Co-culture Of Fibroblasts Research Articles

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

Electrochemical measurement of respiratory activity for evaluation of fibroblast spheroids containing endothelial cell networks

Vascularized three-dimensional (3D) cultures such as spheroids overcome the problems of insufficient nutrient and oxygen supply and poor cell viability observed with non-vascularized spheroids. However, the capacity of drug penetration may differ with the presence of vascular structures, and needs to be evaluated. Co-culture of endothelial cells (ECs) with other cell types such as fibroblasts is one of the promising methods to engineer vascularized 3D tissue models. At a first step for fabricating vascularized tissues, it is important to prepare EC networks inside the tissue models. Here, we present the first attempt to apply electrochemical measurements of respiratory activity to the fibroblast spheroids containing EC networks, aiming to develop an evaluation technique for vascularized tissue models. The spheroids were exposed to doxorubicin (DOX), an inhibitor of DNA and RNA synthesis, to investigate their drug sensitivity that may differ with the presence of ECs, though rarely studied with regard to respiratory activity. Respiratory activity was measured using two electrochemical devices, a scanning electrochemical microscope (SECM) and a large scale integration (LSI)-based amperometric device. The fibroblast spheroids containing EC networks were constructed with a simple method involving co-culture of normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) at different concentrations. Fluorescence images showed that HUVECs formed partially networked structure in the fibroblast spheroids. Respiratory activity of the spheroids as measured by SECM varied according to the proportion of HUVECs. In particular, the respiratory activity decreased more in spheroids with a larger proportion of HUVECs after DOX treatment, indicating that drug sensitivity of the spheroids became higher upon incorporation of EC networks. The results with the LSI device also corresponded to those with SECM. Overall, the electrochemical measurements provide meaningful information regarding the EC networks and play an important role in the analysis of 3D cell structures.

T-Cell Proapoptotic and Antifibrotic Activity Against Autologous Skin Fibroblasts in vitro Is Associated With IL-17A Axis Upregulation in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) T cells can induce apoptosis of autologous skin fibroblasts in vitro. Th17 cells have been reported to increase in SSc patients, and interleukin-17A (IL-17A) has a profibrotic function. We used a system based on T-cell-autologous fibroblast co-cultures to further investigate a possible role of IL-17A in SSc.Methods: T cells from diffuse SSc patients were co-cultured with autologous skin fibroblasts. IL17A mRNA was assessed by real-time PCR in co-cultured and control T cells, while IL17RA, CXCL1, CCL2, CCL3, COL1A1, COL3A1, CTGF, TGFBR2, and SMAD3 mRNAs were assessed in co-cultured and control fibroblasts. In subset experiments, co-cultures and control cells were treated with either IL-17A or IL-17A plus anti-IL17 receptor monoclonal antibody (α-IL-17RA mAb). Chemokine and procollagen type I (PCI) production was further investigated at the protein level in cell culture supernatants by multiple suspension immunoassay and sandwich ELISA, respectively. Co-cultured and control fibroblasts were also stained with Annexin V and analyzed by flow cytometry.Results: T cell–fibroblast co-cultures overexpressed IL17A and IL17RA. Furthermore, co-cultured fibroblasts upregulated IL-17A targets CXCL1, CCL2, and CCL3, while COL1A1, COL3A1, CTGF, and two key effectors of the TGF-β signaling, TGFBR2 and SMAD3, were found downregulated. Consistently, chemokine concentrations were increased in co-culture supernatants, while PCI levels were reduced, especially after stimulation with ectopic IL-17A. Finally, simultaneous α-IL-17RA mAb treatment restored PCI levels and reduced fibroblast apoptosis in IL-17A-stimulated co-cultures.Conclusion: These data suggest that IL-17A upregulation might play a role in modulating T cell-mediated antifibrotic and proapoptotic effects in co-cultured autologous skin fibroblasts.

Development of endomyocardial fibrosis model using a cell patterning technique: In vitro interaction of cell coculture of 3T3 fibroblasts and RL-14 cardiomyocytes.

