Future perspectives for advancing regulatory science of nanotechnology-enabled health products.

The identification of regulatory challenges for nanotechnology-enabled health products, followed by discussions with the involved stakeholders, is the first step towards a strategic planning of how such challenges can be successfully addressed in the future. In order to better understand whether the identified regulatory needs are sector-specific for health products or might also hinder the progress in other domains, the REFINE consortium reached out to communities representing other sectors that also exploit the potential of nanotechnology, i.e. industrial chemicals, food and cosmetics. Through a series of trans-sectorial workshops, REFINE partners identified common as well as sector-specific challenges and discussed possible ways forward. Potential solutions lie in a more strengthen collaboration between regulatory and research communities resulting in a targeted production and exploitation of academic data for the regulatory decision-making. Furthermore, a coordinated use of knowledge sharing platforms and databases, trans-sectorial standardisation activities and harmonisation of regulatory activities between geographical regions are possible ways forward, in line with the upcoming European political initiatives such as the Chemical Strategy for Sustainability (CSS). Finally, we also discuss the perspectives for further development and sustainability of methods and tools developed in the REFINE project.

Delivering the power of nanomedicine to patients today.

The situation of the COVID-19 pandemic reminds us that we permanently need high-value flexible solutions to urgent clinical needs including simplified diagnostic technologies suitable for use in the field and for delivering targeted therapeutics. From our perspective nanotechnology is revealed as a vital resource for this, as a generic platform of technical solutions to tackle complex medical challenges. It is towards this perspective and focusing on nanomedicine that we take issue with Prof Park's recent editorial published in the Journal of Controlled Release. Prof. Park argued that in the last 15 years nanomedicine failed to deliver the promised innovative clinical solutions to the patients (Park, K. The beginning of the end of the nanomedicine hype. Journal of Controlled Release, 2019; 305, 221-222 [1]. We, the ETPN (European Technology Platform on Nanomedicine) [2], respectfully disagree. In fact, the more than 50 formulations currently in the market, and the recent approval of 3 key nanomedicine products (e. g. Onpattro, Hensify and Vyxeos), have demonstrated that the nanomedicine field is concretely able to design products that overcome critical barriers in conventional medicine in a unique manner, but also to deliver within the cells new drug-free therapeutic effects by using pure physical modes of action, and therefore make a difference in patients lives. Furthermore, the >400 nanomedicine formulations currently in clinical trials are expecting to bring novel clinical solutions (e.g. platforms for nucleic acid delivery), alone or in combination with other key enabling technologies to the market, including biotechnologies, microfluidics, advanced materials, biomaterials, smart systems, photonics, robotics, textiles, Big Data and ICT (information & communication technologies) more generally. However, we agree with Prof. Park that " it is time to examine the sources of difficulty in clinical translation of nanomedicine and move forward ". But for reaching this goal, the investments to support clinical translation of promising nanomedicine formulations should increase, not decrease. As recently encouraged by EMA in its roadmap to 2025, we should create more unity through a common knowledge hub linking academia, industry, healthcare providers and hopefully policy makers to reduce the current fragmentation of the standardization and regulatory body landscape. We should also promote a strategy of cross-technology innovation, support nanomedicine development as a high value and low-cost solution to answer unmet medical needs and help the most promising innovative projects of the field to get better and faster to the clinic. This global vision is the one that the ETPN chose to encourage for the last fifteen years. All actions should be taken with a clear clinical view in mind, " without any fanfare", to focus "on what matters in real life", which is the patient and his/her quality of life. This ETPN overview of achievements in nanomedicine serves to reinforce our drive towards further expanding and growing the maturity of nanomedicine for global healthcare, accelerating the pace of transformation of its great potential into tangible medical breakthroughs.

Measurement of CD73 enzymatic activity using luminescence-based and colorimetric assays.

