Cell-cell direct/indirect communication in metastatic breast cancer as a control point in oncotherapy

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Scalable production of human mesenchymal stromal cell-derived extracellular vesicles under serum-/xeno-free conditions in a microcarrier-based bioreactor culture system

Publication . Fuzeta, Miguel de Almeida; Bernardes, Nuno; Oliveira, Filipa D.; Costa, Ana Catarina; Fernandes-Platzgummer, Ana; Farinha, José Paulo; Rodrigues, Carlos A.V.; Jung, Sunghoon; Tseng, Rong-Jeng; Milligan, William; Lee, Brian; Castanho, Miguel A. R. B.; Gaspar, Diana; Cabral, Joaquim M.S.; da Silva, Cláudia Lobato

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between −15.5 ± 1.6 mV and −19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

2020Journal article

Open access

Novel peptides derived from dengue virus capsid protein translocate reversibly the blood−brain barrier through a receptor-free mechanism

Publication . Neves, Vera; Silva, Frederico Aires da; Morais, Maurício; Gano, Lurdes; Ribeiro, Elisabete; Pinto, Antónia; Aguiar, Sandra; Gaspar, Diana; Fernandes, Célia; Correia, João D. G.; Castanho, Miguel A. R. B.

The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain.

2017Journal article

Restricted access

Structure-stability-function mechanistic links in the anti-measles virus action of tocopherol-derivatized peptide nanoparticles

Publication . Figueira, Tiago Nascimento; Mendonça, Diogo A.; Gaspar, Diana; Melo, Manuel N.; Moscona, Anne; Porotto, Matteo; Castanho, Miguel A. R. B.; Veiga, Ana Salomé

Measles remains one of the leading causes of child mortality worldwide and is re-emerging in some countries due to poor vaccine coverage, concomitant with importation of measles virus (MV) from endemic areas. The lack of specific chemotherapy contributes to negative outcomes, especially in infants or immunodeficient individuals. Fusion inhibitor peptides derived from the MV Fusion protein C-terminal Heptad Repeat (HRC) targeting MV envelope fusion glycoproteins block infection at the stage of entry into host cells, thus preventing viral multiplication. To improve efficacy of such entry inhibitors, we have modified a HRC peptide inhibitor by introducing properties of self-assembly into nanoparticles (NP) and higher affinity for both viral and cell membranes. Modification of the peptide consisted of covalent grafting with tocopherol to increase amphipathicity and lipophilicity (HRC5). One additional peptide inhibitor consisting of a peptide dimer grafted to tocopherol was also used (HRC6). Spectroscopic, imaging, and simulation techniques were used to characterize the NP and explore the molecular basis for their antiviral efficacy. HRC5 forms micellar stable NP while HRC6 aggregates into amorphous, loose, unstable NP. Interpeptide cluster bridging governs NP assembly into dynamic metastable states. The results are consistent with the conclusion that the improved efficacy of HRC6 relative to HRC5 can be attributed to NP instability, which leads to more extensive partition to target membranes and binding to viral target proteins.

2018Journal article

Restricted access

Challenging metastatic breast cancer with the natural defensin PvD1

Publication . Figueira, Tiago N.; Oliveira, Filipa D.; Almeida, Inês; Mello, Érica O.; Gomes, Valdirene M.; Castanho, Miguel A. R. B.; Gaspar, Diana

Metastatic breast cancer is a very serious life threatening condition that poses many challenges for the pharmaceutical development of effective chemotherapeutics. As the therapeutics targeted to the localized masses in breast improve, metastatic lesions in the brain slowly increase in their incidence compromising successful treatment outcomes overall. The blood-brain-barrier (BBB) is one important obstacle for the management of breast cancer brain metastases. New therapeutic approaches are in demand for overcoming the BBB's breaching by breast tumor cells. In this work we demonstrate the potential dual role of a natural antimicrobial plant defensin, PvD1: it interferes with the formation of solid tumors in the breast and concomitantly controls adhesion of breast cancer cells to human brain endothelial cells. We have used a combination of techniques that probe PvD1's effect at the single cell level and reveal that this peptide can effectively damage breast tumor cells, leaving healthy breast and brain cells unaffected. Results suggest that PvD1 quickly internalizes in cancer cells but remains located in the membrane of normal cells with no significant damage to its structure and biomechanical properties. These interactions in turn modulate cell adhesiveness between tumor and BBB cells. PvD1 is a potential template for the design of innovative pharmacological approaches for metastatic breast cancer treatment: the manipulation of the biomechanical properties of tumor cells that ultimately prevent their attachment to the BBB.

2017Journal article

Open access

Plant defensin PvD1 modulates the membrane composition of breast tumour-derived exosomes

Publication . Skalska, Julia; Oliveira, Filipa; Figueira, Tiago N.; Mello, Érica O.; Gomes, Valdirene M.; McNaughton-Smith, Grant; Castanho, Miguel A. R. B.; Gaspar, Diana

One of the most important causes of failure in tumour treatment is the development of resistance to therapy. Cancer cells can develop the ability to lose sensitivity to anti-neoplastic drugs during reciprocal crosstalk between cells and their interaction with the tumour microenvironment (TME). Cell-to-cell communication regulates a cascade of interdependent events essential for disease development and progression and can be mediated by several signalling pathways. Exosome-mediated communication is one of the pathways regulating these events. Tumour-derived exosomes (TDE) are believed to have the ability to modulate TMEs and participate in multidrug resistance mechanisms. In this work, we studied the effect of the natural defensin from common bean, PvD1, on the formation of exosomes by breast cancer MCF-7 cells, mainly the modulatory effect it has on the level of CD63 and CD9 tetraspanins. Moreover, we followed the interaction of PvD1 with biological and model membranes of selected composition, by biophysical and imaging techniques. Overall, the results show that PvD1 induces a dual effect on MCF-7 derived exosomes: the peptide attenuates the recruitment of CD63 and CD9 to exosomes intracellularly and binds to the mature exosomes in the extracellular environment. This work uncovers the exosomemediated anticancer action of PvD1, a potential nutraceutical agent.

2019Journal article

Open access