Metabolic Viability Research Articles

Lipid Priming of Adipose Mesenchymal Stromal Cells with Docosahexaenoic Acid: Impact on Cell Differentiation, Senescence and the Secretome Neuroregulatory Profile.

Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential. Comprehensive dose-response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays. Priming with 40µM DHA for 72h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs. These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.

The macrophage migration inhibitory factor/CD74 axis in traumatic spinal cord injury: lessons learned from animal and human studies.

Traumatic spinal cord injury (SCI) is a severe condition leading to long-term impairment of motor, sensory, and autonomic functions. Following the initial injury, a series of additional events is initiated further damaging the spinal cord. During this secondary injury phase, both an inflammatory and immune modulatory response are triggered that have damaging and anti-inflammatory properties, respectively. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) and its receptor CD74 have been extensively studied in traumatic SCI. MIF expression is increased in spinal cord tissue after experimental SCI, mainly in astrocytes and microglia, as well as in the plasma of SCI patients. Functionally, MIF and CD74 were shown to regulate astrocyte viability, proliferation and cholesterol metabolism, microglia migration, and neuronal viability. Moreover, inhibition of the MIF/CD74 axis improved the functional recovery of SCI animals. We provide a detailed overview of studies analyzing the role of MIF and CD74 in traumatic SCI. We describe results from animal studies, using rat and mouse models for SCI, and human studies. Furthermore, we propose a new path for investigation, focused on B cells, that might lead to a better understanding of how MIF and CD74 contribute to the secondary injury cascade following traumatic SCI.

Boosting succinic acid production of Yarrowia lipolytica at low pH through enhancing product tolerance and glucose metabolism

BackgroundSuccinic acid (SA) is an important bio-based C4 platform chemical with versatile applications, including the production of 1,4-butanediol, tetrahydrofuran, and γ-butyrolactone. The non-conventional yeast Yarrowia lipolytica has garnered substantial interest as a robust cell factory for SA production at low pH. However, the high concentrations of SA, especially under acidic conditions, can impose significant stress on microbial cells, leading to reduced glucose metabolism viability and compromised production performance. Therefore, it is important to develop Y. lipolytica strains with enhanced SA tolerance for industrial-scale SA production.ResultsAn SA-tolerant Y. lipolytica strain E501 with improved SA production was obtained through adaptive laboratory evolution (ALE). In a 5-L bioreactor, the evolved strain E501 produced 89.62 g/L SA, representing a 7.2% increase over the starting strain Hi-SA2. Genome resequencing and transcriptome analysis identified a mutation in the 26S proteasome regulatory subunit Rpn1, as well as genes involved in transmembrane transport, which may be associated with enhanced SA tolerance. By further fine-tuning the glycolytic pathway flux, the highest SA titer of 112.54 g/L to date at low pH was achieved, with a yield of 0.67 g/g glucose and a productivity of 2.08 g/L/h.ConclusionThis study provided a robust engineered Y. lipolytica strain capable of efficiently producing SA at low pH, thereby reducing the cost of industrial SA fermentation.Graphical abstract

ENZYMES AND METABOLITES OF ENDOGENOUS CARBON MONOXIDE IN THE FETOPLACENTAL SYSTEM DURING PHYSIOLOGICAL GESTATION AND PLACENTAL DYSFUNCTION

Introduction. Prenatal ontogenesis largely depends on the functional and metabolic viability of the placenta and the entire fetoplacental complex. That is why the dysfunction of the placenta due to the changes in cellular regulation processes will lead to pregnancy complications and impaired fetal develop-ment. An important component of the system of intra- and intercellular regulators is the gas transmitter – carbon monoxide. Its role in cellular signaling and communication reactions, and at the same time, insufficient data of its metabolism, justifies the need to conduct research on this gas transmitter during complicated pregnancy. The aim of the work. To study the features of the metabolism of the active cellular mediator - the gas transmitter carbon monoxide in different objects of the fetoplacental system during physiological pregnancy and placental dysfunction. Material and methods. The research materials included placental tissue, amnion and amniotic fluid. The work uses spectrophotometric methods, enzyme immunoassay, and ion exchange chromatography. Results. Multidirectional changes in the activity of the studied enzymes (heme oxygenase and histidine decarboxylase), the content of the substrate of His-tidine reaction and the product of reaction of Histamine are established in case of placental disfunction. There is a certain correlation between the discov-ered studied components. Indicators of carbon monoxide metabolism can be markers for predicting postnatal damage – cerebral disorders in newborns. Conclusions. The identified injuries are obviously important links in the chain of disorders accompanied by the development of placental dysfunction.

