Co-culture Of Fibroblasts Research Articles

P0209 Unraveling the microbial dynamics in Crohn’s Disease-associated Fibrosis: insights from RNA-seq and Spatial Transcriptomics

Abstract Background In Crohn’s disease (CD), over 50% of cases develop fibrosis, often resulting in strictures. Despite advances in anti-inflammatory therapies, these complications persist due to chronic inflammation, which causes tissue injury and disrupts mucosal functions. Recent studies highlight microbiota dysbiosis as a potential inflammation-independent driver of fibrosis in CD 1,2, but specific microbial signatures across fibrosis stages remain underexplored. Methods We performed a meta-analysis of RNA-seq data from fibrotic CD-associated and healthy tissues. Cells, including immune, fibroblast, endothelial, and epithelial populations, were isolated via flow cytometry (FACS) and analyzed at the transcriptomic level. Co-culture experiments were conducted to explore cellular-bacterial interactions in fibrosis progression, and fibrosis markers (alpha-SMA, vimentin, and FAP) in fibroblasts were evaluated through immunofluorescence (IF). Results Gene ontology analysis revealed activation of antimicrobial immune processes, indicating a targeted response to microbiota during fibrosis progression. Microbiota analysis showed an increased abundance of Roseomonas mucosa in CD patients compared to controls across multiple cell types (endothelial, epithelial, fibroblast, and immune cells). Moreover, fibroblast co-cultures with Roseomonas mucosa (50 MOI/cell) displayed elevated fibrosis markers in IF compared to unstimulated controls. Conclusion Our findings suggest that Roseomonas mucosa, or its products, may modulate the transcriptional state of mucosal cells, including fibroblasts, contributing to intestinal fibrosis. To further elucidate the role of bacterial proteins in the progression from healthy to fibrotic tissue, we are performing spatial transcriptomics analysis on ileo-cecal resection tissues, followed by RNA-scope with bacteria-specific probes. This integrative approach aims to map the temporal progression of fibrosis in relation to microbiota, offering deeper insights into its development.

Modeling Fibroblast-Cardiomyocyte Interactions: Unveiling the Role of Ion Currents in Action Potential Modulation.

Fibrotic cardiomyopathy represents a significant pathological condition characterized by the interaction between cardiomyocytes and fibroblasts in the heart, and it currently lacks an effective cure. In vitro platforms, such as engineered heart tissue (EHT) developed through the co-culturing of cardiomyocytes and fibroblasts, are under investigation to elucidate and manipulate these cellular interactions. We present the first integration of mathematical electrophysiological models that encapsulate fibroblast-cardiomyocyte interactions with experimental EHT studies to identify and modulate the ion channels governing these dynamics. Our findings resolve a long-standing debate regarding the effect of fibroblast coupling on cardiomyocyte action potential duration (APD). We demonstrate that these seemingly contradictory outcomes are contingent upon the specific properties of the cardiomyocyte to which the fibroblast is coupled, particularly the relative magnitudes of the fast Na+ and transient outward K+ currents within the cardiomyocyte. Our results emphasize the critical importance of detailed ionic current representation in cardiomyocytes for accurately predicting the interactions between cardiomyocytes and fibroblasts in EHT. Surprisingly, complex ion channel-based models of fibroblast electrophysiology did not outperform simplified resistance-capacitance models in this analysis. Collectively, our findings highlight the promising potential of synergizing in vitro and in silico approaches to identify therapeutic targets for cardiomyopathies.

Engineering Granular Hydrogels without Interparticle Cross-Linking to Support Multicellular Organization.

Advancing three-dimensional (3D) tissue constructs is central to creating in vitro models and engineered tissues that recapitulate biology. Materials that are permissive to cellular behaviors, including proliferation, morphogenesis of multicellular structures, and motility, will support the emergence of tissue structures. Granular hydrogels in which there is no interparticle cross-linking exhibit dynamic properties that may be permissive to such cellular behaviors. However, designing granular hydrogels that lack interparticle cross-linking but support cellular self-organization remains underexplored relative to granular systems stabilized by interparticle cross-linking. In this study, we developed a polyethylene glycol-based granular hydrogel system, with average particle diameters under 40 μm. This granular hydrogel exhibited bulk stress-relaxing behaviors and compatibility with custom microdevices to sustain cell cultures without degradation. The system was studied in conjunction with cocultures of endothelial cells and fibroblasts, known for their spontaneous network formation. Cross-linking, porosity, and cell-adhesive ligands (such as RGD) were manipulated to control system properties. Toward supporting cellular activity, increased porosity was found to enhance the formation of cellular networks, whereas RGD reduced network formation in the system studied. This research highlights the potential of un-cross-linked granular systems to support morphogenetic processes, like vasculogenesis and tissue maturation, offering insights into material design for 3D cell culture systems.

