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Azithromycin (AZM) is a macrolide which is effective in the treatment of several airway diseases induced by bacterial or environmental insults. We have previously shown barrier enhancing effects of AZM on airway epithelium (AE). In order to analyze barrier enhancing effects of AZM in vivo we have challenged mice with inhalation of sulphur dioxide (SO2) to induce AE barrier failure. Mice were exposed to 50-400 ppm SO2 gas for 0.5-4 hours and monitored up to 7 days before bronchoalveolar lavage fluid (BALF) collection and analysis. AE barrier function was evaluated by measuring human serum albumin (HSA) leakage into BALF. HSA was injected into the tail vein of mice one hour prior to sacrifice, BALF was then harvested and HSA concentration measured by ELISA. AZM administered prior to exposure to SO2 resulted in a reduction of HSA, indicating protective effects of AZM on the AE. Increased intracellular vacuolization and lamellar body (LB) formations were seen in AZM treated animals. Glutathione-S-Transferases (GSTs), a class of enzymes that help maintain cellular integrity, were reduced in SO2 treated mice. Mass spectrometry analysis of BALF from SO2 exposed mice revealed that treating the mice with AZM lead to higher concentrations of GSTs in BALF, suggesting a link between AZM treatment and a reduction in GST concentration. In conclusion, we demonstrated a protective effect of AZM on the barrier integrity of the AE, possibly through stabilizing the intracellular microenvironment and by facilitating formation of LBs, providing further insight to its effectiveness in the treatment of airway diseases.

Publisher's version (útgefin grein), Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases., We thank Inovotion for their work and support in the CAM assays.

Publisher's version (útgefin grein), Primary myelofibrosis (PMF) is a Ph-negative myeloproliferative neoplasm (MPN), characterized by advanced bone marrow fibrosis and extramedullary haematopoiesis. The bone marrow fibrosis results from excessive proliferation of fibroblasts that are influenced by several cytokines in the microenvironment, of which transforming growth factor-β (TGF-β) is the most important. Micromechanics related to the niche has not yet been elucidated. In this study, we hypothesized that mechanical stress modulates TGF-β signalling leading to further activation and subsequent proliferation and invasion of bone marrow fibroblasts, thus showing the important role of micromechanics in the development and progression of PMF, both in the bone marrow and in extramedullary sites. Using three PMF-derived fibroblast cell lines and transforming growth factor-β receptor (TGFBR) 1 and 2 knock-down PMF-derived fibroblasts, we showed that mechanical stress does stimulate the collagen synthesis by the fibroblasts in patients with myelofibrosis, through the TGFBR1, which however seems to be activated through alternative pathways, other than TGFBR2., Patric Teodorescu was financed from an internal grant of the School of Doctoral Studies?Iuliu Hatieganu University (PCD 2018-2021). Ciprian Tomuleasa was also supported by two national research grants of the Romanian Government: the first one awarded for Frontiers Research Projects 2018-2022 (grant number PN-III-P4-ID-PCCF-2016-112), awarded to the Babes Bolyai University in collaboration with the Ion Chiricuta Oncology Institute Cluj Napoca; and the second one awarded for Young Research Teams 2020-2022 (grant number PN-III-P1-1.1-TE-2019-0271), awarded to the Iuliu Hatieganu University of Medicine and Pharmacy Cluj Napoca. Ciprian Tomuleasa, Bobe Petrushev and Delia Dima were also financed by an international collaborative grant of the European Economic Space between Romania and Iceland 2020-2022 (grant number 19-COP-0031).

Azithromycin (AZM) is a broad-spectrum antibiotic widely used to treat infections. AZM also has been shown to have anti-inflammatory and immunomodulatory functions unrelated to its antibacterial activity that contribute to the effectiveness of this drug in chronic respiratory diseases. The mechanisms behind these beneficial effects are not yet fully elucidated. We have previously shown that AZM enhances barrier integrity of bronchial epithelial cells and directs them towards epidermal differentiation. In this study, we analyzed the effect of AZM pre-treatment of human bronchial and alveolar derived cell lines on mechanical stress in a cyclical pressure air-liquid interface device (CPAD) that models the disruption of the epithelial barrier with increased inflammatory response in lung tissue, which is associated with ventilator-induced lung injury (VILI). Immunostaining and electron microscopy showed that barrier integrity of the epithelium was compromised by cyclically stressing the cells but maintained when cells had been pre-treated with AZM. Lamellar body formation was revealed in AZM pre-treated cells, possibly further supporting the barrier-enhancing effects. RNA sequencing showed that the inflammatory response was attenuated by AZM treatment before cyclical stress. YKL-40, an emerging inflammatory marker, increased both due to cyclical stress and upon AZM treatment. These data confirm the usefulness of the CPAD to model ventilator-induced lung injury and suggest that AZM has barrier protective and immunomodulatory effects, attenuating the inflammatory response during mechanical stress, and might therefore be lung protective during mechanical ventilation. The model could be used to assess further drug candidates that influence barrier integrity and modulate inflammatory response.

