When Susanne Gabrielsson first learned about exosomes, her eyes widened. In 1999, she’d just started a postdoctoral fellowship at the Curie Institute in Paris. Gabrielsson, an immunologist now at Sweden’s Karolinska Institute, thumbed through paper after paper describing which types of cells release these tiny blobs of information. 

“I realized that since they were released from many cells, they probably would be released from all cells,” she said. “And that they would be a new means of communication.”

In the early 1980s, biologists pointed the nascent electron microscope at red blood cells, hoping to understand how they mature (1). They made a startling discovery: The cells ejected bits of material enveloped in small membranes. Later, a different team of biochemists working independently called them “exosomes.”

Exosomes, or extracellular vesicles (EV), are tiny — about the size of a virus. Cells eject exosomes from their surfaces. They generally carry proteins or RNA and are enveloped by a lipid bilayer. Researchers first assumed that exosomes were a waste disposal system, but they’re now known to play a critical role in cell-to-cell communication both locally and throughout the body. 

As the vesicle bops around the interstitial space between cells, it may fuse with a neighboring cell. Like a virus, the exosome enters and spills biomolecular information into the new cell. They are carrier pigeons at the microscopic scale.

Exosomes level up information transfer compared to single molecules like hormones. They can deliver more information through a variety of biomolecules and in a targeted way through homing proteins on their surfaces, according to Gabrielsson. 

“What was really fascinating for me was when I saw that they could induce immune responses, especially T cell responses,” she said. “That opened up the possibility both that they must have a really big role in the immune system, but also that you could possibly use them for therapy.” 

From immune response to cancer progression and even neurodegeneration, scientists have linked exosomes to a diverse range of biological processes and pathologies. Gabrielsson and others wondered if exosomes could reinvigorate immune checkpoint inhibitors for cancer therapy. These treatments often fail because tumors dominate their environments. They resist immune attack, grow faster than healthy neighboring cells, and influence those cells to promote more cancer. Cancers such as melanoma that mutate quickly are particularly prone to resisting these kinds of treatment.

Researchers have begun to ask how they can hijack this communication network. Since exosomes can haul cargo that stimulates the immune system, they could potentially protect patients when immune checkpoint inhibitors stop working.

Researchers hope to exploit exosomes for everything from drug delivery to diagnosis, especially for melanomas. To researchers like Gabrielsson, exosomes could be a game changing medical tool; they’ve made significant progress toward treating skin cancers in preclinical animal models. They’re finding in this pursuit that exosomes can both stimulate immune responses and suppress them. The wave of research opens a promising avenue for hitting melanoma where it hurts.

A ‘Canary’ for Cancers

Cancerous cells weaponize exosomes to resist drugs in at least four ways, according to recent studies of skin, colon, brain, breast, and blood cancers (2). In at least 60 different tumor cell lines, cells will sequester chemotherapy drugs like doxorubicin and eject them via EV (3). Exosomes sport molecules on their surfaces that can stick to cancer-fighting antibodies, reducing immune response, cancer sensitivity, or both (4). They can bind to and inhibit immune cells.

Tumors transfer drug dodging skills from drug resistant cells to sensitive ones by loading exosomes with microRNA recipes for making defensive molecules. Cancer stem cells can also produce EV in response to treatment. These will influence surrounding tumors to escape immune attack, metastasize, and resist drugs. 

Melanomas are particularly tricky. They begin in melanocytes and can metastasize and evade the immune system. Chemotherapy is not efficient for melanomas because they’re so quick to mutate and grow resistance (5). Once resistant, melanomas shed more exosomes during chemotherapy, and exosomes help the cancer cells access blood vessels, allowing them to spread throughout the body more easily (6,7). Researchers have found that drug-resistant cells can share resistance via exosomes with the cells around them as well (6).

“In some patients, you have several of these escape mechanisms working simultaneously,” Gabrielsson said, adding that these defenses can appear in different parts of the tumor or in metastases across the body. 

While exosomes maintain a daunting power to make melanomas more deadly, some scientists are now developing exosome inhibitors, hoping to resist the resistance (8).

Intercepting the message

Immunome, a Pennsylvania-based biotech company, specializes in finding targets for cancer drugs. “The human immune system has evolved over a large amount of time to identify targets for antibodies,” said Matthew Robinson, an antibody researcher and the company’s chief technology officer. Immunome researchers analyze samples from human patients’ tumors and lymph nodes to learn what the body naturally tries to target as it fights a cancer like melanoma. Essentially, they job-shadow the immune system. 

Initially, the team assumed that studying human immune responses to cancers would reveal a huge variety of targets. They were wrong. Instead, many of the targets were involved in membrane function, pointing to a potential role for exosomes.

“It's almost as if the immune response of these patients is seeing those exosomes [from cancers] and trying to fight back against what they're doing,” he said. “That's why we started to think about exosomes as potential targets.”

One target in the company’s sights is a protein called epsin 1. They noticed epsin 1 while analyzing the B cells of a patient fighting head and neck cancer. This protein dots the inner surface of the membranes of healthy cells and helps shuttle in proteins that promote growth. “You can think of them as antennas,” Robinson said. 

It's almost as if the immune response of these patients is seeing those exosomes [from cancers] and trying to fight back against what they're doing. 
- Matthew Robinson, Immunome

Immunome’s data revealed that epsin 1 appears on the outside of some cancerous cells. Since the protein is bound to the membrane, that means it winds up on the exosomes that the tumor ejects too. This external epsin 1 distinction between the “bad guys” and “good guys” enables the Immunome researchers to home in on the right targets that redirect the immune response specifically to tumors and the exosomes they produce. 

