Biology subjects

Ding, L.

Publications and source records attributed to Ding, L..

At least 127 records · Page 7Linked to original sources

bioRxiv2020

Cellular Anatomy of the Mouse Primary Motor Cortex

An essential step toward understanding brain function is to establish a cellular-resolution structural framework upon which multi-scale and multi-modal information spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, through a collaborative effort from the Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based description of one brain structure - the primary motor cortex upper limb area (MOp-ul) of the mouse. Applying state-of-the-art labeling, imaging, computational, and neuroinformatics tools, we delineated the MOp-ul within the Mouse Brain 3D Common Coordinate Framework (CCF). We defined over two dozen MOp-ul projection neuron (PN) types by their anterograde targets; the spatial distribution of their somata defines 11 cortical sublayers, a significant refinement of the classic notion of cortical laminar organization. We further combine multiple complementary tracing methods (classic tract tracing, cell type-based anterograde, retrograde, and transsynaptic viral tracing, high-throughput BARseq, and complete single cell reconstruction) to systematically chart cell type-based MOp input-output streams. As PNs link distant brain regions at synapses as well as host cellular gene expression, our construction of a PN type resolution MOp-ul wiring diagram will facilitate an integrated analysis of motor control circuitry across the molecular, cellular, and systems levels. This work further provides a roadmap towards a cellular resolution description of mammalian brain architecture.

neuroscience↗

bioRxiv2020

High-throughput whole-brain mapping of rhesus monkey at micron resolution

Whole-brain mesoscale mapping of primates has been hindered by large brain size and the relatively low throughput of available microscopy methods. Here, we present an integrative approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed, large-scale, volumetric fluorescence microscopy, capable of completing whole-brain imaging of a rhesus monkey at 1 {micro}m x 1 {micro}m x 2.5 {micro}m voxel resolution within 100 hours. A progressive strategy is developed for high-efficiency, long-range tracing of individual axonal fibers through the dataset of hundreds of terabytes, establishing a "Serial sectioning and clearing, 3-dimensional Microscopy, with semi-Automated Reconstruction and Tracing" (SMART) pipeline. This system supports effective connectome-scale mapping of large primates that reveals distinct features of thalamocortical projections of the rhesus monkey brain at the level of individual axonal fibers.

neuroscience↗

bioRxiv2020

Broad-spectrum, patient-adaptable inhaled niclosamide-lysozyme particles are efficacious against coronaviruses in lethal murine infection models

Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, the oral formulation produces systemic drug levels that are too low to inhibit SARS-CoV-2. As an alternative, direct delivery of NIC to the respiratory tract as an aerosol could target the primary site of for SARS-CoV-2 acquisition and spread. We have developed a niclosamide powder suitable for delivery via dry powder inhaler, nebulizer, and nasal spray through the incorporation of human lysozyme (hLYS) as a carrier molecule. This novel formulation exhibits potent in vitro and in vivo activity against MERS-CoV and SARS-CoV-2 and may protect against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to CoV infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure wide-spread utilization

pharmacology and toxicology↗

bioRxiv2020

Prevailing role of mucosal immunoglobulions and B cells in teleost skin immune responses to bacterial infection

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent immunoglobulin (Ig) in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, here we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT-but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B-cells and specific IgT titers respectively within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection, and that because of the large surface of the skin, its SALT probably represents a prominent IgT inductive site in fish.

immunology↗

bioRxiv2020

Single-cell analysis identifies TCF4 and ID3 as a molecular switch of mammary epithelial stem cell differentiation.

It is well known that the expansion of the mammary epithelium during the ovarian cycles in female mammals is supported by the transient increase in mammary epithelial stem cells (MaSCs). However, dissecting the molecular mechanisms that govern MaSC function and differentiation is poorly understood due to the lack of standardized methods for their identification and isolation. The development of robust single-cell mRNA sequencing () technologies and the computational methods to analyze them provides us with novel tools to approach the challenge of studying MaSCs in a completely unbiased way without. Here, we have performed the largest scRNA-seq analysis of individual mammary epithelial cells (~70,000 cells). Our study identified a distinct cell population presenting molecular features of MaSCs. Importantly, further purification and additional in-depth single-cell analysis of these cells revealed that they are not a fully homogenous entity. Instead, we identified three subpopulations representing early stages of lineage commitment. By tracking their molecular evolution through single-cell network analysis we found that one of these subpopulations represents bipotent MaSCs from which luminal and basal lineages diverge. Importantly, we also confirmed the presence of these cells in human mammary glands. Finally, through expression and network analysis studies, we have uncovered transcription factors that are activated early during lineage commitment. These data identified E2-2 (Tcf4) and ID3 as a potential molecular switch of mammary epithelial stem cell differentiation.

