Fibroblast Motility Research Articles

Epigenetic memory of radiotherapy in dermal fibroblasts impairs wound repair capacity in cancer survivors

Radiotherapy (RT), a common cancer treatment, unintentionally harms surrounding tissues, including the skin, and hinders wound healing years after treatment. This study aims to understand the mechanisms behind these late-onset adverse effects. We compare skin biopsies from previously irradiated (RT+) and non-irradiated (RT−) sites in breast cancer survivors who underwent RT years ago. Here we show that the RT+ skin has compromised healing capacity and fibroblast functions. Using ATAC-seq, we discover altered chromatin landscapes in RT+ fibroblasts, with THBS1 identified as a crucial epigenetically primed wound repair-related gene. This is further confirmed by single-cell RNA-sequencing and spatial transcriptomic analysis of human wounds. Notably, fibroblasts in both murine and human post-radiation wound models show heightened and sustained THBS1 expression, impairing fibroblast motility and contractility. Treatment with anti-THBS1 antibodies promotes ex vivo wound closure in RT+ skin from breast cancer survivors. Our findings suggest that fibroblasts retain a long-term radiation memory in the form of epigenetic changes. Targeting this maladaptive epigenetic memory could mitigate RT’s late-onset adverse effects, improving the quality of life for cancer survivors.

The effects of glycoside-rich green extract from Centella asiatica (L.) Urban on wound healing and anti-aging activity

BackgroundCentella asiatica (L.) Urban extracts contain triterpenoid compounds, namely, glycosides (madecassoside and asiaticoside) and aglycones (madecassic acid and asiatic acid), that possess skincare properties. The extract with high amounts of active components could guarantee the quality of extracts for application in the cosmeceutical industry. PurposeThis study investigated the dermatological benefits of glycoside-rich green extracts from C. asiatica and different triterpenoid combinations. MethodsSix C. asiatica green extracts with different triterpenoid contents were divided into two groups: glycoside-rich extracts (GRCs), which contained >10 % glycosides, and glycoside-low extracts (GLCs), which contained <10 % glycosides. The GRCs, the GLCs, and the triterpenoid components of the selected extract were diluted with 1 % DMSO to concentrations of 0.1, 1, 10, and 100 µg/ml for cell viability, cell migration and procollagen type I C-peptide (PIP) production assays. For the in vitro antiglycation assay, all the samples were diluted with 2 % ethanol to concentrations of 0.5, 0.75, and 1 mg/ml before testing. The best green extract at the optimum concentration was selected, and its activity was compared with that of its triterpenoid components. ResultsAt 10 µg/ml, GRCs promoted wound healing by stimulating fibroblast and keratinocyte motility and increasing collagen production. The main pharmacologically active compounds involved in wound healing were glycosides (madecassoside and asiaticoside). Regarding antiaging activity, 1 mg/ml GRC had a stronger antiglycation effect than combined or individual triterpenoid compounds. Therefore, the antiaging activity of GRC may be mainly attributable to compounds other than triterpenoids. ConclusionC. asiatica green extract containing >10 % glycosides has high development potential for use in the cosmeceutical industry due to its ability to promote wound healing and its antiaging properties.

Deciphering the Dysregulating IGF-1–SP1–CD248 Pathway in Fibroblast Functionality during Diabetic Wound Healing

Reduced fibroblast activity is a critical factor in the progression of diabetic ulcers. CD248, a transmembrane glycoprotein prominently expressed in activated fibroblasts, plays a pivotal role in wound healing. However, the role of CD248 in diabetic wound healing and the CD248 regulatory pathway remains largely unexplored. Our study shows that CD248 expression is significantly reduced in skin wounds from both diabetic patients and mice. Single-cell transcriptome data analyses reveal a marked reduction of CD248-enriched secretory-reticular fibroblasts in diabetic wounds. We identify insulin-like growth factor-1 (IGF-1) as a key regulator of CD248 expression through the Akt/mTOR signaling pathway and the Sp1 transcription factor. Overexpression of CD248 enhances fibroblast motility, elucidating the underrepresentation of CD248-enriched fibroblasts in diabetic wounds. Immunohistochemical staining of diabetic wound samples further confirm low SP1 expression and fewer CD248-positive secretory-reticular fibroblasts. Further investigation reveals that elevated tumor necrosis factor α (TNFα) levels in diabetic environment promotes IGF-1 resistance, and inhibiting IGF-1-induced CD248 expression. In summary, our findings underscore the critical role of the IGF1-SP1-CD248 axis in activating reticular fibroblasts during wound-healing processes. Targeting this axis in fibroblasts could help develop a therapeutic regimen for diabetic ulcers.

