Biology subjects

Ali, A.

Publications and source records attributed to Ali, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Comparative analysis of plasma and bone marrow nutrient levels in pediatric B-ALL patients

Nutrient availability in the tumor microenvironment is a key determinant of cancer progression and therapeutic response, yet the physiological nutrient environment for most cancers is poorly understood. In this study, we investigated nutrient levels in pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients across different subtypes undergoing chemotherapy, focusing on both bone marrow and circulation. Our analysis revealed distinct differences in nutrient profiles between leukemic and healthy plasma and among B-ALL subtypes, with hyperdiploid B-ALL exhibiting pronounced alterations in arginine and asymmetric dimethylarginine metabolism. Bone marrow and blood plasma exhibited largely similar metabolite profiles, even after chemotherapy, indicating these environments are metabolically comparable. Comparisons with renal cell carcinoma and non-small cell lung cancer highlighted a unique enrichment of tricarboxylic acid cycle intermediates in the circulation of B-ALL patients. These findings provide a comprehensive view of nutrient dynamics in pediatric B-ALL and identify metabolic alterations that could guide biomarker discovery and new therapeutic strategies.

cancer biology↗

bioRxiv2025

Splicing Retention and Enhancer Divergence Govern the Evolutionary Fate of Ohnologues Following Whole-Genome Duplication in Rainbow Trout

Whole-genome duplication (WGD) produces gene duplicates--known as ohnologues--that expand evolutionary potential while posing challenges for regulatory coordination and functional balance. While divergence in gene expression has been widely studied post-WGD, the long-term evolutionary dynamics of alternative splicing (AS) and its regulatory basis remain unresolved. Here, we investigate the evolution of AS in rainbow trout (Oncorhynchus mykiss), a species that underwent a salmonid-specific WGD [~]100 million years ago. Using a high-quality genome assembly, transcriptome data across six tissues, and ChIP-seq profiling of histone modifications, we classify ohnologue pairs based on their expression divergence, splicing complexity, and epigenetic signatures. We find that most ohnologues are retained through conservation, with a gradual reduction in AS diversity over time. Contrary to earlier models, a substantial fraction of ohnologues follows an independent splicing model, maintaining splicing complexity similar to their unduplicated ancestors. Strikingly, we show that enhancer-associated histone marks, particularly H3K27ac, diverge significantly between neofunctionalized and independently splicing gene pairs, implicating enhancer rewiring as a key driver of regulatory and functional divergence. These findings reveal that AS evolution after WGD is shaped by both selective pressures and epigenetic modulation, challenging assumptions of rapid splicing loss and highlighting the independent model as a dominant long-term fate. Our results provide a unified framework for understanding how splicing and regulatory landscapes evolve following genome duplication, with broad implications for vertebrate genome evolution and functional innovation.

genomics↗

bioRxiv2025

Cells stably expressing shRNA against MYO10 display altered cell motility

Myosin-X (MYO10) is an actin-based motor protein involved in cytoskeletal dynamics, membrane interactions, and integrin-mediated adhesion. To investigate MYO10s cellular roles, we generated MYO10 knockdown (MYO10KD) HeLa and COS7 cell lines using lentiviral shRNA. Compared to wild-type cells, both MYO10KD lines showed reduced proliferation and impaired cell migration in wound assays. There were fewer edge filopodia in HeLa cells. Additionally, MYO10KD cells demonstrated increased spreading on laminin-coated substrates, suggesting altered integrin activation and cytoskeletal linkage. Our results reinforce MYO10s importance in cell proliferation, adhesion, and migration; these MYO10KD lines provide an accessible cell culture model for further study of MYO10.

cell biology↗

bioRxiv2025

Intergenerational Conditioning via Intermittent Parental Hypoxia Confers Stroke Resilience in Offspring

