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Additional file 2: Supplementary Fig. S1. Altered miR-146a expression influences BRAF/MEKi sensitivity and apoptosis in melanoma. A) Boxplots representing IC50 values of 6 matched PLX4032-resistant (R) and sensitive (S) melanoma cell lines to PLX4032 (vemurafenib), BRAFi (dabrafenib), MEKi (trametinib), to the combined treatment BRAF/MEKi, and to sTRAIL-induced apoptosis. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032, BRAFi and MEKi in melanoma cell lines (Spearman analysis). C) Forced expression of miR-146a (+m-miR-146a) in LM16R cell line and in LM69 and LM70 short term cultures increased the effects of PLX4032 treatment as shown by reduced cell growth and increased cell cytotoxicity and apoptosis, evaluated by CCK8, LDH and caspase 8 and 3/7 activity. D) Inhibition of miR-146a expression (i-miR-146a) in LM16 cells increased cell proliferation and decreased the release of LDH and the apoptosis rate as evaluated by CCK8, LDH and caspase 3/7 activity. E) Overexpression of miR-146a (m-miR-146a) upon PLX4032, BRAF/MEKi and sTRAIL treatments in LM47R cells increased cell cytotoxicity and apoptosis, as evaluated by LDH and caspase 8 and 3/7 activity. Data are plotted compared to scrambled control. *: p < 0.05, **: p < 0.01, ***: p < 0.0001 by Student’s unpaired t test. Supplementary Fig. S2. COX2 inhibition increases sensitivity to PLX4032 and to BRAF/MEKi and reduces PGE2 release. A) Relative luciferase activity after co-transfection of PTGS2 3’UTR luciferase reporter vector or control vector with miR-146a mimic or mimic negative control. Experiment was performed in LM16R cells. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032 in melanoma cell lines (Spearman analysis). C) Western blot analyses showing downregulation of COX2 after transfection with siRNA against COX2 (siCOX2) compared to scrambled control (S). COX2 protein levels were downregulated to 15%, as determined by quantification of the signal and expressed as the ratio of COX2/Actin intensity. Experiment was performed with LM47R cells. D) Silencing of COX2 by siRNA transfection enhanced cell growth inhibition and increased cell cytotoxicity and apoptosis upon treatment with PLX4032 compared to scrambled control. E) PGE2 release in culture supernatants following 24 h treatment with the COX2 inhibitors celecoxib and NS398, alone or in combination with BRAF/MEKi. F) Combined treatment with celecoxib increased cell growth inhibition and cytotoxicity by PLX4032. ●: interaction index = 1. G) Dose-dependent effect of celecoxib alone (5, 10, 20, 40, 50 and 80 μM) and combined with PLX4032 (3 μM) on cell growth. ●: interaction index = 1 at the dose of 50 μM of celecoxib combined with PLX4032. P values were calculated by Student’s unpaired t test in A, D and F, by one-way ANOVA followed by Bonferroni correction in E, and by two-way ANOVA followed by Bonferroni correction in G. *: p < 0.05, **: p < 0.01, ***: p < 0.0001. Supplementary Fig. S3. miR-146a expression levels are inversely correlated with levels of target genes. Analysis of correlation between miR-146a expression and its target genes EGFR, CCL2 and BCL2L1 in metastatic melanoma specimens. The rp and p values resulting from Pearson analysis are shown. Supplementary Fig. S4. Dynamic culture in bioreactor preserves TME architecture of 3D tumor explants. Representative histological images of H&E-stained 3D tumor sections after 3 days culture in bioreactor. The marked areas in the left panels are shown at higher magnification in the right panels to display the preserved cellularity and tumor tissue histo-architecture. Scale bar 80 μM. Supplementary Fig. S5. miR-146a overexpression increases drug sensitivity in 3D cultures of tumor explants. A) Modulation of direct and indirect miR-146a target genes upon transfection of miR-146a mimic (m-miR-146a) compared to scrambled-transfected control (S) in 3D cultures of BRAF-mutated melanoma tumors from naïve patients. AU: arbitrary units. B) Decreased staining for Ki67 and pERK proliferating cells and increased Caspase 3 positive cells in 3D cultures upon treatment with BRAF/MEKi and transfection with miR-146a (m-miR-146a) or scrambled control (S). Scale bar 10 um.

