapid in vivo validation of HDAC inhibitor-based treatments using neuroblastoma zebrafish xenografts. The study outlines a zebrafish neuroblastoma yolk sac model specifically designed to evaluate both the effectiveness and toxicity of histone deacetylase (HDAC) inhibitors.

HDAC inhibitors are drugs that target specific enzymes involved in gene regulation. This study tested broad-spectrum HDAC inhibitors as standalone treatments and combined them with doxorubicin, a well-known chemotherapy drug.

But why on Zebrafish? The zebrafish model provides a rapid and efficient means of testing these treatments, offering valuable insights into their potential use in combating neuroblastoma. This model allows for assessing drug efficacy and helps understand the associated toxicities quickly, making it a powerful tool for developing new anti-cancer therapies.

In the study, fish larvae were implanted with fluorescently labelled, well-established neuroblastoma cell line (SK-N-BE(2)-C) and patient samples (HD-N33, NB-S-124) to grow tumours. Non-cancerous cells (VH7 fibroblasts) were utilized to verify that tumour progression in zebrafish was specific to tumour cells. The engraftment of human cells into fish larvae was confirmed by immunohistochemistry (IHC) staining on zebrafish sections injected with neuroblastoma cells (SK-N-BE). This was achieved using a STEM121 antibody that reacts specifically with a human cytoplasmic protein. The findings showed that pediatric tumour cells survive and grow in the zebrafish model at rates like those observed in human tumours.

Before testing drug efficacy in zebrafish xenografts, optimal drug concentrations and maximal tolerated doses (MTD) were determined. Toxicity tests were conducted by treating fish larvae cells for three days without tumour cell injection to identify the maximum tolerated dose that did not cause observable morbidity, changes in morphology, or severe aberrations in larval behaviour. and lethal dose (LD) for each compound. To find optimal drug concentrations, larvae with xenografted tumour cells were incubated with increasing drug doses 24 hours post-implantation to the maximally tolerated dose (MTD). The relative IC50 values were then determined based on changes in tumour mass volume.

To evaluate the treatment, SK-N-BE(2)- cells were used to test the broad-spectrum HDAC inhibitors, including panobinostat, vorinostat, and tubastatin A, both alone and combined with doxorubicin. The partial response rate (PR) was measured to see how well different drug combinations work to shrink tumours in the zebrafish model. Here’s what they found: Doxorubicin combined with panobinostat resulted in a 23% PR, Doxorubicin combined with tubastatin A showed a 31% PR, and Doxorubicin combined with vorinostat achieved the best result with a 36% PR.

To test the effectiveness of the HDAC inhibitor treatment, they monitored the tumour growth using a confocal microscope before and 48 hours after giving the drug. The test revealed that a 48-hour treatment of SK-N-BE (2)-C zebrafish xenografts with vorinostat and doxorubicin alone, `and in combination, increased cell death. The combination of these two drugs was the most effective, causing a significant increase in cancer cell death (apoptosis) by decreasing cell proliferation, as indicated by reduced PPH3 marker and activating the number of Cleaved caspase-3 (Figure 1).

**Figure 1:** Treatment for 48 h with Vorinostat, doxorubicin, or a combination of both increased the amount of cleaved caspase-3 and reduced mitotic tumour cells. Adapted from *Pharmaceuticals* **2020**, 13(11), 345

In essence, this study validates the use of HDAC inhibitors in treating neuroblastoma and paves the way for broader applications of zebrafish models in cancer research. As we look to the future, these innovative models could significantly enhance our ability to develop effective cancer therapies, making strides towards better treatments and, ultimately, more effective cures.

Written by Rabia Saleem

#JournalClub with Ronja: What can we learn from other cancers?

In this row of journal club blog posts, I’ve decided to look at this study: A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy.

