Myofibroblast Phenotype Research Articles

Radiation-induced morphea of the breast – characterization and treatment of fibroblast dysfunction with repurposed mesalazine

Radiation-induced morphea (RIM) is a rare complication of radiotherapy presenting as inflammatory fibrosis, most commonly reported in breast cancer patients. As underlying disease mechanisms are not well understood, targeted therapies are lacking. Since fibroblasts are the key mediators of all fibroproliferative diseases, this study aimed to characterize patient-derived fibroblasts to identify therapeutic targets. We studied primary human control and RIM-fibroblasts on a functional and molecular basis, analyzed peripheral blood and tissue samples and conducted, based on our findings, a treatment attempt in one patient. In RIM, we identified a distinct myofibroblast phenotype reflected by increased alpha-smooth-muscle-actin (αSMA) expression, reduced proliferation and migration rates, and overexpression of osteopontin (OPN). Our RNA sequencing identified aberrant Myc activation as a potential disease driver in RIM fibroblasts, similar to previous findings in systemic sclerosis. Treatment with the anti-inflammatory drug mesalazine reversed the myofibroblast phenotype by targeting Myc. Based on these findings, a patient with RIM was successfully treated with mesalazine, resulting in reduced inflammation and pain and tissue softening, while serum OPN was halved. The present study provides a comprehensive characterization of RIM fibroblasts, suggests a disease-driving role for Myc, demonstrates promising antifibrotic effects of mesalazine and proposes OPN as a biomarker for RIM.

Oxidative Stress, Glutaredoxins, and Their Therapeutic Potential in Posterior Capsular Opacification.

Posterior capsular opacification (PCO) is the most common long-term complication of cataract surgery. Traditionally, the pathogenesis of PCO involves the residual lens epithelial cells (LECs), which undergo transdifferentiation into a myofibroblast phenotype, hyperproliferation, matrix contraction, and matrix deposition. This process is driven by the marked upregulation of inflammatory and growth factors post-surgery. Recently, research on the role of redox environments has gained considerable attention. LECs, which are in direct contact with the aqueous humour after cataract surgery, are subjected to oxidative stress due to decreased levels of reduced glutathione and increased oxygen content compared to contact with the outer fibre layer of the lens before surgery. In this review, we examine the critical role of oxidative stress in PCO formation. We also focus on glutaredoxins (Grxs), which are antioxidative enzymes produced via deglutathionylation, their protective role against PCO formation, and their therapeutic potential. Furthermore, we discuss the latest advancements in PCO therapy, particularly the development of advanced antioxidative pharmacological agents, and emphasise the importance and approaches of anti-inflammatory and antioxidant treatments in PCO management. In conclusion, this review highlights the significant roles of oxidative stress in PCO, the protective effects of Grxs against PCO formation, and the potential of anti-inflammatory and antioxidant therapies in treating PCO.

Immunocytochemistry assessment of vocal fold regeneration after cell-based implant in rabbits.

Cell-based outer vocal fold replacement (COVR) offers a potential treatment for severe vocal fold scarring or cancer reconstruction. Previous work in rabbits using human adipose-derived stem cells (ASC) in fibrin suggested that a hybrid structure emerged within 2 months, containing both implanted and host cells. This project uses immunocytochemistry to better define the phenotypic fate of implanted cells and features of the extracellular environment. Immunocytochemistry was performed on sections collected from rabbits 2 months after COVR implantation or scar surgery. Cellular targets included human leukocyte antigen (HLA), CD31, and smooth muscle actin (SMA). HLA was present in all implanted sections and was used to identify human cells. In adjacent sections, HLA-positive cells were identified expressing CD31. SMA was not identified in the same cells as HLA. These markers were also present in injured vocal folds not receiving COVR. SMA protein content did not differ according to treatment. Implanted human ASC persist in rabbit vocal folds. Some appear to express CD31, an endothelial marker. Smooth muscle actin, a marker of myofibroblast phenotype, was present in all sections regardless of treatment, and was not identified in hASC. Host cells also infiltrate the structure, producing a hybrid host-graft vocal fold.