In vitro comparison of zinc-based, chlorhexidine, and essential oil mouth rinses.

Chlorhexidine (CHX)-based mouth rinses are frequently prescribed following periodontal surgeries. A more recently available brand of zinc-based mouth rinses advertises one of its mouth rinses as a substitute for chlorhexidine. The purpose of this study was to evaluate, in vitro, the effects of this brand of zinc-based mouth rinses on cell survival, cell motility, and gene expression of human gingival fibroblasts (HGFs). HGFs were exposed to essential oil (EO), CHX, and three types of one brand of zinc-based mouth rinses designed to treat breath malodor (ZnA), dry mouth (ZnB), and gingivitis (ZnC). Each mouth rinse was tested over a range of concentrations for its effects on HGF survival and motility. Gene expression of cytokines, interleukins, and growth factors were evaluated via reverse transcriptase-polymerase chain reaction (RT-PCR), as a means to assess potential influences on inflammation and wound healing. Cell survival was significantly decreased for CHX and ZnC at 10% dilutions (p<0.05). For all time points, cells exposed to ZnC displayed the greatest reduction in cell motility (p<0.05). The various mouth rinses examined differentially altered the expression of growth factor transcripts. ZnC particularly enhanced the expression of BMP-2 and FGF-2. ZnC was more cytotoxic and inhibited cell motility to a greater extent than any of the other mouth rinses. Therefore, using ZnC as an alternative to CHX could potentially have negative effects on wound healing after periodontal surgery. However, further investigation is required to confirm the clinical relevance of these in vitro findings. One type of zinc-based mouth rinse designed to replace chlorhexidine (often prescribed after oral surgeries) demonstrated the greatest oral cell death and reduction in cell movement when compared to other zinc-based mouth rinses. These zinc-based mouth rinses also reduced the amounts of proteins involved in regulating inflammation, potentially reducing the destruction of bone holding the teeth in place. They also changed the amounts of several molecules involved in tissue healing. It is unknown if this will speed or slow the healing of the soft tissues of the mouth.

ELFN1 is a new extracellular matrix (ECM)-associated protein

AimsThe ELFN1, discovered in 2007, is a single-pass transmembrane protein. Studies conducted thus far to elucidate the function of the Elfn1 have been limited only to animal studies. These studies have reported that ELFN1 is a universal binding partner of metabotropic glutamate receptors (mGluRs) in the central nervous system and its functional deficiency has been associated with the pathogenesis of neurological and neuropsychiatric diseases. In 2021, we described the first disease-associated human ELFN1 pathogenic gene mutation. Severe joint laxity, which was the most striking finding of this new disease and was clearly seen in the patients since early infancy, showed that the ELFN1 may have a possible function in the connective tissue besides the nervous system. Here, we present the first experimental evidence of the extracellular matrix (ECM)-related function of the ELFN1. Materials and methodsPrimary skin fibroblasts were isolated from the skin biopsies of ELFN1 mutated patients and healthy foreskin donors. For the clinical trial in a dish, in vitro ECM and DEM (decellularized ECM) models were created from skin fibroblasts. All the in vitro models were comparatively characterized and analyzed. Key findingsThe mutation in the ELFN1 signal peptide region of patients resulted in a severe lack of ELFN1 expression and dramatically altered the characteristic morphology and behavior (growth, proliferation, and motility) of fibroblasts. SignificanceWe propose that ELFN1 is involved in the cell-ECM attachment, and its deficiency is critical enough to cause a loss of cell motility and soft ECM stiffness.