Cardiac functions can be altered by changes in the microstructure of the heart, i.e., remodeling of the cardiac tissue, which may activate pathologies such as hypertrophy, dilation, or cardiac fibrosis. Cardiac fibrosis can develop due to an excessive deposition of extracellular matrix proteins, which are products of the activation of fibroblasts. In this context, the anatomical–histological change may interfere with the functioning of the cardiac tissue, which requires specialized cells for its operation. The purpose of the present study was to determine the cellular interactions and morphological changes in cocultures of 3T3 fibroblasts and RL-14 cardiomyocytes via the generation of a platform an in vitro model. For this purpose, a platform emulating the biological characteristics of endomyocardial fibrosis was generated using a cell patterning technique to study morphological cellular changes in compact and irregular patterns of fibrosis. It was found that cellular patterns emulating the geometrical distributions of endomyocardial fibrosis generated morphological changes after interaction of the RL-14 cardiomyocytes with the 3T3 fibroblasts. Through this study, it was possible to evaluate biological characteristics such as cell proliferation, adhesion, and spatial distribution, which are directly related to the type of emulated endomyocardial fibrosis. This research concluded that fibroblasts inhibited the proliferation of cardiomyocytes via their interaction with specific microarchitectures. This behavior is consistent with the histopathological distribution of cardiac fibrosis; therefore, the platform developed in this research could be useful for the in vitro assessment of cellular microdomains. This would allow for the experimental determination of interactions with drugs, substrates, or biomaterials within the engineering of cardiac tissues.

Senescent Cell-Secreted Netrin-1 Modulates Ageing-Related Disorders by Recruiting Sympathetic Fibres

Cellular senescence is implicated in several lines of ageing-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibres (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SF densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cell-induced recruitment of SFs contributes to an elevation in SFs density. Clearing senescent cells by senolytics (Dasatinib(D)+ Quercetin (Q)) improved osteoarthritis (OA), thereby reducing the SF infiltration in the superficial zone of articular cartilage. Mechanistically, senescent cells recruited SFs by secreting the axon guidance cue netrin-1. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective β receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective β receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/-mice) by reducing hepatic sympathetic nerve activity (SNA). We conclude that senescent cell-secreted netrin-1 mediates SFs outgrowth and leads to elevated SNA, which contributes to ageing-related disorders. This suggests that clearing senescent cells is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced ageing-related pathologies. Funding Statement: Our work was supported by the National Natural Science Foundation of China (grant number 81771505 and 81773079), and the Fundamental Research Funds for the Higher Education of Tianjin (Grant Number 2018KJ079). Declaration of Interests: The authors stated that no potential conflicts of interest were disclosed. Ethics Approval Statement: All experiments involving the handling of mice were approved by the animal ethics committee of Peking University Health Science Centre. The human tissue samples were obtained with informed consent, and the study was approved by the Clinical Research Ethics Committee.

Human Induced Pluripotent Stem Cell-Derived Non-Cardiomyocytes Modulate Cardiac Electrophysiological Maturation Through Connexin 43-Mediated Cell-Cell Interactions.

The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%–90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

Regulation of connexin 43 by interleukin 1β in adult rat cardiac fibroblasts and effects in an adult rat cardiac myocyte: fibroblast co-culture model

Connexin 43 expression (Cx43) is increased in cardiac fibroblasts (CFs) following myocardial infarction. Here, potential mediators responsible for increasing Cx43 expression and effects of differential CF phenotype on cardiac myocyte (CM) function were investigated. Stimulating adult rat CFs with proinflammatory mediators revealed that interleukin 1β (IL-1β) significantly enhanced Cx43 levels through the IL-1β pathway. Additionally, IL-1β reduced mRNA levels of the myofibroblast (MF) markers: (i) connective tissue growth factor (CTGF) and (ii) α smooth muscle actin (αSMA), compared to control CFs. A co-culture adult rat CM:CF model was utilised to examine cell-to-cell interactions. Transfer of calcein from CMs to underlying CFs suggested functional gap junction formation. Functional analysis revealed contraction duration (CD) of CMs was shortened in co-culture with CFs, while treatment of CFs with IL-1β reduced this mechanical effect of co-culture. No effect on action potential rise time or duration of CMs cultured with control or IL-1β-treated CFs was observed. These data demonstrate that stimulating CFs with IL-1β increases Cx43 and reduces MF marker expression, suggesting altered cell phenotype. These changes may underlie the reduced mechanical effects of IL-1β treated CFs on CD of co-cultured CMs and therefore have an implication for our understanding of heterocellular interactions in cardiac disease.

Microfluidic vascular-bed devices for vascularized 3D tissue engineering: tissue engineering on a chip.