In this study, researchers developed an advanced model to simulate the environment surrounding pancreatic cancer cells. Using a specialized hydrogel matrix, they encapsulated pancreatic cancer cells, patient-derived stromal cells (non-cancerous cells that influence tumour behaviour), and immune cells. Within this matrix, the cells grew and formed spheroids, closely resembling the structure of tumours in the body. By fine-tuning the hydrogel’s properties, they controlled the stiffness and adhesion, optimizing conditions for cell growth and interaction, thereby enhancing the model’s resemblance to real-life pancreatic cancer. The researchers tested this model to evaluate its effectiveness in assessing new treatments, particularly immunotherapies. They treated the 3D cultures with a combination of immune and chemotherapy drugs and monitored the cells’ responses (See Figure). Notably, they focused on the novel drug ADH-503. Their findings revealed that the model accurately mirrored the responses observed in actual pancreatic cancer patients, confirming its validity for preclinical drug testing.

Furthermore, they explored the impact of these treatments on the secretion of cytokines—proteins crucial for immune regulation and tumour progression. They observed changes in the levels of specific cytokines (IL6 and IL8), indicating that the treatments could alter the tumour microenvironment and potentially improve therapeutic outcomes.

Figure 1 Multicellular spheroids of pancreatic cancer, patient-derived fibroblasts and immune cells stained for cell nuclei(blue), cytoskeleton and proliferating cells. From left to right, (i) untreated control, (ii) ADH-503 and immunotherapeutic, (iii) ADH-503 and chemotherapeutics and (iv) ADH-503 and both immuno- and chemotherapeutics. Modified from *Adv Healthcare Materials, Volume: 12, Issue: 14, First published: 23 November 2022, DOI: (10.1002/adhm.202201907)*

Overall, this study highlights the utility of their model for testing new therapies and gaining insights into the complex interactions within pancreatic tumours. It provides a robust platform for further research to develop more effective treatments for pancreatic cancer. While the study primarily focuses on pancreatic cancer, its findings and methodology have significant relevance to neuroblastoma research. Like pancreatic cancer, neuroblastoma is a solid tumour with a complex microenvironment that influences its growth and response to therapy. Thus, the model developed in this study, which accurately mimics the tumour microenvironment and allows for the testing of immunotherapies and combination treatments, could be adapted for neuroblastoma research. More directly relevant is the combination therapy of ADH-503 with immunotherapy and chemotherapy, underscoring the potential for this approach in treating other types of solid tumours like neuroblastoma. For my project, specifically, this study is helpful because it shows the relevance of immunotherapeutics on the immune cells present and their behaviour, which I plan to investigate for neuroblastoma in the future.

Written by Ronja Struck

Class 2024: Congratulations to Ciara, Ellen and Rabia!

Massive congratulations on the official moulding of PhD and MSc by Research to our promising young scientists: Rabia Saleem, Dr Ciara Gallagher and Dr Ellen King! Great accomplishments!

Three different journeys, with two through the COVID-19 pandemic. The full range of ups and downs. Who said that the PhD is a straight line? It has never been. It is more like the Irish weather: some days are sunny and bright, and some have scattered showers, gale winds and stormy snow, with sunshine developing elsewhere. The journey was spiced up with publications, conferences, travels, days out and fundraising events with the team.

It is a proud moment for me as well. 🙂 Three PhD and one MSc by Research students graduated within the last 12 months.

Of note, Ellen was behind our Twitter activities in the past, making our team visible!

Wish you all the best of luck on your new adventure!

Olga Piskareva

How things work in science: Gene editing technology

Few advancements in biomedical sciences hold as much promise for revolutionising cancer research as CRISPR-Cas9. This ground-breaking gene-editing tool has sparked a wave of innovation, offering precision and efficiency in manipulating the human genome in the fight against cancer.

Now, what is it? CRISPR is basically an acronym for a very long name Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 or CRISPR-Cas9 for short. It was found in simple organisms such as archaea and bacteria. Interestingly, this is a component of bacterial immune systems that can cut DNA. So, this feature was proposed for use as a gene editing tool, a kind of precise pair of molecular scissors that can cut a target DNA sequence. So, the CRISPR-Cas9 scissors allow us to precisely edit the DNA sequence of living organisms by adding in (knock-in) or removing (knockout) a gene of interest.

For cancer research, for example, the CRISPR-Cas9 scissors can be used to introduce therapeutic genes or correct mutations associated with cancer predisposition syndromes. Meanwhile, those scissors can also disrupt genes involved in treatment resistance, sensitising cancer cells to existing therapies.

