Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation.

OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

Mouse Models of Human Gastric Cancer Subtypes With Stomach-Specific CreERT2-Mediated Pathway Alterations.

BACKGROUND & AIMS: Patterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes. METHODS: We searched databases to identify genes with unique expression in the stomach epithelium, resulting in the identification of Anxa10. We generated mice with tamoxifen-inducible Cre recombinase (CreERT2) in the Anxa10 gene locus. We created 3 mouse models with alterations in pathways that characterize the chromosomal instability (CIN) and the genomically stable (GS) subtypes of human gastric cancer: Anxa10-CreERT2;KrasG12D/+;Tp53R172H/+;Smad4fl/f (CIN mice), Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Smad4fl/fl (GS-TGBF mice), and Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Apcfl/fl (GS-Wnt mice). We analyzed tumors that developed in these mice by histology for cell types and metastatic potential. We derived organoids from the tumors and tested their response to chemotherapeutic agents and the epithelial growth factor receptor signaling pathway inhibitor trametinib. RESULTS: The gastric tumors from the CIN mice had an invasive phenotype and formed liver and lung metastases. The tumor cells had a glandular morphology, similar to human intestinal-type gastric cancer. The gastric tumors from the GS-TGFB mice were poorly differentiated with diffuse morphology and signet ring cells, resembling human diffuse-type gastric cancer. Cells from these tumors were invasive, and mice developed peritoneal carcinomatosis and lung metastases. GS-Wnt mice developed adenomatous tooth-like gastric cancer. Organoids derived from tumors of GS-TGBF and GS-Wnt mice were more resistant to docetaxel, whereas organoids from the CIN tumors were more resistant to trametinib. CONCLUSIONS: Using a stomach-specific CreERT2 system, we created mice that develop tumors with morphologic similarities to subtypes of human gastric cancer. These tumors have different patterns of local growth, metastasis, and response to therapeutic agents. They can be used to study different subtypes of human gastric cancer.

Human gastric cancer modelling using organoids.

OBJECTIVE: Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. DESIGN: Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. RESULTS: Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. CONCLUSION: We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing.

Modulation of RAB5A early endosome trafficking in response to KRas mediated macropinocytic fluxes in pancreatic cancer cells.

KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC. Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC. In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking.

Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies.

During surgery, rapid and accurate histopathological diagnosis is essential for clinical decision making. Yet the prevalent method of intra-operative consultation pathology is intensive in time, labour and costs, and requires the expertise of trained pathologists. Here we show that biopsy samples can be analysed within 30 min by sequentially assessing the physical phenotypes of singularized suspended cells dissociated from the tissues. The diagnostic method combines the enzyme-free mechanical dissociation of tissues, real-time deformability cytometry at rates of 100-1,000 cells s-1 and data analysis by unsupervised dimensionality reduction and logistic regression. Physical phenotype parameters extracted from brightfield images of single cells distinguished cell subpopulations in various tissues, enhancing or even substituting measurements of molecular markers. We used the method to quantify the degree of colon inflammation and to accurately discriminate healthy and tumorous tissue in biopsy samples of mouse and human colons. This fast and label-free approach may aid the intra-operative detection of pathological changes in solid biopsies.

Matrix stiffness mechanosensing modulates the expression and distribution of transcription factors in Schwann cells.

After peripheral nerve injury, mature Schwann cells (SCs) de-differentiate and undergo cell reprogramming to convert into a specialized cell repair phenotype that promotes nerve regeneration. Reprogramming of SCs into the repair phenotype is tightly controlled at the genome level and includes downregulation of pro-myelinating genes and activation of nerve repair-associated genes. Nerve injuries induce not only biochemical but also mechanical changes in the tissue architecture which impact SCs. Recently, we showed that SCs mechanically sense the stiffness of the extracellular matrix and that SC mechanosensitivity modulates their morphology and migratory behavior. Here, we explore the expression levels of key transcription factors and myelin-associated genes in SCs, and the outgrowth of primary dorsal root ganglion (DRG) neurites, in response to changes in the stiffness of generated matrices. The selected stiffness range matches the physiological conditions of both utilized cell types as determined in our previous investigations. We find that stiffer matrices induce upregulation of the expression of transcription factors Sox2, Oct6, and Krox20, and concomitantly reduce the expression of the repair-associated transcription factor c-Jun, suggesting a link between SC substrate mechanosensing and gene expression regulation. Likewise, DRG neurite outgrowth correlates with substrate stiffness. The remarkable intrinsic physiological plasticity of SCs, and the mechanosensitivity of SCs and neurites, may be exploited in the design of bioengineered scaffolds that promote nerve regeneration upon injury.

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response.

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1ß and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1ß and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.

A switch in pdgfrb+ cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord.

In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM.

Novel drug candidates for the treatment of metastatic colorectal cancer through global inverse gene-expression profiling.

Drug-induced gene-expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer, for which there is a pressing need to find novel antimetastatic compounds. We computationally predicted three novel and still unknown compounds against colorectal cancer: citalopram (an antidepressant), troglitazone (an antidiabetic), and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation, and migration and in a subcutaneous mouse model. We found evidence that the mode of action of these compounds may be through inhibition of TGFß signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of colorectal cancer. This study proposes citalopram as a potential therapeutic option for patients with colorectal cancer, illustrating the potential of systems pharmacology.