Background and AimsIntergenerational disease transmission, where parental exposures or experiences influence disease susceptibility in offspring, may represent a crucial layer of stroke risk that extends beyond genetics alone. Environmental conditioning, such as intermittent sub-lethal hypoxia, can induce adaptive protective stress responses in the brain. However, whether such parental conditioning enhances offspring resilience to cerebral ischaemia remains unclear. This study investigates whether intermittent hypoxia in parents acts as an intergenerational conditioning stimulus, conferring resilience to ischaemic stroke in offspring, and explores associated molecular mechanisms. MethodsMale and female Balb/C mice (F0) were exposed to intermittent hypoxia (8% O2, 2 hours every other day, 16 cycles) prior to mating. To confirm that intermittent hypoxia induced neuroprotection in the parental generation, a separate cohort of F0 mice underwent transient middle cerebral artery occlusion (tMCAO). Offspring (F1) were generated from hypoxia-exposed F0 breeders and divided into four groups: biparental hypoxia, paternal hypoxia, maternal hypoxia, and normoxic controls. Adult F1 offspring also underwent tMCAO to model ischaemic stroke. Infarct volume and brain swelling were assessed 48 hours post-ischaemia. In a subgroup of F1 offspring, tandem mass tag (TMT)-based proteomic analysis of injured brain tissue was performed post-stroke to identify molecular pathways associated with neuroprotection. ResultsParental intermittent hypoxia significantly reduced infarct size and swelling in F0 mice. These protective effects were inherited by F1 offspring, with biparental exposure producing the greatest reduction in infarct volume, followed by maternal-only and paternal-only groups, and exhibiting sex-specific differences. Proteomic profiling revealed distinct treatment and lineage clusters. Key pathways implicated in offspring neuroprotection included metabolic regulation, immune signalling, cytoskeletal organisation, and cell survival, notably involving PI3K-Akt and EGFR pathways. ConclusionsIntermittent hypoxia in parents acts as an intergenerational conditioning stimulus, conferring offspring resilience to ischaemic stroke. This neuroprotective phenotype is supported by coordinated molecular adaptations in key pathways involved in survival and stress response. These findings highlight the potential for parental environmental conditioning to shape stroke outcomes in offspring, opening new avenues for therapeutic exploration.

neuroscience↗

bioRxiv2025

Reprogramming of Osimertinib-Resistant EGFR-mutant NSCLC: The Pyruvate-Acetaldehyde-Acetate Pathway As a Key Driver of Resistance

Osimertinib (Osi) resistance remains a significant challenge in EGFR mutant non-small-cell lung cancer (NSCLC). This study investigates the metabolic reprogramming associated with Osi resistance, identifying key metabolic vulnerabilities that may be targeted for therapeutic intervention. Employing the EGFR-mutant H1975 parental (Par) cell line and its Osi-resistant (OsiR) counterpart, we integrated transcriptomics, metabolomics, nuclear and mitochondrial genomics, functional assays and bioanalytical techniques, as well as advanced 3D imaging to comprehensively define the resistant phenotype. We found that OsiR cells exhibit mitochondrial dysfunction, including impaired oxidative phosphorylation (OXPHOS), mitochondrial DNA mutations, and altered mitochondrial gene expression. To describe this systems-level characterization, we introduce the concept of mitochondromics, a comprehensive profiling of mitochondrial genomic, transcriptomic, structural, and functional changes contributing to therapeutic resistance. Metabolomic profiling revealed a significant accumulation of glycolytic intermediates (lactate, pyruvate, acetate, and acetaldehyde) in the extracellular medium, indicating a shift toward glycolysis and activation of alternative metabolic pathways, including the Warburg effect. Notably, we identified the pyruvate-acetaldehyde-acetate (PAA) pathway as a functionally repurposed metabolic route that facilitates NADPH production, which is critical for antioxidant defense and anabolic processes in OsiR cells. Additionally, although the pentose phosphate pathway (PPP) is not the primary source of NADPH in OsiR cells, it plays a supporting role in biosynthesis, contributing to the production of amino acids, nucleotides, and vitamins. Altered expression of enzymes involved in glycolysis, the TCA cycle, and both oxidative and non-oxidative arms of the PPP further supports an adaptive metabolic network promoting cell growth and resistance to Osi. This study reveals a complex metabolic reprogramming in Osi-resistant EGFR-mutant NSCLC, where a newly identified role for the PAA pathway, alongside integrated mitochondromic alterations emerges as key driver of resistance. These insights uncover potential metabolic vulnerabilities of Osi-resistant tumors and provide a foundation for developing therapeutic strategies to counteract resistance and improve osimertinib efficacy. Targeting these metabolic pathways may offer promising avenues for overcoming resistance in clinical settings.