Abstract A039: Repeat RNA mediated disruption of cellular plasticity in pancreatic cancer

Abstract Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications for tumor cell state and the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we employed the NanoString CosMx™ spatial molecular imaging (SMI) platform, which utilizes a 1,000-plex RNA panel with custom repeat RNA probes targeting long interspersed nuclear element 1 (LINE-1) retrotransposon ORF1 and ORF2, HSATII satellite (SAT) repeat, and two human endogenous retroviruses (HERV-K and HERV-H) in 46 primary tumors. This analysis revealed correlations of high repeat RNA expression with alterations in the epithelial state of PDAC cells and the myofibroblast phenotype in cancer-associated fibroblasts (CAFs). The loss of cellular identity observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs in PDAC and CAF cell culture models points to cell-cell intercommunication involving these viral-like elements. Differences in the PDAC and CAF response are driven by distinct innate immune signaling pathways through interferon regulatory transcription factor 3 (IRF3). Altogether, our data indicate that cell context-specific viral-like responses driven by tumor cells have broad impacts on single-cell heterogeneity within cancer cells and the pancreatic cancer microenvironment. Citation Format: Eunae You, Patrick Danaher, Chenyue Lu, Siyu Sun, Luli Zou, Ildiko Phillips, Alexandra Rojas, Natalie Ho, Yuhui Song, Michael Raabe, Katherine Xu, Peter Richieri, Hao Li, Natalie Aston, Rebecca Porter, Bidish Patel, Linda Nieman, Nathan Schurman, Briana Hudson, Khrystyna North, Sarah Church, Vikram Deshpande, Andrew Liss, Tae Kim, Yi Cui, Youngmi Kim, Benjamin Greenbaum, Martin Aryee, David Ting. Repeat RNA mediated disruption of cellular plasticity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A039.

The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is almost entirely resistant to conventional chemotherapy and radiation therapy. A significant factor in this resistance appears to be the dense desmoplastic stroma, which contains various cancer-associated fibroblast (CAF) populations. However, our understanding of the communication between tumor cells and CAFs that contributes to this aggressive malignancy is still developing. Recently, we used an advanced three-dimensional heterospecies, heterospheroid co-culture model to investigate the signaling between human pancreatic tumor Panc1 cells and mouse pancreatic stellate cells (mPSCs) through global expression profiling. Upon discovering that CCN1 was significantly upregulated in Panc1 cells during co-culture, we decided to explore the role of CCN1 using CRISPR-Cas9 knockout technology. Panc1 cells lacking CCN1 showed reduced differentiation and decreased sensitivity to gemcitabine, primarily due to lower expression of genes involved in gemcitabine transport and metabolism. Additionally, we observed that stimulation with TGF-β1 and lysophosphatidic acid increased CCN1 expression in Panc1 cells and induced a shift in mPSCs towards a more myofibroblastic CAF-like phenotype.

CGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFβ1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFβ1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Activin A Is a Master Regulator of Phenotypic Switch in Adipose Stromal Cells Initiated by Activated Immune Cell-Secreted Interleukin-1β.

Prolonged tissue ischemia and inflammation lead to organ deterioration and are often accompanied by microvasculature rarefaction, fibrosis, and elevated systemic Activin A (ActA), the level of which frequently correlates with disease severity. Mesenchymal stromal cells are prevalent in the perivascular niche and are likely involved in tissue homeostasis and pathology. This study investigated the effects of inflammatory cells on modulation of phenotype of adipose mesenchymal stromal cells (ASC) and the role of ActA in this process. Peripheral blood mononuclear cells were activated with lipopolysaccharide (activated peripheral blood mononuclear cells [aPBMC]) and presented to ASC. Expression of smooth muscle/myofibroblast markers, ActA, transforming growth factors beta 1-3 (TGFβ1-3), and connective tissue growth factor (CTGF) was assessed in ASC. Silencing approaches were used to dissect the signaling cascade of aPBMC-induced acquisition of myofibroblast phenotype by ASC. ASC cocultured with aPBMC or exposed to the secretome of aPBMC upregulated smooth muscle cell markers alpha smooth muscle actin (αSMA), SM22α, and Calponin I; increased contractility; and initiated expression of ActA. Interleukin (IL)-1β was sufficient to replicate this response, whereas blocking IL-1β eliminated aPBMC effects. ASC-derived ActA stimulated CTGF and αSMA expression in ASC; the latter independent of CTGF. Induction of αSMA in ASC by IL-1β or ActA-enriched media relied on extracellular enzymatic activity. ActA upregulated mRNA levels of several extracellular matrix proteins in ASC, albeit to a lesser degree than TGFβ1, and marginally increased cell contractility. In conclusion, the study suggests that aPBMC induce myofibroblast phenotype with weak fibrotic activity in perivascular progenitors, such as ASC, through the IL-1β-ActA signaling axis, which also promotes CTGF secretion, and these effects require ActA extracellular enzymatic processing.