CD73 is a membrane-anchored ectoenzyme that degrades extracellular AMP into adenosine, a potent immunosuppressive factor. In physiological conditions, induction of the CD73-adenosine pathway acts as natural feedback mechanism to prevent excessive immune reactions and subsequent tissue damage. In the past few years, the CD73-adenosine pathway has emerged as a major immunosuppressive mechanism by which multiple types of cancer evade anti-tumor immunity. Research from our group and others have established that blocking the CD73-adenosine pathway represents a promising approach to improve cancer immunotherapy. In this context, an increasing number of research laboratories are becoming interested in CD73 biology and in the development/characterization of CD73 inhibitors. Implementation of simple, rapid and HTS-compatible assays to evaluate CD73 enzymatic active is a critical step for any laboratory willing to study the CD73-adenosine pathway. Over the years, we developed, optimized or adapted various methodologies to assess CD73 enzymatic activity using in vitro assays. In this chapter, we describe two different in vitro assays adapted to the measurement of CD73 enzymatic activity. Both assays are simple, robust, HTS-compatible and can be used in a cell-based fashion.

CD73 is associated with poor prognosis in high-grade serous ovarian cancer.

The cell surface nucleotidase CD73 is an immunosuppressive enzyme involved in tumor progression and metastasis. Although preclinical studies suggest that CD73 can be targeted for cancer treatment, the clinical impact of CD73 in ovarian cancer remains unclear. In this study, we investigated the prognostic value of CD73 in high-grade serous (HGS) ovarian cancer using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with shorter disease-free survival and overall survival in patients with HGS ovarian cancer. Furthermore, high levels of CD73 expression in ovarian tumor cells abolished the good prognosis associated with intraepithelial CD8(+) cells. Notably, CD73 gene expression was highest in the C1/stromal molecular subtype of HGS ovarian cancer and positively correlated with an epithelial-to-mesenchymal transition gene signature. Moreover, in vitro studies revealed that CD73 and extracellular adenosine enhance ovarian tumor cell growth as well as expression of antiapoptotic BCL-2 family members. Finally, in vivo coinjection of ID8 mouse ovarian tumor cells with mouse embryonic fibroblasts showed that CD73 expression in fibroblasts promotes tumor immune escape and thereby tumor growth. In conclusion, our study highlights a role for CD73 as a prognostic marker of patient survival and also as a candidate therapeutic target in HGS ovarian cancers.

Phosphorylation of DEP-1/PTPRJ on threonine 1318 regulates Src activation and endothelial cell permeability induced by vascular endothelial growth factor.

The protein tyrosine phosphatase DEP-1/PTPRJ positively regulates Src family kinases and critical biological functions in endothelial and hematopoietic cells. Phosphorylation of DEP-1 on Y1311/Y1320 mediates the association and activation of Src, and promotes Src-dependent angiogenic responses including endothelial cell permeability. We have identified T1318 as a phosphorylated residue proximal to Y1320. The aim of this study was to determine if T1318 phosphorylation exerts a regulatory role over the function of DEP-1. We show that phosphorylation of DEP-1 on Y1320 was reduced when T1318 was mutated. This led to the decreased association of DEP-1 T1318A with Src, and defective Src activation in both HEK 293T and VEGF-stimulated endothelial cells. Consistent with these findings, VEGF-induced tyrosine phosphorylation of VE-cadherin, its association to ß-arrestin1/2, and cell permeability were impaired in cells expressing DEP-1 T1318A. Conversely, expression of the phosphomimetic mutant DEP-1 T1318E constitutively enhanced the phosphorylation of Y1320 and VE-cadherin over that induced by WT DEP-1, and resulted in increased VEGF-dependent permeability. DEP-1 T1318 is part of a CK2 consensus phosphorylation site and was identified as a CK2 substrate. Modulation of CK2 expression or activity in endothelial cells regulated T1318 phosphorylation, and correlated with the status of Y1320 phosphorylation, Src activation, and cell permeability. CK2-dependent phosphorylation of DEP-1 T1318 promotes Y1320 phosphorylation and Src activation upon VEGF stimulation. Phosphorylation of T1318 is thus part of a regulatory mechanism that channels the activity of DEP-1 towards Src to allow its optimal activation and the promotion of endothelial cell permeability.

Anti-CD73 therapy impairs tumor angiogenesis.

