The RCSI Cancer Bioengineering Group hosted an in-person event during the National PPI Festival 2024 to share their childhood cancer research and connect with the public and patients.
We welcomed members of the public, family members of children with cancer, researchers, clinicians, and patient/community organisations on October 17th. Our past lab members and students paid a visit, too! Our group shared ongoing research on neuroblastoma biology and finding new treatments. Prof Cormac Owens from CHI brought us through the journey of clinical trials in neuroblastoma patients. We heard the heartbreaking story of the brave young man who lost his life to neuroblastoma and his parents who never gave up. This truly inspirational family founded a charity – the Conor Foley Neuroblastoma Cancer Research Foundation, to support curiosity-driven and translationally-focused research. The Foleys know very well how important it is to return happy days to kids and their families.
As a new PhD student, I’m incredibly excited to dive into cancer research, and what better way to kick off this journey than by exploring 3D models to study neuroblastoma metastasis? Neuroblastoma is one of the most common childhood cancers, and about 50% of patients have metastatic disease at diagnosis. Understanding how these cells spread is key to developing better therapies, which is why this recent study by Gavin et al. (2021) caught my eye.
So, what did the researchers do? They used something called patient-derived xenografts (PDX) and cell lines to grow organoids (tiny mini-tumors) in a 3D extracellular matrix (ECM). This ECM mimics the environment these cells would encounter in the body, which is super important because cells behave very differently in 3D than in the typical 2D Petri dishes. It’s like giving the cells an entire landscape to explore rather than just a flat road—suddenly, they have mountains to climb and valleys to cross, allowing them to behave much more like they would inside the body!
One of the coolest things about this study is how the neuroblastoma cells developed various invasion strategies based on their environment. Some stayed in tightly knit groups, while others decided to go full-on lone wolf, sending out long, thin projections to explore the surrounding matrix. These cells are smart-adapting to different ECM compositions like Matrigel (which is rich in laminin and collagen), made them change their behaviour entirely. It’s like they’re navigating an obstacle course, with each new challenge requiring a different tactic!
Let’s Talk Actin Filaments!
Now, this is where it gets super cool (and nerdy in the best way!). The images captured by confocal microscopy are stunning. They show actin filaments—the internal skeleton of the cells—as they help the cancer cells move and invade new areas. The actin filaments form these amazing, intricate networks that shape the cells and allow them to stretch and invade. It’s almost like watching tiny construction workers build bridges and tunnels as they move forward. Check out this confocal image showing the red filaments—how awesome is that?!
It’s the second round of journal club blog posts, and this time around, we’ll be looking at papers published by this very lab. I’ll be focussing on the paper in which the cell line I am currently working with (KellyCis83) was developed: “The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro” This research addresses a critical challenge in cancer treatment: drug resistance, of course focusing on neuroblastoma, pediatric cancer notorious for its aggressive nature and poor prognosis that this lab has been studying for years.
Neuroblastoma is typically treated with cisplatin, a potent chemotherapy drug that induces DNA damage in cancer cells, leading to their death. The issue is that over time and particularly during relapse, some neuroblastoma cells develop resistance to cisplatin, rendering the treatment ineffective. Understanding the mechanisms behind this resistance is crucial for developing new therapeutic strategies.
In this study, neuroblastoma cell lines resistant to cisplatin were created by gradually exposing the cells to increasing drug concentrations over 6 months. This approach mimics the clinical scenario where tumours are exposed to chemotherapy over an extended period, eventually leading to resistance. The resistant cell lines were then characterized to uncover the molecular changes that had occurred alongside or as part of the increased drug-resistance.
The cisplatin-resistant neuroblastoma cells exhibited significant disruptions in their cell cycle regulation, as highlighted by the most altered pathways identified by mass spectrometry. Cisplatin typically causes DNA damage that halts the cell cycle, leading to cell death. However, the researchers found upregulated pathways in resistant cells that allowed these cells to bypass this damage-induced arrest. One key finding was the identification of Vimentin upregulation in the upstream regulator analysis. Vimentin is a marker typically associated with epithelial-to-mesenchymal transition (EMT).
