Producing sustainable microfluidic devices on a large scale has become a trend in the biomedical field. However, the transition from laboratory prototyping to large-scale industrial production poses several challenges due to the gap between academia and industry. In this context, prototyping with a mass production approach could be the novel strategy necessary to bridge academic research to the market. Here, the performance of polymer inserts to produce PMMA microfluidic devices using the microinjection moulding process is presented. Inserts were fabricated with an additive manufacturing process: material jetting technology. The importance of the inserts’ orientation on the printing plate in order to produce samples with more uniform thickness and lower roughness has been demonstrated using a flat cavity insert. In addition, preliminary tests were carried out on microstructured inserts with inverted channels of various cross-section shapes (semi-circular or trapezoidal) and widths (200 or 300 µm) in order to investigate the microstructures’ resistance during the moulding cycles. The best geometry was found in the channel with the trapezoidal cross-section with a width equal to 300 µm. Finally, a preliminary microfluidic test was performed to demonstrate the devices’ workability.

Pancreatic neuroendocrine neoplasms (PanNENs) are the second most common malignancy of the pancreas. Surgery remains the only curative treatment for localized disease. For patients with inoperable advanced or metastatic disease, few targeted therapies are available, but their efficacy is unpredictable and variable. Exploiting prior knowledge on pathogenetic processes involved in PanNEN tumorigenesis, we tested buparlisib (PI3K inhibitor) and ribociclib (CDK4/6 inhibitor), as single agents or in combination, in different preclinical models. First, we used cell lines representative of well-differentiated (INS-1E, NT-3) and poorly differentiated (BON-1) PanNENs. The combination of buparlisib with ribociclib reduced the proliferation of 2D and 3D spheroid cultures more potently than the individual drugs. Buparlisib, but not ribociclib, induced apoptosis. The anti-proliferative activity of the drugs correlated with downstream target inhibition at mRNA and protein levels. We then tested the drugs on primary islet microtissues from a genetic PanNET animal model (Men1-defective mice) and from wild-type mice: the drug combination was effective against the former without altering islet cell physiology. Finally, we treated PanNET patient-derived islet-like 3D tumoroids: the combination of buparlisib with ribociclib was effective in three out of four samples. Combined targeting of PI3K and CDK4/6 is a promising strategy for PanNENs spanning various molecular and histo-pathological features.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients’ outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

We investigated the design and characterization of a Lab-On-a-Chip (LoC) cell detection system primarily designed to support immunotherapy in cancer treatment. Immunotherapy uses Chimeric Antigen Receptors (CARs) and T Cell Receptors (TCRs) to fight cancer, engineering the response of the immune system. In recent years, it has emerged as a promising strategy for personalized cancer treatment. However, it requires bioreactor-based cell culture expansion and manual quality control (QC) of the modified cells, which is time-consuming, labour-intensive, and prone to errors. The miniaturized LoC device for automated QC demonstrated here is simple, has a low cost, and is reliable. Its final target is to become one of the building blocks of an LoC for immunotherapy, which would take the place of present labs and manual procedures to the benefit of throughput and affordability. The core of the system is a commercial, on-chip-integrated capacitive sensor managed by a microcontroller capable of sensing cells as accurately measured charge variations. The hardware is based on standardized components, which makes it suitable for mass manufacturing. Moreover, unlike in other cell detection solutions, no external AC source is required. The device has been characterized with a cell line model selectively labelled with gold nanoparticles to simulate its future use in bioreactors in which labelling can apply to successfully engineered CAR-T-cells. Experiments were run both in the air—free drop with no microfluidics—and in the channel, where the fluid volume was considerably lower than in the drop. The device showed good sensitivity even with a low number of cells—around 120, compared with the 10⁷ to 10⁸ needed per kilogram of body weight—which is desirable for a good outcome of the expansion process. Since cell detection is needed in several contexts other than immunotherapy, the usefulness of this LoC goes potentially beyond the scope considered here.