Biology subjects

Gurung, S.

Publications and source records attributed to Gurung, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Epithelial stem cells in the premenopausal human vagina

The vagina undergoes physiologic changes across the menstrual cycle, pregnancy, birth and menopause. Most women will experience vaginal dysfunction at some point during their lives and treatment options are limited. The cyclic regeneration of the vaginal epithelium during each menstrual cycle suggests it is a stem-cell based tissue; however, human vaginal epithelial stem cells (veSCs) have not been identified. We utilized in vitro colony forming and organoid assays to confirm that cells in the human vaginal epithelium have self-renewal and differentiation potential. Specifically, we determined that stem cell activity resides in the ITGA6+ and NGFR+ fractions of the basal epithelium. We performed single-cell RNA sequencing to identify the distinct cellular compartments of the full thickness human vagina, including spatially distinct populations comprising layers of the stratified vaginal epithelium. Markers of these cells within the vaginal epithelium were validated by immunohistochemistry. CD9+ITGA6+NGFR+ and CD9+ITGA6+NGFR- cells were capable of efficient colony formation but only the CD9+ITGA6+NGFR+ fraction produced organoids containing basal, intermediate and superficial layers of the vaginal epithelium. We characterized the premenopausal human vagina at single cell resolution, validated markers and assays to test veSC developmental potential, and identified putative stem cells that may open new avenues for treating vaginal dysfunction.

cell biology↗

bioRxiv2025

From blood to pluripotency: Fibrocytes as a reprogrammable somatic cell source for bovine iPSCs

Fibrocytes represent a distinct somatic cell type derived from peripheral blood leukocytes, first described as spindle-shaped adherent cells with dual hematopoietic and mesenchymal features. Although fibrocytes were identified in early descriptive studies across several mammalian systems, most of this work predated modern molecular approaches, and the cells remain incompletely defined at the molecular level and have not previously been derived or characterized in cattle. Seeking somatic cells that could be collected aseptically and reproducibly under field conditions for reprogramming to pluripotency, we recognized fibrocytes as a practical and previously unexplored candidate population. Here, we establish a reproducible method for fibrocyte derivation and expansion from adult bovine blood and define their molecular identity using transcriptomic and network analyses. Principal component and differential expression analyses revealed extensive immune, inflammatory, metabolic, and stress-responsive pathways that distinguished fibrocytes from fibroblasts. Upstream regulator analysis identified a fibrocyte-restricted transcriptional network governed by SPI1, IRF5/IRF7, NFKBIZ, PRDM1, CIITA, and MAFB, supporting a monocyte-derived origin and indicating some retention of hematopoietic lineage memory despite acquisition of mesenchymal features. Optimized fibrocyte medium (FbC; dexamethasone, ascorbate, PDGF-BB, EGF, A83-01, CHIR99021) supported stable proliferation and selectively enhanced cytoskeletal and matrix-constructive programs while attenuating inflammatory tone. When reprogrammed with polycistronic OCT4-SOX2-KLF4-cMYC and SV40 large T antigen, fibrocytes generated induced pluripotent stem cell (iPSC) colonies exhibiting defining molecular and morphological features of pluripotency. These findings establish fibrocytes as a field-adaptable, stably expandable, and reprogrammable somatic cell type with practical applications in induced pluripotent stem cell generation, genetic preservation, and reproductive biotechnology.

cell biology↗

bioRxiv2025

Jam2 Signaling Functions Downstream of Hand2 To Initiate The Formation Of Organ-Specific Vascular Progenitors In Zebrafish

The mechanisms regulating the formation of organ-specific vasculature are still poorly understood. We have previously identified a population of late-forming endothelial progenitor cells in zebrafish embryos (termed secondary vascular field, SVF), which emerge from the lateral plate mesoderm after 24 hpf stage, when blood circulation has already been initiated. Here, we investigate the functional role of SVF cells and the molecular mechanisms that govern the emergence of these SVF cells and their contribution to vasculature. We found that the bHLH transcription factor Hand2 and junctional adhesion molecule Jam2b are expressed in the SVF-forming region and are required for the emergence of SVF cells in zebrafish embryos. Time-lapse imaging and jam2b:Cre-based lineage tracing showed that SVF cells serve as the major source of the intestinal vasculature, including the supraintestinal artery (SIA) and subintestinal vein (SIV), which are subsequently remodeled to provide blood flow to many internal organs. To analyze the functional role of jam2b and the related jam2a gene in vascular development, we generated double maternal-zygotic jam2a; jam2b mutants, which display a greatly reduced number of SVF cells and show defects in the development of the intestinal vasculature. Further analysis showed that hand2 functions in the SVF-forming region upstream of jam2b and is required to induce expression of the transcription factor etv2/etsrp, a known master regulator of vasculogenesis. In summary, our results identify new roles for Jam2 signaling and Hand2 function in the emergence of organ-specific vascular progenitors. The presence of similar progenitors in mammalian embryos suggests that this mechanism is evolutionarily conserved.