cancer biology↗

bioRxiv2025

Proteomic characterization of major fish allergy responsive protein parvalbumins in Hilsa (Tenualosa ilisha): A commercially important fish in Southeast Asia

The protein parvalbumin (PRV)-beta (PRVB) is the primary cause of food allergies to bony fish. Although PRVB is a well-characterized protein in many bony fishes, little is known about the Hilsa, an anadromous fish with great economic importance and predominantly found in Southeast Asia. In this study, we characterized the Hilsa PRV utilizing various proteomic approaches in response to two major riverine habitats and developmental stages. Unique peptide sets corresponding to three PRV isoforms were identified in Hilsa muscle tissues. Label-free quantitative proteomic analysis coupled with ELISA revealed higher levels of PRVB in young fish compared to adults, irrespective of their riverine habitats. A comparative quantitative analysis of PRVB further demonstrated that Hilsa had less PRVB than other commonly consumed freshwater fish species. Multiple reaction monitoring (MRM)-based targeted proteomic approach showed the potential of PRV as a marker protein for allergen quantitation and authenticating the presence of Hilsa in a complex freshwater fish mixture. Our findings collectively offer fundamental knowledge on Hilsa PRVs for further investigation on the food safety and quality evaluation of Hilsa fish.

biochemistry↗

bioRxiv2025

Increased Genetic Protection Against Alzheimer's Disease in Centenarians

We constructed a polygenic protective score specific to Alzheimers disease (AD PPS) based on the current literature among the participants enrolled in five studies of healthy aging and extreme longevity in the US, Europe, and Asia. This AD PPS did not include variants on Apolipoprotein E (APOE) gene. Comparisons of AD PPS in different data sets of healthy agers and centenarians showed that centenarians have stronger genetic protection against AD compared to individuals without familial longevity. The current study also shows evidence that this genetic protection increases with increasingly older ages in centenarians (centenarians who died before reaching age 105 years, semi-supercentenarians who reached age 105 to 109 years, and supercentenarians who reached age 110 years and older). However, the genetic protection was of modest size: the average increase in AD PPS was approximately one additional protective allele per 5 years of gained lifetime. Additionally, we show that the higher AD PPS was associated with better cognitive function and decreased mortality. Taken together, this analysis suggests that individuals who achieve the most extreme ages, on average, have the greatest protection against AD. This finding is robust to different genetic backgrounds with important implications for universal applicability of therapeutics that target this AD PPS.

genetics↗

bioRxiv2025

Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

The breast peritumor microenvironment (pTME) is increasingly recognized as a mediator of breast cancer progression and treatment resistance. However, if and how growth-induced tumor compressive forces (i.e., solid stresses) influence the breast pTME remains unclear. Here we show using instant fluorescence lifetime imaging microscopy (FLIM)--a frequency-domain FLIM system capable of simultaneous image acquisition and instantaneous data processing--that breast tumor-mimicking in vitro compression promotes metabolic changes in stromal cells found in the breast pTME. Namely, compression shifts NIH3T3 fibroblasts and differentiated 3T3-L1 (d3T3-L1) adipocytes toward a more glycolytic state, while it promotes increased oxidative phosphorylation in 3T3-L1 undifferentiated adipocytes. The gold-standard Seahorse extracellular flux assay fails to capture these changes, underscoring the superior sensitivity of instant FLIM in detecting metabolic shifts. We validate these phenotypic findings at the transcriptomic level via RNA sequencing, confirming that compressed fibroblasts downregulate oxidative phosphorylation and upregulate glycolysis compared to uncompressed controls. We further demonstrate that compression induces mitochondrial dysregulation in undifferentiated adipocytes, driven in part by upregulated mitophagy and disrupted fusion dynamics. Finally, we confirm that these stromal cell types recapitulate these distinct metabolic states in human breast cancer patient samples, consistent with our in vitro findings. By elucidating mechano-metabolic interactions occurring at the tumor-host interface, these results will inform the development of innovative mechano-metabolic reprogramming treatment strategies to improve breast cancer patient survival.

