Microfluidic studies for monitoring the metastatic cascade and cancer-immune cells interaction

Metastases are the primary cause of death in cancer patients. Small animal models are helping dissecting some key features of the metastatic cascade but many bio-mechanic details remains difficult to analyze in vivo. For this reason a series of tools for performing systematic analysis of vascular permeability, tissue architecture, blood flow, biochemical stimuli and inflammation were produced in the last decade. Particularly relevant for this field is the use of microfluidic chips allowing to include in vitro models a vascular component. During my PhD, I applied this novel technologies to replicate in vitro key steps in the metastatic cascade and cancer-immune cell interaction with a focus on the establishment of microfluidics for metastasis. More specifically I used 3 different microfluidic chips: i) a single-channel microfluidic chip allowing to study CTCs adhesion and rolling inside a small capillary; ii) a double-channel microfluidic chip, composed by an upper and a lower channels mimicking the vascular and extravascular compartments; the channels are laterally connected by an array of micro pillars acting as a vascular membrane; iii) a three channel device composed by a central 3D culture of tumor cells embedded into a collagen matrix flanked by 2 channels connected to the former by a series of trapezoidal pillars. The two lateral compartments are used to simulate the vascular and stromal environment respectively. In the text we show how the aforementioned microfluidic devices can efficiently recapitulate in vitro multiple key steps of cancer metastatic cascade and some of the most important interactions between immune-cancer cell interactions

A microfluidic platform with permeable walls for the analysis of vascular and extravascular mass transport

Considerable advances have been made in microfluidic devices and their applications since the development of soft lithographic techniques [1]. We developed a PDMS based double channel chip consisting of two microfluidic channels that mimic the vascular and extravascular compartments. The two channels are designed to be confined by sidewalls and connected by a membrane composed by arrays of pillars constituting a permeable vascular wall [2]. The inner surface of the vascular channel is uniformly coated with Human Umbilical Vein Endothelial Cells (HUVEC) resulting in well-controlled 3D model of blood vessel with endothelial barrier functions. In Figure.1A and B, the photolithographic, etching, and replica molding steps needed for realizing double-channel chips are presented together with an image (right) of the vascular channel after cell seeding and self-organization in a tubular shape. The extravascular compartment can be integrated with tumor cells of different type, potentially organized in a 3D fashion inside an extracellular matrix or with extracellular matrix components. The integration of the two compartments allow us to study the transport and permeation of therapeutic molecules, nanomedicines and cells through the endothelial barrier and the efficacy of the administered treatment. Other applications such as modeling of metastatic cell and leucocytes adhesion and migration across the endothelial barrier allow us to characterize cell extravasation from the vascular bed. The vascular transport and subsequent adhesion dynamics of nano-constructs and cells to the vascular channel are also predicted using a 3D computational framework based on coupling Lattice Boltzmann (LB) and Immersed Boundary (IB) methods. The fluid solver for the incompressible Navier-Stokes equations is based on the three dimensional D3Q19 Lattice-Boltzmann Method. The dynamics of deformable nano-constructs and cells is simulated through a neo-Hookean membrane constitutive model coupled iteratively with the fluid (Figure.1C). The combination of microfluidic chips and computational modeling provides a formidable tool for boosting our understanding on disease development and drug delivery. Please click Additional Files below to see the full abstract

A permeable on-chip microvasculature for assessing the transport of macromolecules and polymeric nanoconstructs.

Abstract Hypothesis The selective permeation of molecules and nanomedicines across the diseased vasculature dictates the success of a therapeutic intervention. Yet, in vitro assays cannot recapitulate relevant differences between the physiological and pathological microvasculature. Here, a double-channel microfluidic device was engineered to comprise vascular and extravascular compartments connected through a micropillar membrane with tunable permeability. Experiments The vascular compartment was coated by endothelial cells to achieve permeability values ranging from ~0.1 μm/sec, following a cyclic adenosine monophosphate (cAMP) pre-treatment (25 μg/mL), up to ~2 μm/sec, upon exposure to Mannitol, Lexiscan or in the absence of cells. Fluorescent microscopy was used to monitor the vascular behavior of 250 kDa Dextran molecules, 200 nm polystyrene nanoparticles (PB), and 1,000 × 400 nm discoidal polymeric nanoconstructs (DPN), under different permeability and flow conditions. Findings In the proposed on-chip microvasculature, it was confirmed that permeation enhancers could favor the perivascular accumulation of ~200 nm, in a dose and time dependent fashion, while have no effect on larger particles. Moreover, the microfluidic device was used to interrogate the role of particle deformability in vascular dynamics. In the presence of a continuous endothelium, soft DPN attached to the vasculature more avidly at sub-physiological flows (100 μm/sec) than rigid DPN, whose deposition was larger at higher flow rates (1 mm/sec). The proposed double-channel microfluidic device can be efficiently used to systematically analyze the vascular behavior of drug delivery systems to enhance their tissue specific accumulation

A 3D pancreatic tumor model to study T cell infiltration.

Human T cell infiltration across the endothelium in a 3-dimensional pancreatic tumor model in relation to activation and cellular components

Inhibition of adhesion, migration and of α5β1 integrin in the HCT-116 colorectal cancer cells treated with the ruthenium drug NAMI-A

NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug characterised by the selective activity against tumour metastases. Previously we have shown the influence of the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with α5β1 integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour cells on the extracellular matrix proteins. The fibronectin receptor α5β1 integrin is likely involved in the anti-adhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular matrix. Mechanistically, NAMI-A decreases the α5β1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5β1 integrin activation. These effects were validated by siRNA-induced knock down of the α5 integrin subunit and/or by the use of specific blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of α5β1 integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell functions such as adhesion and migration

Nanoformulated Zoledronic Acid Boosts the Vδ2 T Cell Immunotherapeutic Potential in Colorectal Cancer

Aminobisphosphonates, such as zoledronic acid (ZA), have shown potential in the treatment of different malignancies, including colorectal carcinoma (CRC). Yet, their clinical exploitation is limited by their high bone affinity and modest bioavailability. Here, ZA is encapsulated into the aqueous core of spherical polymeric nanoparticles (SPNs), whose size and architecture resemble that of biological vesicles. On Vδ2 T cells, derived from the peripheral blood of healthy donors and CRC patients, ZA-SPNs induce proliferation and trigger activation up to three orders of magnitude more efficiently than soluble ZA. These activated Vδ2 T cells kill CRC cells and tumor spheroids, and are able to migrate toward CRC cells in a microfluidic system. Notably, ZA-SPNs can also stimulate the proliferation of Vδ2 T cells from the tumor-infiltrating lymphocytes of CRC patients and boost their cytotoxic activity against patients’ autologous tumor organoids. These data represent a first step toward the use of nanoformulated ZA for immunotherapy in CRC patients

Erythrocyte-Inspired Discoidal Polymeric Nanoconstructs Carrying Tissue Plasminogen Activator for the Enhanced Lysis of Blood Clots

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