In vitro 3D co-culture of mesenchymal stromal cells and Hodgkin Lymphoma cells on Collagen Scaffolds

PURPOSE. Conventional 2D culture systems do not consider the importance of tissue architecture that is particularly relevant since tissue microenvironment deeply contribute to determine the outcome of anti-cancer treatments. In this study we aimed to set up and use an in vitro 3D models for Hodgkin lymphoma (HL) to evaluate the activity of specific ADAM10 inhibitors LT4 and MN8, alone or in combination with the anti-CD30 ADC brentuximab-vedotin (Bre-Ved). METHODS. Three different 3D culture models were set up: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells), LN-derived de-cellularized matrices and collagen sponges repopulated with both LN-MSC and HL cells. RESULTS AND DISCUSSION. In these 3D systems LT4 and MN8 reduced the size of mixed spheroids and intracellular ATP content. In addition, sCD30 and TNF\u3b1 shedding was limited by LT4 and MN8 that not only interfered with HL cell growth, but also enhanced the anti-lymphoma effect of Bre-Ved. This effect was evident at low and ineffective doses of Bre-Ved as well, indicating a possible synergistic scheme to potentiate ADC-based lymphoma therapy. CONCLUSIONS. Both direct and combined anti-lymphoma effect of ADAM10 inhibitors with Bre-Ved can be studied in in vitro 3D model recapitulating features of LN microenvironment and leading to ADC effects improvement. For this reason, scaffolds may represent a new promising tool to reproduce LN architecture and useful for the study of pharmacological response

AB012. Transcriptional and chromatin profiling reveals the molecular architecture and druggable vulnerabilities of thymic epithelial tumors (TETs)

Thymic epithelial tumors (TETs) have been profiled to the present moment mainly through several analyses of FFPE samples. Despite the leap forward brought by the TCGA, several questions remain still unsolved. Among these, TETs are characterized by a strong component of immune infiltrate which makes the transcriptomic analyses conducted so far scarcely interpretable to profile stromal subpopulations constitutive of the tumor. Furthermore, rarely correspondent healthy tissue is available due to the lipomatous atrophy of aged thymi. Therefore, the recent report of (I) isolation, (II) propagation (III) and characterization of human thymic epithelial cells (TECs) and their capacity to reconstitute the functional organ ex vivo and in vivo, represents a novel approach to study the biology of both healthy and neoplastic thymi. Human thymic biopsies (both healthy and neoplastic) were digested and plated on a lethally irradiated murine feeder layer. Both RNA-Seq and CUTANDTAG were performed on cultivated TECs at different passages. Cultured TECs were injected with human thymic interstitial cells into rat decellularized scaffolds and cultivated for 10–12 days. sc-RNA Seq is currently being performed on both healthy and neoplastic thymic mini-organs and their correspondent primary tissues. Here show that we successfully cultivated a cohort of 21 clonogenic TECs in vitro including adult neoplastic TECs, their non-tumoral counterpart and pediatric TECs. We show that at the transcriptome level each class of TECs clusters independently and that neoplastic TECs belong to the same cloud independently from thymoma histotype. Around 1,400 differentially expressed genes (DEGs) can be found when comparing adult neoplastic and non-neoplastic counterpart, among which around 70 are transcription factors. Importantly, we prove for the first time that clonogenic TECs derived from TETs can repopulate a decellularized rat scaffold and recreate a 3D architecture mimicking the primary tumor. This work demonstrates that this culture system allows the expansion of clonogenic TECs from both tumor samples and their non-tumoral counterpart. Those cells, when transplanted into decellularized thymi, reproduce the architecture of the primary tissue, showing that TETs contain progenitor/stem epithelial cells. We are currently characterizing TECs at the transcriptomic and epigenomic level with aim of identifying new druggable targets prior to clinical trials

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 6520 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 64 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 64 0.0001) downregulated expression. On the other hand, Insr mRNA (P 64 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

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

SIMPLE SUMMARY: Alterations of the composition and architecture of the extracellular matrix (ECM), leading to increased stiffness, is known to condition development, invasiveness and severity of neoplasms. In this study, we report increased lymph node (LN) stiffness in human lymphomas, measured by LN elastometry or by computerized imaging of bioptic specimens. Stiffness matched to lymphoma histotype and grading. The enzyme lysyl oxidase (LOX) is involved in the rise of collagen cross-linking in Hodgkin lymphomas, while altered architecture, shown by scanning electron microscopy and polarized light microscopy is involved in advanced follicular lymphomas. Based on these data, digital pathology may help in the staging of lymphomas, and lysyl oxidase may represent a target for therapy in Hodgkin lymphomas. ABSTRACT: 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 β-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

The Veterans Affairs Low-Vision Visual Functioning Questionnaire-48 (VA LV VFQ-48): Performance of the Italian version

The Veterans Affairs Low-Vision Visual Functioning Questionnaire-48 is among the most validated tools to collect patient-reported outcomes in a low-vision population. We have aimed to conduct a pilot validation of the Italian version of the Veterans Affairs Low-Vision Visual Functioning Questionnaire-48

Inhibitors of A Disintegrin And Metalloproteinases-10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect

none16Shedding of A Disintegrin And Metalloproteinases (ADAM10) substrates, like TNFα 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 influence the outcome of anti-cancer treatments, we set up new three-dimensional (3D) culture systemsto verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed).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: 1) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase (LDH) release as a cell damage hallmark; 2) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFα shedding; 3) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; 4) ADAM10 inhibitors enhance the antilymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for direct and combined anti-lymphoma effect of ADAM10 inhibitors with BtxVed, leading to improvement of ADC effects; this is documented in 3D models recapitulating features of LN microenvironment, that can be proposed as reliable tool for antilymphoma drug testing.openPece, Roberta; Tavella, Sara; Costa, Delfina; Varesano, Serena; Camodeca, Caterina; Cuffaro, Doretta; Nuti, Elisa; Rossello, Armando; Alfano, Massimo; D'Arrigo, Cristina; Galante, Denise; Ravetti, Jean-Louis; Gobbi, Marco; Tosetti, Francesca; Poggi, Alessandro; Zocchi, Maria RaffaellaPece, Roberta; Tavella, Sara; Costa, Delfina; Varesano, Serena; Camodeca, Caterina; Cuffaro, Doretta; Nuti, Elisa; Rossello, Armando; Alfano, Massimo; D'Arrigo, Cristina; Galante, Denise; Ravetti, Jean-Louis; Gobbi, Marco; Tosetti, Francesca; Poggi, Alessandro; Zocchi, Maria Raffaell