Repurposing nitrofurantoin as a stimulant of fibroblast extracellular matrix repair for the treatment of emphysema

Emphysema is a respiratory disease that causes the progressive loss of lung extracellular matrix (ECM) organisation, subsequently undermining lung integrity and reducing lung function. Fibroblasts must constantly repair damage to the lungs to preserve lung health, however, fibroblast ECM repair is reduced during emphysema, causing ECM damage to outweigh fibroblast ECM maintenance. Current treatments for emphysema fail to address the root causes of emphysematous progression, highlighting the need for novel methods of treating emphysema. Nitrofurantoin is a broad-spectrum antibiotic indicated for the treatment of urinary tract infections that also displays potential as a novel avenue of emphysema treatment. Nitrofurantoin is known to potentially cause fibrotic effects that could be repurposed to increase fibroblast repair and outweigh the progressive ECM damage of the emphysematous lung. Therefore, this study examined the effects of nitrofurantoin treatment on primary human lung fibroblasts derived from emphysema patients to determine if the drug holds potential as a novel treatment for emphysema. Nitrofurantoin was shown to stimulate migration and alter fibroblast morphology by increasing cell area and reducing roundness, suggesting that it could induce an ECM-repair primed phenotype in fibroblasts. Interestingly, nitrofurantoin treatment did not alter collagen-IV, perlecan, periostin or tenascin-C deposition, though fibronectin deposition was significantly upregulated at a higher dosage (20 μg/mL). This study highlighted the nitrofurantoin induced changes to fibroblast motility and morphology that facilitate ECM repair. Thus, nitrofurantoin induced pulmonary fibrosis could be caused by a change in cell phenotype that subsequently upregulates ECM repair, indicating its potential as a treatment for emphysema.

Serum deprivation protein response intervenes in the proliferation, motility, and extracellular matrix production in keloid fibroblasts by blocking the amplification of TGF-β1/SMAD signal cascade via ERK1/2

Keloid formation has been linked to abnormal fibroblast function, such as excessive proliferation and extracellular matrix (ECM) production. Serum deprivation protein response (SDPR) is a crucial regulator of cellular function under diverse pathological conditions, yet its role in keloid formation remains unknown. The current work investigated the function of SDPR in regulating the proliferation, motility, and ECM production of keloid fibroblasts (KFs), as well as to decipher the mechanisms involved. Analysis of RNA sequencing data from the GEO database demonstrated significant down-regulation of SDPR in KF compared to normal fibroblasts (NFs). This down-regulation was also observed in clinical keloid specimens and isolated KFs. Overexpression of SDPR suppressed the proliferation, motility, and ECM production of KFs, while depletion of SDPR exacerbated the enhancing impact of TGF-β1 on the proliferation, motility, and ECM production of NFs. Mechanistic studies revealed that SDPR overexpression repressed TGF-β/Smad signal cascade activation in KFs along with decreased levels of phosphorylated Samd2/3, while SDPR depletion exacerbated TGF-β/Smad activation in TGF-β1-stimulated NFs. SDPR overexpression also repressed ERK1/2 activation in KFs, while SDPR depletion exacerbated ERK1/2 activation in TGF-β1-stimulated NFs. Inhibition of ERK1/2 abolished SDPR-depletion-induced TGF-β1/Smad activation, cell proliferation, motility, and ECM production in NFs. In conclusion, SDPR represses the proliferation, motility, and ECM production in KFs by blocking the TGF-β1/Smad pathway in an ERK1/2-dependent manner. The findings highlight the role of SDPR in regulating abnormal behaviors of fibroblasts associated with keloid formation and suggest it as a potential target for anti-keloid therapy development.