Immunome recently combined an epsin-1-targeting molecule, IMM20059, with a checkpoint inhibitor called atezolizumab. Atezolizumab targets PD-L1, which helps melanoma cells evade the immune system. 

“The only exosomes that are going to have epsin 1 on them are going to be the tumor ones. So, we may be able to help to sweep those out of the microenvironment,” Robinson said. In a recent study of mice with melanoma, the treatment did just that (9). IMM20059 enhanced the efficacy of atezolizumab in cells that normally don’t respond to checkpoint inhibitors. 

“It was very gratifying to see it work,” he said. “And completely surprising in the sense that prior to finding this patient's antibody, we would have never even thought to try and combine the two.”

Giving cancer a taste of its own medicine

While Immunome focuses on targeting specific proteins involved in exosome-mediated communication between cancer cells, Gabrielsson and her team use the exosome as a treatment itself. They administer exosomes as delivery vehicles for drugs that excite multiple different immune cells to attack melanoma.

Gabrielsson works with melanomas because they have many mutations, which make them somewhat easier to target. Each mutation makes the cancerous cells stand out more easily from healthy ones, “so it's easy to find antigens to load on these exosomes,” she said.

Recently, she and her group demonstrated this approach in mice (10). The researchers extracted exosomes from mouse bone marrow and loaded them with messages of their own: α-galactosylceramide and ovalbumin. α-galactosylceramide activates natural killer (NK) cells and natural killer T (NKT) cells, essentially priming them to destroy things, while the exosomes’ ovalbumin activates T cells. (The team intentionally chose mice with ovalbumin-expressing melanomas to be certain that they’d have a target for their proof-of-concept.)

At least four types of immune cells get the message: T cells, NK cells, NKT cells, and B cells. “They start proliferating and start to produce cytokines and other factors that will kill the tumor,” she said. As with Immunome’s treatment, this appears to weaken the tumor’s defenses, and when the researchers combined their exosomes with the conventional immune checkpoint inhibitors anti-PD-1 and anti-PD-L1 drugs, the treatment hit even harder.

The more we learn about what is in them and what is needed for a good immune response, then we could, more and more, move into completely artificial [exosomes] which would make [treatment] even easier. 
- Susanne Gabrielsson, the Karolinska Institute

“We saw a very good response in itself with EV,” Gabrielsson said. “There is an increased T cell activation if we combine the two therapies, and that is really promising for future human studies.” She added that in some cases when a patient’s melanoma is resistant to checkpoint inhibitors, just α-galactosylceramide and ovalbumin-loaded exosomes might work well enough to treat the cancer.

This approach has implications beyond cancers that are sensitive to ovalbumin-loaded EV and beyond melanomas, too. Therapeutic exosomes don’t have to originate from the tumor or even from the patient. Gabrielsson believes that the field will move toward producing artificial exosomes (11). What matters is the message inside. 

“The more we learn about what is in them and what is needed for a good immune response, then we could, more and more, move into completely artificial [exosomes] which would make [treatment] even easier,” she said.

Knowing that cancer cells communicate via exosomes not only makes cancers easier to drug, but it also facilitates diagnosing them. “They really have potential for biomarker discovery,” Gabrielsson said. Cancer exosomes could answer a handful of questions simultaneously: Why did this cell produce it? What is the message? And to what cell are they delivering it? Doctors could look for proteins, mRNA, microRNA, or cell free DNA, before cancer has even started to spread. 

This means that clinicians would get better at diagnosing cancers early. “Instead of doing a lot of biopsies at different sites when you start feeling pain and when it's often too late, you could screen people earlier and find this when the tumors are not noticed in other ways,” she said.

For now, Gabrielsson is focused on exosomes’ potential as powerful therapeutics for melanoma and other cancers. “Being able to manipulate them, to load them with certain cargo, and using different targeting methods to really pinpoint certain cells to take them up — it's still only in the beginning,” she said.

References

1. Harding, C.V., Heuser, J.E., and Stahl, P.D. Exosomes: Looking back three decades and into the future. J Cell Biol   200, 367–371 (2013)
2. Musi, A. and Bongiovanni, L. Extracellular Vesicles in Cancer Drug Resistance: Implications on Melanoma Therapy. Cancers  15, 1074-2023 (2023).
3. Shedden, K. et al. Expulsion of small molecules in vesicles shed by cancer cells: association with gene expression and chemosensitivity profiles. Cancer Research  63, 4331-4337 (2003).
4. Fontana, F. et al. Extracellular Vesicles: Emerging Modulators of Cancer Drug Resistance. Cancers  13, 749 (2021). 
5. Wagstaff, W. et al. Melanoma: Molecular genetics, metastasis, targeted therapies, immunotherapies, and therapeutic resistance. Genes & Diseases  9 , 1608-1623 (2022).
6. Andrade, L.N.d.S. et al. Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy. Scientific Reports  9, 14482 (2019).
7. Braeuer, R.R. et al. Why is melanoma so metastatic? Pigment Cell Melanoma Res  27, 19-36 (2014).
8. Vella, L.J. et al. Intercellular Resistance to BRAF Inhibition Can Be Mediated by Extracellular Vesicle–Associated PDGFRβ. Neoplasia  19 (2017).
9. Richards, K. et al. Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells. Oncogene  36, 1770–1778 (2017).
10. Dowling, J. et al. IMM20059, a novel anti-EPN1 antibody, in combination with atezolizumab significantly enhances tumor regression in the B16.F10 syngeneic melanoma model compared to anti-PD-L1 monotherapy. Journal for ImmunoTherapy of Cancer  10 (2022).
11. Hiltbrunner, S. et al. Exosomal cancer immunotherapy is independent of MHC molecules on exosomes. Oncotarget  7, 38707-38717 (2016).