MULTI-OMIC INTEGRATION REVEAL INTRA-TUMOUR HETEROGENEITY AND IDENTIFY DISULfiRAM AND COPPER AS A SYNERGISTIC THERAPY IN PAEDIATRIC EPENDYMOMA

Abstract AIMS Ependymoma (EPN) is the second most malignant paediatric brain tumour. The PF-A subgroup, associated with a hypoxic microenvironment, has a dismal survival rate of 50% with clinical trials failing to demonstrate a significant survival advantage from chemotherapy to date. As the lack of genomic alterations makes it difficult to uncover novel therapeutic targets, we present a multi-omic integration investigating whether spatially-distinct tumour microenvironments represent targetable metabolic niches in PF-A EPN. METHOD Surgical sampling of spatially-distinct regions was performed on eight PF-A patients. Metabolites and RNA were simultaneously extracted from the same cellular population and analysed using liquid chromatography-mass spectrometry (LC-MS) and RNAseq. Multi-omic integration was performed using Metscape3 and functional assays evaluating proliferation, invasion and siRNA-mediated knockdown were performed in 2D and 3D models of intra-tumour region patient-derived cell lines. Selected single and drug combination therapy was performed in normoxia and hypoxia, and synergy assessed using the Chou-Talalay method. RESULTS Multi-omic integration revealed 124 dysregulated metabolic pathways, encompassing 156 genes and 49 metabolites with the largest number of interactions occurring in the gluconeogenesis pathway. Each anatomical region also presented at least one unique gene-metabolite interaction demonstrating heterogeneity within and across PF-A EPN tumours. Four selected drug targets (Disulfiram, Vandetanib, Mildronate and FBP1 inhibitor) based on metabolically relevant genes (ALDH3A1, FMO3, BBOX1, FBP1) showed impairment of metabolic viability in vitro, with limited chemosensitivity observed in control human cerebellar astrocytes. The addition of Cu2+ significantly sensitized cells to Disulfiram, particularly under hypoxic conditions. This synergistic combination significantly impaired invasion in both normoxia and hypoxia. CONCLUSION This is the first instance where multi-omic data integration and intra-tumour heterogeneity has been investigated for paediatric EPN revealing novel therapeutic targets in the context of gene-metabolite correlations. Drug tolerability will be further assessed in vivo using the previously characterised PF-A EPN orthotopic mouse xenograft MAF-928.

Linear energy transfer dependent variation in viability and proliferation along the Bragg peak curve in sarcoma and normal tissue cells

Objective.The energy deposition of photons and protons differs. It depends on the position in the proton Bragg peak (BP) and the linear energy transfer (LET) leading to a variable relative biological effectiveness (RBE). Here, we investigate LET dependent alterations on metabolic viability and proliferation of sarcoma and endothelium cell lines following proton irradiation in comparison to photon exposure.Approach.Using a multi-step range shifter, each column of a 96-well plate was positioned in a different depth along four BP curves with increasing intensities. The high-throughput experimental setup covers dose, LET, and RBE changes seen in a treatment field. Photon irradiation was performed to calculate the RBE along the BP curve. Two biological information out of one experiment were extracted allowing a correlation between metabolic viability and proliferation of the cells.Main results.The metabolic viability and cellular proliferation were column-wise altered showing a depth-dose profile. Endothelium cell viability recovers within 96 h post BP irradiation while sarcoma cell viability remains reduced. Highest RBE values were observed at the BP distal fall-off regarding proliferation of the sarcoma and endothelial cells.Significance.The high-throughput experimental setup introduced here (I) covers dose, LET, and RBE changes seen in a treatment field, (II) measures short-term effects within 48 h to 96 h post irradiation, and (III) can additionally be transferred to various cell types without time consuming experimental adaptations. Traditionally, RBE values are calculated from clonogenic cell survival. Measured RBE profiles strongly depend on physical characteristics such as dose and LET and biological characteristics for example cell type and time point. Metabolic viability and proliferation proofed to be in a similar effect range compared to clonogenic survival results. Based on limited data of combined irradiation with doxorubicin, future experiments will test combined treatment with systemic therapies applied in clinics e.g. cyclin-dependent inhibitors.