In this report, we describe a microfluidic vascular-bed (micro-VB) device providing a platform for 3D tissue engineering with vascular network formation. The micro-VB device allows functional connections between endothelial capillaries of heterogeneous sections (5-100μm in diameter) and artificial plastic tubes or reservoirs (1-10mm in diameter). Moreover, the micro-VB device can be installed in a standard 100mm-diameter Petri dish. Endothelial networks in 3D engineered tissues were obtained by cellular self-assembly on the device, after co-culturing of human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) in fibrin gel. Endothelial capillary connection between vascularized tissues and microfluidic channels, mimicking arteries and veins, was confirmed by perfusion of fluorescent microspheres. The micro-VB devices were compatible with the use of commercially available culture dishes and did not require the involvement of additional equipment. Thus, these micro-VB devices are expected to substantially improve the routine application of 3D tissue engineering to regenerative medicine.

Abstract C114: Cancer cell induced activation of associated fibroblasts is PDGFR-mediated

Abstract Background: Recent studies have shown that tumorigenesis is not a cell autonomous process and is actively shaped by the tumor microenvironment (TME). The effect of cancer associated fibroblasts (CAFs), a critical component of the TME, on the growth, progression and drug sensitivity of the tumor itself is well studied. However, there is limited understanding about key signaling pathways activated in CAFs by cancer cells (CCs). HYPOTHESIS: CCs and CAFs talk to each other in a bi-directional manner wherein CCs alter signaling in CAFs which in turn feeds back and affects oncogenic signaling networks and behavior of CCs. In this study, we aim to better understand this bi-directional signaling at a systems level. Methods: For this, we first characterized differential tyrosine phosphoproteomes of EGFR mutant PC9 lung cancer cells and WI-38 lung fibroblasts in co-culture using mass spectrometry-based phosphoproteomics (pY). Cell type specific proteome labeling by amino acid precursors (CTAP) was used to distinguish two proteomes in co-culture. To follow up on data from the CTAP pY experiment, fibroblasts were treated with conditioned medium (CM) collected from PC9 cells and analyzed for changes in effectors of tyrosine kinase (TK) signaling, such as phosphorylation of ERK and/or AKT. Treatment with a panel of TK inhibitors coupled with chemical proteomics and siRNA approaches were used to identify the upstream kinase responsible for these signaling changes. Migration and proliferation assays were set up in the Incucyte system to study phenotypic consequences of these cancer cell-induced signaling alterations in fibroblasts. Results: The CTAP pY data shows differential regulation of receptor tyrosine kinase (RTK) signaling by co-culture in both CCs and CAFs. We focused our attention first on using a CM system to explore which potential ligands can mediate some of the CC-induced changes in kinase signaling detected in fibroblasts from the pY experiment. We found that PC9 CM induces a transient but robust increase in AKT and ERK phosphorylation in both normal as well as patient-derived lung fibroblasts. This activation was blocked by TKIs Sunitinib, Cabozantinib and Foretinib. Chemical proteomics and subsequent siRNA experiments show that the cancer cell CM-mediated AKT activation in fibroblasts is driven by PDGFR. CM-induced PDGFR activation results in activation of fibroblasts as evidenced by its increased migration/invasion capacity that can be blocked by the PDGFR inhibitor Sunitinib. We have also developed a PDGFRB/pY100 proximity ligation assay (PLA) that would allow us to annotate active PDGFR signaling complexes in situ. Conclusion: Overall, our data shows that CCs transiently trigger PDGFR-mediated TK signaling in fibroblasts thereby activating them. We next aim to understand the effect of this activation on oncogenic signaling and behavior of CCs. A better understanding of this bi-directional crosstalk may allow identification of targeted therapies against activation of CAFs that support tumor growth. Citation Format: Anurima Majumder, Martial Boutchueng-Djidjou, Jae-Young Kim, Natalia Sumi, Uwe Rix, Eric B Haura. Cancer cell induced activation of associated fibroblasts is PDGFR-mediated [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C114. doi:10.1158/1535-7163.TARG-19-C114

Verteporfin- and sodium porfimer-mediated photodynamic therapy enhances pancreatic cancer cell death without activating stromal cells in the microenvironment.

