#JournalClub with Alysia: How exosomes can promote cancer spread?

Hi, it’s Alysia, here to give you some insight on exosomes’ impact on tumor metastatic niche formation! I found an interesting paper related to my work that I want to share with you: Tumour-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming.

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!

Written by Alysia Scott

#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

What Can VHI Women’s Mini Marathon Do For You?

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.

Written by Olga Piskareva

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

Walking the Wicklow Way, 87/129 km

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!

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 a new Dr in the house: Dr Ellen King

Huge congrats to a newly minted Dr Ellen King!  She passed her PhD viva on April 9. This is a testimony to your dedication, strong will and hard work. May this PhD be the beginning of many more successful endeavours, Ellen!

We thank examiners Prof Sally-Ann Cryan (RCSI) and Prof Joanne Lysaght (TCD) for the time and expertise they provided.

We also thank the RCSI PhD Programme for their generous support!

From left to right: Prof Joanne Lysaght, Dr Ellen King, Dr Olga Piskareva & Prof Sally-Ann Cryan

How things work in science: targeting cell components.

How do researchers study cells? How do we get the nitty gritty?

We use many methods to tag and chase various cell components. One of my favourites is fluorescent microscopy. It allows the use of nearly all spectrum of colours from blue to purple in one go. However, we prefer to narrow it down to 2-3 colours and avoid their overlap.

How does it work? First, we use DAPI or Hoescht, which are blue fluorescent dyes used to stain DNA. This way, we tag the nucleus of the cell. Then, we tag a protein of interest. In our case, it was MYCN, a protein that acts as a transcription factor. MYCN amplification is associated with poor prognosis in neuroblastoma. As a transcription factor, it binds to genomic DNA and is located in the nucleus. We used a specific antibody that was labelled with a green fluorescent dye. Look at the image below. The green colour pattern overlaps with the blue colour. Then, we tagged the cytoskeleton, a complex of various proteins that hold the cell architecture and dynamics. We used phalloidin with red fluorescence. It is a highly selective bicyclic peptide and a popular choice for staining actin filaments.

Neuroblastoma organoids stained with DAPI, Phalloidin and anti-MYCN antibody. This work was done during the Fulbright journey to Ewald’s Lab at Johns Hopkins

Now, we can enjoy visualising cells and test different research questions. For example, how do cells respond to a drug? Or how do neuroblastoma cells spread?

Written by Olga Piskareva

How things work in science: Scaffolding

At the Cancer Bioengineering Group, we use different types of scaffolds to mimic the 3D structure of tumours outside the body. We use these scaffolds to test new therapeutics and understand the tumour microenvironment. But I bet you didn’t think we had this in common with spiders?

Spiders make their webs by producing silk from specialized glands in their abdomen. They release the silk through spinnerets located at the back of their abdomen, then use their legs to manipulate the silk strands into intricate patterns, depending on the species and purpose of the web.

The process of web building begins with a scaffold. The specialized glands that spiders use are called spinnerets, and they produce liquid silk proteins that solidify into a thread when they come into contact with air. Using their many legs, spiders can manipulate the threads by changing the speed and tension they enforce on the silk, thus controlling thickness, stickiness and strength. They first lay a framework of non-sticky threads, known as scaffolding. And layer by layer, different species of spiders will add their own artistic sticky silk design to the scaffold depending on their aim. Take the deadly redback spider for example, these guys have a utilitarian approach to web building relying on their webs mainly for shelter and capturing prey. As such, they don’t put much effort into producing irregular and messy homes. In comparison, the orb-weaving spider produces “Mona Lisa”-like designs, with complex geometric patterns and intricate designs. The differences in effort seem to come from the environments in which the webs are located, with the redbacks choosing more sheltered environments and thus not needing much strength to their webs. Whereas orb-weaving spiders are more adapted to a range of environments, from forests to grasslands to urban gardens. So, while the redback gets a lot of attention for their neurotoxic venom, they need to step up their artistic skills to match that of their orb-weaving colleagues.

The redback spider and its webs are reminiscent of an aggressive tumour, which is erratic, dangerous, and unpredictable. We want to find the “anti-venom” for such tumours so we can wipe them out for good.

Watch this amazing web-building timelapse by BBC Earth.

Written by Ellen King

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)