Microfluidics, the science and engineering of manipulating small amount of fluids within micrometre-sized devices, has a strongly multidisciplinary nature and is at the basis of the Lab-on-Chip (LoC) technology. Thanks to advancements in polymer engineering and to the development of soft lithography techniques, LoCs have become a valuable tool for biological applications, giving the possibility to mimic human patho-physiology by growing different cell populations in complex 3D microenvironment with physiologically relevant chemical and mechanical features. Such devices are widely employed in cancer research, as they allow to reproduce the heterogeneity of the tumour microenvironment (TME), a complex entity composed of several cell populations, contributing to tumour progression through several mechanisms, such as the impairment of dendritic cells (DC) capacity to trigger a proper immune response. In this context, microfluidic devices are valuable platforms to test innovative immunotherapeutic strategies, aimed at restoring an effective host immune response against cancer. For instance, the combination of the histone deacetylase inhibitor romidepsin (R) and IFN-alpha (I) has been demonstrated to be very effective in inducing the immunogenic cancer cells death while enhancing DCs capacity to uptake tumour antigens to trigger a proper immune response. Hence, aim of this project is to realise a microfluidic platform reproducing specific aspects of the TME, namely the tumour-vasculature interface, in order to evaluate the effect of RI on the recruitment of DCs to the tumour site, across the endothelial layer. Physiological levels of shear stress will be guaranteed by perfusing a constant flow within the vasculature, thus reproducing in vivo-like conditions, to which DCs are naturally exposed. DCs migratory behaviour and efficiency in crossing the endothelial layer will be characterised in order to assess the effectiveness of RI in enhancing DCs-mediated immune response.
The possibility to mimic human body physiopathology in close and adjustable systems makes microfluidics a versatile tool for biomedical research, and its introduction in cancer research has represented a great breakthrough.
In fact, this technique provides versatile platforms with customisable geometry to reconstitute the heterogeneity of the TME, creating different compartmentalisation patterns which replicate the convoluted 3D architecture of TME. This allows the inclusion of complex physiological characteristics which cannot be reproduced in traditional 2D culture models [1]. At the same time, microfluidic systems offer a significant advantage over in vivo models as well, reproducing the characteristics of a specific tissue microenvironment "isolated" from the rest of the organism, thus constituting valid alternative to animal models, which, in some cases, might not even be the most appropriate models for drug testing [2].
Furthermore, microfluidic platforms are suited to parallelisation and automation, as well as to carry out high-throughput quantitative analyses. Their production is low cost in terms of materials and fabrication methods, while their small dimension reduces the employment of expensive reagents (culture medium, molecules, drugs) [3, 4].
These key features, along with their suitability for real-time monitoring and high-resolution imaging, have rendered these systems particularly advantageous for testing immunotherapy approaches. Immunotherapeutic studies carried out on microfluidic platforms have widely aimed at characterising CTLs activity within the TME in order to restore or boost it [5, 6].
However, DCs-based immunotherapy has considerably taken hold as a valid and very safe alternative to T-cell immunotherapy, thanks to the pivotal role of this cell population in activating anti-tumour immune responses [7]. In this respect, microfluidics has a great potential to faithfully reproduce DCs interaction with the tumour and its microenvironment, favouring the optimisation of DCs-based immunotherapy. In particular, within this project we intend to analyse the effectiveness of a combinatorial treatment (RI, combining epigenetic and immune therapy) within a physiological context, involving not only the tumour mass but also the tumour-associated vasculature. In fact, in order to reach the tumour site, migratory DCs, which are a circulating subset of the DC population, need to interact with the endothelium to cross it. To our knowledge, this is the first attempt to reproduce and characterise this complex dynamics in presence of a constant laminar flow, which exert hydrodynamic forces, such as shear stress, comparable to in vivo conditions. In fact, flow-induced shear stress has already been proven to crucially affect the morphology and the stability of the mature endothelial layer lining the microchannel [8]. Accordingly, such experimental layout allows to recreate the flow conditions physiologically experienced by DCs, which might also affect their behaviour. In this respect, the quantitative analyses of the parameters related to their migration (trajectory, velocity, number of DCs able to reach the tumour site across the endothelial barrier) will provide important information related to DCs capability to respond to these in vivo-like conditions and to the given treatment. Therefore, our microfluidic platform is useful to test the efficacy of the combinatorial therapy not only in mediating DCs detection of the tumour-associated antigens, but also regarding RI effectiveness in the recruitment of DCs to the tumour site, in such complex dynamic conditions.
For these characteristics, such technology is perfectly suited for pre-clinical studies aimed at testing these cutting-edge therapies and, in the future, upon standardisation of analysis protocol and optimisation of its high-throughput potential, it is likely to be employed even for clinical analyses, allowing the direct loading of patients-derived cell within the devices [3].
[1] M. Shang, R.H. Soon, C.T. Lim, B.L. Khoo, J. Han, Lab Chip, 2019, 19, 369-386
[2] K. Herrmann, F. Pistollato, M.L. Stephens, ALTEX - Alternatives to animal experimentation, 2019, 36, 343-352
[3] A. Boussommier-Calleja, R. Li, M.B. Chen, S.C. Wong, R.D. Kamm, Trends Cancer, 2016, 2, 6-19
[4] M. Mehling, S. Tay, Curr. Opin. Biotechnol., 2014, 25, 95-102
[5] G. Adriani, A. Pavesi, A.T. Tan, A. Bertoletti, J.P. Thiery, R.D. Kamm, Drug Discov. Today., 2016, 21(9):1472-1478
[6] V. Kumar, S. Varghese, Adv. Healthc. Mater., 2019, 8, 1801198
[7] J. Constantino, C. Gomes, A. Falcão, B.M. Neves, M.T. Cruz, Immunol Res., 2017, 65(4):798-810
[8] G. Silvani, C. Scognamiglio, D. Caprini, L. Marino, M. Chinappi, G. Sinibaldi, G. Peruzzi, M.F. Kiani, C.M. Casciola, Small, 2019, 15, 1905375