Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

PURPOSE: Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. METHODS: Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. RESULTS: CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss. CONCLUSION: Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Magnetic Nanoparticle-Mediated Heating for Biomedical Applications.

Magnetic nanoparticles, especially superparamagnetic nanoparticles (SPIONs), have attracted tremendous attention for various biomedical applications. Facile synthesis and functionalization together with easy control of the size and shape of SPIONS to customize their unique properties, have made it possible to develop different types of SPIONs tailored for diverse functions/applications. More recently, considerable attention has been paid to the thermal effect of SPIONs for the treatment of diseases like cancer and for nanowarming of cryopreserved/banked cells, tissues, and organs. In this mini-review, recent advances on the magnetic heating effect of SPIONs for magnetothermal therapy and enhancement of cryopreservation of cells, tissues, and organs, are discussed, together with the non-magnetic heating effect (i.e., high Intensity focused ultrasound or HIFU-activated heating) of SPIONs for cancer therapy. Furthermore, challenges facing the use of magnetic nanoparticles in these biomedical applications are presented.

Cold-Responsive Nanoparticle Enables Intracellular Delivery and Rapid Release of Trehalose for Organic-Solvent-Free Cryopreservation.

Conventional cryopreservation of mammalian cells requires the use of toxic organic solvents (e.g., dimethyl sulfoxide) as cryoprotectants. Consequently, the cryopreserved cells must undergo a tedious washing procedure to remove the organic solvents for their further applications in cell-based medicine, and many of the precious cells may be lost or killed during the procedure. Trehalose has been explored as a nontoxic alternative to traditional cryoprotectants. However, mammalian cells do not synthesize trehalose or express trehalose transporters in their membranes, and the lack of an approach for the efficient intracellular delivery of trehalose has been a major hurdle for its use in cell cryopreservation. In this study, a cold-responsive polymer (poly(N-isopropylacrylamide-co-butyl acrylate)) is utilized to synthesize nanoparticles for the encapsulation and intracellular delivery of trehalose. The trehalose-laden nanoparticles can be efficiently taken up by mammalian cells. The nanoparticles quickly and irreversibly disassemble upon cold treatment, enabling the controlled and rapid release of trehalose from the nanoparticles inside cells. The latter is confirmed by an evident increase in cell volume upon cold treatment. This rapid cold-triggered intracellular release of trehalose is crucial to developing a fast protocol to cryopreserve cells using trehalose. Cells with intracellular trehalose delivered using the nanoparticles show comparable postcryopreservation viability compared to that of cells treated with DMSO, eliminating the need for the tedious and cell-damaging washing procedure required for using the DMSO-cryopreserved cells in vivo. This cold-responsive nanoparticle may greatly facilitate the use of trehalose as a nontoxic cryoprotectant for banking cells and tissues to meet their high demand by modern cell-based medicine.

NANOPARTICLE-MEDIATED DELIVERY OF CRYOPROTECTANTS FOR CRYOPRESERVATION.

Nanotechnology research has continued to garner interest and is investigated across a number of fields and industries, ranging from water treatment to clinical and biomedical applications. In biomedical research, for example, polymeric nanoparticles can be leveraged for controlled delivery of drugs and chemical compounds into cells. In cryobiological applications, polymeric nanoparticles can be utilized to deliver cryoprotectants (CPAs) and other protective agents, particularly those impermeable to the cell membrane, into cells to study their effects on cells during cooling down and warming back and at low temperatures. This perspective will discuss how polymeric nanoparticles have been used in cryobiology, with particular focus on how delivery systems have been specifically developed for low temperature applications and the potential for these systems going forward.

Enhancing POLST Completion in a Hospital Setting: An Interdisciplinary Approach.