developmental biology↗

bioRxiv2020

Nuclear GSK-3β and Oncogenic KRas Promote Expansion of Terminal Duct Cells and the Development of Intraductal Papillary Mucinous Neoplasm

Intraductal papillary mucinous neoplasm (IPMN) represents one type of pancreatic ductal adenocarcinoma (PDA) precursor lesion, however its cell-of-origin remains unclear. Here we describe a new mouse model in which pancreas-specific Cre activation of a nuclear glycogen synthase kinase-3{beta} transgene is combined with oncogenic KRas (referred to as KNGC). KNGC mice show accumulation of neoplastic ductal cells at 4-weeks that progressively develop into IPMN with low-grade dysplasia in advanced age. RNA-sequencing identified expression of several terminal duct cell lineage genes including Agr2 and Aqp5. Interestingly, Aqp5, a water channel, was found to be required for the development of IPMN lesions in KNGC mice. Staining of human IPMN samples indicates that these preneoplastic lesions also arise from expansion of the terminal duct population. Altogether, these data highlight the utility of the KNGC model for understanding the biology of IPMN and potential utility in defining predictive biomarkers of IPMN - PDA development. Statement of significanceUnderstanding the cell-of-origin of IPMN is crucial to developing early detection methods that specifically target aggressive precursors of PDA. This work, using a novel mouse model, identifies Aqp5-modulated development of Agr2+ terminal ducts that could potentially serve as a clinical biomarker for IPMN.

cancer biology↗

bioRxiv2020

LINE-1 expression in cancer correlates with DNA damage response, copy number variation, and cell cycle progression

ABSTRACTRetrotransposons are genomic DNA sequences that are capable of copying themselves to new genomic locations via RNA intermediates; LINE-1 is the only retrotransposon that remains autonomous and active in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues, but LINE-1 is active in many cancers. Recent studies using LINE-1 constructs indicate that host cells activate a DNA damage response (DDR) to repair retrotransposition intermediates and resolve conflicts between LINE-1 and DNA replication. Using multi-omic data from the CPTAC project, we found correlations between LINE-1 expression and ATM-MRN-SMC DDR signalling in endometrial cancer and between LINE-1 and the ATR-CHEK1 pathway in p53 wild type breast cancer. This provides evidence that conflicts between LINE-1 and DNA replication occur in at least some human cancers. Furthermore, LINE-1 expression in these cancers is correlated with the total amount of copy number variation genome wide, indicating that, when active in cancer, pointing to a direct impact of LINE-1 associated DNA damage on genome structure. We also find that, in endometrial and ovarian cancer, LINE-1 expression is correlated with the expression of genes that drive cycle progression including E2F3, PLK1 and Aurora kinase B. This study provides evidence, supporting recent work in model cell lines, of a LINE-1/DDR connection in human tumors and raises the possibility of additional interactions between LINE-1 and the cell cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Predicting transdermal fentanyl delivery using mechanistic simulations for tailored therapy