EMT is a process where epithelial cells acquire mesenchymal, fibroblast-like properties, including enhanced motility and apoptosis resistance. The link between EMT and cancer progression is well-established, as EMT not only facilitates metastasis but also contributes to drug resistance. In the context of neuroblastoma, the upregulation of Vimentin and dysregulation of related EMT proteins found in two of the resistant cell lines (specifically SNAI1 and TWIST1) suggests that these cells are not only evading cisplatin-induced cell cycle arrest but are also acquiring more aggressive, invasive characteristics. This links back to their findings on invasiveness, which showed greater levels in the two resistant cell lines that also had greater changes in EMT-related proteins (Figure 1).
Figure 1 A Relative invasiveness of the parental cell lines compared to the cisplatin resistant daughter cell lines. Graphed data represent mean values ± SD of three independent experiments. Asterisks indicate statistical significance obtained using a paired Student’s t-test. * p < 0.05, ** p < 0.01, ***p < 0.001, n = 3 for all experiments. B The fold change in protein expression of drug resistant cells compared to their parental counterparts was quantified by densitometric analysis of two biological repeat experiments, normalised against endogenous control ACTB. (Adapted from (Piskareva et al., 2015).
Understanding the role of EMT in cisplatin-resistance opens up new avenues for therapeutic intervention. Targeting EMT-related pathways Vimentin could potentially restore the sensitivity of these resistant neuroblastoma cells to cisplatin, by targeting the evasive mechanisms the cells developed to bypass the cell-cycle disruption. Such therapies would offer a new strategy to tackle drug-resistant relapse cases, which currently have very poor outcomes.
Overall, this study provides a valuable model for investigating drug resistance in neuroblastoma and highlights the crucial role of EMT and its associated pathways in finding ways to treat drug-resistant tumours. As we continue to explore these avenues, these models will serve us as a strong foundation facilitating the research currently taking place in our lab towards finding such combination therapies and hopefully improving outcomes for children battling this devastating cancer in the future.
Cancer is the 2nd most common cause of death among children after accidents.
Childhood cancer is an umbrella term for many other types of this disease. Every September, many charities, researchers and parents of children with cancer work hard to raise awareness of this cancer. You may learn more about kids with cancer, their loving families, the doctors and caregivers who look after them and treat them, the young survivors of cancer and those kids and teens who lost their battle, and the scientists who work hard to find a way to stop childhood cancer.
This year, our research team will run the Pub Quiz on September 18th, 2024, in honour of Childhood Cancer Awareness Month. All donations will go to the Conor Foley Neuroblastoma Research Foundation (CFNRF).
If you would like to get involved in this amazing challenge and help us raise vital funds for childhood cancers, you can contribute to our fundraising page:
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 Pharmaceuticals2020, 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.
Exosomes have been a groundbreaking field of research due to their hypothesized ability to create a pre-metastatic niche through non-cancerous cell manipulation. They achieve this by relaying oncogenes and proteins from cancer cells in their molecular “cargo”. In this study, Morrissey et al. examined the role of tumor-derived exosomes (TDEs) in increasing programmed death ligand-1 (PDL-1) expression in macrophages. In pre-metastatic tumour microenvironments, there is an increase in inflammation and an immunosuppressive response, thus promoting tumorigenesis. PDL-1 is increased by TDEs manipulating the signalling of toll-like receptor 2 (TLR2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), which increases lactate production and glucose uptake in an event called the “Warburg Effect”, which is common in tumour microenvironments. TDEs taken from lung cancer samples were injected into mice, and PDL-1 expression levels of macrophages were higher than in healthy lung samples.
To accomplish this, Lewis lung carcinoma tumour cells (derived from mice) labelled with a green fluorescent protein (LLC-GFP+) were injected into mice with tumours; seven days later, they were injected with either non-GFP LLC exosomes or control mouse lung epithelial cell exosomes (MLE-12). The mice injected with non-GFP LLC exosomes had no metastatic difference compared to the control mice. However, the mice injected with LLC-GFP+ exosomes had an increase in micro-metastases, which was confirmed using fluorescent microscopy to observe LLC-GFP+ occurrence. With confocal microscopy, they observed an increase in PDL-1 expression in lung tissue compared to the control mice. These findings suggest that PDL-1 expression is preferentially increased in lung tumour tissue by injecting LLC-GFP+ exosomes.