EXECUTIVE SUMMARY: With increased therapeutic capabilities in healthcare today, many patients with multiple progressive comorbidities are living longer. They experience recurrent hospitalizations and often undergo procedures that are not aligned with their personal goals. That is why it is essential to discuss and document healthcare preferences prior to an acute event when significant interventions could occur, especially for patients with serious and progressive illness. Completion of an advance directive and a physician order for life-sustaining treatment (POLST) supports provision of goal-concordant care. Further, for patients who have do not attempt resuscitation (DNAR) orders or are diagnosed with advanced dementia, having a POLST is essential. This may be best accomplished with hospitalization discharge plans. Our 896-bed academic medical center, Cedars-Sinai Medical Center, launched a quality initiative in 2015 to complete POLSTs for patients being discharged with DNAR status or with dementia returning to a skilled nursing facility. As part of interdisciplinary progression of care rounds, emphasis was placed on those patients for whom POLST completion was indicated. Proactive, facilitated discussions with patients, family members, and attending physicians were initiated to support POLST completion. The completed forms were then uploaded to the electronic health record. Individual units and physicians received regular feedback on POLST completion rates, and the data were later shared at medical staff quality improvement meetings.During the initiative, POLST completion rates for DNAR patients discharged alive rose from 41% in fiscal year (FY) 2014 to 75% in FY 2019. Similar improvement was seen for patients with dementia discharged to skilled nursing facilities, regardless of code status (rising from 14% in FY 2014 to 54% in FY 2019). Subsequently, we have expanded our efforts to include early discussion and completion of these advanced care planning documents for patients recently diagnosed with high mortality cancers (ovarian, pancreatic, lung, glioblastoma), focusing on the completion of advanced care planning documentation and palliative care referrals.

Intracellular Delivery of Trehalose for Cell Banking.

Advances in stem cell technology and regenerative medicine have underscored the need for effective banking of living cells. Cryopreservation, using very low temperatures to achieve suspended animation, is widely used to store or bank cells for later use. This process requires the use of cryoprotective agents (CPAs) to protect cells against damage caused by the cooling and warming process. However, current popular CPAs like DMSO can be toxic to cells and must be thoroughly removed from cells before they can be used for research or clinical applications. Trehalose, a nontoxic sugar found in organisms capable of withstanding extreme cold or desiccation, has been explored as an alternative CPA. The disaccharide must be present on both sides of the cellular membrane to provide cryo-protection. However, trehalose is not synthesized by mammalian cells nor has the capability to diffuse through their plasma membranes. Therefore, it is crucial to achieve intracellular delivery of trehalose for utilizing the full potential of the sugar for cell banking. In this review, various methods that have been explored to deliver trehalose into mammalian cells for their banking at both cryogenic and ambient temperatures are surveyed. Among them, the nanoparticle-mediated approach is particularly exciting. Collectively, studies in the literature demonstrate the great potential of using trehalose as the sole CPA for cell banking, to facilitate the widespread use of living cells in modern medicine.

Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

A social network analysis approach to assess COVID19-related disruption to substance use treatment and informal social interactions among people who use drugs in Scotland.

AIMS: To assess the extent of Coronavirus-related disruption to health and social care treatment and social interactions among people with lived or living experience of substance use in Scotland, and explore potential reasons for variations in disruption. DESIGN: Cross sectional mixed methods interview, incorporating a social network 'egonet interview' approach asking about whether participants had interactions with a range of substance use, health, social care or third sector organisations, or informal social interactions. SETTING: Five Alcohol and Drug Partnership Areas in Scotland. PARTICIPANTS: 57 (42% women) participants were involved in the study, on average 42 years old. MEASUREMENTS: Five-point Likert scale reporting whether interactions with a range of services and people had gotten much better, better, no different (or no change), worse, or much worse since COVID19 and lockdown. Ratings were nested within participants (Individuals provided multiple ratings) and some ratings were also nested within treatment service (services received multiple ratings). The nested structure was accounted for using cross classified ordinal logistic multilevel models. FINDINGS: While the overall average suggested only a slight negative change in interactions (mean rating 2.93), there were substantial variations according to type of interaction, and between individuals. Reported change was more often negative for mental health services (Adjusted OR = 0.93 95% CI 0.17,0.90), and positive for pharmacies (3.03 95% CI 1.36, 5.93). The models found between-participant variation of around 10%, and negligible between-service variation of around 1% in ratings. Ratings didn't vary by individual age or gender but there was variation between areas. CONCLUSIONS: Substance use treatment service adaptations due to COVID19 lockdown led to both positive and negative service user experiences. Social network methods provide an effective way to describe complex system-wide interaction patterns, and to measure variations at the individual, service, and area level.