Transdermal drug delivery is a key technology for administering drugs. However, most devices are "one-size-fits-all", even though drug diffusion through the skin varies significantly from person-to-person. For next-generation devices, personalization for optimal drug release would benefit from an augmented insight into the drug release and percutaneous uptake kinetics. Our objective was to quantify the changes in transdermal fentanyl uptake with regards to the patients age and the anatomical location where the patch was placed. We also explored to which extent the drug flux from the patch could be altered by miniaturizing the contact surface area of the patch reservoir with the skin. To this end, we used validated mechanistic modeling of fentanyl diffusion, storage, and partitioning in the epidermis to quantify drug release from the patch and the uptake within the skin. A superior spatiotemporal resolution compared to experimental methods enabled in-silico identification of peak concentrations and fluxes, and the amount of stored drug and bioavailability. The patients drug uptake showed a 36% difference between different anatomical locations after 72 h, but there was a strong interpatient variability. With aging, the drug uptake from the transdermal patch became slower and less potent. A 70-year-old patient received 26% less drug over the 72-h application period, compared to an 18-year-old patient. Additionally, a novel concept of using micron-sized drug reservoirs was explored in silico. These reservoirs induced a much higher local flux ({micro}g cm-2 h-1) than conventional patches. Up to a 200-fold increase in the drug flux was obtained from these small reservoirs. This effect was mainly caused by transverse diffusion in the stratum corneum, which is not relevant for much larger conventional patches. These micron-sized drug reservoirs open new ways to individualize reservoir design and thus transdermal therapy. Such computer-aided engineering tools also have great potential for in-silico design and precise control of drug delivery systems. Here, the validated mechanistic models can serve as a key building block for developing digital twins for transdermal drug delivery systems.

bioengineering↗

bioRxiv2020

One-Step Rapid Quantification of SARS-CoV-2 Virus Particles via Low-Cost Nanoplasmonic Sensors in Generic Microplate Reader and Point-of-Care Device

The spread of SARS-CoV-2 virus in the ongoing global pandemics has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemics. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. We demonstrate that we can detect as few as 30 virus particles in one step within 15 minutes and can quantify the virus concentration linearly in the range of 103 vp/ml to 106 vp/ml. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.

bioengineering↗

bioRxiv2020

Genus-wide characterization of bumblebee genomes reveals variation associated with key ecological and behavioral traits of pollinators

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary lifecycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships while incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. These high-quality genomic resources capture natural genetic and phenotypic variation across bumblebees, offering new opportunities to advance our understanding of their remarkable ecological success and to identify and manage current and future threats.

genomics↗

bioRxiv2020

Tumor-on-a-chip platform to interrogate the role of macrophages in tumor progression

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor "M1"-type to protumor "M2"-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolve as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell to cell dynamics at high spatial and temporal resolution. Using our recently developed micro-physiological "tumor-on-a-chip" (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells while the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polorized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of chemokines CXCL9, CXCL10, and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA-sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of MMP7 and ANGPT2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment. Insight BoxMacrophages in the tumor microenvironment are key determinants of tumor behavior and clinical outcome. The macrophage subset composition and its functional impact change as tumors progress or during treatment, but adequate models to study this are lacking. We developed a tumor-on-a-chip model of perfused 3D tumor growth to probe the impact of defined macrophage subsets. Our data is consistent with previously described macrophage activity in the tumor microenvironment, and provides potential new molecular targets. Herein, we demonstrate feasibility of probing immuno-oncology questions in a 3D in vitro microenvironment and at a spatiotemporal resolution.

cancer biology↗

bioRxiv2020

Electronic blood vessel

Advances in bioelectronics have great potential to address unsolvable biomedical problems in the cardiovascular system. By using poly(L-lactide-co-{varepsilon}-caprolactone) (PLC) that encapsulates the liquid metal to make flexible and bio-degradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelization process through electrical stimulation and can enable controlled gene delivery into specific part of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency three months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of vascular tissue-machine interface.

bioengineering↗

bioRxiv2020

Human IgG cell neutralizing monoclonal antibodies block SARS-CoV-2 infection

The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development.

immunology↗

bioRxiv2020

Impaired state-dependent potentiation of GABAergic synaptic currents triggers seizures in an idiopathic generalized epilepsy model