Evaluating the Reparative Potential of Secretome from Patient-Derived Induced Pluripotent Stem Cells during Ischemia–Reperfusion Injury in Human Cardiomyocytes

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

Comparative investigation of exosome extraction from rat bone marrow mesenchymal stem cells using three different methodologies

AbstractExosomes have been identified as crucial mediators in numerous physiological and pathological processes, emerging as a focal point of scientific inquiry. This study aims to compare three methods for isolating exosomes from rat bone marrow mesenchymal stem cells: ultracentrifugation (UC), ultrafast separation system (EXODUS), and commercial precipitation kit (EXO‐kit). First, the investigation compared exosomal morphology, particle size distribution, and expression of marker proteins. Subsequently, the RNA content, protein concentration, and purity of exosomes were evaluated. Finally, the impact of these exosomes on cellular metabolic viability and migration capacity was assessed. Results indicated that exosomes exhibited spherical or elliptical membrane structures, and most of the exosomes extracted by the three methods were in the range of 30to 200 nm. UC‐extracted exosomes demonstrated the least impurities and clearest background, followed by EXODUS‐extracted exosomes, and lastly EXO‐kit‐extracted exosomes. The EXO‐kit‐extracted exosomes yielded the highest RNA and protein content, whereas those isolated through UC exhibited superior purity. Furthermore, exosomes extracted from EXODUS and EXO‐kit methods effectively enhanced the metabolic viability and migratory ability of osteoblast precursor cells compared to UC‐extracted exosomes. In conclusion, each of the three methodologies presents advantages and limitations. Therefore, the selection of an appropriate exosome extraction technique should be based on specific experimental objectives and requirements.

Merging Metabolic Modeling and Imaging for Screening Therapeutic Targets in Colorectal Cancer.

Cancer-associated fibroblasts (CAFs) play a key role in metabolic reprogramming and are well-established contributors to drug resistance in colorectal cancer (CRC). To exploit this metabolic crosstalk, we integrated a systems biology approach that identified key metabolic targets in a data-driven method and validated them experimentally. This process involved a novel machine learning-based method to computationally screen, in a high-throughput manner, the effects of enzyme perturbations predicted by a computational model of CRC metabolism. This approach reveals the network-wide effects of metabolic perturbations. Our results highlighted hexokinase (HK) as the crucial target, which subsequently became our focus for experimental validation using patient-derived tumor organoids (PDTOs). Through metabolic imaging and viability assays, we found that PDTOs cultured in CAF-conditioned media exhibited increased sensitivity to HK inhibition, confirming the model predictions. Our approach emphasizes the critical role of integrating computational and experimental techniques in exploring and exploiting CRC-CAF crosstalk.

Bacterial Cellulose In Vitro Uptake by Macrophages, Epithelial Cells, and a Triculture Model of the Gastrointestinal Tract.

Bacterial cellulose (BC) has a long-standing human consumption history in different geographies without any report of adverse effects. Despite its unique textural and functional properties, the use of BC in food products in Europe is still restricted due to concerns over its nanosize. Here, we evaluated the potential uptake of celluloses (from plant and microbial sources, processed using different blenders) by macrophages (differentiated THP-1 cells) and human intestinal epithelial cells (Caco-2 and HT29-MTX cells) without (coculture) or with (triculture) Raji-B cells. A carbohydrate-binding module coupled to a green fluorescent protein was employed to observe cellulose in the cell cultures by confocal laser scanning microscopy and stimulated emission depletion microscopy. The methodology demonstrated excellent sensitivity, allowing detection of single nanocrystals within cells. All celluloses were taken up by the macrophages, without significantly compromising the cell's metabolic viability. The viability of the cocultures was also not affected. Furthermore, no internalization was observed in the triculture cell model that was exposed 24 h to BC and Avicel LM310. When (rarely) detected, cellulose particles were found on the apical side of the membrane. Overall, the obtained results suggest that BC should not be absorbed into the human gut.