Evaluating transport conditions of conventional, widely used EDTA blood tubes for gene expression analysis in comparison to expensive, specialized PAXgene tubes in preparedness for radiological and nuclear events.

PURPOSE: Gene expression (GE) analysis of a radio-sensitive gene set (FDXR, DDB2, WNT3, POU2AF1) has been introduced in the last decade as an early and high-throughput prediction tool of later developing acute hematologic radiation syndrome (H-ARS) severity. The use of special tubes for RNA extraction from peripheral whole blood (PAXgene) represent an established standard in GE studies, although uncommonly used in clinics and not immediately available in the quantities needed in radiological/nuclear (R/N) incidents. On the other hand, EDTA blood tubes are widely utilized in clinical practice. MATERIAL AND METHODS: Using blood samples from eleven healthy donors, we investigated GE changes associated with delayed processing of EDTA tubes up to 4 h at room temperature (RT) after venipuncture (simulating delays caused by daily clinical routine), followed by a subsequent transport time of 24 h at RT, 4 °C, and -20 °C. Differential gene expression (DGE) of the target genes was further examined after X-irradiation with 0 Gy and 4 Gy under optimal transport conditions. RESULTS: No significant changes in DGE were observed when storing EDTA whole blood samples up to 4 h at RT and subsequently kept at 4 °C for 24 h which is in line with expected DGE. However, other storage conditions, such as -20 °C or RT, decreased RNA quality and/or (significantly) caused changes in DGE exceeding the known methodological variance of the qRT-PCR. CONCLUSION: Our data indicate that the use of EDTA whole blood tubes for GE-based H-ARS severity prediction is comparable to the quality of PAXgene tubes, when processed ≤ 4 h after venipuncture and the sample is transported within 24 hours at 4 °C.

Controlled Ice Nucleation With a Sand-PDMS Film Device Enhances Cryopreservation of Mouse Preantral Ovarian Follicles.

Ovarian follicle cryopreservation is a promising strategy for fertility preservation; however, cryopreservation protocols have room for improvement to maximize post-thaw follicle viability and quality. Current slow-freezing protocols use either manual ice-seeding in combination with expensive programmable-rate freezers or other clinically incompatible ice initiators to control the ice-seeding temperature in the extracellular solution, a critical parameter that impacts post-cryopreservation cell/tissue quality. Previously, sand has been shown to be an excellent, biocompatible ice initiator, and its use in cryopreservation of human induced pluripotent stem cells enables high cell viability and quality after cryopreservation. This study applies sand as an ice initiator to cryopreserve multicellular microtissue, preantral ovarian follicles, using a simple slow-freezing protocol in the mouse model. Ovarian follicles cryopreserved using the sand partially embedded in polydimethylsiloxane (PDMS) film to seed ice in the extracellular solution exhibit healthy morphology, high viability, and the ability to grow similarly to fresh follicles in culture post-thaw. This sand-based cryopreservation strategy can facilitate convenient ovarian follicle cryopreservation using simple equipment, and this study further demonstrates the translatability of this strategy to not only single cells but also multicellular tissues.

The Influence of Computed Tomography Contrast Agent on Radiation-Induced Gene Expression and Double-Strand Breaks.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07-0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03-0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Liquid Biopsy in Whole Blood for Identification of Gene Expression Patterns (mRNA and miRNA) Associated with Recurrence of Glioblastoma WHO CNS Grade 4.

