Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Anthropometric and glucometabolic changes in an aged mouse model of lipocalin-2 overexpression.

BACKGROUND: Lipocalin-2 (LCN2) is widely expressed in the organism with pleiotropic roles. In particular, its overexpression correlates with tissue stress conditions including inflammation, metabolic disorders, chronic diseases and cancer. OBJECTIVES: To assess the effects of systemic LCN2 overexpression on adipose tissue and glucose metabolism. SUBJECTS: Eighteen-month-old transgenic mice with systemic LCN2 overexpression (LCN2-Tg) and age/sex-matched wild-type mice. METHODS: Metabolic cages; histology and real-time PCR analysis; glucose and insulin tolerance tests; ELISA; flow cytometry; microPET and serum analysis. RESULTS: LCN2-Tg mice were smaller compared to controls but they ate (P = 0.0156) and drank (P = 0.0057) more and displayed a higher amount of visceral adipose tissue. Furthermore, LCN2-Tg mice with body weight ≥20 g showed adipocytes with a higher cell area (P < 0.0001) and altered expression of genes involved in adipocyte differentiation and inflammation. In particular, mRNA levels of adipocyte-derived Pparg (P ≤ 0.0001), Srebf1 (P < 0.0001), Fabp4 (P = 0.056), Tnfa (P = 0.0391), Il6 (P = 0.0198), and Lep (P = 0.0003) were all increased. Furthermore, LCN2-Tg mice displayed a decreased amount of basal serum insulin (P = 0.0122) and a statistically significant impaired glucose tolerance and insulin sensitivity consistent with Slc2a2 mRNA (P ≤ 0.0001) downregulated expression. On the other hand, Insr mRNA (P ≤ 0.0001) was upregulated and correlated with microPET analysis that demonstrated a trend in reduced whole-body glucose consumption and MRGlu in the muscles and a significantly reduced MRGlu in brown adipose tissue (P = 0.0247). Nevertheless, an almost nine-fold acceleration of hexokinase activity was observed in the LCN2-Tg mice liver compared to controls (P = 0.0027). Moreover, AST and ALT were increased (P = 0.0421 and P = 0.0403, respectively), which indicated liver involvement also demonstrated by histological staining. CONCLUSIONS: We show that LCN2 profoundly impacts adipose tissue size and function and glucose metabolism, suggesting that LCN2 should be considered as a risk factor in ageing for metabolic disorders leading to obesity.

Osteoblast adhesion and response mediated by terminal -SH group charge surface of SiOxCy nanowires.

Robust cell adhesion is known to be necessary to promote cell colonization of biomaterials and differentiation of progenitors. In this paper, we propose the functionalization of Silicon Oxycarbide (SiOxCy) nanowires (NWs) with 3-mercaptopropyltrimethoxysilane (MPTMS), a molecule containing a terminal -SH group. The aim of this functionalization was to develop a surface capable to adsorb proteins and promote cell adhesion, proliferation and a better deposition of extracellular matrix. This functionalization can be used to anchor other structures such as nanoparticles, proteins or aptamers. It was observed that surface functionalization markedly affected the pattern of protein adsorption, as well as the in vitro proliferation of murine osteoblastic cells MC3T3-E1, which was increased on functionalized nanowires (MPTMS-NWs) compared to bare NWs (control) (p < 0.0001) after 48 h. The cells showed a better adhesion on MPTMS-NWs than on bare NWs, as confirmed by immunofluorescence studies on the cytoskeleton, which showed a more homogeneous vinculin distribution. Gene expression analysis showed higher expression levels for alkaline phosphatase and collagen I, putative markers of the osteoblast initial differentiation stage. These results suggest that functionalization of SiOxCy nanowires with MPTMS enhances cell growth and the expression of an osteoblastic phenotype, providing a promising strategy to improve the biocompatibility of SiOxCy nanowires for biomedical applications.

Lysyl-Oxidase Dependent Extracellular Matrix Stiffness in Hodgkin Lymphomas: Mechanical and Topographical Evidence.

PURPOSE: The biochemical composition and architecture of the extracellular matrix (ECM) is known to condition development and invasiveness of neoplasms. To clarify this point, we analyzed ECM stiffness, collagen cross-linking and anisotropy in lymph nodes (LN) of Hodgkin lymphomas (HL), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL), compared with non-neoplastic LN (LDN). METHODS AND RESULTS: We found increased elastic (Young's) modulus in HL and advanced FL (grade 3A) over LDN, FL grade 1-2 and DLBCL. Digital imaging evidenced larger stromal areas in HL, where increased collagen cross-linking was found; in turn, architectural modifications were documented in FL3A by scanning electron microscopy and enhanced anisotropy by polarized light microscopy. Interestingly, HL expressed high levels of lysyl oxidase (LOX), an enzyme responsible for collagen cross-linking. Using gelatin scaffolds fabricated with a low elastic modulus, comparable to that of non-neoplastic tissues, we demonstrated that HL LN-derived mesenchymal stromal cells and HL cells increased the Young's modulus of the extracellular microenvironment through the expression of LOX. Indeed, LOX inhibition by ß-aminopropionitrile prevented the gelatin stiffness increase. CONCLUSIONS: These data indicate that different mechanical, topographical and/or architectural modifications of ECM are detectable in human lymphomas and are related to their histotype and grading.