In Vitro Culture of Human Dermal Fibroblasts on Novel Electrospun Polylactic Acid Fiber Scaffolds Loaded with Encapsulated Polyepicatechin Physical Gels.

Polyepicatechin (PEC) in a hydrogel has previously shown promise in enhancing physiological properties and scaffold preparation. However, it remains unclear whether PEC-based fibers can be applied in skin tissue engineering (STE). This study aimed to synthesize and characterize electrospun PEC physical gels and polylactic acid (PLA) scaffolds (PLAloadedPECsub) for potential use as constructs with human dermal fibroblasts (HDFs). PEC was produced through enzymatic polymerization, as confirmed by Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) demonstrated the feasibility of producing PLAloadedPECsub by electrospinning. The metabolic activity and viability of HDFs cocultured with the scaffolds indicate that PLAloadedPECsub is promising for the use of STE.

Platelet and epithelial cell interations can be modeled in cell culture, and are not affected by dihomo-gamma-linolenic acid.

Increasing evidence is implicating roles for platelets in the development and progression of ovarian cancer, a highly lethal disease that can arise from the fallopian tubes, and has no current method of early detection or prevention. Thrombosis is a major cause of mortality of ovarian cancer patients suggesting that the cancer alters platelet behavior. The objective of this study was to develop a cell culture model of the pathological interactions of human platelets and ovarian cancer cells, using normal FT epithelial cells as a healthy control, and to test effects of the anti-platelet dihomo-gamma-linolenic acid (DGLA) in the model. Both healthy and cancer cells caused platelet aggregation, however platelets only affected spheroid formation by cancer cells and had no effect on healthy cell spheroid formation. When naturally-formed spheroids of epithelial cells were exposed to platelets in transwell inserts that did not allow direct interactions of the two cell types, platelets caused increased size of the spheroids formed by cancer cells, but not healthy cells. When cancer cell spheroids formed using magnetic nanoshuttle technology were put in direct physical contact with platelets, the platelets caused spheroid condensation. In ovarian cancer cells, DGLA promoted epithelial-to-mesenchymal (EMT) transition at doses as low as 100 μM, and inhibited metabolic viability and induced apoptosis at doses ≥150 μM. DGLA doses ≤150 μM used to avoid direct DGLA effects on cancer cells, had no effect on the pathological interactions of platelets and ovarian cancer cells in our models. These results demonstrate that the pathological interactions of platelets with ovarian cancer cells can be modeled in cell culture, and that DGLA has no effect on these interactions, suggesting that targeting platelets is a rational approach for reducing cancer aggressiveness and thrombosis risk in ovarian cancer patients, however DGLA is not an appropriate candidate for this strategy.

Phosphoenolpyruvate carboxykinase-2 (PCK2) is a therapeutic target in triple-negative breast cancer.

Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500nM and 3.5-27nM for PCK1 and PCK2, respectively. 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.

Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells.

Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC.