In vitro and in vivo studies on bioactive hydroxyapatite-coated magnesium for glaucoma drainage implant

Given the alarmingly increasing rates of glaucoma worldwide and the lack of satisfactory treatments, there is a dire need to explore more feasible treatment options. Magnesium (Mg) is an essential element in maintaining the functional and structural integrity of vital ocular tissues, but Mg and its alloys are rarely mentioned in ophthalmic applications. Our previous research found that hydroxyapatite-coated Mg (Mg@HA) shows the best biocompatibility and bioactivity, and exhibits the effect of inhibiting fibrosis after filtration surgery in the rabbit model, which is expected to be a promising material for glaucoma drainage device. In this study, we further demonstrated the anti-fibrosis effect of Mg@HA from the molecular signal level and the efficacy of implantation in the rabbit filtration surgery model. In vitro experiments showed the surface modification of Mg affects the adhesion behavior and the reorganization of cytoskeleton of Human Western blot analysis and immunofluorescence found that Mg@HA regulates the adhesion and motility of human Tenon's capsule fibroblasts mainly by down-regulating the phosphorylation of Smad2 and Smad3 in the canonical transforming growth factor-beta (TGF - β) signaling pathway. By observing and recording the condition of filtering blebs and intraocular pressure after surgery, the effectiveness of Mg@HA applied in the rabbit filtration surgery model was further evaluated. In conclusion, the application of hydroxyapatite-coated Mg in the eye has good biocompatibility and has the potential to resist postoperative glaucoma filtration fibrosis, which may be mediated by the regulation of the TGFβ/Smad signaling pathway.

Endoplasmic Reticulum Stress Inducer Dithiothreitol Affects the Morphology and Motility of Cultured Human Dermal Fibroblasts and Fibrosarcome HT1080 Cell Line

Some inducers of endoplasmic reticulum (ER) stress can affect the motility of normal and tumor cells. However, it is unknown what mechanisms mediate this effect and whether it is a consequence of ER stress. The aim of our work was to study the effect of the ER stress inducer dithiothreitol (DTT) on morphological features reflecting the locomotor properties of cells, as well as directly on the migratory properties of cultured human dermal fibroblasts and fibrosarcoma HT1080 cells. We have shown that DTT causes disruption of the organization of actin cytoskeleton in both types of cells, which is accompanied by a change in the cell surface and shape of cells, as well as a decrease in their spreading area. In addition, a decrease in the number of focal contacts was observed in dermal fibroblasts. DTT also reduced the motility of dermal fibroblasts and fibrosarcoma cells. To analyze cell motility and determine the moment of its change, we developed a method which showed that the change in the migratory properties of fibrosarcoma cells cultured with DTT began earlier than in dermal fibroblasts. Thus, activation of ER stress by DTT is accompanied by a change in the organization of the actin cytoskeleton and motility in normal and tumor human cells. Consequently, ER stress triggered by various inducers with different mechanisms of action affects the motility of normal and tumor cells, which must be taken into account when developing antitumor drugs that cause cell death through activation of ER stress.

SEMA7a primes integrin α5β1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5β1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

A novel integrated experimental and computational approach to unravel fibroblast motility in response to chemical gradients in 3D collagen matrices.

Fibroblasts play an essential role in tissue repair and regeneration as they migrate to wounded areas to secrete and remodel the extracellular matrix. Fibroblasts recognize chemical substances such as growth factors, which enhance their motility towards the wounded tissues through chemotaxis. Although several studies have characterized single-cell fibroblast motility before, the migration patterns of fibroblasts in response to external factors have not been fully explored in 3D environments. We present a study that combines experimental and computational efforts to characterize the effect of chemical stimuli on the invasion of 3D collagen matrices by fibroblasts. Experimentally, we used microfluidic devices to create chemical gradients using collagen matrices of distinct densities. We evaluated how cell migration patterns were affected by the presence of growth factors and the mechanical properties of the matrix. Based on these results, we present a discrete-based computational model to simulate cell motility, which we calibrated through the quantitative comparison of experimental and computational data via Bayesian optimization. By combining these approaches, we predict that fibroblasts respond to both the presence of chemical factors and their spatial location. Furthermore, our results show that the presence of these chemical gradients could be reproduced by our computational model through increases in the magnitude of cell-generated forces and enhanced cell directionality. Although these model predictions require further experimental validation, we propose that our framework can be applied as a tool that takes advantage of experimental data to guide the calibration of models and predict which mechanisms at the cellular level may justify the experimental findings. Consequently, these new insights may also guide the design of new experiments, tailored to validate the variables of interest identified by the model.