developmental biology↗

bioRxiv2025

Metabolic modulation of intratumoral cholesterol with gut microbiota for the treatment of colorectal cancer

Excess cholesterol is positively correlated with colorectal cancer (CRC). Current therapeutic strategies for modulating cholesterol levels in CRC are limited and often come with complications. Here, we demonstrated that microbiome shunting of intestinal cholesterol to anticancer metabolites is a safe and applicable therapeutic approach for CRC. By screening major microbial metabolic products of cholesterol, we found that 4-cholesten-3-one (4-C-3) was selectively depleted in fecal samples and tumor tissues of patients with CRC. 4-C-3 exhibits strong antitumor effects on human CRC cell lines, patient-derived organoids, and patient-derived xenograft (PDX) models. Mechanistically, 4-C-3 suppresses CRC tumorigenesis by dually targeting the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homologue (KRAS). 4-C-3 directly binds to EGFR, blocking its signal transduction by inhibiting the binding of EGFR ligands. Additionally, 4-C-3 inhibits oncogenic KRAS variants, such as KRASG12D, by suppressing nucleotide exchange activity and effector engagement. By targeting both EGFR and KRAS, 4-C-3 reduces primary resistance to anti-EGFR therapies caused by KRAS mutations in CRC. As a proof-of-concept study, we showed that delivery of 4-C-3 by Oscillibacter ruminantium, a 4-C-3- producing commensal bacterium that is reduced in CRC patients, or a nonpathogenic Escherichia coli strain engineered to specifically convert intestinal cholesterol into 4-C-3, removed intratumoral cholesterol and led to rapid tumor regression in multiple models of CRC. These results suggest that microbial therapies for restoring intestinal cholesterol homeostasis represent a new therapeutic avenue for CRC.

cancer biology↗

bioRxiv2025

Neutrophil derived microvesicles induce endothelial cell dysfunction associated with atherosclerotic plaque erosion.

Atherosclerosis is a major cause of death globally. It is characterised by the development of fibro-fatty lesions in the artery wall that can impede blood flow and lead to myocardial infarction. Historically, research has focused primarily on plaque rupture. However, the importance of erosion of the endothelium leading to thrombosis in plaques with lower lipid content, fewer inflammatory cells and a thick fibrous cap has emerged. Neutrophils have recently been implicated in plaque erosion through the induction of endothelial cell dysfunction. Neutrophils produce 0.1-1m extracellular vesicles (microvesicles) from their cell membrane that are linked with atherosclerotic plaque progression. The hypothesis that neutrophil microvesicles affect plaque erosion through driving endothelial cell dysfunction and platelet adhesion was investigated. Peripheral blood neutrophils and platelets were isolated from healthy subjects and neutrophils stimulated with native LDL to induce microvesicle release. These microvesicles were found to contain proteases capable of degrading extracellular matrix proteins. Incubation of human coronary artery endothelial cells with neutrophil microvesicles lead to an increase in apoptosis and detachment, and a decrease in migration and proliferation in the endothelial cells. Moreover, microvesicles induced an increase in both platelet P-selectin expression and platelet-endothelial cell interaction. This study demonstrates the propensity of neutrophil microvesicles to promote functions within human coronary artery endothelial cells that may predispose the endothelium to erosion and thrombosis and identifies a potential link between a known risk factor for atherosclerosis, elevated LDL cholesterol, and the production of neutrophil microvesicles. These findings highlight the need for further research to better understand the effects of neutrophil microvesicles in atherosclerosis.

cell biology↗

bioRxiv2023

Timp2 loss-of-function mutation and TIMP2 treatment in murine model of NSCLC: modulation of immunosuppression and oncogenic signaling