.The goal of our study was to determine the susceptibility of different pancreatic cell lines to clinically applicable photodynamic therapy (PDT). The efficacy of PDT of two different commercially available photosensitizers, verteporfin and sodium porfimer, was compared using a panel of four different pancreatic cancer cell lines, PANC-1, BxPC-3, CAPAN-2, and MIA PaCa-2, and an immortalized non-neoplastic pancreatic ductal epithelium cell line, HPNE. The minimum effective concentrations and dose-dependent curves of verteporfin and sodium porfimer on PANC-1 were determined. Since pancreatic cancer is known to have significant stromal components, the effect of PDT on stromal cells was also assessed. To mimic tumor–stroma interaction, a co-culture of primary human fibroblasts or human pancreatic stellate cell (HPSCs) line with PANC-1 was used to test verteporfin-PDT-mediated cell death of PANC-1. Two cytokines (TNF- and IL-) were used for stimulation of primary fibroblasts (derived from human esophageal biopsies) or HPSCs. The increased expression of smooth muscle actin (-SMA) confirmed the activation of fibroblasts or HPSC upon treatment with TNF- and IL-. Cell death assays showed that both sodium porfimer- and verteporfin-mediated PDT-induced cell death in a dose-dependent manner. However, verteporfin-PDT treatment had a greater efficiency with lower concentration than sodium porfimer-PDT in the PANC-1 incubated with stimulated fibroblasts or HPSC. Moreover, activation of stromal cells did not affect the treatment of the pancreatic cancer cell lines, suggesting that the effects of PDT are independent of the inflammatory microenvironment found in this two-dimensional culture model of cancers.

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 μm microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

CD147 self-regulates matrix metalloproteinase-2 release in gingival fibroblasts after coculturing with U937 monocytic cells.

Cluster of differentiation 147 (CD147) is a multifunctional glycoprotein that functions as an inducer of matrix metalloproteinase (MMP) expression in fibroblasts. Synergistically enhanced MMP-2 expression was recently observed in the coculture of human gingival fibroblasts (HGFs) and U937 human monocytic cells; however, the responsible mechanisms have not yet been fully established. The aim of this study was to evaluate the release of soluble CD147 in HGFs after coculturing with U937 cells and its functional effect on the enhancement of MMP-2 expression in HGFs. Enzyme-linked immunosorbent assay was used to determine the amount of CD147 protein in media, whereas real-time polymerase chain reaction was performed to evaluate the mRNA levels of CD147 and MMP-2 in HGFs and U937 cells. The enzyme activities of MMP-2 released from cells were examined by zymography. Transwell coculturing and conditioned media treatments were selected to rule out the effect of direct contact of HGFs and U937 cells. The protein and mRNA expression of CD147 in HGFs were enhanced after transwell coculturing with U937 cells and exposure to U937-conditioned medium. MMP-2 enzyme activities in HGFs were also significantly increased by the coculturing methods. Administration of exogenous CD147 enhanced MMP-2 expression in HGFs, whereas treatment with cyclosporine-A, which inhibited CD147 expression, reduced U937-enhanced MMP-2 expression in HGFs. CD147 can interact with fibroblasts to stimulate the expression of MMPs associated with periodontal extracellular matrix degradation. This study has demonstrated that CD147 released from fibroblasts might play a role in monocyte-enhanced MMP-2 expression in HGFs.

CD73 complexes with emmprin to regulate MMP-2 production from co-cultured sarcoma cells and fibroblasts

BackgroundInteraction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts.MethodsCD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays.ResultsStimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells.ConclusionThis study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Cardiac regeneration using human‐induced pluripotent stem cell‐derived biomaterial‐free 3D‐bioprinted cardiac patch in vivo

One of the leading causes of death worldwide is heart failure. Despite advances in the treatment and prevention of heart failure, the number of affected patients continues to increase. We have recently developed 3D-bioprinted biomaterial-free cardiac tissue that has the potential to improve cardiac function. This study aims to evaluate the in vivo regenerative potential of these 3D-bioprinted cardiac patches. The cardiac patches were generated using 3D-bioprinting technology in conjunction with cellular spheroids created from a coculture of human-induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and endothelial cells. Once printed and cultured, the cardiac patches were implanted into a rat myocardial infarction model (n = 6). A control group (n = 6) without the implantation of cardiac tissue patches was used for comparison. The potential for regeneration was measured 4 weeks after the surgery with histology and echocardiography. 4 weeks after surgery, the survival rates were 100% and 83% in the experimental and the control group, respectively. In the cardiac patch group, the average vessel counts within the infarcted area were higher than those within the control group. The scar area in the cardiac patch group was significantly smaller than that in the control group. (Figure S1) Echocardiography showed a trend of improvement of cardiac function for the experimental group, and this trend correlated with increased patch production of extracellular vesicles. 3D-bioprinted cardiac patches have the potential to improve the regeneration of cardiac tissue and promote angiogenesis in the infarcted tissues and reduce the scar tissue formation.