Figure A. Schematic of experimental design. Figure B. Micro-metastatic burden quantified by LLC-GFP% in the lungs of s.c. GFP-LLC tumour-bearing mice treated intravenously (i.v.) with MLE-12 or LLC exosomes. Representative dot plots of GFP expression in the lungs and summarized data of LLC-GFP% are shown. Each dot represents data from one mouse. *p < 0.05, one-way ANOVA with multiple comparisons. Figure C. Confocal microscopic analysis of lung GFP+ micro-metastases from primary s.c. LLC tumor-bearing mice. Figure D. Confocal microscopic analysis of PD-L1 expression in LLC Exo- or LLC Exo+ -treated lungs. Adapted from Morrissey et al.
Researchers also examined the clinical significance of human TDEs and their manipulation of macrophages in the lymph nodes of non-small-cell lung cancer (NSCLC) patients. They found that there was increased expression of PDL-1 in the lymph nodes of these patients. To further study this, the researchers treated macrophages with either MLE-12 or LLC exosomes and then put them in a co-culture with OVA transgenic T-cells (a type of immune cell). The T-cells had a decrease in cell proliferation, which was later reversed by adding neutralizing α-PD-1 to the co-culture. This led them to think that PDL-1 had an inhibitory effect on T-cell function through macrophage stimulation. In summary, this study found that TDEs have an impact on PDL-1 expression found in tumour tissue and pre-metastatic tissue, resulting in creating an immunosuppressed environment for cancer cell growth. In my project, I’m interested in what cargo exosomes can carry from parental tumour cells to non-cancerous cells when creating a pre-metastatic niche. Knowing that there has been documented manipulation of the cellular makeup by exosomes encourages me to continue my research using neuroblastoma cells to elucidate the oncogenic markers relayed by exosomes. This research is very exciting, and I hope to shed some light on exosome impact in neuroblastoma tumour microenvironments with my own work!
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.
I have never been a runner. In my head, the word ‘marathon’ was linked to professional athletes and the Olympics or Athletics competitions. I could not imagine anyone doing a race as long as one gets guts. However, once I found the courage and motivation to explore my body’s limits. It was probably our team fundraising ‘Hell&Back’ challenge in 2019 that ignited that courage. We did many! Virtual VHI MiniMarathon, the Dublin Mountain Way in a Day, Cyclothone, to name a few.
Eventually, I decided to do it on my own in 2024. I booked the VHI Mini Marathon 2024 entry and started my training. Having many fitness apps at my disposal let me pick the right training plan. 10K sounded manageable. The fitness watch kept all my training records, so I got a very good understanding of my body potential and limits to do 10K.
On that sunny day, hundreds and hundreds of women were getting to the starting line. Each set their own ambition and target. I had three: 1) jogging from start to finish, 2) finishing within 1hr and 20 min, and 3) supporting the Conor Foley Neuroblastoma Cancer Research Foundation.
So, June 2nd was fast approaching, and I injured my calf during the Wicklow hike. However, giving up was not an option, but healing was required. So, I decided to stop running for a week and gradually get back on track.
The atmosphere was cheering and empowering, and the sun was bright and hot. The green ‘wave’ began their race at about 12:45. Running in a big company was easy. It is less doubtful if you are in a group of allies. Distance and time flew. I was sinking into the diversity and variety of running women and supporters along the road, enjoying every minute. Some took over me, and I took over some. I stuck to my training pace to ensure my power and energy were not draining quickly under the sun. My fitness watch counted the distance, heart bit and steps, showing that I was slightly slower on average than during my training. I decided not to break the limits and kept running, listening to my body. Somewhere at the back of my head, I hoped to speed up at the end, if any. Unfortunately for me, the final 1-2 km were up the hill, so the slop ate my efforts.
