Spontaneous NLRP3 inflammasome-driven IL-1-ß secretion is induced in severe COVID-19 patients and responds to anakinra treatment.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained. OBJECTIVES: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1ß, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes. METHODS: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line. RESULTS: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1ß in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation. CONCLUSIONS: These results provide further evidence that IL-1ß targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through α5ß1 Integrin.

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5ß1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5ß1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5ß1 modulates the expression/activity of another integrin, α2ß1, involved in the cell adhesion to collagen I. These results suggest that α5ß1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2ß1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5ß1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.

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

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.

The desmoplastic nature of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) prevents the infiltration of T cells and the penetration of chemotherapeutic drugs, posing a challenge to the validation of targeted therapies, including T cell immunotherapies. We present an in vitro 3D PDAC-TME model to observe and quantify T cell infiltration across the vasculature. In a three-channel microfluidic device, PDAC cells are cultured in a collagen matrix in the central channel surrounded, on one side, by endothelial cells (ECs) to mimic a blood vessel and, on the opposite side, by pancreatic stellate cells (PSCs) to simulate exocrine pancreas. The migration of T cells toward the tumor is quantified based on their activation state and TME composition. The presence of EC-lining drastically reduces T cell infiltration, confirming the essential role of the vasculature in controlling T cell trafficking. We show that activated T cells migrate ∼50% more than the not-activated ones toward the cancer cells. Correspondingly, in the absence of cancer cells, both activated and not-activated T cells present similar migration toward the PSCs. The proposed approach could help researchers in testing and optimizing immunotherapies for pancreatic cancer.

Modulating the Distant Spreading of Patient-Derived Colorectal Cancer Cells via Aspirin and Metformin.

Although screening has reduced mortality rates for colorectal cancer (CRC), about 20% of patients still carry metastases at diagnosis. Postsurgery chemotherapy is toxic and induces drug resistance. Promising alternative strategies rely on repurposing drugs such as aspirin (ASA) and metformin (MET). Here, tumor spheroids were generated in suspension by primary CRCs and metastatic lymph nodes from 11 patients. These spheroids presented a heterogeneous cell population including a small core of CD133+/ESA+ cancer stem cells surrounded by a thick corona of CDX2+/CK20+ CRC cells, thus maintaining the molecular hallmarks of the tumor source. Spheroids were exposed to ASA and/or MET at different doses for up to 7 days to assess cell growth, migration, and adhesion in three-dimensional assays. While ASA at 5 mM was always sufficient to mitigate cell migration, the response to MET was patient specific. Only in MET-sensitive spheroids, the 5 mM ASA/MET combination showed an effect. Interestingly, CRCs from diabetic patients daily pretreated with MET gave a very low spheroid yield due to reduced cancer cell survival. This study highlights the potential of ASA/MET treatments to modulate CRC spreading.

Two-Channel Compartmentalized Microfluidic Chip for Real-Time Monitoring of the Metastatic Cascade.

Metastases are the primary cause of death in cancer patients. Small animal models are helping in dissecting some key features in the metastatic cascade. Yet, tools for systematically analyzing the contribution of blood flow, vascular permeability, inflammation, tissue architecture, and biochemical stimuli are missing. In this work, a microfluidic chip is designed and tested to replicate in vitro key steps in the metastatic cascade. It comprises two channels, resting on the same plane, connected via an array of rounded pillars to form a permeable micromembrane. One channel acts as a vascular compartment and is coated by a fully confluent monolayer of endothelial cells, whereas the other channel is filled with a mixture of matrigel and breast cancer cells (MDA-MB-231) and reproduces the malignant tissue. The vascular permeability can be finely modulated by inducing pro-inflammatory conditions in the tissue compartment, which transiently opens up the tight junctions of endothelial cells. Permeability ranges from 1 µm/s (tight endothelium) to 5 µm/s (TNF-α at 50 ng/mL overnight) and up to ∼10 µm/s (no endothelium). Fresh medium flowing continuously in the vascular compartment is sufficient to induce cancer cell intravasation at rates of 8 cells/day with an average velocity of ∼0.5 µm/min. On the other hand, the vascular adhesion and extravasation of circulating cancer cells require TNF-α stimulation. Extravasation occurs at lower rates with 4 cells/day and an average velocity of ∼0.1 µm/min. Finally, the same chip is completely filled with matrigel and the migration of cancer cells from one channel to the other is monitored over a region of about 400 µm. Invasion rates of 12 cells/day are documented upon TNF-α stimulation. This work demonstrates that the proposed compartmentalized microfluidic chip can efficiently replicate in vitro, under controlled biophysical and biochemical conditions, the multiple key steps in the cancer metastatic cascade.