Jennifer Doudna and Emmanuelle Charpentier have won the 2020 Nobel Prize in Chemistry “for the development of a method for genome editing.”. A nice accompanying piece was published in The Conversation, highlighting the history of these scissors and the politics behind it.

Jennifer Doudna explains this revolutionary genetic engineering tool in a TED lecture. However, she warns:

“All of us have a huge responsibility to consider carefully both the unintended consequences as well as the intended impacts of a scientific breakthrough.”

I hope you enjoyed it!

Witten by Rabia Saleem

Congratulations to Dr Ciara Gallagher!

Huge congrats to a newly minted Dr Ciara Gallagher! She defended her PhD on March 8 – International Women’s Day. Your enthusiasm and perseverance are truly fascinating! May this be the stepping stone towards a brighter future, Ciara!

We thank examiners Dr Marie McIlroy (RCSI) and Prof Jan Škoda (Masaryk Uni) for the time and expertise they provided.

We also thank the Irish Research Council for their generous support!

Dr Ciara Murphy (Chair), Dr Olga Piskareva (Supervisor), Dr Ciara Gallagher, Prof Jan Skoda (examiner), Dr Marie McIlroy (Examiner)

How things work in Science: Classifiers

For our next little series introducing a different thing in science and how it works every week, I decided to focus on classifiers. With artificial intelligence becoming more and more prominent in our daily lives as of late, I thought this would be a good lead into the explicitly science-focused topics to come. So, what is a classifier? How does it work? And why does it matter?

At their core, classifiers are algorithms designed to categorize input data into predefined classes or categories. They learn patterns and relationships from labelled training data to make predictions on new, unseen data.

Once features are extracted, identified and quantified from labelled or annotated input data, mathematical models are employed for pattern recognition and predictions.

These models can range from simple decision trees to complex neural networks, each with its own strengths and weaknesses.

Training these models is an iterative process. That means to produce one good classifier, lots of classifiers were created in the process: Every time the pattern recognition is run, the annotated data is categorised by the classifier and compared to the annotation class. Prediction errors are corrected, and performance is optimised. This whole process is one iteration. How many iterations are required for a well-trained classifier varies widely and is largely dependent on the input data and application. For my tissue classifiers, it took up to 20,000 iterations.

Classifiers use these models to categorise unseen data into categories the user-defined at the start. In the figure, you can see my annotated histological slides from which the classifier extracted patterns to then classify the rest of the slide and entirely unseen slides into tumour (red), stroma (green) and background (blue) classes.

From identifying fraudulent transactions, filtering out junk mail, targeted advertising, and facial recognition to unlock your phone or diagnosing diseases, classifiers play a vital role in automating decision-making processes and driving advancements across a wide range of industries. Keep your eyes peeled, and you can find more classifiers in action all around you.

Written by Ronja Struck

Women in Science: Rosalind Franklin

On a blog post series of Women in Science by a Cancer Bioengineering lab, you didn’t think you were going to get around reading about Rosalind Franklin, did you? In recent years, she finally started to receive the acknowledgement she is owed, placing her all the way up there in terms of famous scientists with Marie Curie and Albert Einstein.

As mentioned by Ellen last week, there were only ever 13 women to win the Nobel Prize in Physiology or Medicine. But in 1962, it was erroneously bestowed upon three men for the scientific breakthrough of a woman. This blog post would just as well fit into a true crime in science series.

All down to misogyny and a single piece of evidence: Photograph 51, an X-ray crystallography of the structure of DNA viewed perpendicular to the DNA fibre axis, revealing the double helix structure.

You will likely all remember James Watson and Francis Crick from biology classes in school. You were probably taught that they figured out the structure of DNA.

But at the same time, they worked together in Cambridge, Franklin was working together with her PhD student Raymond Gosling at Kings College in London. Forced to work alongside Maurice Wilkins, who did not take well to her confident, goal-oriented ways, which led her to criticise her well-respected peers and dared to interrupt and correct them.

Leading to her downfall in the race with Watson and Crick was that Franklin complied with her understanding of scientific ethics, and rather than rushing to publish her findings, she sought to verify them and replicate the findings in Photograph 51.

