International expert group collaboration for developing an adverse outcome pathway for radiation induced leukemia.

PURPOSE: The concept of the adverse outcome pathway (AOP) has recently gained significant attention as to its potential for incorporation of mechanistic biological information into the assessment of adverse health outcomes following ionizing radiation (IR) exposure. This work is an account of the activities of an international expert group formed specifically to develop an AOP for IR-induced leukemia. Group discussions were held during dedicated sessions at the international AOP workshop jointly organized by the MELODI (Multidisciplinary European Low Dose Initiative) and the ALLIANCE (European Radioecology Alliance) associations to consolidate knowledge into a number of biological key events causally linked by key event relationships and connecting a molecular initiating event with the adverse outcome. Further knowledge review to generate a weight of evidence support for the Key Event Relationships (KERs) was undertaken using a systematic review approach. CONCLUSIONS: An AOP for IR-induced acute myeloid leukemia was proposed and submitted for review to the OECD-curated AOP-wiki (aopwiki.org). The systematic review identified over 500 studies that link IR, as a stressor, to leukemia, as an adverse outcome. Knowledge gap identification, although requiring a substantial effort via systematic review of literature, appears to be one of the major added values of the AOP concept. Further work, both within this leukemia AOP working group and other similar working groups, is warranted and is anticipated to produce highly demanded products for the radiation protection research community.

Adverse outcome pathway: a path toward better data consolidation and global co-ordination of radiation research.

BACKGROUND: The purpose of toxicology is to protect human health and the environment. To support this, the Organisation for Economic Co-operation and Development (OECD), operating via its Extended Advisory Group for Molecular Screening and Toxicogenomics (EAGMST), has been developing the Adverse Outcome Pathway (AOP) approach to consolidate evidence for chemical toxicity spanning multiple levels of biological organization. The knowledge transcribed into AOPs provides a structured framework to transparently organize data, examine the weight of evidence of the AOP, and identify causal relationships between exposure to stressors and adverse effects of regulatory relevance. The AOP framework has undergone substantial maturation in the field of hazard characterization of chemicals over the last decade, and has also recently gained attention from the radiation community as a means to advance the mechanistic understanding of human and ecological health effects from exposure to ionizing radiation at low dose and low dose-rates. To fully exploit the value of such approaches for facilitating risk assessment and management in the field of radiation protection, solicitation of experiences and active cooperation between chemical and radiation communities are needed. As a result, the Radiation and Chemical (Rad/Chem) AOP joint topical group was formed on June 1, 2021 as part of the initiative from the High Level Group on Low Dose Research (HLG-LDR). HLG-LDR is overseen by the OECD Nuclear Energy Agency (NEA) Committee on Radiation Protection and Public Health (CRPPH). The main aims of the joint AOP topical group are to advance the use of AOPs in radiation research and foster broader implementation of AOPs into hazard and risk assessment. With global representation, it serves as a forum to discuss, identify and develop joint initiatives that support research and take on regulatory challenges. CONCLUSION: The Rad/Chem AOP joint topical group will specifically engage, promote, and implement the use of the AOP framework to: (a) organize and evaluate mechanistic knowledge relevant to the protection of human and ecosystem health from radiation; (b) identify data gaps and research needs pertinent to expanding knowledge of low dose and low dose-rate radiation effects; and (c) demonstrate utility to support risk assessment by developing radiation-relevant case studies. It is envisioned that the Rad/Chem AOP joint topical group will actively liaise with the OECD EAGMST AOP developmental program to collectively advance areas of common interest and, specifically, provide recommendations for harmonization of the AOP framework to accommodate non-chemical stressors, such as radiation.

Effects of Chronic Low-Dose Internal Radiation on Immune-Stimulatory Responses in Mice.

The Linear-No-Threshold (LNT) model predicts a dose-dependent linear increase in cancer risk. This has been supported by biological and epidemiological studies at high-dose exposures. However, at low-doses (LDR ≤ 0.1 Gy), the effects are more elusive and demonstrate a deviation from linearity. In this study, the effects of LDR on the development and progression of mammary cancer in FVB/N-Tg(MMTVneu)202Mul/J mice were investigated. Animals were chronically exposed to total doses of 10, 100, and 2000 mGy via tritiated drinking water, and were assessed at 3.5, 6, and 8 months of age. Results indicated an increased proportion of NK cells in various organs of LDR exposed mice. LDR significantly influenced NK and T cell function and activation, despite diminishing cell proliferation. Notably, the expression of NKG2D receptor on NK cells was dramatically reduced at 3.5 months but was upregulated at later time-points, while the expression of NKG2D ligand followed the opposite trend, with an increase at 3.5 months and a decrease thereafter. No noticeable impact was observed on mammary cancer development, as measured by tumor load. Our results demonstrated that LDR significantly influenced the proportion, proliferation, activation, and function of immune cells. Importantly, to the best of our knowledge, this is the first report demonstrating that LDR modulates the cross-talk between the NKG2D receptor and its ligands.

