Pathophysiological role and therapeutic potential of extracellular vesicles in cancer

Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles that are endogenously generated through various biogenesis pathways within most cellular entities. Subsequently, they are released into the extracellular milieu to facilitate intercellular communication. They are composed of diverse bioactive molecules with important roles in physiological and pathological states. Over the past few decades, the therapeutic potential of EVs has garnered significant interest in the drug delivery field. However, deepened understanding of EV biology and further technological advances are needed to bridge the gap between research and clinical translation. In this thesis, we address these challenges and investigate EVs as novel biomedical agents.

EVs are crucial components of physiological processes and disease development. Sensitive visualisation techniques are needed to better understand their function as therapeutic agents. In paper I, a bioluminescent labelling system was developed to track EVs in vitro and in vivo. The system uses genetic modifications to enable the encapsulation of sensitive luciferase-variants in EVs. The system was used in vivo to enable highly sensitive detection of EV distribution pattern. Exogenously administered EVs were found to rapidly distribute within different organs, with a preference for the spleen, lung, and liver.

In addition to endogenously engineered EVs for in vivo tracking, exogenously engineered EVs can be utilised as promising drug delivery platforms. However, cargo loading is often insufficient, requiring improved EV loading approaches. In paper II, we developed an optimised cargo loading method using electroporation. An optimised protocol was designed to load EVs with doxorubicin, which increased cargo loading, EV recovery, and drug potency by 190-fold over free doxorubicin.

Owing to their potential to cross biological barriers, transport bioactive cargo, and targetability, EVs can be exploited as delivery vehicles for targeting of therapeutics. EVs were used as delivery vectors in paper III by coating their surfaces with an Fc domain-specific antibodybinding moiety. These Fc-EVs were then decorated with various IgG antibodies and targeted to cells of interest. In vitro and in vivo antibody targeting studies showed the broad potential of this technology for cancer therapy. The platform efficiently targeted EVs to cancer cells, including HER2 and PD-L1 positive cells. As proof of concept, Fc-EVs with PD-L1 antibody accumulate in tumour tissue and, when loaded with doxorubicin, reduce tumour burden, and increase survival in melanoma-bearing mice.

Despite significant EV engineering advances, we have a limited understanding of the biology of tumour-derived extracellular vesicles (tEVs). In paper IV, we investigated the role of in vitrogenerated melanoma-derived EVs as indirect communicators in tumour-induced haematopoiesis dysregulation. The tEVs, which contain high levels of angiogenic factors like VEGF, osteopontin, and tissue factor, were found to cause splenomegaly, extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and anaemia in syngeneic mice. These findings suggest that tEVs dysregulate haematopoiesis during the immune escape phase of cancer immunoediting, making them potential targets for overcoming immune evasion and restoring normal haematopoiesis.

To summarise, the tools generated in this thesis, including the ability to detect EVs in vivo, effective cargo loading, display antibody binding moieties on EV surfaces for targeting, and understanding the pathophysiological role of tEVs, contribute to the advancement of EVs for biomedical purposes, and clinical translation down the line.

List of scientific papers

I. Gupta, Dhanu*, Xiuming Liang*, Svetlana Pavlova, Oscar P. B. Wiklander, Giulia Corso, Ying Zhao, Osama Saher, Jeremy Bost, Antje M. Zickler, Andras Piffko, Cecile L. Maire, Franz L. Ricklefs, Oskar Gustafsson, Virginia Castilla Llorente, Manuela O. Gustafsson, R. Beklem Bostancioglu, Doste R. Mamand, Daniel W. Hagey, André Görgens, Joel Z. Nordin, and Samir EL Andaloussi. 2020. Quantification of Extracellular Vesicles in Vitro and in Vivo Using Sensitive Bioluminescence Imaging. Journal of Extracellular Vesicles. 9(1). *These authors contributed equally.
https://doi.org/10.1080/20013078.2020.1800222

II. Lennaárd, Angus J.*, Doste R. Mamand*, Rim Jawad Wiklander, Samir E. L. Andaloussi, and Oscar P. B. Wiklander. 2022. Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin. Pharmaceutics. 14(1). *These authors contributed equally.
https://doi.org/10.3390/pharmaceutics14010038

III. Oscar P. B. Wiklander&, Doste R. Mamand&, Dara K. Mohammad Wenyi Zheng, Rim Jawad Wiklander, Taras Sych, Antje M. Zickler, Dhanu Gupta, Xiuming Liang, Heena Sharma, Andrea Lavado, Jeremy Bost, Giulia Corso, Angus J. Lennaárd, Manuchehr Abedi-Valugerdi, Imre Mäger, Erdinc Sezgin, Joel Z. Nordin, André Görgens, Samir EL Andaloussi. Targeted Therapy by Antibody Displaying Extracellular Vesicles. [Submitted]

IV. Doste R. Mamand, Safa Bazaz, Dara K. Mohammad, Xiuming Liang, Carsten Mim, Susanne Gabrielsson, Oscar Wiklander, Manuchehr Abedi-Valugerdi, Samir El-Andaloussi. Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting. Running title: Induction of haematopoiesis dysregulation by melanoma cell derived EVs. [Manuscript]