The Di Vizio Laboratory investigates the molecular mechanisms of tumor progression to advanced disease in prostate and lung cancers, amongst others, with a significant focus on intercellular communication between tumor and stromal cells. Their current research aims to profile Large Oncosomes (LOs) and other cancer-derived Extracellular Vesicles (EVs). LOs are cancer-derived EVs originating from shedding membrane blebs and are associated with advanced disease. The Di Vizio lab uses advanced techniques to understand the internalization mechanism of LOs in recipient cells and identify novel biomarkers for liquid biopsy approaches.
The value of studying EVs in cancer disease and metastasis
Professor Di Vizio has a long-standing interest in studying cancer, particularly its aggressiveness, to better understand how tumors progress to advanced and metastatic disease. Her research group started about 15 years ago at Harvard University in Boston. There, she was granted an NCI Pathway to Independence Award to investigate the functional role of LOs in tumor progression, with a focus on tumor microenvironment responses both at the primary site and in other parts of the body. One of their long-standing hypotheses is that LOs contribute to cancer metastasis.
EVs, including LOs, are very small, yet they play a crucial role in cell-to-cell communication. EVs are present in all biological fluids and play a part in both physiological and pathological processes. However, the mechanisms of EV and LO biogenesis and release remain largely unclear, as is the role of LOs in tumor aggressiveness and metastasis. This requires more detailed research to better detect and understand LOs and how their molecular cargo is packaged, transferred, and processed within target cells. While it has become clear how important it is to understand how EVs and LOs impact recipient cell biology, it is still unclear if the transfer of mitochondrial material from LOs to the recipient cell changes its metabolism or whether the recipient cell can become more cancerous.
Recent work at the Di Vizio lab is also exploring mechanisms whereby the immune system responds against nucleic acid cargo contained within LOs. They believe that LOs will emerge as key regulators of metastasis, as they are enriched in metabolic enzymes and nucleic acids that might stimulate nucleic acid sensors in the tumor microenvironment.
Excitingly, the team started a ten-year project using multi-omics to profile LOs. They are using tools like mass spectrometry and next-generation sequencing to identify markers that could be interrogated for liquid biopsy approaches. The Di Vizio lab is soon kick-starting a project to test this in large patient cohorts, working in collaboration with Professor Francesca Demichelis (University of Trento) and Professor Shannon Stott (Massachusetts General Hospital, Harvard Medical School).
With a large team of experts in different fields, including mechanical engineering, biology, genomics, and transcriptomics, they hope to develop a blood test to identify patients requiring aggressive treatments early on. Similarly, if such liquid biopsy tests were to be available, it could spare excessive treatment to patients who don’t need it or be used to monitor patient responses. In addition, the team has recently been awarded funding to explore how targeting or blocking LOs might influence tumor progression. This could benefit patients and provide improved disease prognosis.
Super-resolution imaging to enhance EV research in cancer progression
Like many EV research labs, the Di Vizio team combines multiple technologies and methods to characterize LO and EV populations, including proteomics, RNA sequencing (both from bulk and at the single-EV level), MRPS to determine particle size, flow cytometry, and diffraction-limited confocal microscopy. However, current methods often lack the sensitivity and resolution to image and provide advanced sample analysis.
Typically, the small size of EVs (30 - 1000 nm in diameter) and even some LOs (1-10 micrometers in diameter) make them challenging to study using conventional light microscopy. Studying the molecular cargo contained within LOs and the uptake mechanisms by target cells requires high sensitivity and the ability to quantify markers at the single vesicle level to characterize population heterogeneity and identify key molecular signatures. This was the motivation for Professor Di Vizio to apply for ONI’s 2024 EV competition and gain access to an ONI Nanoimager for three months to apply super-resolution microscopy on their LO and EV research.
Super-resolution microscopy enables researchers to image beyond the limits of conventional optical microscopy and to examine biological specimens from tissues to cells and nanoparticles at the nanometer scale.
The team is excited to get their hands on the Nanoimager super-resolution microscope because it will enable them to not only visualize LOs with 15 nm resolution but also quantify the molecules associated with them, both at the single vesicle and single molecule level. Their three-month EV competition prize is timely for the Di Vizio team, after they just concluded a large effort to identify LO markers. The team has optimized reagents, such as antibodies for specific targets, which they are now ready to test using the Nanoimager microscope for full vesicle characterization. They will also continue to study different aspects of large oncosome function, such as their biogenesis and shedding from cells, the immune response to cancer, and the functional role of mitochondria in LOs.
“...the Nanoimager super-resolution microscope will enable them to not only visualize large onocomes with 15 nm resolution but also quantify the molecules associated with them, both at the single vesicle and single molecule level.”
Current challenges and opportunities in the EV field
There are different challenges that EV researchers are currently facing. Among them is the necessity to meticulously perform a range of techniques to purify and validate EV sample identity, integrity, and characteristics. This results in the usage and, often, loss of precious EV samples. Thus, methods for EV characterisation that use minimal amounts of sample while maintaining analytical rigor offer an added benefit to researchers in the field. Another challenge in the field is that the different model systems behave in completely distinct manners when it comes to EVs and intercellular communication, creating a need to understand and develop better models that better recapitulate biological EV questions.
On the other hand, the EV field is filled with opportunities. Despite the incredible amount of research that has been carried out in the past over 15 years, there are still many unknown and exciting mechanisms to be uncovered relating to EVs, extracellular communication, and nanoparticles in general. The EV field is also a highly collaborative community, and scientific collaboration strengthens the level of research, shapes new partnerships and projects, improves grant funding applications, and increases technology usage.
The Di Vizio lab members highlight the importance of having a group of collaborators with whom to share expertise, concerns, reagents, and even perform inter-laboratory validations. They have collaborated with several teams over the last two decades, including Professor Michael Freeman at Cedar-Sinai Medical Center, Professor Francesca Demichelis at the University of Trento in Italy, Professor Shannon Stott at the Massachusetts General Hospital, Professor Kendall Van Keuren-Jensen at the National Institutes of Health in Bethesda, Professor Leonora Balaj also the Massachusetts General Hospital; and several International Society for Extracellular Vesicles (ISEV) members and industrial partners, such as Dr John Nolan (The Scintillon Research Institute), George Daaboul (Everest Biolabs), Professor Andries Zijlstra (Vanderbilt University Medical Center), and several others.
Professor Di Vizio and her lab are extremely excited to attend the upcoming 2025 ISEV meeting in Vienna (Austria), share some of their latest work, and get feedback from the community.
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