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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

mCherry contains a fluorescent protein isoform that interferes with its reporter function

Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

molecular biology↗

bioRxiv2021

Structural mechanism for bi-directional actin crosslinking by T-plastin

To fulfill the cytoskeletons diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction. One sentence summaryCryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.

biophysics↗

bioRxiv2021

CRISPR knockouts of pmela and pmelb engineered a golden tilapia by regulating relative pigment cell abundance

Premelanosome protein (pmel) is a key gene for melanogenesis in vertebrates. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study we found that most fishes have two pmel genes arising from the teleost-specific whole genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9 gene editing. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypo-pigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants, without effects on RPE pigmentation. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a strong golden body color without bars in the trunk. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, while both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates. Author SummaryMelanophores, the most common pigment cell type, have been studied for nearly 150 years. Many genes are involved in melanoblast migration, melanophore differentiation, and melanin biosynthesis. Pmel is fundamental for melanosome development by directing melanin biosynthesis and melanosome phase transition. Specifically, PMEL can form a fibrillar structure within the melanosome upon which melanin is deposited. We identified two pmel genes in Nile tilapia arising from the teleost-specific whole genome duplication. Disruption of either pmela or pmelb in tilapia leads to significant hypo-pigmentation. PMEL disrupted fish showed not only a reduction in melanin and tiny melanophores, but also a significant increase in the number of xanthophores, and even guanine-filled melanophores, which led to a golden tilapia with hypo-pigmented RPE. Our study confirmed the role of pmel in melanin biosynthesis and maturation, and also highlighted its effects on melanophore number and size. These results provide new insights into pigment cell biology and will help us better understand the mechanisms of color patterning in teleosts. Knockout of both pmela and pmelb provide a new strategy for engineering a golden tilapia, which might provide a foundation for developing new strains in the tilapia industry.

genetics↗

bioRxiv2021

imageseg: an R package for deep learning-based image segmentation

O_LIConvolutional neural networks (CNNs) and deep learning are powerful and robust tools for ecological applications. CNNs can perform very well in various tasks, especially for visual tasks and image data. Image segmentation (the classification of all pixels in images) is one such task and can for example be used to assess forest vertical and horizontal structure. While such methods have been suggested, widespread adoption in ecological research has been slow, likely due to technical difficulties in implementation of CNNs and lack of toolboxes for ecologists. C_LIO_LIHere, we present R package imageseg which implements a workflow for general-purpose image segmentation using CNNs and the U-Net architecture in R. The workflow covers data (pre)processing, model training, and predictions. We illustrate the utility of the package with two models for forest structural metrics: tree canopy density and understory vegetation density. We trained the models using large and diverse training data sets from a variety of forest types and biomes, consisting of 3288 canopy images (both canopy cover and hemispherical canopy closure photographs) and 1468 understory vegetation images. C_LIO_LIOverall classification accuracy of the models was high with a Dice score of 0.91 for the canopy model and 0.89 for the understory vegetation model (assessed with 821 and 367 images, respectively), indicating robustness to variation in input images and good generalization strength across forest types and biomes. C_LIO_LIThe package and its workflow allow simple yet powerful assessments of forest structural metrics using pre-trained models. Furthermore, the package facilitates custom image segmentation with multiple classes and based on color or grayscale images, e.g. in cell biology or for medical images. Our package is free, open source, and available from CRAN. It will enable easier and faster implementation of deep learning-based image segmentation within R for ecological applications and beyond. C_LI

ecology↗

bioRxiv2021

Directing Min protein patterns with advective bulk flow

The Min proteins constitute the best-studied model system for pattern formation in cell biology. We theoretically predict and experimentally show that the propagation direction of in vitro Min protein patterns can be controlled by a hydrodynamic flow of the bulk solution. We find downstream propagation of Min wave patterns for low MinE:MinD concentration ratios, upstream propagation for large ratios, but multistability of both propagation directions in between. Whereas downstream propagation can be described by a minimal model that disregards MinE conformational switching, upstream propagation can be reproduced by a reduced switch model, where increased MinD bulk concentrations on the upstream side promote protein attachment. Our study demonstrates that a differential flow, where bulk flow advects protein concentrations in the bulk, but not on the surface, can control surface-pattern propagation. This suggests that flow can be used to probe these features and to constrain mathematical models for pattern-forming systems.