Betrayed by her colleague Wilkins after ample tensions over the years. He passes her priceless finding to the competition, allowing Watson and Crick to model the double helix, publish a breakthrough paper and relegate her to a methods paper in the same issue of the nature journal.

Tragically, her young death at 37 from ovarian cancer prevented her from witnessing the Nobel Prize being awarded for discoveries in the molecular structure of DNA four years later. Which in 1962 was not to be awarded posthumously. Instead, her reputation for years was dominated by the more than unflattering recollections in James Watson’s biography. The book clued the public into the crimes the three men committed. All the while tarnishing Franklin’s name, portraying wildly misogynistic images and downplaying her indisputable contribution to science. Only after society as a whole changed its views on women and misogyny did perceptions of Rosalind Franklin and James Watson finally get corrected.

Today, Rosalind Franklin’s legacy stands as a symbol of tenacity, intellect, and an unyielding spirit that will inspire generations to come. It is about time that science books get rewritten to remind us that those are the virtues we should hold in high regard. Rather than the yearning for glory and a legacy that we see in the pressure to publish, the chase of impact factors never intended to rank journals and scientists but as a tool for librarians and the impossible climb through academia, forcing impossible expectations on principal investigators to take on endless students and responsibilities. Let’s take this opportunity to refocus and make sure it’s the pursuit of knowledge and answering questions that drive science forward that determine our decisions.

Written by Ronja Struck

Navigating Time and Tasks in the PhD Journey – The Struggles of Autonomy

Embarking on a PhD is an exhilarating endeavour. It offers the freedom to structure one’s own time. But this autonomy can be a double-edged sword; while providing a sense of flexibility and leisure, it also presents challenges in managing time effectively, prioritising tasks, and maintaining a productive schedule. In the context of a PhD, self-discipline and efficient planning quickly become the guiding stars of success.

The absence of rigid working hours requires a strong sense of self-motivation and discipline to stay on track. Without proper time management, it’s easy to fall into the trap of leisurely indulgence, neglecting the essential tasks and milestones that shape the PhD journey. Never before did I appreciate nagging parents, teachers or just people to which you could outsource motivation and feedback as easily. In a PhD, you’re on your own. You’re the only one who truly cares that what you’re working on is getting done. Done well and done at the right time. There is your supervisor, of course, and maybe collaborators. But it is not their job to stand behind you and say have you done this yet or that yet. They don’t see how much work you do or don’t do in a day. No one tells you to get off your arse when you’ve just stared at a blank screen for 20 minutes, and no one tells you to give it a rest when a simple problem turns out to be far more time-consuming and exhausting than expected because things still need to be kept moving. In the end, you can only rely on yourself to tell you whether you have worked enough or not. No one else knows. That can be extremely motivating and similarly defeating when you feel like you’ve done nothing but work for a couple of weeks and the results still aren’t there, so it seems like it doesn’t make a difference.

To conquer the time management challenge, prioritisation becomes paramount. As a PhD student, the spectrum of tasks can quickly seem overwhelming. Between different avenues and tasks that would progress your project, keeping up with writing, creating figures for adjacent projects, producing posters and presentations for conferences, writing blog posts, and making videos for funders and meetings, there always are more things to do in a day than could possibly be crammed in on the most productive of days. Figuring out how to manage urgency and importance becomes crucial to staying afloat. Identifying the most critical tasks and allocating time accordingly ensures progress and prevents the accumulation of unfinished work.

Maintaining a reasonable schedule becomes a balancing act. Especially when you pepper a couple of meetings in the very early morning because your collaborators are in a different time zone. And yet creating and adhering to a schedule is the foundation of effective time management. Despite the constant changes and different requirements, I find it helps immensely to establish a routine to cultivate discipline and maintains an easy overview over the week to allow myself to check what has been achieved and how long it took, so I can gauge how much more I need to do or whether I get to relax and leave half an hour early another day. It is crucial to strike a balance between focused research, data analysis, writing, and personal well-being. Regularly reassessing and readjusting the schedule as priorities shift guarantees that all aspects of the PhD journey receive the attention they deserve.