Idiopathic generalized epilepsy(IGE) patients have genetic causes and their seizure onset mechanisms particularly during sleep remain elusive. Here we proposed that sleep-like slow-wave oscillations(0.5 Hz SWOs) potentiated excitatory or inhibitory synaptic currents in layer V cortical pyramidal neurons from wild-type(wt) mouse ex vivo brain slices. In contrast, SWOs potentiated excitatory, not inhibitory, currents in cortical neurons from heterozygous(het) knock-in(KI) IGE mice(GABAA receptor {gamma}2 subunit Gabrg2Q390X mutation), creating an imbalance between evoked excitatory and inhibitory currents to effectively prompt neuronal action potentials. Similarly, more physiologically similar up/down-state(present during slow-wave sleep) induction in cortical neurons could potentiate excitatory synaptic currents within slices from wt/het Gabrg2Q390X KI mice. Consequently, SWOs or up/down-state induction in vivo (using optogenetic method) could trigger epileptic spike-wave discharges(SWDs) in het Gabrg2Q390X KI mice. To our knowledge, this is the first operative mechanism to explain why epileptic SWDs preferentially happen during non-REM sleep or quiet-wakefulness in human IGE patients.

neuroscience↗

bioRxiv2020

Aquaporin mediating stomatal closure is associated with water conservation under mild water deficit

O_LIContradictory results indicate that aquaporins might facilitate the diffusion of both water and H2O2 during abscisic acid (ABA) triggered stomatal closure. Here, we tested whether maize plasma membrane PIP2;5 aquaporin regulates stomatal closure under water deficit or ABA treatment in intact plants, detached leaves, and peeled epidermis. C_LIO_LITranspiration, stomatal conductance and aperture, as well as reactive oxygen species (ROS) in stomatal complexes were studied in maize lines deregulated in PIP2;5 gene expression, under water deficit and/or ABA treatments. C_LIO_LIIn well-watered conditions, the PIP2;5 overexpressing (OE) plants transpired more than the wild-type plants (WT), while no significant difference in transpiration was observed between pip2;5 KO and WT plants. Upon mild-water deficit or low ABA concentration treatment, the transpiration and stomatal conductance decreased more in PIP2;5 OE, and less in pip2;5 KO lines, in comparison with WT plants. Using isolated epidermis, ABA treatment induced faster stomatal closing in PIP2;5 OE lines compared to the WT, while pip2;5 KO stomata were ABA insensitive. These phenotypes were associated with guard cell ROS accumulation. C_LIO_LITogether, these data indicate that maize PIP2;5 regulates early stomatal closure for water conservation upon a water deficit environment. C_LI

plant biology↗

bioRxiv2020

Ancestry-Specific Predisposing Germline Variants in Cancer

BackgroundCancer risk differs across ancestries and these differences may result from differing prevalence of inherited genetic predisposition. Yet, most germline genomic studies performed to date have focused on individuals of European ancestry. Ancestry-specific analyses of germline genomes are required to inform cancer genetic risk and prognosis for each ancestral group. Here, we investigate potentially germline pathogenic variants in cancer predisposition genes (CPG) and their somatic effects in patients across diverse ancestral backgrounds. MethodsWe performed a retrospective analysis of germline genomic data of 9,899 patients from 33 cancer types generated by The Cancer Genome Atlas (TCGA) project along with matching somatic genomic and transcriptomic data. By collapsing pathogenic and likely pathogenic variants to the gene level, we analyzed the association between variants in CPGs and cancer types within each ancestry. We also identified ancestry- specific predisposing variants and their associated somatic two-hit events and gene expression levels. ResultsRecent genetic ancestry analysis classified the cohort of 9,899 cancer cases into individuals of primarily European, (N = 8,184, 82.7%), African (N = 966, 9.8%), East Asian (N = 649, 6.6%), South Asian (N=48, 0.5%), Native/Latin American (N=41, 0.4%), and admixed (N=11, 0.1%) ancestries. In the African ancestry, we discovered a potentially novel association of BRCA2 in lung squamous cell carcinoma (OR = 41.4 [95% CI, 6.1-275.6]; FDR = 0.002) along with the previously identified association of BRCA2 in ovarian serous cystadenocarcinoma (OR=8.5 [95% CI, 1.5-47.4]; FDR=0.045). Similarly, in the East Asian ancestry, we discovered one previously known association of BRIP1 in stomach adenocarcinoma (OR=12.8 [95% CI, 1.8-90.84]; FDR=0.038). Rare variant burden analysis further identified 7 suggestive associations for cancer-gene pairs in African ancestry individuals previously well described in European ancestry including SDHB in pheochromocytoma and paraganglioma, ATM in prostate adenocarcinoma, VHL in kidney renal clear cell carcinoma, FH in kidney renal papillary cell carcinoma, and PTEN in uterine corpus endometrial carcinoma. Loss of heterozygosity was identified for 7 out of the 15 African ancestry carriers of predisposing variants. Further, tumors from the SDHB or BRCA2 carriers showed simultaneous allelic specific expression and low gene expression of their respective affected genes; and FH splice-site variant carriers showed mis-splicing of FH. ConclusionWhile several predisposing genes are shared across patients, many pathogenic variants are found to be ancestry-specific and trigger somatic effects. Analysis of larger diverse ancestries genomic cohorts are required to pinpoint ancestry- specific genetic predisposition to inform personalized diagnosis and screening strategies.