biochemistry↗

bioRxiv2025

Transcriptomic profiling of a novel gastric implantation model identifies mechanisms and pathways that drive implantation into explanted human peritoneum

Epithelial cancers such as stomach and ovary cancer tend to metastasize to the peritoneum, often leading to intractable disease and poor survival. Currently, the mechanisms that enable gastric cancer cells to penetrate the mesothelium, and to implant, invade, and survive in the peritoneal niche are poorly understood. To investigate these mechanisms, we developed a novel human peritoneal explant model. Briefly, fresh peritoneal tissue samples from abdominal surgery patients were cultured on top of a layer of GFP-labeled human gastric adenocarcinoma cells (AGS); 2% of these cells implanted into the peritoneum. The transcriptomic profile of the implanted AGS cells was compared to the profile of AGS cells that failed to implant using RNA sequencing. Differentially expressed genes were enriched significantly (fold change>2) for genes that enable cell adhesion, motility, and membrane depolarization. We compared this list of genes with a previously identified peritoneal metastasis whole exome sequencing dataset (SRP043661). Upon further analysis based on subcellular localization, cell adhesion and cytoskeletal organization, we found nine core "peritoneal implantation" genes. We functionally validated these genes with CRISPR knockout and assessed peritoneal implantation and invasion using the human peritoneal explant model described above. From these data we identified ADAM12 as a key player of peritoneal metastasis. Knock out of ADAM12 significantly impaired peritoneal metastasis in vivo and ex vivo. Exploration of three publicly available independent datasets indicated that ADAM12 is indeed clinically relevant in peritoneal metastasis. To explore the role of ITGA{beta}1 in mediating cell-matrix interactions in the presence of ADAM12, we performed ITGA{beta}1 pull-down assays followed by mass spectrometry analysis in ADAM12 WT cells. ADAM12 KO cells show a marked disruption of the ITGA{beta}1 interactome in GCa cells. Key cytoskeletal proteins such as MYH14, MYH10, MYH9, ACTA2, SPTN1 and TPM1-TPM4 were found to be interactors with ITGA{beta}1 in ADAM12 WT cells but not ADAM12 KO cells. Our approach and the new data identify a distinct peritoneal metastasis gene set that facilitates implantation and invasion of gastric cancer cells within the peritoneum. Disruption of these pathways with peritoneal-directed therapies has the potential to improve survival in patients with high-risk primary gastric cancer.

cancer biology↗

bioRxiv2025

Zebrafish optic nerve injury results in systemic retinal ganglion cell dedifferentiation