Abstract A030: Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer

Abstract Cancer-associated fibroblasts (CAFs) are a heterogenous cell population that can promote esophageal cancer progression through multiple molecular mechanisms including increased cancer cell proliferation and migration, immunosuppression, and therapeutic resistance. Despite the divergence of CAFs from normal fibroblasts with regards to phenotype and functions, CAFs rarely exhibit widespread genetic alterations. Instead, it is proposed that CAFs are regulated at the epigenetic level. Our objective is to delineate this epigenetic regulation of CAFs in esophageal cancer. Ultimately, we aim to identify the epigenetic regulatory complexes that mediate CAF heterogeneity and pro-tumorigenic functions. To this end, we completed RNA-seq and ATAC-seq on fibroblasts isolated from esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (EAC) and non-cancerous esophageal tissue. Enrichment analysis identified the polycomb repressive complex 2 (PRC2) as a potential regulator of ESCC and EAC CAFs. PRC2 catalyzes the methylation (mono-, di- or tri-) of lysine 27 on histone 3 (H3K27me1/2/3) resulting in transcriptional repression. Inhibition of two key proteins of PRC2 (EZH2 and SUZ12) through either pharmacological or genetic modulation, altered expression of multiple CAF phenotype related proteins and inhibited CAF functions including CAF proliferation. Ongoing studies, using co-cultures of fibroblasts and tumor cells, seek to identify if inhibition of PRC2 can prevent the initial transition of normal esophageal fibroblasts into CAFs. Importantly, we are conducting in vivo studies to determine if selective PRC2 inhibition in CAFs can inhibit esophageal tumor progression with specific focus on primary tumor growth and metastatic spread. In summary, initial studies have identify that PRC2 complex mediates CAF phenotype and functions, and we aim to establish the larger impact of these CAF specific effects on esophageal cancer. Citation Format: Karen J. Dunbar, Raul Navaridas Fernandez de Bobadil, Katherine M. Cunningham, Emily Esquea, Gizem Efe, Anil K. Rustgi. Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A030.

Abstract C024: MPLA+IFNg-activated tumor-associated macrophages alter fibroblast composition

Abstract In a previous study, we developed a strategy to reprogram tumor-associated macrophages (TAMs) and induce a collaborative innate-adaptive immune response by combining monophosphoryl lipid A (MPLA) with IFNγ. However, we also observed downregulation of extracellular matrix (ECM)-associated genes in MPLA+IFNγ-treated mammary tumors, indicating that the combination treatment impacts the tumor microenvironment beyond just the immune cells. In this study, we investigated how MPLA+IFNg treatment modulates the tumor ECM, with a particular focus on fibroblasts-the major ECM-producing cells. Using scRNA-seq to profile mammary carcinomas, we identified six macrophage populations (C1q+, Ccr2+, Spp1+, Lyz+, mt+ and ki67+ TAMs) and two fibroblast populations (Cd34+ and a-SMA+ fibroblasts) in MMTV-PyMT tumor. MPLA+IFNγ treatment increased the numbers and percentages of Lyz+ TAMs and Cd34+ fibroblasts, while the numbers and percentages of Spp1+ TAMs and α-SMA+ fibroblasts were decreased by the treatment. Thus, MPLA+IFNγ treatment induced significant changes in both macrophage and fibroblast populations. Further analysis revealed an unusual gene expression pattern in Lyz+ TAMs. Mgst1 and Gng12 expression was detected in Lyz+ TAMs at levels comparable to those in fibroblasts, despite these genes typically being expressed in fibroblasts but not in macrophages of normal human tissues. However, to our surprise, MPLA+IFNγ treatment did not significantly increase Mgst1 and Gng12 expression of mono-cultured TAMs or bone marrow-derived macrophages, suggesting that these genes were not directly upregulated by MPLA+IFNγ treatment. Instead, the Mgst1 and Gng12 transcripts may have originated from engulfed fibroblasts. In addition, following MPLA+IFNγ treatment, more iNOS+ macrophages (which we previously identified as tumoricidal macrophages) were found in very close proximity to α-SMA+ fibroblasts. To directly test whether MPLA+IFNg-treated TAMs kill and engulf fibroblasts, we performed ex vivo co-cultures of TAMs and fibroblasts. Following MPLA+IFNγ treatment, TAMs effectively killed and engulfed the fibroblasts, whereas minimal fibroblast engulfment was observed in the control co-cultures. Our data suggest that tumoricidal MPLA+IFNγ-treated TAMs can regulate the ECM of tumors by targeting and eliminating the major ECM-producing cells, the fibroblasts. Ongoing studies are determining the functional consequences of the TAM-mediated regulation of fibroblasts and ECM composition. Citation Format: Lijuan Sun, Bodu Liu, Yixin Zhao, Mikala Egeblad. MPLA+IFNg-activated tumor-associated macrophages alter fibroblast composition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C024.