Green Tea Polyphenol (-)-Epicatechin Pretreatment Mitigates Hepatic Steatosis in an In Vitro MASLD Model.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), is becoming more prominent globally due to an increase in the prevalence of obesity, dyslipidemia, and type 2 diabetes. A great deal of studies have proposed potential treatments for MASLD, with few of them demonstrating promising results. The aim of this study was to investigate the potential effects of (-)-epicatechin (EPI) on the development of MASLD in an in vitro model using the HepG2 cell line by determining the metabolic viability of the cells and the levels of PPARα, PPARγ, and GSH. HepG2 cells were pretreated with 10, 30, 50, and 100 μM EPI for 4 h to assess the potential effects of EPI on lipid metabolism. A MASLD cell culture model was established using HepG2 hepatocytes which were exposed to 1.5 mM oleic acid (OA) for 24 h. Moreover, colorimetric MTS assay was used in order to determine the metabolic viability of the cells, PPARα and PPARγ protein levels were determined using enzyme-linked immunosorbent assay (ELISA), and lipid accumulation was visualized using the Oil Red O Staining method. Also, the levels of intracellular glutathione (GSH) were measured to determine the level of oxidative stress. EPI was shown to increase the metabolic viability of the cells treated with OA. The metabolic viability of HepG2 cells, after 24 h incubation with OA, was significantly decreased, with a metabolic viability of 71%, compared to the cells pretreated with EPI, where the metabolic viability was 74-86% with respect to the concentration of EPI used in the experiment. Furthermore, the levels of PPARα, PPARγ, and GSH exhibited a decrease in response to increasing EPI concentrations. Pretreatment with EPI has demonstrated a great effect on the levels of PPARα, PPARγ, and GSH in vitro. Therefore, considering that EPI mediates lipid metabolism in MASLD, it should be considered a promising hepatoprotective agent in future research.

Enhancing [177Lu]Lu-DOTA-TATE therapeutic efficacy in vitro by combining it with metronomic chemotherapeutics

BackgroundPeptide receptor radionuclide therapy (PRRT) uses [177Lu]Lu-[DOTA0-Tyr3]octreotate ([177Lu]Lu-DOTA-TATE) to treat patients with neuroendocrine tumours (NETs) overexpressing the somatostatin receptor 2A (SSTR2A). It has shown significant short-term improvements in survival and symptom alleviation, but there remains room for improvement. Here, we investigated whether combining [177Lu]Lu-DOTA-TATE with chemotherapeutics enhanced the in vitro therapeutic efficacy of [177Lu]Lu-DOTA-TATE.ResultsTransfected human osteosarcoma (U2OS + SSTR2A, high SSTR2A expression) and pancreatic NET (BON1 + STTR2A, medium SSTR2A expression) cells were subjected to hydroxyurea, gemcitabine or triapine for 24 h at 37oC and 5% CO2. Cells were then recovered for 4 h prior to a 24-hour incubation with 0.7–1.03 MBq [177Lu]Lu-DOTA-TATE (25 nM) for uptake and metabolic viability studies. Incubation of U2OS + SSTR2A cells with hydroxyurea, gemcitabine, and triapine enhanced uptake of [177Lu]Lu-DOTA-TATE from 0.2 ± 0.1 in untreated cells to 0.4 ± 0.1, 1.1 ± 0.2, and 0.9 ± 0.2 Bq/cell in U2OS + SSTR2A cells, respectively. Cell viability post treatment with [177Lu]Lu-DOTA-TATE in cells pre-treated with chemotherapeutics was decreased compared to cells treated with [177Lu]Lu-DOTA-TATE monotherapy. For example, the viability of U2OS + SSTR2A cells incubated with [177Lu]Lu-DOTA-TATE decreased from 59.5 ± 22.3% to 18.8 ± 5.2% when pre-treated with hydroxyurea. Control conditions showed no reduced metabolic viability. Cells were also harvested to assess cell cycle progression, SSTR2A expression, and cell size by flow cytometry. Chemotherapeutics increased SSTR2A expression and cell size in U2OS + SSTR2A and BON1 + STTR2A cells. The S-phase sub-population of asynchronous U2OS + SSTR2A cell cultures was increased from 45.5 ± 3.3% to 84.8 ± 2.5%, 85.9 ± 1.9%, and 86.6 ± 2.2% when treated with hydroxyurea, gemcitabine, and triapine, respectively.ConclusionsHydroxyurea, gemcitabine and triapine all increased cell size, SSTR2A expression, and [177Lu]Lu-DOTA-TATE uptake, whilst reducing cell metabolic viability in U2OS + SSTR2A cells when compared to [177Lu]Lu-DOTA-TATE monotherapy. Further investigations could transform patient care and positively increase outcomes for patients treated with [177Lu]Lu-DOTA-TATE.