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.

Deciphering the relative contribution of vascular inflammation and blood rheology in metastatic spreading.

Vascular adhesion of circulating tumor cells (CTCs) is a key step in cancer spreading. If inflammation is recognized to favor the formation of vascular "metastatic niches," little is known about the contribution of blood rheology to CTC deposition. Herein, a microfluidic chip, covered by a confluent monolayer of endothelial cells, is used for analyzing the adhesion and rolling of colorectal (HCT-15) and breast (MDA-MB-231) cancer cells under different biophysical conditions. These include the analysis of cell transport in a physiological solution and whole blood over a healthy and a TNF-α inflamed endothelium with a flow rate of 50 and 100 nl/min. Upon stimulation of the endothelial monolayer with TNF-α (25 ng/ml), CTC adhesion increases from 2 to 4 times whilst cell rolling velocity only slightly reduces. Notably, whole blood also enhances cancer cell deposition from 2 to 3 times, but only on the unstimulated vasculature. For all tested conditions, no statistically significant difference is observed between the two cancer cell types. Finally, a computational model for CTC transport demonstrates that a rigid cell approximation reasonably predicts rolling velocities while cell deformability is needed to model adhesion. These results would suggest that, within microvascular networks, blood rheology and inflammation contribute similarly to CTC deposition, thereby facilitating the formation of metastatic niches along the entire network, including the healthy endothelium. In microfluidic-based assays, neglecting blood rheology would significantly underestimate the metastatic potential of cancer cells.

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

Tissue plasminogen activator (tPA) is the sole approved therapeutic molecule for the treatment of acute ischemic stroke. Yet, only a small percentage of patients could benefit from this life-saving treatment because of medical contraindications and severe side effects, including brain hemorrhage, associated with delayed administration. Here, a nano therapeutic agent is realized by directly associating the clinical formulation of tPA to the porous structure of soft discoidal polymeric nanoconstructs (tPA-DPNs). The porous matrix of DPNs protects tPA from rapid degradation, allowing tPA-DPNs to preserve over 70% of the tPA original activity after 3 h of exposure to serum proteins. Under dynamic conditions, tPA-DPNs dissolve clots more efficiently than free tPA, as demonstrated in a microfluidic chip where clots are formed mimicking in vivo conditions. At 60 min post-treatment initiation, the clot area reduces by half (57 ± 8%) with tPA-DPNs, whereas a similar result (56 ± 21%) is obtained only after 90 min for free tPA. In murine mesentery venules, the intravenous administration of 2.5 mg/kg of tPA-DPNs resolves almost 90% of the blood clots, whereas a similar dose of free tPA successfully recanalizes only about 40% of the treated vessels. At about 1/10 of the clinical dose (1.0 mg/kg), tPA-DPNs still effectively dissolve 70% of the clots, whereas free tPA works efficiently only on 16% of the vessels. In vivo, discoidal tPA-DPNs outperform the lytic activity of 200 nm spherical tPA-coated nanoconstructs in terms of both percentage of successful recanalization events and clot area reduction. The conjugation of tPA with preserved lytic activity, the deformability and blood circulating time of DPNs together with the faster blood clot dissolution would make tPA-DPNs a promising nanotool for enhancing both potency and safety of thrombolytic therapies.

Inhibition of adhesion, migration and of α5ß1 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 α5ß1 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 α5ß1 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 α5ß1 integrin expression, and reduces FAK (Focal Adhesion Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in α5ß1 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 α5ß1 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.