Carcinoma-Associated Fibroblasts Accelerate Growth and Invasiveness of Breast Cancer Cells in 3D Long-Term Breast Cancer Models.

Background/Objectives: Carcinoma-associated fibroblasts (CAFs), a prominent cell type in the tumor microenvironment (TME), significantly contributes to cancer progression through interactions with cancer cells and other TME components. Consequently, targeting signaling pathways driven by CAFs has potential to yield new therapeutic approaches to inhibit cancer progression. However, the mechanisms underlying their long-term interactions with cancer cells in vivo remains poorly understood. Methods: To address this, we developed a three-dimensional (3D) parallel coculture model of human triple-negative breast cancer (TNBC) cells and CAFs using our innovative TAME devices. This model allowed for the analysis of TNBC paracrine interactions via their secretome over extended culture periods (at least 70 days). Results: Using TNBC cell lines (MDA-MB-231, MCF10.DCIS, and HCC70), we found that TNBC spheroids in 3D parallel cocultures with CAFs exhibited more pronounced invasive finger-like outgrowths than those in cocultures of TNBC cells and normal fibroblasts (NFs) over a period of 50-70 days. We also established that the CAF-derived secretome affects TNBC migration towards the CAF secretome region. Additionally, we observed a preferential migration of CAFs, but not NFs, toward TNBC spheroids. Conclusions: Overall, our results suggest that paracrine interactions between TNBC cells and CAFs enhance TNBC invasive phenotypes and promote reciprocal migration.

Design and self-assembly of new peptides and peptide bolaamphiphiles as antioxidant biocomposite scaffolds for potential tissue regeneration applications – a replica modeling and experimental study

ABSTRACT In this work, five new peptides derived from natural resources and two peptide bolaamphiphiles were designed. The self-assembling ability of the peptides and the bolaamphiphiles, as well as their predicted antioxidant activity was examined computationally. In particular, replica modeling molecular dynamics studies were carried out at three different temperatures. Results showed that the bolaamphiphiles as well as three of the peptides efficiently formed spherical or fibrous assemblies, particularly at physiological temperatures. In addition, stacking interactions and hydrogen bonds played a critical role in assembly formation. Furthermore, molecular docking studies with extracellular matrix proteins such as the triple helix motif of collagen and the fibronectin (III) motif of tenascin-X displayed binding interactions with the peptides and the bolaamphiphiles. The most optimal peptide bolaamphiphile WMYGGGWMY-CO-NH-(CH2)4-YMWGGGYMW was then synthesized in the laboratory and its ability to form functional scaffolds upon binding to collagen and tenascin-X was examined. The scaffolds were bioprinted with co-cultures of fibroblasts and keratinocytes. The cells not only proliferated over time but also showed strong adherence and spreading within the matrix. Thus, the peptides and the bolaamphiphiles studied in this work, may be potentially developed as scaffold components for tissue regeneration applications.

3D Bioprinting a Novel Skin Co-Culture Model Using Human Keratinocytes and Fibroblasts.