Navigating the realm of a PhD requires a delicate dance between self-motivation and effective time management. While the allure of autonomy can be tempting, the importance of prioritising tasks and maintaining a schedule cannot be understated. By striking a balance between work and personal well-being, the PhD journey can be transformed into a harmonious symphony of progress and achievement. Well, that’s the idea anyway.

As you embark on your own PhD adventure, you realise every day that time is a precious resource, and effective management is the compass that guides you toward success.

Written by Ronja Struck

So this is science..?

Had you told me before I started my PhD that I’d rushedly be writing a blog post on a bus in Bergamo, and it’s all part of my project, I certainly would have laughed and figured sure, maybe as a one-time exception if I find out something fascinating. But no, this is my second conference abroad this year, out of five in the past 4 months. My view on science and what is important to conduct good science has significantly changed since then, though. I have a ton of data from my secondment to Vilnius, but it is not all analysed yet. There are a number of decisions left to be made before my project becomes fully rounded and provides useful conclusions that I could share with people. But conferences serve another purpose. If everyone was only there to present their finished project, who would they present them to? At the current stage of my research, exchanging ideas, receiving feedback and seeing what others do helps immensely to provide perspective and both motivate me to do more and do better, inspire me to find new angles and also to relax and understand the bigger picture your project is a part, rather than getting bogged down by the day-to-day issues that so easily cloud your mind in everyday routine (as far as a PhD allows for routine…). In this way, conferences can shape a project, inform analyses and provide far more than an excuse to be out of the office.

Even more enjoyable are, of course, conferences when they’re held in such beautiful places! I’d never been to Barcelona or Milan. While I have no intention of making the cultural metropolises of Athlone and Limerick pale in comparison, it does feel different when adding an afternoon of sightseeing, includes a couple of centuries-old towns that look like they fell out of a fairy tale and churches built in the 13 hundreds in 20 degrees in March rather than freezing your fingers off after just an hour outside or seeing some trees and an old pub. I never thought science would facilitate me seeing the world, but I am delighted that it does. And while I never would have expected it before, I can now appreciate the value of presenting your project halfway to ensure that it’s the best it could have been when it’s done.

Presented my project at the European Association of Cancer Research Conference on National Pathology because I was awarded the Organisation of European Cancer Institutes (OECI) travel grant. So, I could enjoy some of the stunning views in Bergamo and even visit Milan.

Written by Ronja Struck

Work-Life Balance as a final year PhD student

Hi everyone,

If you haven’t met me before, my name is Ciara. I’m a final year PhD student in the Cancer Bio-engineering group. My research focuses on using a 3D model of neuroblastoma to uncover pathways that cause cancer cells to invade other parts of the body, in a process we term ‘metastatic spread’. As a whole, I really enjoy my research and have a keen interest in the topic, making it easy to stay motivated and driven. However, as a final-year student, my lab days can be long and labour-intensive; unfortunately, experiments can sometimes span the weekends. This makes work-life balance hard to achieve. I have come up with three effective strategies that help me manage my workload and still enjoy my PhD work while taking the necessary time to recuperate. I would love to share them with you.

Make a fun recipe – After a stressful day at work, plan a yummy dinner meal. While science experiments can fail, recipes only ever end with a delicious reward of a belly hug after a long day. Making a home-cooked meal helps me take my mind off the day’s stresses and fill myself with nutrients that keep my immune system strong. Check out some of the delicious recipes I’ve made so far.

2. Attend conferences. Conferences are a great way to meet researchers on the same journey as you. They can help keep you motivated and trigger new ideas for your own research. My most recent conference was in Athlone. While I enjoyed presenting my research and listening to talks, I also took some time to make new friends from other universities and explore the history of county Westmeath – for example, having a Guinness in the oldest bar in Ireland (they have a Guinness world record to prove it).

3. Plan a holiday to work towards – There is no better motivation to complete your work than jet-setting off to explore a new country. I aim to take a short science break every 4-6 months. My most recent one was to the snowy Italian mountains for a week of skiing. I came back with lots of fun memories and laughs, feeling ready to launch into another few months of hard work.

Ski hols 2023

Written by Ciara Gallagher