Tag: #JournalClub

#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 Shreya: Modelling Brain Tumour Spread

This article by Krieger et al. discusses the most common form of brain cancer called glioblastoma. Due to its highly aggressive nature, research must be conducted consistently and rapidly to develop new treatments. This has proven challenging due to primary tumours being resected before further research can be done, as well as the lack of current technologies to fully explore relationships between GBM and surrounding brain tissues. This study aimed to study the aforementioned interactions in under 4 weeks, accounting for the rapid progression of the disease in real life.  

GBM cells were first derived from four patients and treated with glutamine, heparin, epidermal and fibroblast growth factors, then underwent a sequence of manipulations, such as second-generation replication lentivirus infection of GBM cells, iPSC line 409b2 inoculation in Aggrewell plates and later manipulation with invasion assays, and scRNA sequencing, which, along with the Aggrewell cells, produced neural progenitor cell spheroids for analysis. Confocal microscopy and the developed image processing algorithm allowed for visualization of these cells following fluoroscopy and depicted consistent growth of tumour cells. There was also the growth of microtubules. Any dissociated organoids were then co-cultured with GBM cells again, promoting interaction between the two. Further analysis revealed the upregulation of 45 genes, including PAX6, GJA1, GPC3, and others involved in cell regulation.  

Credit to Teresa G Krieger, Stephan M Tirier, Jeongbin Park, Katharina Jechow, Tanja Eisemann, Heike Peterziel, Peter Angel, Roland Eils, Christian Conrad, Modeling glioblastoma invasion using human brain organoids and single-cell transcriptomics, Neuro-Oncology, Volume 22, Issue 8, August 2020, Pages 1138–1149

In conclusion, this novel mechanism of analysis of GBM cells using Aggrewell plates provided fruitful results, indicating intricate relationships between GBM cells and organoids, providing crucial insight for treatments by elucidating specific gene expression, heterogeneity of cells, and offering new targets based on ligand-receptor interactions. The particular relevance of this study to my work is regarding the usage of Aggrewell plates, which I am currently studying to determine how best to keep cells growing successfully within the wells. This article proves the usability and efficiency of Aggrewell and establishes its crucial role in the realm of brain cancer treatment research.  

Written by Shreya Sankar

#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

#JournalClub: Anti-Cancer Immunotherapy

Hi there, Federica here! In the fast-paced world of scientific research, staying informed about the latest studies and breakthroughs is crucial. It enables researchers to build upon existing knowledge, avoid redundant efforts, and discover new directions for their work. That’s why we’ve started a new series of blog posts highlighting recent papers and explaining their significance for our research.

Recently, a fascinating study explored an innovative method to boost the effectiveness of cancer immunotherapy: “A combination of a TLR7/8 agonist and an epigenetic inhibitor suppresses triple-negative breast cancer through triggering anti-tumour immune“.

The researchers investigated a combination of immune checkpoint blockade (ICB) and other drugs to turn “immune-cold” tumours (which evade the immune system) into “immune-hot” tumours (which the immune system can attack). They developed a special delivery system using nanoparticles called metal-organic frameworks (MOFs). These nanoparticles were loaded with two types of drugs—a TLR7/8 agonist and an epigenetic inhibitor (BRD4 inhibitor). To make the nanoparticles even more effective, they were coated with vesicles from the cancer cells themselves. This coating helps the nanoparticles specifically target cancer cells.

But how does it work?

The nanoparticles are designed to find and enter triple-negative breast cancer (TNBC) cells. Once inside, the drugs prompt the cancer cells to break apart and release signals that alert the immune system. These signals attract dendritic cells, which then activate CD8+ T cells—the body’s natural cancer fighters. The TLR7/8 agonist further enhances this immune response, making the treatment more powerful.

In both laboratory tests and animal models, this method showed significant promise. It not only slowed down tumour growth but also improved the body’s immune response to cancer. Importantly, the study found that this approach could remodel the tumour environment, making it more hostile to cancer cells. For example, they wanted to verify that their combined delivery system could really boost the body’s ability to fight tumours. They focused on a protein called calreticulin (CRT) that, when it shows up on the surface of tumour cells, helps the immune system spot and remove them. They found that when they used their special delivery system (CM@UN and MCM@UN), the levels of CRT on the surface of tumour cells went way up. This was especially true for the MCM@UN group, showing just how powerful their method was in getting the immune system to attack the tumours.

The original image was published in J Nanobiotechnology. 2024; 22: 296.

So, why is this study important for my work?

The principles of enhancing the immune system’s ability to fight cancer are central to both the research in the study and in my project. Like the nanoparticles in the study, mRNA vaccines can be designed to specifically target cancer cells, ensuring that the treatment reaches its intended destination. Another similarity is how the drugs activate the immune system, which parallels how mRNA vaccines work—by training the immune system to recognise and attack cancer cells.

I find this study really interesting as it sheds light on innovative strategies for cancer treatment and provides valuable insights that can inform and inspire our research on developing mRNA vaccines for childhood neuroblastoma!

Written by Federica Cottone