3D bioprinting can generate the organized structures found in human skin for a variety of biological, medical, and pharmaceutical applications. Challenges in bioprinting skin include printing different types of cells in the same construct while maintaining their viability, which depends on the type of bioprinter and bioinks used. This study evaluated a novel 3D bioprinted skin model containing human keratinocytes (HEKa) and human dermal fibroblasts (HDF) in co-culture (CC) using a high-viscosity fibrin-based bioink produced using the BioX extrusion-based bioprinter. The constructs containing HEKa or HDF cells alone (control groups) and in CC were evaluated at 1, 10, and 20 days after bioprinting for viability, immunocytochemistry for specific markers (K5 and K10 for keratinocytes; vimentin and fibroblast specific protein [FSP] for fibroblasts). The storage, loss modulus, and viscosity properties of the constructs were also assessed to compare the effects of keratinocytes and fibroblasts individually and combined, providing important insights when bioprinting skin. Our findings revealed significantly higher cell viability in the CC group compared to individual keratinocyte and fibroblast groups, suggesting the combined cell presence enhanced survival rates. Additionally, proliferation rates of both cell types remained consistent over time, indicating non-competitive growth within the construct. Interestingly, keratinocytes exhibited a greater impact on the viscoelastic properties of the construct compared to fibroblasts, likely due to their larger size and arrangement. These insights contribute to optimizing bioprinting strategies for skin tissue engineering and emphasize the important role of different cell types in 3D skin models.

Trem2 acts as a non-classical receptor of interleukin-4 to promote diabetic wound healing.

The immunoglobulin superfamily protein Trem2 (triggering receptor expressed on myeloid cells 2) is primarily expressed on myeloid cells where it functions to regulate macrophage-related immune response induction. While macrophages are essential mediators of diabetic wound healing, the specific regulatory role that Trem2 plays in this setting remains to be established. This study was developed to explore the potential importance of Trem2 signalling in diabetic wound healing and to clarify the underlying mechanisms through which it functions. Following wound induction, diabetic model mice exhibited pronounced upregulation of Trem2 expression, which was primarily evident in macrophages. No cutaneous defects were evident in mice bearing a macrophage-specific knockout of Trem2 (T2-cKO), but they induced more pronounced inflammatory responses and failed to effectively repair cutaneous wounds, with lower levels of neovascularization, slower rates of wound closure, decreased collagen deposition following wounding. Mechanistically, we showed that interleukin (IL)-4 binds directly to Trem2, inactivating MAPK/AP-1 signalling to suppress the expression of inflammatory and chemoattractant factors. Co-culture of fibroblasts and macrophages showed that macrophages from T2-cKO mice suppressed the in vitro activation and proliferation of dermal fibroblasts through upregulation of leukaemia inhibitory factor (Lif). Injecting soluble Trem2 in vivo was also sufficient to significantly curtail inflammatory responses and to promote diabetic wound healing. These analyses offer novel insight into the role of IL-4/Trem2 signalling as a mediator of myeloid cell-fibroblast crosstalk that may represent a viable therapeutic target for efforts to enhance diabetic wound healing.

A 3D Epithelial-Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by ongoing inflammation, impaired tissue repair, and aberrant interplay between airway epithelium and fibroblasts, resulting in an altered extracellular matrix (ECM) composition. The ECM is the three-dimensional (3D) scaffold that provides mechanical support and biochemical signals to cells, now recognized not only as a consequence but as a potential driver of disease progression. To elucidate how the ECM influences pathophysiological changes occurring in COPD, in vitro models are needed that incorporate the ECM. ECM hydrogels are a novel experimental tool for incorporating the ECM in experimental setups. We developed an airway wall model by combining lung-derived ECM hydrogels with a co-culture of primary human fibroblasts and epithelial cells at an air-liquid interface. Collagen IV and a mixture of collagen I, fibronectin, and bovine serum albumin were used as basement membrane-mimicking coatings. The model was initially assembled using porcine lung-derived ECM hydrogels and subsequently with COPD and non-COPD human lung-derived ECM hydrogels. The resulting 3D construct exhibited considerable contraction and supported co-culture, resulting in a differentiated epithelial layer. This multi-component 3D model allows the investigation of remodelling mechanisms, exploring ECM involvement in cellular crosstalk, and holds promise as a model for drug discovery studies exploring ECM involvement in cellular interactions.