Mounting evidence suggests that the tissue inhibitor of metalloproteinases-2 (TIMP2) can reduce tumor burden and metastasis. However, the demonstration of such anti-tumor activity and associated mechanisms using in vivo tumor models is lacking. The effects of a Timp2 functional mutation and administration of recombinant TIMP2 were examined in both orthotopic and heterotopic murine models of lung cancer using C57Bl/6 syngeneic Lewis Lung 2-luciferase 2 cells (LL2-luc2) cells. Mice harboring a functional mutation of TIMP2 (mT2) display markedly increased primary lung tumor growth, increased mortality, enriched vasculature, and enhanced infiltration of pro-tumorigenic, immunosuppressive myeloid cells. Treatment with recombinant TIMP2 reduced primary tumor growth in both mutant and wild-type (wt) mice. Comparison of transcriptional profiles of lung tissues from tumor-free, wt versus mT2 mice reveals only minor changes. However, lung tumor-bearing mice of both genotypes demonstrate significant genotype-dependent changes in gene expression following treatment with TIMP. In tumor-bearing wt mice, TIMP2 treatment reduced the expression of upstream oncogenic mediators, whereas treatment of mT2 mice resulted in an immunomodulatory phenotype. A heterotopic subcutaneous model generating metastatic pulmonary tumors demonstrated that daily administration of recombinant TIMP2 significantly downregulates the expression of heat shock proteins, suggesting a reduction of cell-stress responses. In summary, we describe how TIMP2 exerts novel, anti-tumor effects in a murine model of lung cancer and that rTIMP2 treatment supports a normalizing effect on the tumor microenvironment. Our findings show that TIMP2 treatment demonstrates significant potential as an adjuvant in the treatment of NSCLC. One Sentence SummaryTIMP2 treatment compensates for TIMP2 mutation to reveal immunomodulatory and oncogene regulation that inhibits tumor growth and metastatic niche formation.

cancer biology↗

bioRxiv2023

The incidence of movement disorders increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Argininosuccinate lyase is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide biosynthesis cycle. Inherited argininosuccinate lyase deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonaemia and nitric oxide deficiency. Patients present with developmental delay, epilepsy and movement disorders, associated with nitric oxide-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for movement disorders-related symptoms: movement disorders such as ataxia, tremor and dystonia, hypotonia and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an ASA patient with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appears in the 2nd and 3rd decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonaemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorders with functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy. SynopsisMovement disorders-related symptoms in ASA appear in the 2nd and 3rd decades of life, becoming more prevalent with age and shows abnormal neuroimaging features of basal ganglia in ASA patients, not recapitulated in ASA mice.

genetics↗

bioRxiv2023

Macrophage inhibitor clodronate enhances liver transduction of lentiviral but not AAV vectors or mRNA lipid nanoparticles in vivo.

Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases hemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional strategies are rapidly emerging to overcome some inherent AAV limitations, such as non-persistence of episomal transgene in rapidly growing liver and immune response. Integrating lentiviral vectors and non-viral lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at preclinical and clinical stages respectively. Macrophages are first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNPs has been reported. In this study, we assessed the biodistribution of AAV, lentiviral and LNP-mRNA gene therapy following inhibition of tissue macrophages by clodronate liposomes in neonatal and juvenile mice. Juvenile clodronate-treated mice showed significant increase of lentiviral-transduced hepatocytes, and increasing trend of transduction was shown in neonatally-injected mice. In contrast, AAV- and LNP-mRNA-treated neonatal and juvenile animals did not show significant increase of liver biodistribution following clodronate administration. These findings will have translational application for liver-targeting gene therapy programmes.

molecular biology↗

bioRxiv2023

SH2 domain protein E (SHE) and ABL signaling regulate blood vessel size

Blood vessels in different vascular beds vary in lumen diameter, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vascular lumen size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA lumen, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA lumen in she mutants correlated with an increased endothelial expression of claudin 5a and 5b (cldn5a / cldn5b), which encode proteins enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA lumen size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates lumen size during vascular tubulogenesis.

developmental biology↗

bioRxiv2023

Ex vivo primary liver sections recapitulate disease phenotype and therapeutic rescue for liver monogenic diseases

In academic research and the pharmaceutical industry, in vitro single cell line cultures and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have limitations, with incomplete reproduction of pathophysiological characteristics and absence of 3-dimensional architecture with cell lines or the use of live animals brings ethical considerations, limiting the experimental scale and design. The use of precision-cut tissue slices can bridge the gap between these mainstream models as this technique combines the advantages of studying all cell sub-types whilst preserving the tissue-matrix architecture, thereby closely mimicking a mini-organ. Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers. We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.