Paracrine recruitment and activation of fibroblasts by c‐Myc expressing breast epithelial cells through the IGFs/IGF‐1R axis

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr397 , as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion.

A Layer-by-Layer Single-Cell Coating Technique To Produce Injectable Beating Mini Heart Tissues via Microfluidics.

Human induced pluripotent stem cells (hiPSCs) are used as an alternative for human embryonic stem cells. Cardiomyocytes derived from hiPSCs are employed in cardiac tissue regeneration constructs due to the heart's low regeneration capacity after infarction. A coculture of hiPSC-CM and primary dermal fibroblasts is encapsulated in injectable poly(ethylene glycol)-based microgels via microfluidics to enhance the efficiency of regenerative cell transplantations. The microgels are prepared via Michael-type addition of multi-arm PEG-based molecules with an enzymatically degradable peptide as a cross-linker and modified with a cell-adhesive peptide. Cell-cell interactions and, consequently, cell viability are improved by a thin extracellular matrix (ECM) coating formed on the cell surfaces via layer-by-layer (LbL) deposition. The beating strength of encapsulated cardiomyocytes (∼60 BPM) increases by 2-fold compared to noncoated cells. The combination of microfluidics with the LbL technique offers a new technology to fabricate functional cardiac mini tissues for cell transplantation therapies.

Epithelial-fibroblast cross talk aggravates the impact of the nephrotoxin ochratoxin A

BackgroundChronic nephropathies result from different pathogenic agents, including nutritional factors triggering vicious pathophysiological cycles. Ochratoxin A (OTA) is a globally occurring nephrotoxic mycotoxin detectable in a variety of foodstuff and suspected to cause tubulointerstitial damage. The underlying mechanisms are not sufficiently understood, compromising risk assessment. Because crosstalk of proximal tubule cells with fibroblasts is crucial for tubulointerstitial damage, we investigated the effects of OTA in co-culture of these two cell types. MethodsRat renal proximal tubule cells (NRK-52E) and renal fibroblasts (NRK-49F) were exposed to nanomolar OTA concentrations under mono- and/or co-culture conditions for up to 48 h. We determined the impact on inflammation-, EMT- and fibrosis-associated proteins as well as microRNAs by western blot or qPCR, respectively. Alterations in cell morphology were quantitatively assessed. The roles of miRs, COX-2 and ERK1/2 in OTA-induced effects were investigated by specific inhibition. FindingsOnly under co-culture condition, OTA caused an increase of vimentin, fibronectin and miR-21 and a decrease of collagen III, E-cadherin, COX-2 and WISP1 mRNA abundance in NRK-52E cells. In NRK-49F cells, OTA induced an increase of N-cadherin, COX-2, WISP1 in co-culture only. The OTA-induced increase of fibronectin in NRK-52E cells was prevented by simultaneous inhibition of miR-21 and -200a, COX-2 or ERK1/2. The OTA-induced increase of COX-2 in NRK-49F cells was prevented by inhibition of miR-21 and -200a or ERK1/2. InterpretationOur results show that the complete nephropathic potential of nanomolar OTA, leading to EMT, is unveiled when cellular crosstalk is possible. In monoculture, the nephropathic potential is underestimated. Research in contextChronic nephropathies are a severe health burden and the result of different pathogenic mechanisms, including nutritional factors that trigger vicious pathophysiological cycles. Ochratoxin A (OTA) is a ubiquitous, globally occurring nephrotoxic mycotoxin detectable in a variety of foodstuff and suspected to cause tubulointerstitial damage. Because underlying pathomechanisms are unclear, risk assessment is problematic. Crosstalk of proximal tubule cells (the main target of OTA) with fibroblasts is crucial for the development of tubulointerstitial damage. We show that during co-culture of proximal tubule cells and fibroblasts, OTA-induced effects (e.g. epithelial-mesenchymal transition (EMT)) change significantly as compared to monoculture. Our results show that the complete nephropathic potential of OTA is unveiled when cellular crosstalk is possible. In monoculture, the nephropathic potential of OTA is underestimated.

A Visible Light-Cross-Linkable, Fibrin-Gelatin-Based Bioprinted Construct with Human Cardiomyocytes and Fibroblasts.