genomics↗

bioRxiv2020

Single-cell copy number lineage tracing enabling gene discovery

Aneuploidy plays critical roles in genome evolution. Alleles, whose dosages affect the fitness of an ancestor, will have altered frequencies in the descendant populations upon perturbation. Single-cell sequencing enables comprehensive genome-wide copy number profiling of thousands of cells at various evolutionary stage and lineage. That makes it possible to discover dosage effects invisible at tissue level, provided that the cell lineages can be accurately reconstructed. Here, we present a Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) algorithm that infers the evolution history of a cell population based on single-cell copy number (SCCN) profiles. We also present a statistical routine named lineage speciation analysis (LSA), which facilitates discovery of fitness-associated alterations and genes from SCCN lineage trees. We assessed our approaches using a variety of single-cell datasets. Overall, MEDALT appeared more accurate than phylogenetics approaches in reconstructing copy number lineage. From the single-cell DNA-sequencing data of 20 triple-negative breast cancer patients, our approaches effectively prioritized genes that are essential for breast cancer cell fitness and are predictive of patient survival, including those implicating convergent evolution. Similar benefits were observed when applying our approaches on single-cell RNA sequencing data obtained from cancer patients. The source code of our study is available at https://github.com/KChen-lab/MEDALT.

bioinformatics↗

bioRxiv2020

The superoxide dismutase mimetic GC4419 enhances tumor killing when combined with stereotactic ablative radiation

The penta-aza macrocyclic manganese compound GC4419 is in phase 3 clinical trials as a modifier of mucositis in H&N cancer treated by radio-chemotherapy based upon its properties as a superoxide dismutase mimetic. In studies to address the potential for tumor radioprotection, a significant anti-tumor effect was identified in tumors generated from the non-small cell lung cancer (NSCLC) cell line H1299, when GC4419 was combined with radiation. This effect was directly related to the size of the radiation dose as demonstrated by greater efficacy in tumor growth delay when biologically equivalent irradiation regimens using a limited number of dose fractions was substantially more effective compared to regimens where the fraction number was higher and dose per fraction decreased. Furthermore, a TCD50 assay using H1299 tumors that tested the combination of GC4419 with radiation revealed a Dose Enhancement Factor of 1.67. Based upon these results the hypothesis that GC4419 was generating cytotoxic levels of hydrogen peroxide during the superoxide dismutation process. Peroxide flux did increase in cells exposed to GC4419 as did the expression of the oxidative stress markers 4-HNE and 3-NT. H1299 cells that overexpressed catalase were then challenged as tumors by the combination of radiation and GC4419 and the tumoricidal effect was nearly eliminated. The enhanced radiation response was not specific to NSCLC as similar findings were observed in human head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma xenografts. RNA sequencing analysis revealed that GC4419, in addition to generating high levels of hydrogen peroxide in irradiated cells, alters inflammatory and differentiation signaling in the tumor following irradiation. Together, these findings provide abundant evidence that the radioprotector GC4419 has dual functionality and will increase tumor response rates when combined with agents that generate high levels of superoxide like stereotactic ablative body radiotherapy (SAbR). Combining SAbR with GC4419 may be an effective strategy to enhance tumor response in general but may also allow for fully potent radiation doses to tumors that might not necessarily be able to tolerate such doses. The potential for protection of organs at risk may also be exploitable.

cancer biology↗