Abstract A036: The role of hypoxia in CD8+ T cell localization and function in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to existing therapeutic options. This resistance to treatment is partly due to its extensive inflammatory, desmoplastic stromal response, along with a hypoxic microenvironment. Although tumor hypoxia induces adaptive responses in both cancer cells and the surrounding stroma, most studies on the role of hypoxia in tumorigenesis have focused on cancer cell-intrinsic properties. The impact of hypoxia on stromal cells themselves and their interactions with cancer cells has remained largely unknown. We previously showed that inflammatory cancer-associated fibroblasts (iCAFs), which express high levels of cytokines and chemokines, are preferentially located in hypoxic regions of PDAC, and that hypoxia promotes an inflammatory fibroblast phenotype. The iCAF phenotype has been correlated with highly immunosuppressive features of PDAC. These findings and our preliminary observation that CD8+ T cells are largely excluded from hypoxic PDAC regions, raise the possibility that hypoxia suppresses the infiltration and activation of CD8+ T cells by modulating their interactions with inflammatory fibroblasts. By culturing CD8+ T cells under hypoxia with conditioned media from co-cultures of pancreatic cancer cells and fibroblasts, we have found that cancer cells and fibroblasts decrease CD8+ T cell proliferation and cytotoxic activity under low oxygen conditions. We are working to elucidate the mechanism by which hypoxia regulates CD8+ T cell infiltration and activation through cancer cell-fibroblast crosstalk. Citation Format: Ashley M. Mello, Tenzin Ngodup, Katelyn L. Donahue, Marina Pasca Di Magliano, Kyoung Eun Lee. The role of hypoxia in CD8+ T cell localization and function 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 A036.

Abstract A057: Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) originates from precursor lesions, the most common of which are known as pancreatic intraepithelial neoplasia (PanIN). PanIN formation and progression is accompanied by accumulation of a precursor microenvironment (PME) composed of desmoplastic stroma that plays an important role in promoting tumor growth and immune evasion. The crosstalk between tumor cells and stromal cells is an area of active investigation. While PanIN and the PME have been extensively studied in mouse models, their nature in humans have remained elusive as there is no clinical indication to sample healthy pancreas. Through a unique collaboration with the Gift of Life Organ and Tissue donation program, we performed transcriptomic analysis on normal pancreata declined for transplant. We noted that epithelial cells in the PanIN and cancer microenvironment were transcriptomically similar; however, their surrounding stromal fibroblasts were markedly distinct. Given that most precursor lesions remain quiescent while some progress to tumor, we sought out to elucidate how the transcriptional programs in epithelial cells influence fibroblast cell fate. To do so, we developed a 3D ex-vivo organoid-fibroblast coculture system using primary organoid and fibroblast cultures successfully derived from Gift of Life pancreata or PDAC patients undergoing surgical resection. Our coculture system retains histological features of heterogeneity observed in vivo. Using single cell RNA-sequencing analysis of matched fibroblast-organoid cocultures we identified factors that were highly expressed in fibroblasts in tumor cocultures, but not in normal cocultures. We are currently interrogating candidate ligand/receptor interactions in our cocultures to determine the mediators of this epithelial-mediated fibroblast polarization. This model will allow us to gain an understanding about how components of the microenvironment restrain or promote malignant transformation and serve as a great tool to study therapies targeting tumor and stromal cells in a patient specific manner. Citation Format: Padma Kadiyala, Ahmed Elhossiny, Jianhua Liu, Alexander Bray, Katelyn Donahue, Arvind Rao, Jiaqi Shi, Yaqing Zhang, Filip Bednar, Timothy Frankel, Eileen Carpenter, Marina Pasca Di Magliano. Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment [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 A057.

Application of an artificial intelligence for quantitative analysis of endothelial capillary beds in vitro.

The use of endothelial cell cultures has become fundamental to study angiogenesis. Recent advances in artificial intelligences (AI) offer opportunities to develop automated assessment methods in medical research, analyzing larger datasets. The aim of this study was to compare the application of AI with a manual method to morphometrically quantify in vitro angiogenesis. Co-cultures of human microvascular endothelial cells and fibroblasts were incubated mimicking endothelial capillary-beds. An AI-software was trained for segmentation of endothelial capillaries on anti-CD31-labeled light microscope crops. Number of capillaries and branches and average capillary diameter were measured by the AI and manually on 115 crops. The crops were analyzed faster by the AI than manually (3 minutes vs 1 hour per crop). Using the AI, systematically more capillaries (mean 48/mm2 vs 27/mm2) and branches (mean 23/mm2 vs 11/mm2) were counted than manually. Both methods had a strong linear relationship in counting capillaries and branches (r-capillaries = 0.88, r-branches = 0.89). No correlation was found for measurements of the diameter (r-diameter = 0.15). The present AI reduces the time required for quantitative analysis of angiogenesis on large datasets, and correlates well with manual analysis.