How to Keep Myofibroblasts under Control: Culture of Mouse Skin Fibroblasts on Soft Substrates

During the physiological healing of skin wounds, fibroblasts recruited from the uninjured adjacent dermis and deeper subcutaneous fascia layers are transiently activated into myofibroblasts to first secrete and then contract collagen-rich extracellular matrix into a mechanically resistant scar. Scar tissue restores skin integrity after damage but comes at the expense of poor esthetics and loss of tissue function. Stiff scar matrix also mechanically activates various precursor cells into myofibroblasts in a positive feedback loop. Persistent myofibroblast activation results in pathologic accumulation of fibrous collagen and hypertrophic scarring, called fibrosis. Consequently, the mechanisms of fibroblast-to-myofibroblast activation and persistence are studied to develop antifibrotic and prohealing treatments. Mechanistic understanding often starts in a plastic cell culture dish. This can be problematic because contact of fibroblasts with tissue culture plastic or glass surfaces invariably generates myofibroblast phenotypes in standard culture. We describe a straight-forward method to produce soft cell culture surfaces for fibroblast isolation and continued culture and highlight key advantages and limitations of the approach. Adding a layer of elastic silicone polymer tunable to the softness of normal skin and the stiffness of pathologic scars allows to control mechanical fibroblast activation while preserving the simplicity of conventional 2-dimensional cell culture.

The integrin receptor beta7 subunit mediates airway remodeling and hyperresponsiveness in allergen exposed mice

BackgroundFibroblast differentiation to a myofibroblast phenotype is a feature of airway remodeling in asthma. Lung fibroblasts express the integrin receptor α4β7 and fibronectin induces myofibroblast differentiation via this receptor.ObjectivesTo investigate the role of the β7 integrin receptor subunit and α4β7 integrin complex in airway remodeling and airway hyperresponsiveness (AHR) in a murine model of chronic allergen exposure.MethodsC57BL/6 wild type (WT) and β7 integrin null mice (β7 -/-) were sensitized (days 1,10) and challenged with ovalbumin (OVA) three times a week for one or 4 weeks. Similar experiments were performed with WT mice in the presence or absence of α4β7 blocking antibodies. Bronchoalveolar (BAL) cell counts, AHR, histological evaluation, soluble collagen content, Transforming growth factor-β (TGFβ) and Interleukin-13 (IL13) were measured. Phenotype of fibroblasts cultured from WT and β7 -/- saline (SAL) and OVA treated mice was evaluated.ResultsEosinophil numbers were similar in WT vs β7-/- mice. Prolonged OVA exposure in β7-/- mice was associated with reduced AHR, lung collagen content, peribronchial smooth muscle, lung tissue TGFβ and IL13 expression as compared to WT. Similar findings were observed in WT mice treated with α4β7 blocking antibodies. Fibroblast migration was enhanced in response to OVA in WT but not β7 -/- fibroblasts. α-SMA and fibronectin expression were reduced in β7-/- fibroblasts relative to WT.ConclusionsThe β7 integrin subunit and the α4β7 integrin complex modulate AHR and airway remodeling in a murine model of allergen exposure. This effect is, at least in part, explained by inhibition of fibroblast activation and is independent of eosinophilic inflammation.