In this study, fibrin was added to a photo-polymerizable gelatin-based bioink mixture to fabricate cardiac cell-laden constructs seeded with human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) or CM cell lines with cardiac fibroblasts (CF). The extensive use of platelet-rich fibrin, its capacity to offer patient specificity, and the similarity in composition to surgical glue prompted us to include fibrin in the existing bioink composition. The cell-laden bioprinted constructs were cross-linked to retain a herringbone pattern via a two-step procedure including the visible light cross-linking of furfuryl-gelatin followed by the chemical cross-linking of fibrinogen via thrombin and calcium chloride. The printed constructs revealed an extremely porous, networked structure that afforded long-term in vitro stability. Cardiomyocytes printed within the sheet structure showed excellent viability, proliferation, and expression of the troponin I cardiac marker. We extended the utility of this fibrin-gelatin bioink toward coculturing and coupling of CM and cardiac fibroblasts (CF), the interaction of which is extremely important for maintenance of normal physiology of the cardiac wall in vivo. This enhanced "cardiac construct" can be used for drug cytotoxicity screening or unraveling triggers for heart diseases in vitro.

Olfactory Ensheathing Cells Grafted Into the Retina of RCS Rats Suppress Inflammation by Down-Regulating the JAK/STAT Pathway

The inflammatory microenvironment in the retina plays a vital role in the pathogenesis and progression of retinitis pigmentosa (RP). Microglial inflammatory cytokines production leads to gliosis and apoptosis of retinal neurons, and ultimately, visual loss. Cell-based therapies using grafted olfactory ensheathing cells (OECs) have demonstrated modulation of degenerative microenvironments in the central nervous system (CNS), in a number of animal models. However, mechanisms by which grafted OECs can reduce degeneration in the retina are not well understood. In the present study, we set up an in vitro OEC/BV2 microglia co-culture system, and an in vivo royal college of surgeons (RCS) rat model, used cell transplantation, immunohistochemistry, RT-PCR, western blot to explore the mechanisms by which OECs affect expression of pro- or anti-inflammatory cytokines and polarization of M(IL-6) and M(Arg1) type microglial activation in the retina. We found that compared with the LPS (Lipopolysaccharide) and olfactory nerve fibroblast (ONF), the OEC and BV2 co-culture group modulate microglial cytokines releasing toward the anti-inflammation, and away from the pro-inflammation, which was followed by higher IL-4 and IL-10 and lower TNF-a and IL-6 in their expression levels. In vivo, the transplantation group significantly reduced activated resident microglia/infiltrated macrophage, and expression of pro-inflammatory cytokines in RCS rats retina, increased anti-inflammatory cytokines in transplantation area. Additionally, we found that OECs expressed SOCS3 and down-regulated the JAK2/STAT3 (Janus Kinase 2/Signal Transducer and Activator of Transcription 3) pathway. Thirdly, OEC transplantation reduced Caspase-3 expression, protected inner retinal neurons and photoreceptors and therefore, delayed the visual function degeneration. In conclusion, our data suggest that OECs delay retinal degeneration in RP, at least in part through immunomodulation of microglia via the JAK/STAT pathway.

Evaluation of Tumor Cell-Tumor Microenvironment Component Interactions as Potential Predictors of Patient Response to Napabucasin.

Napabucasin is an NAD(P)H:quinone oxidoreductase 1 (NQO1)-bioactivatable small molecule hypothesized to affect multiple oncogenic pathways. In a prespecified, retrospective analysis of the napabucasin phase III CO.23 study, overall survival was longer for napabucasin versus placebo in patients expressing phosphorylated STAT3 (pSTAT3) in tumor cells and cells of the tumor microenvironment (TME). We hypothesized that a connection may exist between NQO1 expression in cancer cells and pSTAT3 in tumor cells and the TME. In 3D spheroid cocultures of cancer cells and cancer-associated fibroblasts, the antitumor activity of napabucasin was NQO1 dependent. The levels of cytokines such as IL6, CXCL10, and GM-CSF were higher in NQO1-positive versus NQO1-deleted cocultures. These differentially secreted cytokines promoted STAT3 phosphorylation in tumor cells and the TME. NQO1-expressing, napabucasin-sensitive tumor cells can modify tumor cells and the TME to promote STAT3 phosphorylation, suggesting that pSTAT3 may be used to identify a subpopulation of patients who would likely respond to napabucasin. IMPLICATIONS: pSTAT3 is a potential biomarker for patient response to the anticancer drug napabucasin.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/7/1429/F1.large.jpg.