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Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4+ T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34+ cells. A fraction of these cells is viable for up to 3 months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4+ or CD34+ primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells.

ABSTRACT FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Colorectal cancer (CRC) is a leading cause of cancer-associated death. In the tumor site, the interplay between effector immune cells and cancer cells determines the balance between tumor elimination or outgrowth. We discovered that the protein TMEM123 is over-expressed in tumour-infiltrating CD4 and CD8 T lymphocytes and it contributes to their effector phenotype. The presence of infiltrating TMEM123+ CD8+ T cells is associated with better overall and metastasis-free survival. TMEM123 localizes in the protrusions of infiltrating T cells, it contributes to lymphocyte migration and cytoskeleton organization. TMEM123 silencing modulates the underlying signaling pathways dependent on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, which are required for synaptic force exertion. Using tumoroid-lymphocyte co-culture assays, we found that lymphocytes form clusters through TMEM123, anchoring to cancer cells and contributing to their killing. We propose an active role for TMEM123 in the anti-cancer activity of T cells within tumour microenvironment.

Summary: Background: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. Methods: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. Findings: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. Interpretation: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. Funding: This work was supported by an unrestricted grant from “Fondazione Romeo ed Enrica Invernizzi” to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project – P.I. Manfrini Nicola.

Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype in vitro. In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid’s capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration.

Lipid accumulation in the liver leads to nonalcoholic fatty liver disease (NAFLD) that is a risk factor for developing hepatocellular carcinoma (HCC). Here, the authors show that activation of the translation initiation factor eIF6 promotes lipid accumulation in the liver and targeting eIF6 in murine models reduces NAFLD and associated HCC.

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.