Form-deprivation myopia promotes sclera M2-type macrophages polarization in mice

PurposeTo explore the phenotype of sclera macrophages in form-deprivation (FD) myopia mice and the effects of M2 macrophage in FD myopia development. MethodsC57BL/6 mice were under 2 weeks of unilateral FD treatment. and they were separated into two groups, including an intraperitoneally injected(IP) vehicle group and Panobinostat (LBH589) (10 mg/kg per body weight) treatment group. All biometric parameters were measured before and after treatments, and the type and density of sclera macrophages were identified by immunofluorescence and RT-qPCR. In vitro, we analyzed the M2 macrophage and primary human sclera fibroblast (HSF) co-culture system by using the transcriptome sequencing method. Gene ontology (GO) and KEGG enrichment analyses were used to pinpoint the biological functions and pathways associated with the identified Differentially Expressed Genes (DEGs). The hub genes were investigated using the STRING database and Cytoscape software and were confirmed using RT-qPCR. ResultsWe found that the M2-type sclera macrophage density and expression increased in FD-treated eyes. The results showed that LBH589 inhibited the M2 macrophage polarization, and reduced FDM development. GO and KEGG analyses revealed that the DEGs were predominantly involved in the synthesis and breakdown of the extracellular matrix (ECM), as well as in pathways related to ECM-receptor interaction and the PI3K-Akt signaling pathway. Five hub genes (FN-1, MMP-2, COL1A1, CD44, and IL6) were identified, and RT-qPCR validated the variation in expression levels among these genes. ConclusionM2 macrophage polarization occurred in the sclera in FDM mice. Panobinostat-mediated inhibition of M2 macrophage polarization may decrease FDM progression, as M2 macrophages are crucial in controlling ECM remodeling by HSFs.

Fibroblast Stromal Support Model for Predicting Human Papillomavirus-Associated Cancer Drug Responses.

Currently, there are no specific antiviral therapeutic approaches targeting Human papillomaviruses (HPVs), which cause around 5% of all human cancers. Specific antiviral reagents are particularly needed for HPV-related oropharyngeal cancers (HPV+OPCs) whose incidence is increasing and for which there are no early diagnostic tools available. We and others have demonstrated that the estrogen receptor alpha (ERalpha) is overexpressed in HPV+OPCs, compared to HPV-negative cancers in this region, and that these elevated levels are associated with an improved disease outcome. Utilizing this HPV+ specific overexpression profile, we previously demonstrated that estrogen attenuates the growth and cell viability of HPV+ keratinocytes and HPV+ cancer cells in vitro. Expansion of this work in vivo failed to replicate this sensitization. The role of stromal support from the tumor microenvironment (TME) has previously been tied to both the HPV lifecycle and in vivo therapeutic responses. Our investigations revealed that in vitro co-culture with fibroblasts attenuated HPV+ specific estrogen growth responses. Continuing to monopolize on the HPV+ specific overexpression of ERalpha, our co-culture models then assessed the suitability of the selective estrogen receptor modulators (SERMs), raloxifene and tamoxifen, and showed growth attenuation in a variety of our models to one or both of these drugs in vitro. Utilization of these SERMs in vivo closely resembled the sensitization predicted by our co-culture models. Therefore, the in vitro fibroblast co-culture model better predicts in vivo responses. We propose that utilization of our co-culture in vitro model can accelerate cancer therapeutic drug discovery.

Para rubber seed oil and its fatty acids alleviate photoaging and maintain cell homeostasis.