cell biology↗

bioRxiv2022

mRNA therapy restores ureagenesis and corrects glutathione metabolism in argininosuccinic aciduria

Argininosuccinate lyase (ASL) is a key enzyme integral to the hepatic urea cycle which is required for ammonia detoxification, and the citrulline-nitric oxide (NO) cycle for NO production. ASL deficient patients present with argininosuccinic aciduria (ASA), an inherited metabolic disease with hyperammonaemia and a chronic systemic phenotype with neurocognitive impairment and chronic liver disease. ASL deficiency as an inherited model of systemic NO deficiency, shows enhanced nitrosative and oxidative stress. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FSPG). Upregulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. hASL mRNA encapsulated in lipid nanoparticles corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis and glutathione metabolism and ameliorating chronic liver disease. We further present [18F]FSPG PET as a novel non-invasive diagnostic tool to assess liver disease and therapeutic efficacy in ASA. These findings support clinical translation of mRNA therapy for ASA.

genetics↗

bioRxiv2022

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates (NHP) fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After [~]8-11 months of maternal WD feeding (mWD), dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in mWD-exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriacea and Prevotellacea were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

developmental biology↗

bioRxiv2022

EARLY AND MID-GESTATION ZIKA VIRUS (ZIKV) INFECTION IN THE OLIVE BABOON (PAPIO ANUBIS) LEADS TO FETAL CNS PATHOLOGY BY TERM GESTATION

Zika virus (ZIKV) infection in pregnancy can produce catastrophic teratogenic damage to the developing fetus including microcephaly and congenital Zika syndrome (CZS). We previously described fetal CNS pathology occurring by three weeks post-ZIKV infection in Olive baboons at mid-gestation, including neuroinflammation, loss of radial glia (RG), RG fibers, neuroprogenitor cells (NPCs) resulting in disrupted NPC migration. In the present study, we explored fetal brain pathologies at term gestation resulting from ZIKV infection during either first or second trimester in the Olive baboon. In all dams, viremia resolved after 7 days post infection (dpi). One first trimester infected dam aborted at 5 dpi. All dams developed IgM and IgG response to ZIKV with ZIKV IgG detected in fetal serum. Substantial placental pathology and inflammation was observed including disruption of syncytiotrophoblast layers, delayed villous maturation, partially or fully thrombosed vessels, calcium mineralization and fibrin deposits. In the uterus, ZIKV was detected in [3/4] first trimester but not in second trimester infected dams. While ZIKV was not detected in any fetal tissue at term, all fetuses exhibited varying degrees of neuropathology. Fetal brains from ZIKV infected dams exhibited a range of gross brain pathologies including irregularities of the major gyri and sulci of the cerebral cortex and cerebellar pathology. Frontal cortices of ZIKV fetuses showed a general disorganization of the six-layered cortex with degree of disorganization varying among the fetuses from the two groups. Frontal cortices from first but not second trimester infections exhibited increased microglia and astrocyte numbers (white matter) in both trimester infections. In the cerebellum, increased microglia were observed in both first and second trimester infected fetuses and decreased oligodendrocyte precursor cell populations in the cerebellar white matter in first trimester infections. In general, our observations are in accordance with those described in human ZIKV infected fetuses.

pathology↗

bioRxiv2021

Single cell profiling of the Timp gene family

Tissue inhibitor of metalloproteinases (TIMPs/Timps) are an endogenous family of widely expressed matrisome-associated proteins that were initially identified as inhibitors of matrix metalloproteinase activity (Metzincin family proteases). Consequently, TIMPs are often considered simply as protease inhibitors by many investigators. However, an evolving list of new metalloproteinase-independent functions for TIMP family members suggests that this concept is outdated. These novel TIMP functions include direct agonism/antagonism of multiple transmembrane receptors, as well as functional interactions with matrisome targets. While the family was fully identified over two decades ago, there has yet to be an in-depth study describing the expression of TIMPs in normal tissues of adult mammals. An understanding of the tissues and cell-types that express TIMPs 1 through 4, in both normal and disease states are important to contextualize the growing functional capabilities of TIMP proteins, which are often dismissed as non-canonical. Using publicly available single cell RNA sequencing data from the Tabula Muris Consortium, we analyzed approximately 100,000 murine cells across eighteen tissues from non-diseased organs, representing seventy-three annotated cell types, to define the diversity in Timp gene expression across healthy tissues. We describe the unique expression profiles across tissues and organ-specific cell types that all four Timp genes display. Within annotated cell-types, we identify clear and discrete cluster-specific patterns of Timp expression, particularly in cells of stromal and endothelial origins. RNA in-situ hybridization across four organs expands on the scRNA sequencing analysis, revealing novel compartments associated with individual Timp expression. These analyses provide evidence of the biological significance of Timp expression in the identified cell sub-types, which are consistent with novel roles in normal tissue homeostasis and changing roles in disease progression. This understanding of the tissues, specific cell types and microenvironment conditions in which Timp genes are expressed adds important physiological context to the growing array of novel functions for TIMP proteins.