Co-targeting Bulk Tumor and CSCs in Clinically Translatable TNBC Patient-Derived Xenografts via Combination Nanotherapy.

Triple-negative breast cancer (TNBC) accounts disproportionally for the majority of breast cancer-related deaths throughout the world. This is largely attributed to lack of a specific therapy capable of targeting both bulk tumor mass and cancer stem cells (CSC), as well as appropriate animal models to accurately evaluate treatment efficacy for clinical translation. Thus, development of effective and clinically translatable targeted therapies for TNBC is an unmet medical need. We developed a hybrid nanoparticles-based co-delivery platform containing both paclitaxel and verteporfin (PV-NP) to target TNBC patient-derived xenograft (PDX) tumor and CSCs. MRI and IVIS imaging were performed on mice containing PDX tumors to assess tumor vascularity and accumulation of NPs. NF-κB, Wnt, and YAP activities were measured by reporter assays. Mice bearing TNBC PDX tumor were treated with PV-NPs and controls, and tumors progression and CSC subpopulations were analyzed. MRI imaging indicated high vascularization of PDX tumors. IVIS imaging showed accumulation of NPs in PDX tumors. In comparison with control-NPs and free-drug combination, PV-NPs significantly retarded tumor growth of TNBC PDX. PV-NPs simultaneously repressed NF-κB, Wnt, and YAP that have been shown to be crucial for cancer growth, CSC development, and tumorigenesis. In conclusion, NPs containing two clinically used drugs concurrently inhibited NF-κB, Wnt, and YAP pathways and exhibited synergic effects on killing TNBC bulk tumor and CSCs. This combination nanotherapy evaluated with a PDX model may lead to an effective treatment of patients with TNBC.

Low-dose radiobiology program at Canadian nuclear laboratories: past, present, and future.

Health risks associated with the exposure of humans to low-dose ionizing radiation are currently estimated using the Linear-No-Threshold model. Over the last few decades, however, this model has been widely criticized for inconsistency with a large body of experimental evidence. Substantial efforts have been made to delineate biological mechanisms and health-related outcomes of low-dose radiation. These include a large DOE-funded Low Dose program operated in the 2000s, as well as the EU funded programs, previously NOTE and DOREMI and currently MELODI. Although not as widely known, the Atomic Energy of Canada Limited (AECL) in Chalk River, operated a low-dose radiobiology program since as early as 1948. The Canadian Nuclear Laboratories (CNL), the successor to AECL since 2015, has expanded this program into new areas making it the world's most robust, centrally coordinated and long-lived research efforts to delineate the biological effects of low-dose radiation. The purpose of this review is to provide a high-level overview of the low-dose radiobiology program maintained at CNL while capturing the historical perspectives. Past studies carried out at CNL have substantially influenced the area of low-dose radiobiology, exemplified by highly cited papers showing delays in spontaneous tumorigenesis in low-dose irradiated mice. The current low-dose research program at CNL is not only addressing a wide range of mechanistic questions about the biological effects of low doses - from genetic to epigenetic to immunological questions - but also moving toward novel areas, such as the dosimetry and health consequences of space radiation and the use of low-dose radiation in cancer therapy and regenerative medicine.

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis.

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3ß) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia.

PURPOSE: The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity. FINDINGS: MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34(+) hematopoietic stem and progenitor cells (HSPCs). Moreover, we observed that AML-derived MSCs had markedly increased adipogenic potential and delayed osteogenic differentiation, while maintaining normal morphology and viability. AML-derived MSCs, however, possessed reduced proliferative capacity and decreased frequency of subendothelial quiescent MSCs compared to controls. CONCLUSION: Our results support the notion of a bone marrow microenvironment characterized by increased propensity toward adipogenesis in AML, which may negatively impact normal hematopoiesis. Larger confirmatory studies are needed to understand the impact of various clinical factors. Novel leukemia treatments aimed at normalizing bone marrow niches may enhance the competitive advantage of normal hematopoietic progenitors over leukemia cells.