IRAK-M Regulates Proliferative and Invasive Phenotypes of Lung Fibroblasts.

Lung fibroblasts play an important role in subepithelial fibrosis, one feature for airway remodeling. IL-1 receptor-associated kinase (IRAK)-M was shown to involve fibrosis formation in airways and lung through regulation of inflammatory responses. IRAK-M is expressed by lung fibroblasts, whether IRAK-M has direct impact on lung fibroblasts remains unclear. In this investigation, we evaluated in vitro effect of IRAK-M on phenotypes of lung fibroblasts by silencing or overexpressing IRAK-M. Murine lung fibroblasts (MLg) were stimulated with house dust mite (HDM), IL-33, and transforming growth factor (TGF) β1. Techniques of small interfering RNA or expression plasmid were employed to silence or overexpress IRAK-M in MLg fibroblast cells. Proliferation, migration, invasiveness, and fibrosis-related events were evaluated. Significant upregulation of IRAK-M expression in MLg cells was caused by these stimuli. Silencing IRAK-M significantly increased proliferation, migration, and invasiveness of lung fibroblasts regardless of stimulating conditions. By contrast, IRAK-M overexpression significantly inhibited proliferation and motility of MLg lung fibroblasts. IRAK-M overexpression also significantly decreased the expression of fibronectin, collagen I, and α-SMA in MLg cells. Under stimulation with TGFβ1 or IL-33, IRAK-M silencing reduced MMP9 production, while IRAK-M overexpression increased MMP9 production. Modulation of IRAK-M expression affected cytokines production, either decreased or increased expression of TNFα and CXCL10 by the cells regardless of stimulation. Our in vitro data reveal that IRAK-M directly impacts on lung fibroblasts through modulation of cellular motility, release of inflammatory, and fibrotic cytokines of lung fibroblasts. These might suggest a new target by regulation of IRAK-M in slowing airway remodeling.

Transmembrane serine protease 2 cleaves nidogen 1 and inhibits extrahepatic liver cancer cell migration and invasion.

We aimed to confirm whether transmembrane serine protease 2 (TMPRSS2) regulates nidogen 1 (NID1) expression in extracellular vesicles (EVs) and metastatic hepatocellular carcinoma (HCC) cells. HCC cells, HUVEC cells, MRC-5 cells, HLE cells, MHCCLM3 cells, MHCC97L cells, H2P cells, H2M cells, as well as LO2 cells were cultured according to providers' instruction and EV models were established by using BALB/cAnN-nu mice to facilitate the verifications. We found that TMPRSS2 expression was inversely correlated with the metastatic potential of HCC cell lines. The expression of TMPRSS2 decreased in a time-dependent manner in tumor-bearing model mice implanted with MHCCLM3 cells compared with uninoculated mice. TMPRSS2 overexpression in MHCCLM3 and MHCC97L cells led to the significant downregulation of NID1 expression in total cell lysates and isolated EVs. In contrast, TMPRSS2 silencing resulted in the elevation of NID1 expression in cells and EVs. Administration of EVs from MHCCLM3 and MHCC97L cells with overexpressed or silenced TMPRSS2 inhibited or strengthened, respectively, the invasion, proliferation, and migration of LO2 tumor cells. EVs derived from MHCCLM3 and MHCC97L cells with overexpressed or depleted TMPRSS2 also deactivated or activated fibroblasts, respectively. These EVs secrete inflammatory cytokines and phosphorylated p65, facilitate the colonization of fibroblasts, and augment fibroblast growth and motility. These findings provide evidence for a new candidate drug targeting tumorigenic EV-NID1 to treat HCC.