cell biology↗

bioRxiv2021

Ultraviolet light-induced collagen degradation inhibits melanoma invasion

Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma1-4. The ageing, sun-exposed dermis accumulates UVR damage5, and older patients develop more melanomas at UVR-exposed sites4,6,7. As fibroblasts are functionally heterogeneous and play key roles in the stromal contribution to cancer8,9, we asked whether UVR modifies dermal fibroblast function. Here we confirmed the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) by UVR-damaged fibroblasts was persistently upregulated to reduce local levels of collagen 1 (COL1A1), and found dermal COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, we show inhibiting extracellular matrix degradation and MMP1 expression restored melanoma invasion to UVR damaged dermis. We confirmed in vitro findings in a cohort of primary cutaneous melanomas of aged humans, showing more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen. We found collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. These data indicate melanomas arising over UVR-damaged, collagen-poor skin of the elderly are less invasive, and this reduced invasion improves survival. Consequently, although UVR increases tumour incidence, it delays primary melanoma invasion by degrading collagen. However, we show melanoma-associated fibroblasts can restore invasion in low-collagen primary tumours by increasing collagen synthesis. Finally, we demonstrate high COL1A1 gene expression is a biomarker of poor outcome across a broad range of primary cancers.

cancer biology↗

bioRxiv2021

Female immunity protects from cutaneous squamous cell carcinoma

PurposeCancer susceptibility and mortality are higher in males, and the mutational and transcriptomic landscape of cancer differs by sex. The current assumption is that men are at higher risk of epithelial cancers as they expose more to carcinogens and accumulate more damage than women. We present data showing women are more protected from aggressive cutaneous squamous cell carcinoma (cSCC) due to strong immune activation. MethodsWe explored clinical and molecular sexual disparity in immunocompetent and immunosuppressed patients (N= 738, N=160) with carcinoma cSCC, in FVB/N mice exposed to equal doses of DMBA, and in human keratinocytes by whole exome sequencing, bulk and single cell RNA sequencing. ResultsWe show cSCC is more aggressive in men, and immunocompetent women develop mild cSCC, later in life. To test if sex drives disparity, we exposed male and female mice to equal doses of carcinogen, and found males present more aggressive, metastatic cSCC than females. Critically, females activate cancer immune-related expression pathways and CD4 and CD8 T cell infiltration independently of mutations. In contrast, males increase the rate of mitoses and proliferation in response to carcinogen. Human female skin and keratinocytes also activate immune-cancer fighting pathways and immune cells at ultraviolet radiation-damaged sites. Critically, a compromised immune system leads to high-risk, aggressive cSCC specifically in women. ConclusionsThis work shows the immune response is sex biased in cSCC, and highlights female immunity offers greater protection than male immunity.

cancer biology↗

bioRxiv2020

Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders.

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unraveled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell penetrating autophagy inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and hyperammonemia, and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.

genetics↗

bioRxiv2020

Impact of sustained TGFβ receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-{beta} receptor (TGF{beta}-R) inhibitor, promotes pharmacological expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGF{beta}-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGF{beta}-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RAR{beta}). Selective RAR{beta} inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture. Significance statementCycling human endometrium is a rich source of adult stem/progenitor cells that could be exploited for clinical purposes. Small molecules, such as A83-01, that modulate cell identity may open new avenues to maintain the functional properties of eMSC upon expansion in culture. By integrating complementary genome-wide profiling techniques, we mapped the dynamic changes in chromatin landscape and gene expression in response to prolonged A83-01 treatment of eMSC. Our findings provide new insights into the mechanisms of action of TGF{beta}-R inhibition that may lead to the development of more targeted pharmacological approaches for MSC expansion.

cell biology↗