Mesenchymal stromal cells from patients with acute myeloid leukemia have altered capacity to expand differentiated hematopoietic progenitors.

The bone marrow microenvironment may be permissive to the emergence and progression of acute myeloid leukemia (AML). Studying interactions between the microenvironment and leukemia cells should provide new insight for therapeutic advances. Mesenchymal stromal cells (MSCs) are central to the maintenance of the hematopoietic niche. Here we compared the functions and gene expression patterns of MSCs derived from bone marrow aspirates of healthy donors and patients with AML. MSCs expanded from AML patients had heterogeneous morphology and displayed a wide range of proliferation capacity compared to MSCs from healthy controls. The ability of AML-MSCs to support the expansion of committed hematopoietic progenitors from umbilical cord blood-derived CD34+ cells may be impaired while the expression of genes associated with maintaining hematopoietic quiescence appeared to be increased in AML-MSCs compared to healthy donors. These results highlight important potential differences in the biologic profile of MSCs from AML patients compared to healthy donors that may contribute to the emergence or progression of leukemia.

A systematic review of preclinical studies on the therapeutic potential of mesenchymal stromal cell-derived microvesicles.

BACKGROUND: The therapeutic potential of mesenchymal stromal cells (MSCs) may be largely mediated by paracrine factors contained in microvesicles (MV) released from intracellular endosomes. A systematic review of controlled interventional animal studies was performed to identify models of organ injury where clinical translation of MSC-derived microvesicle therapy appears most promising as regenerative therapy. METHODS: A total of 190 published articles were identified in our systematic search of electronic databases (MEDLINE, EMBASE, PUBMED). After screening for eligibility, a total of 17 controlled studies testing MSC-derived MVs as therapeutic interventions in animal models of disease underwent comprehensive review, quality assessment, and data extraction. RESULTS: Thirteen studies addressed the regenerative potential following organ injury. Six studies were included on acute kidney injury, 4 on myocardial infarction and reperfusion injury, 1 on hind limb ischemia, 1 on liver injury, and 1 on hypoxic lung injury. Four studies addressed immunological effects of MSC-derived MVs on inhibiting tumor growth. Twelve studies (71%) provided explicit information regarding the number of animals allocated to treatment or control groups. Five studies (29%) randomly assigned animals to treatment or control groups and only 1 study (6%) reported on blinding. Therapeutic intervention involved isolation of exosomes (40-100 nm) in eight studies, while nine studies tested unfractionated microvesicles (<1,000 nm). In studies of tissue regeneration, all 13 reported that treatment with MSC-derived MVs improved at least one major/clinical parameter associated with organ dysfunction. Three of 4 studies evaluating the inhibition of tumor growth reported benefit. CONCLUSIONS: In preclinical studies, the use of MSC-derived MVs is strongly associated with improved organ function following injury and may be useful for inhibiting tumor growth. Improved preclinical study quality in terms of treatment allocation reporting, randomization and blinding will accelerate needed progress towards clinical trials that should assess feasibility and safety of this therapeutic approach in humans.

Greater organ involution in highly proliferative tissues associated with the early onset and acceleration of ageing in humans.

Domination of cell proliferation over cell death is a driving force for carcinogenesis, whereas reduced cell proliferation and increased cell death are characteristic of ageing. We employed published data to estimate representative mean values of cell turnover times for 31 different organs and tissues in adult humans and animals (when data in humans were lacking) as well as functional mass loss for 5 organs, accounting for actual mass loss and tissue conversion to fat, in humans over the adult period, age 25 to 70. We found that greater actual and functional mass loss was significantly associated (P=0.001 and P<0.0001, respectively) with the log of shorter cell turnover times. We propose that this is characteristic of stem cell exhaustion and replicative senescence. In addition, we provide quantitative evidence that, in many organs, involution is evident even in young adults. On the basis of published mass measurements of major organs, by analysis of covariance, we identified examples of significant (P≤0.05), accelerated actual or functional mass loss and ageing from early to late adulthood. We hypothesise and quantitatively demonstrate that functional mass loss accelerates with ageing by incorporating the contribution of actual mass loss, tissue conversion to fatty or fibrous tissue, and the presence of apoptotic, necrotic and senescent cells. We propose that mass loss, linked to replicative senescence, is an evolutionary adaptation that effectively limits cancer in young adults, as mass loss is first apparent soon after the end of the growth period, accelerating in the more elderly as biological conditions deviate away from those prevailing in youth, when the selective pressure on pleiotropic genes is greatest.

Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): proteomic analysis of plasma protein adsorption.

Grafting of methoxypoly(ethylene glycol) (mPEG) to cells and biomaterials is a promising non-pharmacological immunomodulation technology. However, due to the labile nature of cells, surface-plasma interactions are poorly understood; hence, a latex bead model was studied. PEGylation of beads resulted in a density and molecular weight dependent decrease in total adsorbed protein with a net reduction from (159.9±6.4) ng cm(-2) on bare latex to (18.4±0.8) and (52.3±5.3) ng cm(-2) on PEGylated beads (1 mmol L(-1) of 2 or 20 kD SCmPEG, respectively). SDS-PAGE and iTRAQ-MS analysis revealed differential compositions of the adsorbed protein layer on the PEGylated latex with a significant reduction in the compositional abundance of proteins involved in immune system activation. Thus, the biological efficacy of immunocamouflaged cells and materials is mediated by both biophysical obfuscation of antigens and reduced surface-macromolecule interactions.

Immunocamouflage: the biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) (mPEG).

Development of novel approaches for the immunomodulation of donor cells would have significant utility in transfusion and transplantation medicine. Immunocamouflage of cell surfaces by covalently grafted methoxypoly(ethylene glycol) (mPEG) (PEGylation) has emerged as a promising approach. While previous studies demonstrated the in vitro and in vivo efficacy of immunocamouflaged allogeneic blood cells, the biophysical mechanisms of immunoprotection have not been well-defined due to the fragility of intact cells. To overcome this limitation, polystyrene beads (1.2 and 8.0 microm) were used to elucidate the biophysical effects of polymer size, density and linker chemistry on charge camouflage and protein adsorption. These findings were correlated with biological studies using red blood cells and lymphocytes. Charge camouflage of both beads and cells was best achieved with long polymers. However, protein adsorption studies demonstrated an unexpected effect of target size. For 1.2 microm beads, decreased protein adsorption was best achieved with short (2 kDa) polymers whereas long chain (20 kDa) polymers were optimal for 8.0 microm particles. The biophysical findings correlated well with biological immunocamouflage as measured by particle electrophoresis and the inhibition of antibody-antigen (CD3, CD4 and CD28) recognition. Moreover, it was observed that antigen topography (CD28 vs. CD4) was of significance in selecting the appropriate polymer size. The biophysical interactions of PEGylated surfaces and macromolecules involve complex mechanisms dependent on the molecular weight, grafting concentration, target size and surface complexity. Cellular PEGylation strategies must be customized to account for target cell size, membrane complexity and antigen density and height.

Poly(oligo(ethylene glycol)acrylamide) brushes by surface initiated polymerization: effect of macromonomer chain length on brush growth and protein adsorption from blood plasma.

Three hydrolytically stable polyethyleneglycol (PEG)-based N-substituted acrylamide macromonomers, methoxypolyethyleneglycol (350) acrylamide (MPEG350Am) methoxypolyethyleneglycol (750) acrylamide(MPEG750Am) and methoxypolyethyleneglycol (2000)acrylamide (MPEG2000Am) with increasing PEG chain length were synthesized. Surface-initiated aqueous atom transfer radical polymerization (ATRP) using CuCl/1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) catalyst was utilized to generate dense polymer brushes from these monomers via an ester linker group on the surface of model polystyrene (PS) particles. The molecular weight, hydrodynamic thickness, and graft densities of the grafted polymer layers were controlled by changing the reaction parameters of monomer concentration, addition of Cu(II)Cl2, and sodium chloride. The graft densities of surface-grafted brushes decreased with increasing PEG macromonomer chain length, 350 > 750 >> 2000, under similar experimental conditions. The molecular weight of grafts increased with increase in monomer concentration, and only selected conditions produced narrow distributed polymer chains. The molecular weight of grafted polymer chains differs significantly to those formed in solution. The hydrodynamic thicknesses of the grafted polymer layers were fitted to the Daoud and Cotton model (DCM) for brush height on spherical surfaces. The results show that the size of the pendent groups on the polymer chains has a profound effect on the hydrodynamic thickness of the brush for a given degree of polymerization. The new PEG-based surfaces show good protection against nonspecific protein adsorption from blood plasma compared to the bare surface. Protein adsorption decreased with increasing surface density of grafted polymer chains. Poly(MPEG750Am) brushes were more effective in preventing protein adsorption than poly(MPEG350Am) even at low graft densities, presumably due to the increase in PEG content in the grafted layer.