Lectin-Based Study Reveals the Presence of Disease-Relevant Glycoepitopes in Bladder Cancer Cells and Ectosomes.

Bladder cancer is a malignancy that remains a therapeutic challenge and requires the identification of new biomarkers and mechanisms of progression. Several studies showed that extracellular vesicles promote angiogenesis, migration and metastasis, and inhibit apoptosis in bladder cancer. This effect may depend on their glycosylation status. Thus, the aim of this study was to compare glycosylation profiles of T-24 urothelial bladder cancer cells, HCV-29 normal ureter epithelial cells, and ectosomes released by both cell lines using lectin blotting and flow cytometry. Ectosomes displayed distinct total and surface glycosylation profiles with abundance of β-1,6-branched glycans and sialilated structures. Then, it was investigated whether the glycosylation status of the T-24 and HCV-29 cells is responsible for the effect exerted by ectosomes on the proliferation and migration of recipient cells. Stronger proproliferative and promigratory activity of T-24-derived ectosomes was observed in comparison to ectosomes from HCV-29 cells. When ectosomes were isolated from DMJ-treated cells, the aforementioned effects were diminished, suggesting that glycans carried by ectosomes were involved in modulation of recipient cell function. HCV-29- and T-24-derived ectosomes also increased the viability and motility of endothelial HUVEC cells and Hs27 fibroblasts. This supports the hypothesis that ectosomes can modulate the function of various cells present in the tumor microenvironment.

Post-Translational Modifications Evoked by Reactive Carbonyl Species in Ultraviolet-A-Exposed Skin: Implication in Fibroblast Senescence and Skin Photoaging.

Photoaging is an accelerated form of aging resulting from skin exposure to ultraviolet (UV) radiation. UV-A radiation deeply penetrates the dermis and triggers the generation of reactive oxygen species (ROS) which promotes damage to DNA, lipids and proteins. Lipid peroxidation results from the oxidative attack of polyunsaturated fatty acids which generate a huge amount of lipid peroxidation products, among them reactive carbonyl species (RCS) such as α, β-unsaturated hydroxyalkenals (e.g., 4-hydroxynonenal), acrolein or malondialdehyde. These highly reactive agents form adducts on free NH2 groups and thiol residues on amino acids in proteins and can also modify DNA and phospholipids. The accumulation of RCS-adducts leads to carbonyl stress characterized by progressive cellular and tissular dysfunction, inflammation and toxicity. RCS-adducts are formed in the dermis of skin exposed to UV-A radiation. Several RCS targets have been identified in the dermis, such as collagen and elastin in the extracellular matrix, whose modification could contribute to actinic elastosis lesions. RCS-adducts may play a role in fibroblast senescence via the modification of histones, and the sirtuin SIRT1, leading to an accumulation of acetylated proteins. The cytoskeleton protein vimentin is modified by RCS, which could impair fibroblast motility. A better identification of protein modification and carbonyl stress in the dermis may help to develop new treatment approaches for preventing photoaging.

Involvement of Aquaporin 1 in the Motility and in the Production of Fibrillin 1 and Type I Collagen of Cultured Human Dermal Fibroblasts

Aminocarbonyl proteins increase with age in the dermal layer. Gene Chip analysis of mRNA expression in human dermal fibroblasts cultured on collagen gels treated with glyceraldehyde as an aminocarbonyl protein and on untreated collagen gels showed a decrease in the amount of aquaporin 1 (AQP1) mRNA. In this study, we clarified the involvement of AQP1 in collagen gel contraction and the production of fibrillin 1 and type I collagen in cultured human dermal fibroblasts. In the experiment, AQP1 siRNA was transfected into cultured human dermal fibroblasts to deplete AQP1, and the cell motility and contractile activity of the collagen gel were assessed. The production of fibrillin 1 and type I collagen was also examined by RT–qPCR and antibody staining. AQP1 depletion decreased the collagen gel contractile activity, and both the amount of mRNA and the antibody staining of fibrillin 1 and type I collagen decreased. Furthermore, the depletion of AQP1 reduced the levels of F-actin and phosphorylated myosin light chain 2, suggesting their involvement in reductions of the motility and collagen gel contractile activity of fibroblasts. These findings suggest that AQP1 is an important biomolecule for cell motility in human dermal fibroblasts and that decreased motility results in decreased expression of extracellular matrix proteins such as fibrillin 1 and type I collagen.

