Small extracellular vesicles as a potential clinical tool for hard-to-treat cancer therapy

Resumen

Hard-to-treat cancers such as glioblastoma (GBM) and pancreatic ductal adenocarcinoma (PDAC), are characterized by their aggressive nature and resistance to conventional therapies. Despite the standard of care, which includes tumor resection followed by chemotherapy and radiotherapy, patients have a poor prognosis. Therefore, the development of novel therapeutic approaches is necessary. In this sense, the use of small extracellular vesicles (EVs) as delivery systems has become a promising tool for cancer treatment. This system can deliver anticancer drugs directly to tumor cells, improving the efficacy of treatment. In this work, we used small EVs loaded with different drugs to prove their target specificity towards their parental cells and evaluate their potential as an effective treatment for cancer. In addition, we studied the PADI4 interactome and citrullination capacity, its relationship with cancer progression, and its role as a target for cancer therapy. This doctoral thesis is structured as a compendium of six articles published in high-impact journals (Q1) that correspond to each chapter, with an additional chapter of the data pending publication. In Chapter 1, we discussed how small EVs play a crucial role in intercellular communication and have emerged as potential biomarkers and therapeutic tools, particularly in cancer. Additionally, we reviewed the significance of small EVs as biomarkers in various cancer types. Furthermore, we explored the development of chimeric EVs that can be conjugated with nanoparticles, biomolecules, and anti-cancer drugs, showing their potential for targeted cancer therapy. The review also provides an overview of ongoing clinical trials utilizing EVs to improve the prognosis of different cancer types, emphasizing the potential of EV-based therapies to combat these challenging diseases. In Chapter 2, we performed the isolation and characterization of small EVs derived from GBM patient cell lines. We loaded these EVs with temozolomide (TMZ) and EPZ015666 drugs and observed that a minimal amount of the drug is sufficient to affect tumor cells when it is loaded into small EVs. Additionally, GBM-derived small EVs loaded with the drugs showed the potential to induce an antiproliferative effect in pancreatic cancer cells. These findings support the use of GBM-derived small EVs as a promising drug delivery tool for further preclinical and clinical development of GBM treatments. In Chapter 3, we isolated and characterized small EVs derived from a PDAC cell line named RWP-1. We loaded these small EVs with two chemotherapeutic drugs, TMZ and EPZ015666 by the direct incubation method, which proved to be the most effective loading method, requiring minimal drug dosage to affect tumor cells. The antiproliferative effect was tested on different cancer cell lines, revealing greater efficacy of RWP-1 small EVsTMZ compared to RWP-1 small EVsEPZ015666. These findings highlight the potential of RWP-1-derived small EVs as a promising drug delivery tool for PDAC treatment. In Chapter 4, we identified the expression and localization of PADI4, a Ca2+-dependent protein involved in the conversion of arginine to citrulline, in several cancer cell lines, including GBM (GB-39), and pancreatic cancer (RWP-1). Additionally, we studied its relationship with p53. The characterization by immunofluorescence (IF) revealed different subcellular localizations of PADI4 depending on the cancer cell line. In the Westen Blot (WB) we observed different patterns of protein, revealing the possibility that PADI4 might experience alternative splicing. These findings shed light on the functional role of PADI4 in cancer development. In Chapter 5, we performed WB to identify the expression of PADI4 in GBM cell lines. Additionally, we investigated if GSK484, a well-known PADI4 inhibitor, could have an effect on GBM and PDAC cell proliferation. Finally, we tested if GSK484 loaded into small EVs had a higher effect on cell proliferation than the administration of the drug alone. Our results indicated that PADI4 expression was different among several cancer cell lines. Besides, GSK484 effectively decreases the proliferation of GBM and PDAC cell lines when it was loaded into small EVs, with a minimum concentration of the drug. In Chapter 6, we analyzed the interaction between NUPR1 and PADI4 in GBM cell lines. In vitro and in cellulo experiments revealed a strong binding affinity between NUPR1 and PADI4 in the nucleus, suggesting that the NUPR1/PADI4 complex may be essential in DNA repair, metastasis promotion, or citrullination of other proteins. These results highlight the importance of their interaction and their role as a potential target for cancer therapy. Finally, in Chapter 7, we identified the interaction between PADI4 and MDM2. In this work, the PADI4/MDM2 complex was found in the nucleus and cytoplasm of different cancer cell lines. Additionally, we found that their treatment with GSK484 impeded the nuclear binding of these two proteins, suggesting that MDM2 binds to the active site of PADI4. The interaction between MDM2 and PADI4 may lead to MDM2 citrullination, which could be a therapeutic target for cancer treatment. This Thesis revealed the potential use of small EVs derived from hard-to-treat cancer cells as a novel therapeutic approach for the treatment of GBM and PDAC. By loading these EVs with therapeutic drugs, such as TMZ, EPZ015666, and GSK484, we enhanced the drug delivery efficiency, which could reduce the side effects associated with conventional therapies. Furthermore, we explored the role of PADI4 in GBM and PDAC. By elucidating the interactions between PADI4 and its partners, we uncovered potential mechanisms involved in cancer progression and identified new avenues for targeted therapies. This work settles the basis of future personalized medical approaches and provides valuable insights into EVs biology to develop innovative strategies for the treatment of GBM and PDAC based on them as drug delivery systems.