Being a researcher puts every experience in perspective. We tend to analyse the flow of any information, sometimes unconsciously, by asking questions and reflecting. One of the running advantages is that you see an accomplishment right now at the finish line. Your fitness watch provides all the data to plan and complete a given challenge with very good accuracy. It is not about luck. It is about your trust in your body and mind. You are in the driving seat! Delighted with my accomplishment!
So, what is next on my to-do list? Definitely another race, very likely Run in the Dark.
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!
Little did I know about hikers when I moved to Ireland in 2004. Who they are and how they get around. My knowledge was limited to Rosalind Franklin’s love of hiking. I could not even imagine that one day I’d try their shoes.
However, things have changed since then! Spiced by the COVID-19 pandemic and various fundraising activities inspired by my team, my daily walking transformed into regular one-day hiking here and there. Luckily, my spouse shares the same attitude. So, we decided to explore longer walks one day.
The first go was the Dublin Mountain Way (42 km) in a Day. We started in Glensmole-Tallagh on a dry and sunny morning and finished in Shankill in the dark and pouring rain with a short recharge at Johnny Foxes. We were delighted with ourselves and raised the bar.
So, last week, we attempted the Wicklow Way. After studying the route, accommodation options and our fitness, we agreed on three days of walking in the north-to-south direction and 2 nights of sleep in B&Bs. We also monitored the weather forecast to make the most of this adventure. So, May 10-12 were the best. However, accommodation became a quest. Nevertheless, luck was on our side, and we found two nice places: one was near Roundwood, and the second was in Glenmalure.
Early morning of May 10th, we cheerfully started our journey in Marley Park. The day was fab; the topics for a chat were endless. We were walking away from Dublin. My fitness watch counted the steps and kilometres. During the walk, we got a confirmation that our accommodation in Glenmaluer had been upgraded to a room with a shower. Happy days!
37 km later, we arrived at our first B&B. It was actually a fancy hostel where all the guests walked in socks, parking their heavy boots in the lobby. It was the night of aurora borealis, but we did not know about it. We were tired and fell asleep before 11 pm. The next morning, none of the guests shared any pics or insights. Apparently, everyone was knocked down by the long day in Wicklow.
May 11th. Fueled with a tasty Irish breakfast, we said “Goodbye” to our hosts and headed further. While walking slowly to warm up for a long day ahead, I noticed that my calf was strained and walking downhill had become unpleasant. Where did it happen? I had no idea. We reached Glendalough around noon.
The day was warm, the car parks were full, and everyone enjoyed the beauty of Glendalough and the sunshine. We stretched our legs and backs on greens. During our light lunch, we discussed our options: 1) evacuation home or 2) continuing and hoping for the best. I did not give up. But our walking pace considerably slowed down.
We covered another ~15 km from Glendalough to Glenmalure and landed in the Glenmalure Lodge – the healing station for all hikers, cyclers, and bikers alike. People gathered outside, and strangers had cheat-chat sharing their tricks and tips for a better hike. Something adventurous was in the air.
Our friendly host picked us up at the Lodge and drove to their B&B. We stayed late and hoped to catch a glimpse of aurora borealis. The sky was cloudy. We saw nothing. While disappointed, our bodies cried for long rest, and we did not resist.
May 12 was the last leg of our journey. My calf did not improve. We took the shortest option to finish in Aughrim. I doubled the dose of painkillers. Then, we moved tirelessly, enjoying the sunshine and the forest.
This part of the Wicklow Way was mostly foresty. The forest was magical and a bit surreal. We agreed that it is perfect for various fantasy and horror movies. My fitness watch signposted that its battery was low, but it continued to count the steps and kilometres.
Overall, our hike had a fair amount of ups and downs. Some climbs and descents were quite steep before Glendalough. Then, they became more gradual, working well for my injured calf.
The luck was again on our side in Aughrim. We saw a taxi – a rarity in this area. The cheerful driver dropped us at the bus stop in Arklow. In 15 min, we were on the way to Dublin, relaxing and enjoying the peaceful countryside from the bus seat.
Our tally was 87 km in 3 days, fully recharged mind but worn body. Would I do it again? Absolutely!