biophysics↗

bioRxiv2021

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialised parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae, fungi) like some parasites, but then devour their cell contents by phagocytosis. Besides prey recognition and attachment, this complex behaviour involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure. Yet, the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder, and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g. locomotion, cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g. lytic polysaccharide monooxygenases) belong to families that are largely uncharacterised, emphasising the potential of non-fungal micro-eukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behaviour, and new directions for cell biology research on non-model eukaryotes.

microbiology↗

bioRxiv2022

Multivalent interactions drive the Toxoplasma AC9:AC10:ERK7 complex to concentrate ERK7 in the apical cap

The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the MAP kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast-2-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC10:AC9:ERK7 complex and demonstrate that the interactions among them are critical to maintain the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap. IMPORTANCEThe phylum Apicomplexa consists of obligate, intracellular parasites including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections.

microbiology↗

bioRxiv2022

Revised Michaelis-Menten rate law with time-varying molecular concentrations

The Michaelis-Menten (MM) rate law has been the dominant paradigm of modeling biochemical rate processes for over a century with applications in biochemistry, biophysics, cell biology, systems biology, and chemical engineering. The MM rate law and its remedied form stand on the assumption that the concentration of the complex of interacting molecules, at each moment, approaches an equilibrium (quasi-steady state) much faster than the molecular concentrations change. Yet, this assumption is not always justified. Here, we relax this quasi-steady state requirement and propose the generalized MM rate law for the interactions of molecules with active concentration changes over time. Our approach for time-varying molecular concentrations, termed the effective time-delay scheme (ETS), is based on rigorously estimated time-delay effects in molecular complex formation. With particularly marked improvements in protein-protein and protein-DNA interaction modeling, the ETS provides an analytical framework to interpret and predict rich transient or rhythmic dynamics (such as autogenously-regulated cellular adaptation and circadian protein turnover), which goes beyond the quasi-steady state assumption.

biophysics↗

bioRxiv2022

Application of sequential cyclic compression on cancer cells in a flexible microdevice

Mechanical forces shape physiological structure and function within cell and tissue microenvironments, during which cells strive to restore their shape or develop an adaptive mechanism to maintain cell integrity depending on strength and type of the mechanical loading. While some cells are shown to experience permanent plastic deformation after a repetitive mechanical tensile loading and unloading, the impact of such repetitive compression on plastic deformation of cells is yet to be understood. As such, the ability to apply cyclic compression is crucial for any experimental setup aimed at the study of mechanical compression taking place in cell and tissue microenvironments. Here, we demonstrate such cyclic compression using a microfluidic compression platform on live cell actin in SKOV-3 ovarian cancer cells. Live imaging of the actin cytoskeleton dynamics of the compressed cells was performed for varying pressures applied sequentially in ascending order during cell compression. Additionally, recovery of the compressed cells was investigated by capturing actin cytoskeleton and nuclei profiles of the cells at zero time and 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of minute differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. The extent of recovery of compressed cells, inferred by imaging cell membrane bulges and actin cytoskeleton and measuring the shape descriptors of cell nuclei, can give insights into the plasticity of cancer cells. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. As demonstrated in this work, the microfluidic platform could control the strength and duration of cyclic compression, while enabling the observation of morphological, cytoskeletal and nuclear changes and recovery in compressed cells, thus providing a powerful new tool for the study of mechanobiological processes in cancer and cell biology.

bioengineering↗

bioRxiv2022

Plasma cell maintenance and antibody secretion are under the control of Sec22b-mediated regulation of organelle dynamics

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the SNARE Sec22b as a unique and critical regulator of plasma cell maintenance and function. In absence of Sec22b, plasma cells were barely detectable and serum antibody titres were dramatically reduced. Accordingly, Sec22b deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b is indispensable for efficient antibody secretion but also for plasma cell fitness through the regulation of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil a critical role for Sec22b-mediated regulation of plasma cell biology through the control of organelle dynamics.