Innovative Models to Assess Multiple Myeloma Biology and the Impact of Drugs

Multiple Myeloma (MM) develops almost exclusively within the Bone Marrow (BM), highlighting the critical role of the microenvironment in conditioning disease progression and resistance to drugs. Indeed, while the therapeutic armamentarium for MM has significantly improved over the past 20 years, the disease remains ultimately incurable. This failure may depend on the high phenotypic and genetic heterogeneity of MM, but also on the paucity and inadequacy of two-dimensional (2D) conventional preclinical models in reproducing MM within the BM. In the present paper, we provide a brief updated overview on MM BM microenvironment. We then discuss newly developed preclinical models mimicking MM/microenvironment interactions, including three-dimensional (3D), gel-based, in vitro models and a novel ex vivo system of isolated tumor and stromal cells cultured in bioreactor. Potential applications of each model, relative to investigation of MM pathogenic mechanisms and prediction of the best drug/combination for each individual patient will be also evaluated.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

Erdheim-Chester disease (ECD) is a rare histiocytosis, characterized by xanthogranulomatous tissue infiltration by foamy histiocytes. Fibrosis, a histologic hallmark of ECD, is responsible for lesion growth and clinical manifestations. Unraveling molecular fibrotic pathway in ECD would allow the identification of new pharmacologic targets. In this study, we evaluated serum and tissue samples from a large cohort of ECD patients focusing on two major pro-fibrotic mediators, TGF-β1 and chemokine ligand 18 (CCL18). We found a marked increase in CCL18 but not TGF-β1 levels in serum and lesions of ECD patients (p < 0.001), independently of treatment status and consistently over time. Using a linear mathematical model, we also found that elevated CCL18 serum levels correlate with both number and severity of disease localizations. These findings suggest the involvement of CCL18-induced fibrosis in ECD pathogenesis, providing a rationale for exploring CCL18 inhibition as a treatment for progressive fibrosis in ECD.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo. Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis, characterised by tissue infiltration by foamy CD68+ CD1a− histiocytes.1 The disease has pleomorphic clinical manifestations, including long bones and extraskeletal involvement, and may be life-threatening, particularly when heart and central nervous system are affected.1 ECD histiocytes secrete proinflammatory cytokines2 and carry activating mutations along the RAS-RAF-MEK-ERK protein kinase signalling pathway, most commonly the BRAFV600E oncogenic mutation.3 4 Accordingly, patients with ECD have been treated with cytokine inhibitors, including infliximab,1 ,5 and, more recently, with the BRAFV600E inhibitor vemurafenib.6 The latter, however, induces sustained but partial clinical responses and recurrences on discontinuation,6 underlining the need for more effective therapeutic strategies. To identify the outcomes downstream constitutive ERK phosphorylation in ECD histiocytes and their response to small molecule-based inhibition, we performed three-dimensional (3D) culture of tissues from three BRAFV600E-mutated ECD patients in the RCCS bioreactor7 (and online supplementary methods) in the presence/absence of vemurafenib or infliximab, used as control.### Supplementary data [annrheumdis-2018-214432supp001.pdf] All patient samples maintained production of prototypical cytokines and chemokines2 in bioreactor, thus validating this technology also for ECD; moreover, infliximab, and, to a lesser degree, vemurafenib, significantly decreased cyto-chemokines …

Treatment of Erdheim–Chester disease (ECD), a rare non-Langerhans histiocytosis, relies on interferon-α, chemotherapeutic agents such as purine analogs, cytokine-blocking agents and BRAF inhibitors. Since interleukin (IL)-6 levels are elevated in serum and lesions of ECD patients, we evaluated the therapeutic efficacy and safety of IL-6 blockade with tocilizumab. We conducted an open-label, single-arm, phase II, prospective study of tocilizumab in three patients with multisystem ECD and poor tolerance/contraindications to IFN-α. Modifications of symptoms attributed to ECD represented the criteria for evaluation of clinical response. Changes at positron emission tomography scan, computed tomography scan, and magnetic resonance imaging at month 6 represented the main criteria for the evaluation of radiological response. Sustained complete clinical response and partial radiological improvement were observed in two patients, paralleled by modulation of systemic pro-inflammatory mediators. In spite of disease stabilization or improvement at extra-neurological sites, a third patient experienced a radiologic and clinical progression of central nervous system involvement, mirrored by a dramatic increase of circulating IL-6 and related cytokines. These findings indicate that IL-6 inhibition can be effective in ECD, but caution is advisable in patients with neurologic involvement. IL-6 emerges as a central mediator in ECD pathogenesis.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.