CD73 is an ecto-nucleotidase overexpressed in various types of tumors that catabolizes the generation of extracellular adenosine, a potent immunosuppressor. We and others have shown that targeted blockade of CD73 can rescue anti-tumor T cells from the immunosuppressive effects of extracellular adenosine. Another important function of extracellular adenosine is to regulate adaptive responses to hypoxia. However, the importance of CD73 for tumor angiogenesis and the effect of anti-CD73 therapy on tumor angiogenesis remain unknown. In this study, we demonstrated that CD73 expression on tumor cells and host cells contribute to tumor angiogenesis. Our data revealed that tumor-derived CD73 enhances the production of vascular endothelial growth factor (VEGF) by tumor cells that host-derived CD73 is required for in vivo angiogenic responses and that endothelial cells require CD73 expression for tube formation and migration. Notably, the pro-angiogeneic effects of CD73 relied on both enzymatic and non-enzymatic functions. Using a mouse model of breast cancer, we demonstrated that targeted blockade of CD73 with a monoclonal antibody significantly decreased tumor VEGF levels and suppressed tumor angiogenesis in vivo. Taken together, our study strongly suggests that targeted blockade of CD73 can significantly block tumor angiogenesis, and further supports its clinical development for cancer treatment.

Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation.

DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin-associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1-catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)-mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.

Non-redundant roles of the Gab1 and Gab2 scaffolding adapters in VEGF-mediated signalling, migration, and survival of endothelial cells.

Gab1 was previously described as a positive modulator of Akt, Src, ERK1/2, endothelial cell migration, and capillary formation in response to vascular endothelial growth factor (VEGF). However, its involvement in endothelial cell survival, as well as the potential contribution of the other family member Gab2 to signalling and biological responses remained unknown. Here, we show that Gab2 is tyrosine phosphorylated in a Grb2-dependent manner downstream of activated VEGF receptor-2 (VEGFR2), and that it associates with signalling proteins including PI3K and SHP2, but apparently not with the receptor. Similarly to Gab1, over-expression of Gab2 induces endothelial cell migration in response to VEGF, whereas its depletion using siRNAs results in its reduction. Importantly, depletion of both Gab1 and Gab2 leads to an even greater inhibition of VEGF-induced cell migration. However, contrary to what has been reported for Gab1, the silencing of Gab2 results in increased Src, Akt and ERK1/2 activation, slightly reduced p38 phosphorylation, and up-regulation of Gab1 protein levels. Accordingly, re-expression of Gab2 in Gab2-/- fibroblasts leads to opposite results, suggesting that the modulation of both Gab2 and Gab1 expression in these conditions might contribute to the impaired signalling observed. Consistent with their opposite roles on Akt, the depletion of Gab1, but not of Gab2, results in reduced FOXO1 phosphorylation and VEGF-mediated endothelial cell survival. Mutation of VEGFR2 Y801 and Y1214, which abrogates the phosphorylation of Gab1, also correlates with inhibition of Akt. Altogether, these results underscore the non-redundant and essential roles of Gab1 and Gab2 in endothelial cells, and suggest major contributions of these proteins during in vivo angiogenesis.

New role for the protein tyrosine phosphatase DEP-1 in Akt activation and endothelial cell survival.

Functional inactivation of the protein tyrosine phosphatase DEP-1 leads to increased endothelial cell proliferation and failure of vessels to remodel and branch. DEP-1 has also been proposed to contribute to the contact inhibition of endothelial cell growth via dephosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a mediator of vascular development. However, how DEP-1 regulates VEGF-dependent signaling and biological responses remains ill-defined. We show here that DEP-1 targets tyrosine residues in the VEGFR2 kinase activation loop. Consequently, depletion of DEP-1 results in the increased phosphorylation of all major VEGFR2 autophosphorylation sites, but surprisingly, not in the overall stimulation of VEGF-dependent signaling. The increased phosphorylation of Src on Y529 under these conditions results in impaired Src and Akt activation. This inhibition is similarly observed upon expression of catalytically inactive DEP-1, and coexpression of an active Src-Y529F mutant rescues Akt activation. Reduced Src activity correlates with decreased phosphorylation of Gab1, an adapter protein involved in VEGF-dependent Akt activation. Hypophosphorylated Gab1 is unable to fully associate with phosphatidylinositol 3-kinase, VEGFR2, and VE-cadherin complexes, leading to suboptimal Akt activation and increased cell death. Overall, our results reveal that despite its negative role on global VEGFR2 phosphorylation, DEP-1 is a positive regulator of VEGF-mediated Src and Akt activation and endothelial cell survival.