Retinal ganglion cells (RGCs) are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma, optic neuropathies and after ocular trauma results in irreversible loss of vision as RGCs do not regenerate in the human eye. Moreover, there are no FDA approved therapies that prevent RGC death and/or promote RGC survival in the diseased or injured eye. There is a critical need to better understand the molecular underpinnings of neuroprotection to develop effective therapeutic approaches to preserve damaged RGCs. Unlike in mammals, RGCs in zebrafish are resilient to optic nerve injury, even after complete transection of the optic nerve. Here, we leveraged this unique model and utilized single-cell RNA sequencing to characterize RGC responses to injury and identify putative neuroprotective and regenerative pathways. RGCs are heterogeneous and studies in mice have shown that there is differential resiliency across RGC subtypes. Our results demonstrated that all RGC subtypes are resilient to injury in zebrafish. Quantifying changes in gene expression revealed the upregulation of progenitor and regenerative markers in all RGC subtypes after injury as well as distinct early and late phases to the injury response. This shift in gene expression causes injury-responsive RGCs to resemble RGC subtype 3, a low frequency population of endogenous immature RGCs that are normally maintained in the wild-type, uninjured adult retina. A similar but restricted transcriptomic injury response in RGCs of the uninjured contralateral eye was also detected, highlighting a systemic RGC response to unilateral optic nerve injury. Taken together, these results demonstrate that zebrafish RGCs dedifferentiate in response to injury, and this may be a novel mechanism mediating their unique cell survival and regenerative capabilities. Author SummaryRetinal ganglion cells (RGCs) connect the eye to the brain and are essential for vision. Their death in conditions like glaucoma, affecting over 70 million people worldwide, leads to permanent blindness, with no FDA-approved treatments to prevent it. Unlike mammals, zebrafish RGCs are resilient to optic nerve injury. In this study, we used next-generation sequencing technologies to characterize the RGC response to optic nerve injury at the single-cell level. We discovered that all zebrafish RGCs survive damage by temporarily shifting into a less mature state, resembling a rare population of immature RGCs found in uninjured animals. We identified many genes whose expression changes early or late in the injury response as well as a similar but restricted transcriptomic injury response in the uninjured contralateral RGCs, highlighting the systemic RGC response to optic nerve injury. This work is significant because our detailed characterization of RGC responses to optic nerve injury identifies dedifferentiation as an injury response, possibly important for cell survival and axon regrowth. The genes and pathways we identify are potential therapeutic targets to enable RGC survival in the injured or diseased human eye.

neuroscience↗

bioRxiv2025

Refined cell transfer model reveals roles for Ascl2 and Cxcr3 in splenic localization of mouse NK cells during virus infection

Cell transfer experiments complement the rigorous investigation of antiviral and antitumor functions of natural killer (NK) cells. Success in these endeavors is enhanced by expansion of small numbers of input NK cells driven by viral antigens or homeostatic proliferation in immunodeficient hosts. In contrast, analysis of other NK-cell functions, including immunoregulation, are non-proliferative and require an intact immune system in recipient mice. We reveal poor persistence of conventional congenic (CD45.1) BoyJ NK cells following adoptive transfer in comparison to CRISPR-generated CD45.1+ (JAXBoy) NK cells. Reciprocal transfers between C57BL/6 and JAXBoy mice substantially improve seeding and maintenance of donor NK cells. Using this system, we confirm that CXCR3 re-positions NK cells in the white pulp of the spleen after infection, which is vital for immunoregulation. Moreover, we discovered that the transcription factor ASCL2 is required for recruitment of NK cells into the spleen and white pulp. These results provide improved tools and novel insights into NK cell biology. Key pointsO_LIJAXBoy are superior to conventional BoyJ mice for NK cell persistence after transfer. C_LIO_LICXCR3 repositions donor NK cells in T/B-zones of the spleen after infection. C_LIO_LIThe transcription factor ASCL2 is required for NK-cell recruitment to spleen white pulp. C_LI