GBM WHO CNS Grade 4 represents a major challenge for oncology due to its aggressive behavior. Conventional imaging has restrictions in detecting tumor recurrence. This prospective study aims to identify gene-based biomarkers in whole blood instead of isolating exosomes for the early detection of tumor recurrence. Blood samples (n = 33) were collected from seven GBM patients at time points before and after surgery as well as upon tumor recurrence. Four tumor tissue samples were assessed in parallel. Next-generation sequencing (NGS), including mRNA-seq and small RNA-seq, was used to analyze gene expression profiles in blood samples and tumor tissues. A novel filtering pipeline was invented to narrow down potential candidate genes. In total, between 6-93 mRNA and 1-19 small RNA candidates could be identified among the seven patients. The overlap of genes between the patients was minimal, indicating significant inter-individual variance among GBM patients. In summary, this prospective study supports the applicability of gene expression measurements in whole blood for the detection of tumor recurrence. It might provide an alternative to the challenging workflow of liquid biopsy after laborious exosome isolation from whole blood.

Micromechanical characterizations and viscoelastic modeling reveal elastic and viscoelastic heterogeneities in ovarian tissue and the significant viscoelastic contribution to the apparent elastic modulus determined by AFM indentation.

Ovarian follicles develop in a highly regulated mechanical microenvironment and disruptions to the microenvironment may cause infertility. However, the viscoelastic properties of the ovarian tissue are not well studied. Here, we characterize both the elastic and viscoelastic properties of ovarian tissue from both reproductively older and younger domestic cats using atomic force microscopy (AFM) indentation and viscoelastic models of stress relaxation. Importantly, our analyses reveal the apparent elastic modulus obtained from the conventional AFM indentation measurement is significantly higher than the intrinsic elastic modulus and insignificantly different from the equivalent elastic modulus that is the summation of the intrinsic elastic modulus and the viscoelastic contribution to modulus at time 0. Interestingly, the ovarian cortex of both reproductive age groups has a higher apparent/intrinsic modulus than that of the medulla. Furthermore, two different kinetics of stress relaxation are identified with rate constants of ∼1 s and ∼20-40 s, respectively. Moreover, the rate constant of the slow kinetics is significantly different between the cortex and medulla in the reproductively older ovaries. Finally, these mechanical heterogeneities appear to follow the heterogeneous distribution of hyaluronic acid (HA) in the ovary. These findings may be invaluable to the development of biomimetic follicle culture for treating infertility. STATEMENT OF SIGNIFICANCE: This study investigates not only elastic but also the viscoelastic heterogeneity in both reproductively younger and older ovarian tissues for the first time. Further, by combining AFM indentation measurement and viscoelastic modeling, we show the apparent elastic modulus conventionally reported in the literature for AFM indentation measurement is the summation of the intrinsic elastic modulus and a significant viscoelastic contribution to the modulus at time 0. This is an important consideration for others who use this method to quantify biomaterial properties. In addition, the possible connection between the mechanical and compositional heterogeneities is explored. These findings may be invaluable for designing biomaterials to recapitulate the mechanical environment of the ovary and possibly many other organs for biomimetic tissue engineering.

The effects of moderate concentrations of Karenia brevis on stone crab reproduction.

The stone crab, Menippe mercenaria, supports a commercial fishery along Florida's Gulf coast where harmful algae blooms, known as red tides (Karenia brevis) develop. Red tides occur nearly annually and can overlap with the stone crab reproductive season. We determined the impact of moderate red tide (K. brevis) concentrations (∼105 cells L-1) on stone crab embryo development, hatching success, female stress, hatch duration, and larval survival. Crabs and larvae were exposed to a control (no K. brevis) or moderate concentrations of K. brevis. No difference in embryo development or hatching success was observed. Stress was elevated in the K. brevis treatment, resulting in prolonged hatching relative to the control. Larval survival was reduced in K. brevis relative to the control. Moderate concentrations of K. brevis results in sublethal effects on stone crabs and reduces larval survival, suggesting that mitigation that reduces bloom concentrations could provide relief to stone crab populations.

Four Genes Predictive for the Severity of Hematological Damage Reveal a Similar Response after X Irradiation and Chemotherapy.