Para rubber seed oil was indicated for skin dullness and hair loss in regard to its cutaneous beneficial fatty acids. Nonetheless, the oil's potency against photoaging remains unexplored. We proposed that para rubber seed oil could alleviate photoaging. Para rubber seed oil was investigated in cocultures of human HaCaT cells and dermal fibroblasts (HDF). Photoaging protectant efficiency was monitored in terms of IL-6 and IL-8 as well as MMP-1 (collagenase) and MMP-9 (gelatinase) in a comparison with its fatty acid components. Para rubber seed oil standardized in fatty acids was indicated as the promising plant oil for photoaging treatment. Its photoprotection mechanism was demonstrated in the coculture system of keratinocyte and fibroblast cells for the first time. Where the oil and its fatty acid constituents (100 μg/mL) were indicated to be safe and efficiently protect the cocultures against UV damage. The oil significantly (p < 0.001) suppressed UV-induced IL-6, IL-8, MMP-1 and MMP-9 secretions. The revealed photoprotection proficiency was abided by its fatty acids, particularly the unsaturated C18 ones. The oil was indicated on its potential to maintain skin homeostasis and would alleviate senescence ageing in regard to its photoprotection abilities exhibited. Para rubber seed oil is warranted as a new generation of photoaging protectant agent with the profiled safety and efficacy demonstrated in the epidermal coculture system. The findings encourage the development of innovative anti-ageing products containing the oil, which is categorizable as a sustainable specialty material for photoaging treatment.

FFAR-mediated signaling drives migration of pancreatic cancer cells in hypoxic fibroblast co-cultures

The tumor microenvironment (TME) comprises cancer and non-cancerous stromal cells, including fibroblasts. Free fatty acids (FFAs) regulate various biological responses by binding to G protein-coupled FFA receptors (FFARs). In this study, we examined the impact of FFAR1 and FFAR4 on the cell migration of pancreatic cancer PANC-1 cells co-cultured with 3T3 fibroblast cells under hypoxic conditions. PANC-1 cells cultured at 1 % O2 exhibited elevated FFAR1 expression and decreased FFAR4 expression compared to those at 21 % O2. Cell migration of PANC-1 cells was reduced under 1 % O2 conditions. FFAR1 knockdown enhanced PANC-1 cell migration, whereas FFAR4 knockdown inhibited it. Co-culture of PANC-1 cells with 3T3 cells at 1 % O2 significantly increased FFAR4 expression, while FFAR1 expression remained unchanged. To evaluate the effects of FFAR1 and FFAR4 on PANC-1 cell migration in co-culture with 3T3 cells, we conducted a wound healing assay using the Culture-Insert 2 Well. PANC-1 and 3T3 cells were individually seeded into the two wells and incubated at both 21 % and 1 % O2 for 13 h. The cell migration of PANC-1 cells co-cultured with 3T3 cells at 1 % O2 was notably higher compared to 21 % O2. TUG-770 reduced and TUG-891 enhanced the cell migration of PANC-1 cells co-cultured with 3T3 cells under both 21 % and 1 % O2 conditions. These findings suggest that FFAR1 and FFAR4 play important roles in regulating the cell migration of PANC-1 cells co-cultured with 3T3 cells under hypoxic conditions.

Drug-Resistance Biomarkers in Patient-Derived Colorectal Cancer Organoid and Fibroblast Co-Culture System.

Colorectal cancer, the third most commonly occurring tumor worldwide, poses challenges owing to its high mortality rate and persistent drug resistance in metastatic cases. We investigated the tumor microenvironment, emphasizing the role of cancer-associated fibroblasts in the progression and chemoresistance of colorectal cancer. We used an indirect co-culture system comprising colorectal cancer organoids and cancer-associated fibroblasts to simulate the tumor microenvironment. Immunofluorescence staining validated the characteristics of both organoids and fibroblasts, showing high expression of epithelial cell markers (EPCAM), colon cancer markers (CK20), proliferation markers (KI67), and fibroblast markers (VIM, SMA). Transcriptome profiling was conducted after treatment with anticancer drugs, such as 5-fluorouracil and oxaliplatin, to identify chemoresistance-related genes. Changes in gene expression in the co-cultured colorectal cancer organoids following anticancer drug treatment, compared to monocultured organoids, particularly in pathways related to interferon-alpha/beta signaling and major histocompatibility complex class II protein complex assembly, were identified. These two gene groups potentially mediate drug resistance associated with JAK/STAT signaling. The interaction between colorectal cancer organoids and fibroblasts crucially modulates the expression of genes related to drug resistance. These findings suggest that the interaction between colorectal cancer organoids and fibroblasts significantly influences gene expression related to drug resistance, highlighting potential biomarkers and therapeutic targets for overcoming chemoresistance. Enhanced understanding of the interactions between cancer cells and their microenvironment can lead to advancements in personalized medical research..