immunology↗

bioRxiv2025

Far-Ultraviolet Light Causes Direct DNA Damage in Human Lung Cells and Tissues

Far-ultraviolet C (Far-UVC) radiation, with a wavelength range from 200 to 235 nm, is germicidal and holds potential for clinical applications. However, its use against deep-seated and internal infections, such as those affecting the lungs, remains less well established. The safety profile of Far-UVC irradiation requires further investigation across different human tissues. In this study, we utilised a krypton-chloride (KrCl) excimer lamp and a pulsed laser system to examine the effects of Far-UVC irradiation on human lung cells in vitro and primary human tracheal tissue. Primary human tracheal tissue and cells exposed to continuous wave (222 nm) and pulsed 206 nm and 222 nm light at doses of 5, 25, and 50 mJ/cm2 exhibited Deoxyribonucleic acid (DNA) damage, including phosphorylation of {gamma}H2AX (Ser139). The continuous wave and pulsed 222 nm irradiation caused the formation of pyrimidine-pyrimidone (6-4) photoproducts. Irradiated human lung cells demonstrated reduced viability in vitro, and increased lactate dehydrogenase release into the culture medium 48 hours post-irradiation. Our findings reveal that even low doses of Far-UVC (206 nm, 222 nm) light can penetrate monolayers of human lung epithelial cells, causing direct DNA damage in the form of (6-4) photoproducts and DNA double-strand breaks, ultimately leading to cell death.

cell biology↗

bioRxiv2025

AKAP12 variant 1 knockout enhances vascular endothelial cell motility

In this study, we examined the role of AKAP12, in endothelial cell motility, with a specific focus on AKAP12 variants AKAP12v1 and AKAP12v2. Previous work has shown that AKAP12, a multivalent A-kinase anchoring protein that binds to PKA and several other proteins regulating protein phosphorylation, is expressed at low levels in most endothelia in vivo but at higher levels in cells in vitro. Here, we found that AKAP12 expression in endothelial cell (HUVEC) cultures was cell density-dependent, with the expression being highest in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of variants 1 and 2 inhibited cell migration, whereas CRISPR/Cas9 knockout of AKAP12v1 enhanced migration, indicating that the absence of this variant and the presence of AKAP12v2 may shift the signaling pathways. Further analysis using bulk RNA sequencing revealed that the loss of AKAP12v1 affects genes associated with cell migration and intercellular junctions. We propose that AKAP12v1 and AKAP12v2 work together to modulate endothelial cell migration, providing insights into their distinct yet complementary roles in endothelial function and potential implications for cardiovascular health.

cell biology↗

bioRxiv2024

LAG3 marks activated but hyporesponsive NK cells.

Natural Killer (NK) cells are critical for immunosurveillance yet become dysfunctional in contexts such as chronic stimulation by viral infections or cancer. This phenomenon is similar to T cell exhaustion but less well characterized, which limits therapeutic interventions. As shown for T cells, NK cells often display an increased expression of immune checkpoint proteins (ICP) following chronic stimulation, and ICP blockade therapies are currently being explored for several cancer types, which have remarkable patient benefits. Nevertheless, the nature of ICP expression in NK cells is still poorly documented. In this study, we aimed to identify the conditions that lead to and the phenotype of immune checkpoint LAG3 (Lymphocyte-activation gene 3) expressing NK cells. Using various experimental models, we found that LAG3 is expressed by murine NK cells upon activation in different contexts, including in response to cancer and acute viral infections. LAG3 marks a subset of immature, proliferating and activated cells, which, despite activation, have a reduced capacity to respond to a broad range of stimuli. Further characterization also revealed that LAG3+ NK cells exhibit a transcriptional signature similar to that of exhausted CD8+ T cells. Taken together, our results support the use of LAG3 as a marker of dysfunctional NK cells across diverse chronic and acute inflammatory conditions.

immunology↗

bioRxiv2024

Prothrombin prevents fatal T cell-dependent anemia during chronic virus infection of mice