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

Inhibition and reversal of a TGF-β1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors

BackgroundHypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation.MethodsThe transforming growth factor-β1 (TGF-β1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts.ResultsAdipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-β1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue.ConclusionsCultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

Fibroblasts from HPV-negative oropharynx squamous cell carcinomas stimulate the release of osteopontin from cancer cells via the release of IL-6.

HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood. To investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV- OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk. A retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication. HPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV-positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV- OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs. This investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity.

Abstract PO1-25-07: Obesity, breast cancer, and adipose-derived mesenchymal stem/stromal cells: The complex cross-signaling in the tumor microenvironment

Abstract Objective: Breast cancer is the most frequently diagnosed cancer and a common cause of cancer related death in women. The breast cancer microenvironment (TME) is mainly composed of adipose tissue, consisting of adipocytes, adipose-derived mesenchymal stem/stromal cells (bASCs) and immune cells. Interestingly, it is well recognized that obesity, as indicated by increased body mass index (BMI≥30), is associated with an enhanced risk of more aggressive breast cancer (BC) as well as reduced patient survival. Therefore, we asked how BC affects the biological functionality of bASC, how obesity fuels this process, and on the other hand how these cells influence BC cells. Methods: Multiple molecular biological approaches as transcriptomic profiling and diverse functional assays were conducted to characterize at least 5 lean and obese patients derived bASCs samples near the TME (lean/obese-aT) or distant (lean/obese-dT). Afterwards, these cells were used to investigate the interaction with BC cells in 2-dimensional and 3-dimensional cell culture experiments. Moreover, immunohistological analyses were conducted in 16 lean and 16 obese breast cancer patients (grade 3-4). Results: The TME shifts bASCs towards cancer supporting phenotypes into an inflammatory cancer-associated phenotype, whereas ob-aT bASCs are prone to be cancer-educated into a myofibroblastic phenotype with a significant deregulated gene expression, cytokine/chemokine secretion, and reduced differentiation capacity. In corroberation, these BMI dependent de-differentiation processes could be confirmed in breast cancer tissue. Moreover, the cell-cell interaction of BC cells with aT-bASCs increased their proliferation, motility, and chemoresistance. Conclusions: The study found multiple pathways deregulated in bASCs associated with tumor promoting effects. Additionally, it revealed the role of aT-bASCs in promoting breast cancer progression on a functional and molecular level. Citation Format: Andreas Ritter, Christine Solbach, Juping Yuan. Obesity, breast cancer, and adipose-derived mesenchymal stem/stromal cells: The complex cross-signaling in the tumor microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-07.

P311 KO Mice are Protected from Experimentally Induced Lung Fibrosis

Pulmonary fibrosis (PF) is a fatal chronic interstitial lung disease and one of the most irreversible forms of tissue/organ fibrosis. Idiopathic pulmonary fibrosis is a very progressive disease accompanied by inflammatory damage with a median survival of less than 5 years. Etiology of IPF is still elusive with potential reasons being genetic predisposition and environmental exposures including particulates, radiation, and SARS-CoV-2. Recent studies indicate that about 50% of COVID19 associated acute respiratory distress syndrome survivors develop PF. There are very limited treatment options with no curative therapies available for PF apart from invasive lung transplantation. P311 is an 8 kDa protein previously shown to have a key role in lung regeneration. As P311 overexpression in fibroblasts induced myofibroblast phenotype, we hypothesized that P311 has a role in tissue fibrosis. We induced PF by intra-tracheally instilling bleomycin (BL) in P311 knockout (KO) mice and wild type (WT, C57BL/6) control mice and collected lungs after 2-5 weeks. P311 KO mice showed significantly less fibrosis. After intra-tracheal instillation of BL, the lungs of P311 KO mice (D21) had significantly reduced number of myofibroblasts as indicated by reduced SM α-actin expression and collagen deposition as indicated by Masson trichrome staining compared to their WT counterparts. We further observed reduced levels of TGF-β1-3 and Smad-2 and -3 along with decreased epithelial to mesenchymal transdifferentiation markers in the lungs of bleomycin treated P311 KOs compared to WT controls. Further bronchoalveolar lavage fluid (BALF) was collected (D14) and utilized to determine the pulmonary inflammation by determining the total and different immune cell populations in the BALF and lung samples by FACS analysis. Even though we saw a significant increase in the BALF cellularity, we did not observe significant changes in T-cell, macrophage, and neutrophil numbers between WT and P311KOs. However, lung cell isolates showed significantly increased CD3 and CD4 positive cells indicating increased T-cells and helper T-cells in fibrotic P311KO lungs compared to WT lungs. In conclusion, bleomycin administration resulted in lung fibrosis in WT mice, but P311 KO mice are protected. This study was partially supported by research funding to KB, from American Lung Association, DA-196629-N. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2149: Smooth Muscle-derived Resident Vascular Adventitial Progenitor Cell Contribution To Cardiac Fibrosis And Inflammation Is Dependent On KLF4