Proteomic characterisation of prostate cancer intercellular communication reveals cell type-selective signalling and TMSB4X-dependent fibroblast reprogramming

BackgroundIn prostate cancer, the tumour microenvironment (TME) represents an important regulator of disease progression and response to treatment. In the TME, cancer-associated fibroblasts (CAFs) play a key role in tumour progression, however the mechanisms underpinning fibroblast-cancer cell interactions are incompletely resolved. Here, we address this by applying cell type-specific labelling with amino acid precursors (CTAP) and mass spectrometry (MS)-based (phospho)proteomics to prostate cancer for the first time.MethodsReciprocal interactions between PC3 prostate cancer cells co-cultured with WPMY-1 prostatic fibroblasts were characterised using CTAP-MS. Signalling network changes were determined using Metascape and Enrichr and visualised using Cytoscape. Thymosin β4 (TMSB4X) overexpression was achieved via retroviral transduction and assayed by ELISA. Cell motility was determined using Transwell and random cell migration assays and expression of CAF markers by indirect immunofluorescence.ResultsWPMY-1 cells co-cultured with PC3s demonstrated a CAF-like phenotype, characterised by enhanced PDGFRB expression and alterations in signalling pathways regulating epithelial-mesenchymal transition, cytoskeletal organisation and cell polarisation. In contrast, co-cultured PC3 cells exhibited more modest network changes, with alterations in mTORC1 signalling and regulation of the actin cytoskeleton. The expression of the actin binding protein TMSB4X was significantly decreased in co-cultured WPMY-1 fibroblasts, and overexpression of TMSB4X in fibroblasts decreased migration of co-cultured PC3 cells, reduced fibroblast motility, and protected the fibroblasts from being educated to a CAF-like phenotype by prostate cancer cells.ConclusionsThis study highlights the potential of CTAP-MS to characterise intercellular communication within the prostate TME and identify regulators of cellular crosstalk such as TMSB4X.

Abstract 3861: Microscope-based sorting of highly motile cancer-associated fibroblasts using a photoconvertible fluorescent protein

Abstract In the tumor microenvironment, cancer-associated fibroblasts (CAFs) are known to play key roles in promoting cancer cell invasion and cancer progression. CAFs are also highly invasive compared with normal fibroblasts in a 3-D co-culture system with lung cancer cells. Due to CAF heterogeneity, there are various differences in cell motility even within a CAF population. To classify CAFs according to their motility, we named less motile CAFs as “static CAFs” and more motile CAFs as “motile CAFs”. To separate and isolate these static CAFs and motile CAFs from a 3-D co-culture system, CAFs were transfected with a green-to-red photoconvertible fluorescent protein, Dendra2. Dendra2-transfected CAFs at specific areas (i.e., static or motile CAFs) were UV-irradiated using a confocal microscope. CAFs with red fluorescence were then isolated by flow cytometry and were subjected to RNA sequencing. In gene ontology and gene set enrichment analyses, two types of CAFs showed significant differences in global gene expression patterns. These data suggest that heterogeneous CAFs with different motility have distinct roles in the regulation of various signaling pathways in the tumor microenvironment. Citation Format: Sieun Lee, Young-Ho Ahn. Microscope-based sorting of highly motile cancer-associated fibroblasts using a photoconvertible fluorescent protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3861.

MicroRNA-138 Abates Fibroblast Motility With Effect on Invasion of Adjacent Cancer Cells.

BackgroundRecent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells.MethodsExpression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion.ResultsExpression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway.ConclusionsDespite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.

Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI β, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell–extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.