Role of inflammatory populations and their intermediates in post-ischemic adverse remodeling in rat model of myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía- Fondos FEDER Background Compelling evidence demonstrated the critical role of inflammation in ventricular adverse remodeling after acute myocardial infarction. However, it remains unclear how inflammatory cell population and their intermediates participate in the progress of the disease. Purpose This study aims to elucidate the role of inflammatory population in cardiac remodeling after myocardial infarction in a rat model, focusing on their role in fibrosis. Methods We used a surgical procedure by transient ligation of the left descendant coronary artery, to evoke ischemia-reperfusion (I/R) in rats. We performed FACS analysis to evaluate counts of neutrophils and two monocytes’ subsets: CD43lowHis48high (classical monocytes), and CD43highHis48low (non-classical monocytes), which are the most pro-inflammatory subset in peripheral blood. Subsequently, we performed immunofluorescence to detect pro-inflammatory M1 and M2 macrophages (CD68+ and CD163+) in the rat infarcted myocardium. Moreover, we sorted the two monocyte subsets and performed genes and microRNAs Array. Results FACS analysis of neutrophils and monocytes subsets showed an increased level of these cells in the rat model of myocardial infarction, one week after the post-ischemic event. In parallel, the infiltration of M1 macrophages was increased in the myocardium until 1 week after surgery, indicating a long extravasation of inflammatory cells after the ischemic event. Moreover, co-culture of fibroblasts and cardiomyocytes with intermediates released by inflammatory cells subjected to I/R increased the expression of fibrotic and apoptotic genes. In addition, the analysis of genes’ Array by GSEA indicated that CD43highHis48low monocytes had an enrichment score in pathways associated with angiogenesis, fibrosis and inflammation, while CD43lowHis48high population exhibited an enrichment in cytokines and inflammation, apoptosis and fibrosis. Finally, we found significant overexpression of microRNAs, for instance, miR-27a-3p, miR-29b-3p, miR-30e-5p and miR-194-5p in CD43highHis48low 24h after the I/R procedure. Those miRNAs are predicted to modulate pro-fibrotic genes. Conclusions The level of inflammatory cell populations is increased in I/R rat model. Moreover, these cells infiltrate in the myocardium likely to change the transcriptome of resident cells towards a pro-apoptotic and pro-fibrotic phenotype.

Potential role of IGF-1R in the interaction between orbital fibroblasts and B lymphocytes: an implication for B lymphocyte depletion in the active inflammatory phase of thyroid-associated ophthalmopathy

BackgroundThyroid eye disease (TED) is an inflammatory process involving lymphocyte-mediated immune response and orbital tissue damage. The anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies produced by B lymphocytes are involved in the activation of orbital fibroblasts and the inflammatory process of orbital tissue damage in TED. The purpose of this study was to explore the role of IGF-1R in the mechanistic connection between orbital fibroblasts and B lymphocytes in TED.MethodsOrbital fibroblasts sampled from orbital connective tissues and peripheral B lymphocytes isolated from peripheral blood, which were obtained from 15 patients with TED and 15 control patients, were co-cultured at a ratio of 1:20. The level of IGF-1R expression in orbital fibroblasts was evaluated by flow cytometry and confocal microscopy. Transient B lymphocyte depletion was induced with anti-CD20 monoclonal antibody rituximab, while the IGF-1R pathway was blocked by the IGF-1R binding protein. The expression levels of interleukin-6 (IL-6) and regulated upon activation, normal T cell expressed and secreted (RANTES) in the co-culture model were quantified via ELISA.ResultsIGF-1R expression was significantly elevated in TED orbital fibroblasts compared to that of controls. A 24-h co-culture of orbital fibroblasts with peripheral B lymphocytes induced elevated expression levels of IL-6 and RANTES in each group (TED patients and controls), with the highest levels occurring in TED patients (T + T group). Rituximab and IGF-1R binding protein significantly inhibited increased levels of IL-6 and RANTES in the co-culture model of TED patients.ConclusionsIGF-1R may mediate interaction between orbital fibroblasts and peripheral B lymphocytes; thus, blocking IGF-1R may reduce the local inflammatory response in TED. Rituximab-mediated B lymphocyte depletion played a role in inhibiting inflammatory responses in this in vitro co-culture model, providing a theoretical basis for the clinical application of anti-CD20 monoclonal antibodies in TED.