Evasion of antitumor immunity and immunotherapy by melanoma extracellular vesicles

Información del proyecto

EVasion

Identificador del acuerdo de subvención: 101109429

DOI

10.3030/101109429

Proyecto cerrado

Fecha de la firma de la CE 3 Mayo 2023

Fecha de inicio 1 Junio 2023

Fecha de finalización 31 Mayo 2025

Financiado con arreglo a

• Marie Skłodowska-Curie Actions (MSCA)

Coste total

Sin datos

Aportación de la UE

€ 172 618,56

Coordinado por

FUNDACAO D. ANNA DE SOMMER CHAMPALIMAUD E DR. CARLOS MONTEZ CHAMPALIMAUD
Portugal

Periodic Reporting for period 1 - EVasion (Evasion of antitumor immunity and immunotherapy by melanoma extracellular vesicles)

Período documentado: 2023-06-01 hasta 2025-05-31

Resumen del contexto y de los objetivos generales del proyecto

Melanoma is the deadliest form of skin cancer. In 2022, around 331,722 people were diagnosed with melanoma, and approximately 58,667 died from the disease. When detected early, melanoma is highly treatable, with a 5-year relative survival rate exceeding 99%. However, if it spreads to distant parts of the body (known as metastatic melanoma), the prognosis worsens significantly. In such cases, the 5-year relative survival rate drops to about 35%, meaning that only 35% of patients with advanced melanoma are expected to live for at least five years after diagnosis.
Immunotherapy, which helps the immune system fight diseases, has shown promising results in treating metastatic melanoma. Immune checkpoint inhibitors (ICI) are a type of immunotherapy that is currently used for the treatment of advanced melanoma. However, only around 50% of patients respond to ICI treatment. To improve outcomes, it is critical to understand why some patients respond to these therapies while others do not.
Extracellular vesicles (EVs) are very small particles released by all cells. They carry and transfer biomolecules (e.g. proteins, lipids, nucleic acids, and metabolites) throughout the body. In recent years, EVs have been shown to also act as protein decoys, by binding and sequestering soluble proteins, preventing them from interacting with their intended targets.
Previous research from our group found that EVs released by melanoma cells carry receptors for interferon-gamma (IFNγ). IFNγ plays a pivotal role in the body’s immune response to cancer, leading to tumor cell killing and to activation of immune cells. Importantly, defects in the IFNγ signaling pathway have been linked to resistance to ICI therapy. Since EVs derived from tumor cells are known to promote tumor growth and metastases, we hypothesized that they modulate the bioavailability and activity of IFNγ, contributing to tumor development, metastases formation, and resistance to ICI therapy.
Our research has the potential to unveil a novel cancer immune evasion mechanism mediated by EVs. Understanding the mechanisms behind antitumor resistance is essential for developing alternative or complementary treatment strategies to improve response to ICIs and other cancer therapies. This work could also help identify biomarkers that predict how patients will respond to treatment, paving the way for more personalized and effective cancer therapies. Importantly, our findings may also apply to other types of cancer beyond melanoma.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

We confirmed that EVs derived from melanoma, breast cancer and pancreatic cancer cell lines carry receptors for IFNγ, known as interferon-gamma receptor (IFNGR).
Additionally, we showed that IFNγ binds less effectively to EVs derived from melanoma cells that express lower levels of IFNGR (IFNGR⁻ EVs, generated by IFNGR1-knockdown), than to EVs derived from wild-type melanoma cells (IFNGR+ EVs). This confirms that melanoma EVs bind IFNγ, at least in part, via IFNGR.
To understand whether the IFNGR on EVs influences tumor growth and metastases formation, we treated mice bearing melanoma tumors with IFNGR+ EVs, IFNGR- EVs, or PBS (used as control). The tumor volume and metastatic spread to the draining lymph node and lungs did not significantly differ across treatment groups. However, in tumors from both EV-treated groups, we observed an increase in the percentage of immune cells, including CD8⁺ T cells and macrophages, compared to tumors from PBS-treated animals. These results suggest that EVs promote immune cell recruitment to the tumor microenvironment; however, the effect appears to be independent of IFNGR levels on the EVs.
We hypothesized that we could not distinguish the specific effects of IFNGR+ EVs vs IFNGR- EVs due to the continued production of IFNGR+ EVs by the melanoma cell line itself. To address that, we generated a melanoma cell line with reduced EV secretion (EV-inhibited cells) by knocking down RalB, a protein involved in exosome biogenesis.
We found that IFNγ was more cytotoxic to EV-inhibited cells than to wild-type melanoma cells, suggesting that EVs protect melanoma cells from IFNγ-mediated cytotoxicity in vitro. Importantly, we also observed that IFNGR⁺ EVs, but not IFNGR⁻ EVs, enhanced in vivo tumor growth, compared to PBS. However, the proportion of tumor and immune cells within the tumors was not significantly different between treatment groups.
To evaluate whether IFNGR⁺ EVs influence the effectiveness of ICI therapy, we treated tumor-bearing mice with PBS, IFNGR⁺ EVs alone, anti-PD-L1 antibody alone, or the combination of IFNGR⁺ EVs and anti-PD-L1. As expected, anti-PD-L1 treatment slightly reduced tumor growth, relatively to PBS. However, the combination therapy also decreased tumor volume, when comparing with PBS, suggesting that melanoma-derived IFNGR⁺ EVs do not compromise the efficacy of ICI therapy.
Altogether, our findings support the hypothesis that IFNGR on EVs is required to promote tumor growth and that EVs can protect tumor cells from the cytotoxic effects of IFNγ. However, further studies are needed to determine whether IFNGR on EVs influences ICI therapy outcomes and whether it could serve as a biomarker to stratify patients based on their likelihood of responding to treatment.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

In vitro data show that melanoma cells producing reduced levels of EVs are more affected by the cytotoxic effects of IFNγ compared to wild-type cells that produce normal levels of EVs. Additionally, we demonstrated that IFNγ binds to EVs, and that this binding is at least partially mediated by the presence of IFNGR on EVs. In vivo data show that IFNGR on EVs is required to promote tumor growth. Altogether, our data suggest that IFNGR⁺ EVs enhance tumor growth by binding to IFNγ, likely neutralizing its effect by preventing IFNγ from interacting with IFNGR on tumor cells. This research reveals a novel cancer immune evasion mechanism mediated by EVs.
However, we observed that only a very small percentage of intravenously administered EVs reach the tumor. To enhance the effect of EVs and possibly increase the distinction between IFNGR⁺ EV and IFNGR⁻ EV treatments, intra-tumoral administration of EVs should be considered.
Further research is also needed to determine whether IFNGR on EVs affects the response to ICI therapy. We conducted a single experiment using anti-PD-L1 therapy and did not compare the effects between IFNGR⁺ EVs and IFNGR⁻ EVs. It is also possible that the dosage of EVs and antibody used influenced the outcome; therefore, a titration assay is necessary.
To assess whether levels of IFNγ bound to circulating melanoma EVs can serve as a predictor of patient response to ICI therapy, additional patient samples must be analyzed.
Understanding this mechanism allows for the development of alternative or complementary strategies to improve ICI response or other suboptimal cancer therapies, by targeting EVs. One potential approach could involve using molecules that inhibit EV biogenesis, either alone or in combination with ICIs.

IFNγ-decoy by IFNGR⁺ EVs