immunology↗

bioRxiv2022

Lysosomes are Required for Early Dorsal Signaling in the Xenopus Embryo

Lysosomes are the digestive center of the cell and play important roles in human disease, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of {beta}{square}catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at 64-cell stage, long before zygotic transcription starts. Expansion of the multivesicular body (MVB) compartment with low-dose Hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist Lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) Bafilomycin A1 inhibitor at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) also interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 mRNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is essential for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation. SignificanceThe dorsal axis of the vertebrate Xenopus embryo is established by an early Wnt signal generated by a rotation of the cortex of the egg towards the opposite side of the sperm entry point. In this study, we report that lysosomal Cathepsin D becomes activated on the dorsal marginal zone of the embryo already at the 64-cell stage, and that this asymmetry is enhanced by increasing Wnt signaling levels. We present experiments showing that lysosome activity, macropinocytosis, and multivesicular body formation are required for the dorsal signal provided maternally in the egg, and for twinning by microinjected huluwa and Wnt8 mRNA. The results indicate that the cell biology of lysosomes plays a fundamental role in vertebrate development.

developmental biology↗

bioRxiv2022

A reference single-cell regulomic and transcriptomic map of cynomolgus monkeys

Non-human primates (NHP) are attractive laboratory animal models that accurately reflect both developmental and pathological features of humans. Here we present a compendium of cell types from the cynomolgus monkey Macaca fascicularis (denoted as Monkey Atlas) using both single-cell chromatin accessibility (scATAC-seq) and RNA sequencing (scRNA-seq) data at the organism-wide level. The integrated cell map enables in-depth dissection and comparison of molecular dynamics, cell-type composition and cellular heterogeneity across multiple tissues and organs. Using single-cell transcriptomic data, we inferred pseudotime cell trajectories and cell-cell communications to uncover key molecular signatures underlying their cellular processes. Furthermore, we identified various cell-specific cis-regulatory elements and constructed organ-specific gene regulatory networks at the single-cell level. Finally, we performed a comparative analysis of single-cell landscapes among mouse, cynomolgus monkey and human, and we showed that cynomolgus monkey has significantly higher degree of cell-type similarity to human than mouse. Taken together, our study provides a valuable resource for NHP cell biology.

bioinformatics↗

bioRxiv2022

Replacing bar graphs of continuous data with more informative graphics: Are we making progress?

Recent work has raised awareness about the need to replace bar graphs of continuous data with informative graphs showing the data distribution. The impact of these efforts is not known. This observational meta-research study examined how often scientists in different fields use various graph types, and assessed whether visualization practices have changed between 2010 and 2020. We developed and validated an automated screening tool, designed to identify bar graphs of counts or proportions, bar graphs of continuous data, bar graphs with dot plots, dot plots, box plots, violin plots, histograms, pie charts, and flow charts. Papers from 23 fields (approximately 1,000 papers/field/year) were randomly selected from PubMed Central and screened (n=227,998). F1 scores for different graphs ranged between 0.83 and 0.95 in the internal validation set. While the tool also performed well in external validation sets, F1 scores were lower for uncommon graphs. Bar graphs are more often used incorrectly to display continuous data than they are used correctly to display counts or proportions. The proportion of papers that use bar graphs of continuous data varies markedly across fields (range in 2020: 4%-58%), with high rates in biochemistry and cell biology, complementary and alternative medicine, physiology, genetics, oncology and carcinogenesis, pharmacology, microbiology and immunology. Visualization practices have improved in some fields in recent years. Fewer than 25% of papers use flow charts, which provide information about attrition and the risk of bias. This study highlights the need for continued interventions to improve visualization and identifies fields that would benefit most.

scientific communication and education↗

bioRxiv2022

Novel Murine models of Mania and Depression

Neuropathological mechanisms triggering manic syndrome or manic episodes in bipolar disorder remain poorly characterised, as the research progress is severely limited by the paucity of appropriate animal models. Here we developed a novel manic mice model by combining a series of chronic unpredictable rhythm disturbances (CURD), which include disruption of circadian rhythm, sleep deprivation, exposure to cone light, with subsequent interference of followed spotlight, stroboscopic illumination, high temperature stress, noise disturbance and foot shock. To validate this novel manic model, we used multiple behavioural and cell biology approaches comparing the CURD-model with healthy controls and depressed mice. The depression model was created by an exposure to an improved chronic unpredictable mild stress, which we defined as chronic unpredictable mild restraint (CUMR). A novel manic mice model induced by environmental stressors and free from genetic or pharmacological interventions will benefit research into pathological mechanisms of mania.

animal behavior and cognition↗

bioRxiv2022

Microgravity enhances the phenotype of Arabidopsis zigzag-1 and reduces the Wortmannin-induced vacuole fusion in root cells