Radiological and especially nuclear accidents and incidents pose a threat to populations. In such events, gene expression (GE) analysis of a set of 4 genes (FDXR, DDB2, POU2AF1, WNT3) is an emerging approach for early and high-throughput prediction of the later manifesting severity degrees of the hematological acute radiation syndrome (H-ARS). Validation of this gene set on radiation victims is difficult since these events are rare. However, chemotherapy (CTX) is widely used e.g., breast cancer patient treatment and pathomechanisms, as well as blood cell count changes are comparable among both exposure types. We wondered whether GE changes are similarly deregulated after CTX, which would be interpreted as a confirmation of our already identified gene set for H-ARS prediction after irradiation. We examined radiation-induced differential GE (DGE) of our gene set as a positive control using in vitro whole blood samples from ten healthy donors (6 females, 4 males, aged: 24-40 years). Blood was incubated in vitro for 8 h after X irradiation with 0 and 4 Gy (1 Gy/min). These data were compared with DGE measured in vivo in blood samples of 10 breast tumor CTX patients (10 females, aged: 39-71 years) before and 4 days after administration of cyclophosphamide and epirubicin. RNA was isolated, reverse transcribed and quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to assess DGE of FDXR, DDB2, POU2AF1 and WNT3 relative to the unexposed samples using TaqMan assays. After X irradiation, we found a significant upregulation (irrespective of sex) with mean fold changes of 21 (P < 0.001) and 7 (P < 0.001) for FDXR and DDB2 and a significant down-regulation with mean fold changes of 2.5 (P < 0.001) and 2 (P = 0.005) for POU2AF1 and WNT3, respectively. After CTX, a similar pattern was observed, although mean fold changes of up-regulated FDXR (6-fold, P < 0.001) and DDB2 (3-fold, P < 0.001) as well as down-regulated POU2AF1 (1.2-fold, P = 0.270) and WNT3 (1.3-fold, P = 0.069) appeared lower corresponding to less altered blood cell count changes observed after CTX compared to historic radiation exposure data. However, a subpopulation of CTX patients (n = 6) showed on average a significant downregulation of POU2AF1 (1.8-fold, P = 0.04) and WNT3 (2.1-fold, P = 0.008). In summary, the pattern of up-regulated GE changes observed in all CTX patients and down-regulated GE changes observed in a subgroup of CTX patients appeared comparable with an already identified gene set predictive for the radiation-induced H-ARS. This underlines the significance of in vivo GE measurements in CTX patients, employed as a surrogate model to further validate already identified radiation-induced GE changes predictive for the H-ARS.

Ex-vivo dose response characterization of the recently identified EDA2R gene after low level radiation exposures and comparison with FDXR gene expression and the γH2AX focus assay.

OBJECTIVE: Recently, promising radiation-induced EDA2R gene expression (GE) changes after low level radiation could be shown. Stimulated by that, in this study, we intended to independently validate these findings and to further characterize dose-response relationships in comparison to FDXR and the γH2AX-DNA double-strand break (DSB) focus assay, since both assays are already widely used for biodosimetry purposes. MATERIALS AND METHODS: Peripheral blood samples from six healthy human donors were irradiated ex vivo (dose: ranging from 2.6 to 49.7 mGy). Subsequently, the fold-differences relative to the sham irradiated reference group were calculated. Radiation-induced changes in GE of FDXR and EDA2R were examined using the quantitative real-time polymerase-chain-reaction (qRT-PCR). DSB foci were quantified in 100 γH2AX + 53BP1 immunostained cells employing fluorescence microscopy. Examinations were performed at single time points enabling sufficient detection of both endpoints. RESULTS: A significant increase in EDA2R GE relative to the unexposed control was observed in the range of 2.6 mGy (1.6-fold, p = .045) to 5.4 mGy (2.2-fold, p = .0002), whereas the copy numbers increased linearly up to 13.1-fold at 49.7 mGy. On the contrary, FDXR upregulation (2.2-fold) became significant after a 22.6 mGy exposure (p ≤ .02) and increased linearly up to 4-fold at 49.7 mGy. A significant increase in radiation-induced foci (relative to unexposed, RIF-fd) was observed after 11.3 mGy (RIF-fd: 1.5 ± 0.5, p ≤ .03), while the foci increased linearly up to 3-fold at 49.7 mGy. From this, the FDXR and RIF-fd slopes have shown comparability, while the EDA2R slope was five times higher. Nevertheless, the coefficient of variation (CV) of EDA2R was about 30% higher than for RIF-fd. CONCLUSION: Higher radiation-induced EDA2R GE changes and a lower radiation detection level compared to RIF-fd and FDXR GE changes examined under optimal conditions ex vivo on human samples appear promising. Yet, our results represent just the beginning of further studies to be conducted in animal models for further time- and dose-dependent evaluation and additional examinations on radiologically examined patients to evaluate the impact of confounder, such as age, sex, social behavior, or diseases.