109Pd/109mAg in-vivo generator in the form of nanoparticles for combined β- - Auger electron therapy of hepatocellular carcinoma

BackgroundConvenient therapeutic protocols for hepatocellular carcinoma (HCC) are often ineffective due to late diagnosis and high tumor heterogeneity, leading to poor long-term outcomes. However, recently performed studies suggest that using nanostructures in liver cancer treatment may improve therapeutic effects. Inorganic nanoparticles represent a unique material that tend to accumulate in the liver when introduced in-vivo. Typically, this is a major drawback that prevents the therapeutic use of nanoparticles in medicine. However, in HCC tumours, this may be advantageous because nanoparticles may accumulate in the target organ, where the leaky vasculature of HCC causes their accumulation in tumour cells via the EPR effect. On the other hand, recent studies have shown that combining low- and high-LET radiation emitted from the same radionuclide, such as 161Tb, can increase the effectiveness of radionuclide therapy. Therefore, to improve the efficacy of radionuclide therapy for hepatocellular carcinoma, we suggest utilizing radioactive palladium nanoparticles in the form of 109Pd/109mAg in-vivo generator that simultaneously emits β− particles and Auger electrons.ResultsPalladium nanoparticles with a size of 5 nm were synthesized using 109Pd produced through neutron irradiation of natural palladium or enriched 108Pd. Unlike the 109Pd-cyclam complex, where the daughter radionuclide diffuses away from the molecules, 109mAg remains within the nanoparticles after the decay of 109Pd. In vitro cell studies using radioactive 109Pd nanoparticles revealed that the nanoparticles accumulated inside cells, reaching around 50% total uptake. The 109Pd-PEG nanoparticles exhibited high cytotoxicity, even at low levels of radioactivity (6.25 MBq/mL), resulting in almost complete cell death at 25 MBq/mL. This cytotoxic effect was significantly greater than that of PdNPs labeled with β− (131I) and Auger electron emitters (125I). The metabolic viability of HCC cells was found to be correlated with cell DNA DSBs. Also, successful radioconjugate anticancer activity was observed in three-dimensional tumor spheroids, resulting in a significant treatment response.ConclusionThe results indicate that nanoparticles labeled with 109Pd can be effectively used for combined β− - Auger electron-targeted radionuclide therapy of HCC. Due to the decay of both components (β− and Auger electrons), the 109Pd/109mAg in-vivo generator presents a unique potential in this field.

Bioprinted keratinocyte and stem cell laden constructs for skin tissue engineering

Treating large-scale skin wounds continues to be a significant therapeutic challenge. Oftentimes, insufficient autologous material is available to achieve full coverage. Since biofabrication offers the chance of reproducible and precise large-scale production, the aim was to develop a customized 3D bioprinted skin construct containing immortalized HaCaT keratinocytes and ADSC to test the principal feasibility of this approach. Keratinocytes in different hydrogels containing varying concentrations of alginate, fibrin, collagen, gelatin, gelatin methacryloyl (GelMA) and hyaluronic acid as well as on a prefabricated collagen-elastin-matrix were cultured for a period of seven days. The metabolic activity of cultured keratinocytes was then evaluated during the co-cultivation of HaCaT and ADSC in a transwell model. The metabolic activity in all groups increased over the test period. The hydrogels containing alginate/hyaluronic acid/gelatin (Alg/HA/Gel) and GelMA demonstrated a good printability and the highest diffusion rates. There was no significant difference in pore size when comparing all hydrogels with each other. On the basis of the performed printability and diffusion assays as well as on the SEM analysis and rheological measurements Alg/HA/Gel and GelMA were chosen as hydrogels for the bioprinted 3D model. Fibrin hydrogel was integrated into the biofabricated constructs due to its excellent metabolic activity in the transwell model. As a second step, hydrogel stability, cell survival and metabolic activity in bioprinted 3D models containing keratinocytes and ADSC were evaluated over a period of 14 days. On day 14 the metabolic activity as well as the live cell count within the bioprinted constructs of the cocultured groups was significantly higher compared with day 1. The biofabricated constructs made of GelMA showed a higher cell viability compared with those made of Alg/HA/Gel. Additionally, to evaluate the cell migration out of the constructs, the metabolic activity and viability of the cells on the well bottom, was examined. After 14 days, an average of 50% of the well bottom was covered by HaCaT, which were initially printed in coculture into the constructs. These findings indicate that constructs made of GelMA containing keratinocytes and ADSC may offer a promising therapeutic option in the treatment of large chronic wounds.