Introduction: Melflufen is a lipophilic peptide-conjugated alkylator currently in phase 3 trial for the treatment of late stage multiple myeloma. Due to its lipophilic nature it readily enters cells where it is cleaved by peptidases leading to entrapment and enrichment of hydrophilic alkylator payloads. Since peptidases are often upregulated in many cancer types, melflufen represents an attractive candidate for cancer therapy. Here, we have analysed the efficacy of the known alkylator melphalan and melflufen in isogenic normal and cancerous breast epithelial cell lines D492 and D492HER2 as well as the triple-negative MDA-MB-231 cell line using both 2D and 3D cell cultures and in vivo studies. Methods and results: Cell viability and cell proliferation studies unveiled that all cell lines were affected by melphalan and melflufen in both conditions but to a distinct extent. Melphalan treated cells needed quite high drug dosages to see an effect. In contrast, when treated with melflufen both cancerogenic D492HER2 and MDA-MB-231 lines were more sensitive than the normal-derived D492 line. Immunofluorescence staining showed that the sensitivity was lower in 3D culture compared to 2D culture. Western blot analysis and immunostaining revealed that treatment with melflufen induced profound DNA damage. Through comparison with the commonly used anti-cancer drug doxorubicin in cell viability studies we were able to show that melflufen was significantly more effective in reducing cellular growth. Chicken chorioallantoic membrane (CAM) assays showed comparable effects in vivo. The effect of melflufen in D492HER2 was attenuated if cells were pre-treated with the aminopeptidase inhibitor bestatin confirming the role of aminopeptidases in mediating the effect of melflufen in breast cancer cells. The aminopeptidase CD13 has been shown to facilitate melflufen cleavage. By separating both D492 and D492HER2 cells into CD13high and CD13low subpopulations and subsequently treating them with melflufen, we demonstrated increased melflufen sensitivity in the CD13high compared to CD13low population in D492HER2. Furthermore, siRNA knockdown of CD13 reduced the efficacy of melflufen corroborating its role in melflufen hydrolysis. Interestingly, knockdown of two other aminopeptidases, LAP3 and DPP7, led to similar efficacy reduction, suggesting that other aminopeptidases can facilitate melflufen to melphalan conversion. Conclusion: In this study, we have shown that melflufen is a highly efficient and selective anti neoplastic agent in breast cancer cell lines in-vitro and in-vivo and its efficacy is facilitated by expression of aminopeptidases including CD13, LAP3 and DPP7 indicating that melflufen could be a potent drug in breast cancer cells expressing high level of aminopeptidases. Citation Format: Alexander Schepsky, Gunnhildur A. Traustadottir, Jon Petur Joelsson, Saevar Ingthorsson, Jennifer Kricker, Jon T. Bergthyorsson, Katrina B. Petursdottir, Thorkell Gudjonsson, Ana Slipicevic, Fredrik Lehmann, Thorarinn Gudjonsson. Melflufen, a peptide-conjugated alkylator, shows efficacy in breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5205.

Mechanical ventilation (MV) is a life-saving therapy for critically ill patients, alleviating the work of breathing and supporting adequate gas exchange. However, MV can cause ventilator induced lung injury (VILI) by baro/volu- and atelectrauma, even lead to acute respiratory distress syndrome (ARDS), and substantially augment mortality. There is a need for specific biomarkers and novel research platforms for VILI/ARDS research to study these detrimental disorders and seek ways to avoid or prevent them. Previous in vitro studies on bronchial epithelium, cultured in air-liquid interface (ALI) conditions, have generally utilized static or constant pressure. We have developed a Cyclical Pressure ALI Device (CPAD) that enables cyclical stress on ALI cultured human bronchial cells, with the aim of mimicking the effects of MV. Using CPAD we were able to analyze differentially expressed VILI/ARDS and innate immunity associated genes along with increased expression of associated proteins. CPAD provides an easy and accessible way to analyze functional and phenotypic changes that occur during VILI and may provide a platform for future drug testing.