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the vascular adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. In contrast, loss of Klf4 expression promotes the transition of AdvSca1-SM cells to a myofibroblast phenotype in vascular injury and remodeling. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. Our recent preliminary data confirm the existence of perivascular cardiac AdvSca1-SM cells. Leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation is characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes are recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Very surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protects against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

Targeting PDGF signaling of cancer-associated fibroblasts blocks feedback activation of HIF-1α and tumor progression of clear cell ovarian cancer

Ovarian clear cell carcinoma (OCCC) is a gynecological cancer with a dismal prognosis; however, the mechanism underlying OCCC chemoresistance is not well understood. To explore the intracellular networks associated with the chemoresistance, we analyze surgical specimens by performing integrative analyses that combine single-cell analyses and spatial transcriptomics. We find that a chemoresistant OCCC subpopulation with elevated HIF activity localizes mainly in areas populated by cancer-associated fibroblasts (CAFs) with a myofibroblastic phenotype, which is corroborated by quantitative immunostaining. CAF-enhanced chemoresistance and HIF-1α induction are recapitulated in co-culture assays, which show that cancer-derived platelet-derived growth factor (PDGF) contributes to the chemoresistance and HIF-1α induction via PDGF receptor signaling in CAFs. Ripretinib is identified as an effective receptor tyrosine kinase inhibitor against CAF survival. In the co-culture system and xenograft tumors, ripretinib prevents CAF survival and suppresses OCCC proliferation in the presence of carboplatin, indicating that combination of conventional chemotherapy and CAF-targeted agents is effective against OCCC.

Targeting MetaLnc9/miR-143/FSCN1 axis inhibits oxidative stress and myofibroblast transdifferentiation in oral submucous fibrosis

Background/purposePersistent activation of myofibroblasts is attributed to various dysregulated biological events conferring multiple types of fibrosis diseases, including oral submucous fibrosis (OSF). Although the significance of non-coding RNAs (ncRNAs) in the occurrence of fibrosis has been appreciated, the detailed mechanisms still have not been fully elucidated. The aim of this study was to identify key dysregulated ncRNAs and elucidate their pro-fibrotic mechanisms in promoting myofibroblast activation and the pathological development of OSF. Materials and methodsExpression of non-coding RNAs and mRNAs in OSF cohort was determined using RNA sequencing and qRT-PCR. The molecular axis of pro-fibrotic ncRNAs were exploited via luciferase reporter activity assay and RNA expression rescue experiments. Functional assays, including collagen gel contraction, wound healing ability, cell migration, and reactive oxygen species (ROS) production, were conducted to assess the changes in the myofibroblastic phenotypes of primary human buccal mucosal fibroblasts. ResultsHerein, we found that long non-coding RNA MetaLnc9 was upregulated in OSF specimens and positively associated with several fibrosis markers. Silencing of MetaLnc9 diminished the features of activated myofibroblasts and the production of ROS. We not only showed that MetaLnc9 functioned as a competitive endogenous RNA of microRNA (miR)-143, but also demonstrated that the pro-fibrosis effect of MetaLnc9 on myofibroblast activities was mediated by suppression of miR-143. Moreover, our data showed that fascin actin-bundling protein 1 (FSCN1) was a direct target of miR-143 and positively related to MetaLnc9. ConclusionUpregulation of MetaLnc9 may enhance the activation of myofibroblasts by sponging miR-143 and titrating its inhibitory property on FSCN1.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-β. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.