In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy.

Cancer immunotherapy that deploys the host's immune system to recognize and attack tumors, is a promising strategy for cancer treatment. However, its efficacy is greatly restricted by the immunosuppressive (i.e., immunologically cold) tumor microenvironment (TME). Here, we report an in-situ cryo-immune engineering (ICIE) strategy for turning the TME from immunologically "cold" into "hot". In particular, after the ICIE treatment, the ratio of the CD8+ cytotoxic T cells to the immunosuppressive regulatory T cells is increased by more than 100 times in not only the primary tumors with cryosurgery but also distant tumors without freezing. This is achieved by combining cryosurgery that causes "frostbite" of tumor with cold-responsive nanoparticles that not only target tumor but also rapidly release both anticancer drug and PD-L1 silencing siRNA specifically into the cytosol upon cryosurgery. This ICIE treatment leads to potent immunogenic cell death, which promotes maturation of dendritic cells and activation of CD8+ cytotoxic T cells as well as memory T cells to kill not only primary but also distant/metastatic breast tumors in female mice (i.e., the abscopal effect). Collectively, ICIE may enable an efficient and durable way to leverage the immune system for combating cancer and its metastasis.

Greatly Enhanced CTC Culture Enabled by Capturing CTC Heterogeneity Using a PEGylated PDMS-Titanium-Gold Electromicrofluidic Device with Glutathione-Controlled Gentle Cell Release.

The circulating tumor cells (CTCs, the root cause of cancer metastasis and poor cancer prognosis) are very difficult to culture for scale-up in vitro, which has hampered their use in cancer research/prognosis and patient-specific therapeutic development. Herein, we report a robust electromicrofluidic chip for not only efficient capture of heterogeneous (EpCAM+ and CD44+) CTCs with high purity but also glutathione-controlled gentle release of the CTCs with high efficiency and viability. This is enabled by coating the polydimethylsiloxane (PDMS) surface in the device with a 10 nm gold layer through a 4 nm titanium coupling layer, for convenient PEGylation and linkage of capture antibodies via the thiol-gold chemistry. Surprisingly, the percentage of EpCAM+ mammary CTCs can be as low as ∼35% (∼70% on average), showing that the commonly used approach of capturing CTCs with EpCAM alone may miss many EpCAM- CTCs. Furthermore, the CD44+ CTCs can be cultured to form 3D spheroids efficiently for scale-up. In contrast, the CTCs captured with EpCAM alone are poor in proliferation in vitro, consistent with the literature. By capture of the CTC heterogeneity, the percentage of stage IV patients whose CTCs can be successfully cultured/scaled up is improved from 12.5% to 68.8%. These findings demonstrate that the common practice of CTC capture with EpCAM alone misses the CTC heterogeneity including the critical CD44+ CTCs. This study may be valuable to the procurement and scale-up of heterogeneous CTCs, to facilitate the understanding of cancer metastasis and the development of cancer metastasis-targeted personalized cancer therapies conveniently via the minimally invasive liquid/blood biopsy.