ABSTRACT
Background: Bone marrow mesenchymal stem cells [BM-MSCs] have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs [ESC-MSCs] as an alternative for BM-MSCs. Some of the therapeutic effects of the ESCMSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome.Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs [hESC-MSCs] spheroids on secretion of IL-1Beta, IL-10, and tumor necrosis factor Alpha [TNF-Alpha] from lipopolysaccharide [LPS]-induced peripheral blood mononuclear cells [PBMCs]
Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were nonadherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation
Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-Alpha [301.7 +/- 5.906, p < 0.0001] and IL-1 Alpha [485.2 +/- 48.38, p < 0.001] from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome [TNF-Alpha : 166.6 +/- 8.04, IL-1Beta: 125.2 +/- 2.73]
Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs
ABSTRACT
Background: Breast cancer is a heterogeneous disease characterized by differential responses to targeted and chemotherapeutic agents. Antibody-drug conjugates are one of the promising strategies for the treatment of breast cancer. Monomethyl auristatin E [MMAE] is a highly potent microtubule inhibitor and a common payload used for development of antibody-drug conjugates. The purpose of this study was to investigate the cytotoxic effects of MMAE on breast cancer cell lines
Materials and Methods: MDA-MB-468 and MDA-MB-453 cells were treated with MMAE at various concentrations [1, 10, 100, and 1000 ng/ml], and cytotoxicity was measured after 48 and 72 hours using an MTT assay
Results: Our findings indicated that MMAE possesses dose- and time-dependent cytotoxic activities against human breast cancer cells. The morphological features of the treated cells were supportive of the cytotoxic activity of MMAE. The results of the MTT assay showed that MMAE has a significant cytotoxicity against MDA-MB-468 and, to a lesser degree, MDA-MB-453 cells
Conclusion: MMAE can be used as a highly cytotoxic payload for development of antibody-drug conjugates against breast cancer
ABSTRACT
Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates [ADCs]. Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates
Methods: In the present study, we investigated the effect of various dithiothreitol [DTT] concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively
Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37[degree]C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively
Conclusion: Optimized site specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs
Subject(s)
Oxidation-Reduction , Pharmaceutical Preparations , Trastuzumab , Temperature , Sulfhydryl Compounds , Alkylation , Electrophoresis, Polyacrylamide Gel , DithiothreitolABSTRACT
Background: Mesenchymal stem cells [MSCs] are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells [ESCs] and bone marrow cells after hypoxia and normoxia preconditioning
Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs [bone marrow-derived mesenchymal stromal cells], which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell [PBMC] assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs
Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium
Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BMMSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor alpha, which needs further investigation
Subject(s)
Humans , Human Embryonic Stem Cells , Culture Media, Conditioned/pharmacology , Cell Hypoxia/physiology , Blood Cells , Leukocytes, Mononuclear/physiology , Interleukin-10/metabolismABSTRACT
Background: Monomethyl auristatin E [MMAE] is a synthetic analog of dolastatin 10, a compound originally isolated from the marine mollusk. MMAE, as a highly potent microtubule inhibitor, exerts its potent cytotoxic effect by inhibiting microtubule assembly, tubulin-dependent GTP hydrolysis and microtubes polymerization. This molecule, by itself, lacks the tumor specificity required to elicit therapeutic benefit. Nevertheless, the extremely cytotoxic potential of MMAE could be harnessed in the form of MMAE-antibody conjugates. The aim of the present study was to evaluate the cytotoxic activity of MMAE against breast [SKBR3] and kidney [HEK293] cancer cell lines in an in vitro cell-based assay
Materials and Methods: SKBR3 and HEK293 cells were treated with different concentrations ranging from 0.002048, 0.01024, 0.0512, 0.256, 1.28, 6.4, 32, 160, 800 and 4000 nM of MMAE, and cell viability was determined after 72 hours using an MTT colorimetric assay. The effect of MMAE was regularly monitored by direct observation using an invert microscope
Results: Microscopic observation showed that there was a concentration-dependent increase in cell death
Results from the MTT assay revealed a statistically significant loss of viability [P<0.0001] at concentrations ranging from 0.01024 to 4000 nM in SKBR3 cells, and 0.0512 to 4000 nM in HEK293 cells. Our findings showed that MMAE inhibited the growth of SKBR3 and HEK293 cells in a concentration-dependent manner, with IC50 values of 3.27 +/- 0.42 and 4.24 +/- 0.37 nM, respectively
Conclusion: MMAE was able to significantly inhibit cell growth at nanomolar concentrations, emphasizing its great potential for the development of antibody-drug conjugates