Thrombin promotes the proliferation and function of CD8+ T cells. To test if thrombin prevents exhaustion and sustains antiviral T cell activity during chronic viral infection, we depleted the thrombin-precursor prothrombin to 10% of normal levels in mice prior to infection with the clone 13 strain of lymphocytic choriomeningitis virus. Unexpectedly, prothrombin insufficiency resulted in 100% mortality after infection that was prevented by depletion of CD8+ T cells, suggesting that reduced availability of prothrombin enhances virus-induced immunopathology. Yet, the number, function, and apparent exhaustion of virus-specific T cells were measurably unaffected by prothrombin depletion. Histological analysis of the lung, heart, liver, kidney, spleen, intestine, and brain did not reveal any evidence of hemorrhage or increased tissue damage in low prothrombin mice that could explain mortality. Viral loads were also similar in infected mice regardless of prothrombin levels. Instead, infection of prothrombin-depleted mice resulted in a severe, T cell-dependent anemia associated with increased hemolysis. Thus, thrombin plays an unexpected protective role in preventing hemolytic anemia during virus infection, with potential implications for patients who are using direct thrombin inhibitors as an anticoagulant therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/623028v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@d01b49org.highwire.dtl.DTLVardef@af76ddorg.highwire.dtl.DTLVardef@1a4b6e8org.highwire.dtl.DTLVardef@e44264_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

A simple, cost-effective microfluidic device using a 3D cross-flow T-junction for producing decellularized extracellular matrix-derived microcarriers

Cell therapies using human mesenchymal stromal cells (MSCs) are promising for a wide variety of clinical applications. However, broad-scale clinical translation is limited by conventional culture methods for MSC expansion within 2D tissue-culture flasks. MSC expansion on ECM-derived microcarriers within stirred bioreactor systems offers a promising approach to support MSC growth. Previously, our team established methods for fabricating ECM-derived microcarriers from a variety of decellularized tissue sources using electrospraying techniques. However, these microcarriers are relatively large and have a broad size distribution, which may limit their utility. Smaller and more uniform microcarriers may be favorable for MSC growth within bioreactors and have greater potential to serve as a minimally invasive injectable cell delivery platform. To address these limitations, the current project focused on the development of a new microfluidic-based approach enabling both uniform and small microcarrier production. Using a novel, modified 3D T-junction design, we successfully generated microcarriers using human decellularized adipose tissue (DAT) as the ECM source. Our new cost-effective device produced microbeads that were small and monodisperse, at a range of flow rate combinations and with high production rates. Photo-crosslinking using rose bengal allowed for the generation of microcarriers that were stable following rehydration, with a mean diameter of 196 {+/-} 47 {micro}m. Following methods optimization and microcarrier characterization, in vitro studies confirmed that the new microcarriers supported human adipose-derived stromal cell (hASC) attachment and growth, as well as ECM production, across 14 days within spinner flask bioreactors. Overall, this study demonstrates the feasibility of using our novel, cost-effective, and reusable microfluidics device to generate cell-supportive microcarriers comprised exclusively of ECM in a size range that could be injected via a small gauge needle and were stable in long-term culture.

bioengineering↗

bioRxiv2024

Nanoscale Optical Imaging, Reconstruction, and Spatial Analysis of Whole Mouse Glomeruli

Renal glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate the ultrastructure of thin sections. While these approaches are powerful, they have been limited in their ability to obtain detailed views of the glomeruli as holistic 3D functional units. To fill this knowledge gap, we have developed a novel pipeline for imaging, reconstructing, and analyzing whole mouse glomeruli at 100 nm resolution using super-resolution fluorescence microscopy. This pipeline integrates both manual and machine learning approaches to annotate and analyze glomerular structures. Using this method, we created 18 detailed glomerulus models, from a range of healthy, aged, and model diseased mice, that outline all major structures and cell types. These models have been made publicly accessible in an online repository, providing a valuable resource for further studies. Our results also uncovered a diverse set of novel phenotypes including nuclear enlargement in all glomerular cell types in aging and disease, as well as an aging-related pattern of regional thickening of the Bowmans capsule basement membrane near the tubular-glomerular junction.

physiology↗

bioRxiv2024

Site of breast cancer metastasis is independent of single nutrient levels

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. In this study, we quantify the absolute levels of over 100 nutrients available across multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different organs. We then asked how tumor growth in different tissues relates to nutrient availability and tumor biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. Additionally, we identify purine synthesis as a requirement for tumor growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumor de novo nucleotide synthesis activity. These data suggest that a complex interplay of multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

cancer biology↗