The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity and other conditions on the ISS display root skewing, changes in gene expression and protein abundance that may result in changes in cell wall composition, antioxidant accumulation and modification of growth anisotropy. Systematic studies that address the effects of microgravity on cellular organelles are lacking but altered numbers and sizes of vacuoles have been detected in previous flights. The prominent size of plant vacuoles makes them ideal models to study organelle dynamics in space. Here, we used Arabidopsis zigzag1 (zig-1) as a sensitized genotype to study the effect of microgravity on plant vacuole fusion. Wortmannin was used to induce vacuole fusion in seedlings and a formaldehyde-based fixation protocol was developed to visualize plant vacuole morphology after sample return, using confocal microscopy. Our results indicate that microgravity enhances the zig-1 phenotype by reducing hypocotyl growth and vacuole fusion in some cells. This study demonstrates the feasibility of chemical inhibitor treatments for plant cell biology experiments in space.

plant biology↗

bioRxiv2022

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae.

Role of Horizontal Gene Transfer (HGT) in evolution transcends across the three domains of life. Ammonium transporters are present in all species and therefore offer an excellent paradigm to study protein evolution following HGT. While investigating HGT through complementation assay, we observed that synonymous and nonsynonymous mutations follow an epistastic relationship. As a proxy for HGT, we attempted to complement a mep1mep2mep3{Delta} strain of S. cerevisiae (triple deletion strain) which cannot grow on ammonium as a sole nitrogen source below a concentration of 3 mM, with amtA of D. discoideum. As the wild type amtA did not complement, we isolated two mutant derivatives of amtA that complemented the triple deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions and these substitutions are necessary for its functionality. amtA M2 bears two nonsynonymous and one synonymous substitution, all of which are necessary for functionality. These mutants were then studied at phenotypic, cell biological, and biochemical level. Interestingly, AmtA M1 transports ammonium but does not confer toxicity to methylamine while AmtA M2 transports ammonium as well as confers methylamine toxicity, demonstrating functional diversification. Based on the results presented, we suggest that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions separately. This is because, protein evolution entails an interaction between synonymous and nonsynonymous substitution, which seems to have gone unnoticed thus far. Above observations have significant implications in various facets of biological processes and are discussed in detail. HighlightsO_LIAmmonium transporters (Amts) from bacteria to humans complement in yeast C_LIO_LIAmtA of D. discoideum does not complement yeast defective for ammonium uptake C_LIO_LISynonymous & nonsynonymous mutations are essential for AmtA functionality in yeast C_LIO_LIConformational differences underlie functionality & functional diversification C_LIO_LIProtein evolution entails interaction of synonymous & nonsynonymous mutations C_LI

genetics↗

bioRxiv2022

Kidins220 promotes thymic iNKT cell development by reducing TCR signals, but enhances TCR signals in splenic iNKT cells

The stepwise development of thymic invariant natural killer T (iNKT) cells is controlled by the TCR signal strength. The scaffold protein Kinase D interacting substrate of 220 kDa (Kidins220) binds to the TCR regulating TCR signaling. T cell-specific Kidins220 knock-out (T-KO) mice contain severely decreased iNKT numbers. Very early in iNKT development TCR signals are reduced in the T-KO. In later steps, TCR signaling is increased in the T-KO leading to enhanced apoptosis of iNKT cells. Kidins220s absence affects the iNKT1 subset most as it requires the weakest TCR signals for development. We also show that in iNKT1 development, weak TCR signals promote the progressive loss of CD4. In the periphery, Kidins220 switches its role back to promoting TCR signaling as splenic T-KO iNKT cells produce less cytokines and show reduced TCR signaling after in vivo stimulation with -galactosylceramide. In conclusion, Kidins220 promotes or inhibits TCR signaling depending on the developmental context. summary statementWe demonstrate that the transmembrane scaffold protein Kidins220 switches its role twice in iNKT cell biology: from a positive to a negative regulator of TCR signal strength during thymic development and back to a positive regulator in the periphery.

immunology↗

bioRxiv2022

Metabolic-imaging of human glioblastoma explants: a new precision-medicine model to predict tumor treatment response early

BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy (RT) remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients. MethodsGB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were then performed to characterize tumor cases stratification. ResultsOur functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non Responder tumors were identified. FLIM-based metabolic tumor stratification was well-reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17 gene molecular signature associated with survival. The number of promoter methylated tumors for the MGMT gene was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker. ConclusionsFor the first time, FLIM-based metabolic imaging was used on ex-vivo live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

molecular biology↗