Dual roles of photosynthetic hydrogel with sustained oxygen generation in promoting cell survival and eradicating anaerobic infection

Tissue engineering offers a promising alternative for oral and maxillofacial tissue defect rehabilitation; however, cells within a sizeable engineered tissue construct after transplantation inevitably face prolonged and severe hypoxic conditions, which may compromise the survivability of the transplanted cells and arouse the concern of anaerobic infection. Microalgae, which can convert carbon dioxide and water into oxygen and glucose through photosynthesis, have been studied as a source of oxygen supply for several biomedical applications, but their promise in orofacial tissue regeneration remains unexplored. Here, we demonstrated that through photosynthetic oxygenation, Chlamydomonas reinhardtii (C. reinhardtii) supported dental pulp stem cell (DPSC) energy production and survival under hypoxia. We developed a multifunctional photosynthetic hydrogel by embedding DPSCs and C. reinhardtii encapsulated alginate microspheres (CAMs) within gelatin methacryloyl hydrogel (GelMA) (CAMs@GelMA). This CAMs@GelMA hydrogel can generate a sustainable and sufficient oxygen supply, reverse intracellular hypoxic status, and enhance the metabolic activity and viability of DPSCs. Furthermore, the CAMs@GelMA hydrogel exhibited selective antibacterial activity against oral anaerobes and remarkable antibiofilm effects on multispecies biofilms by disrupting the hypoxic microenvironment and increasing reactive oxygen species generation. Our work presents an innovative photosynthetic strategy for oral tissue engineering and opens new avenues for addressing other hypoxia-related challenges.

Ionic liquid and ultrasound as a fast and innovative combination for improved extraction of Chlorella sorokiniana-derived carotenoids

Chlorella sorokiniana is a microalgae with high amounts of carotenoids, but the extraction methods need to be improved. This work hypothesized that the combination of ionic liquids (ILs) and ultrasound would provide a fast and efficient process for carotenoid extraction, allowing for the reuse of ILs in several extractive cycles. This study aimed to develop an improved ILs/ultrasound-based method for extracting carotenoids from C. sorokiniana. The potential reuse of ILs in five consecutive extraction cycles and the recovery of carotenoids was also examined. Moreover, the antioxidant activity of the carotenoid extracts and their effects on metabolic cell viability and cell death were investigated using HT22 neuronal cells. Initial tests with four ILs and acetone as a control were carried out in an ultrasonic probe. The ILs (1-butyl-3-methylimidazolium chloride [BMIM][Cl]; 1-hexyl-3-methylimidazolium chloride [HMIM][Cl]) were selected for the 25−1 fractional experimental design. The maximized parameters obtained in the experimental design were the following: IL ([HMIM][Cl]), a 1:10 solid-liquid ratio, a 1:4 IL-cosolvent ratio, and two repetitions of extraction with 7.5 min (with an extraction yield of 1.29 mg·g‐1 of dry matter). A total of 11 carotenoids were separated, and nine were identified, the major ones being lutein and β-carotene. [HMIM][Cl] recyclability using resin Amberlite XAD-7HP was efficient for at least five cycles. On average, 91 % of the IL was recovered, and the pigment yield increased by 40 %. The antioxidant activities of the extracts obtained using [HMIM][Cl] and acetone were 1.65 μmol and 2.32 μmol of α-tocopherol, respectively. Both extracts (≤ 4.0 μg·mL‐1) exhibited no significant toxicity to HT22 cells. The proposed method is an innovative and improved approach for carotenoid extraction from C. sorokiniana due to its short extraction times and high process yield. [HMIM][Cl] demonstrated stability in reuse cycles and proved to have the potential for obtaining carotenoids from C. sorokiniana.