3D Extracellular Matrix Regulates the Activity of T Cells and Cancer Associated Fibroblasts in Breast Cancer

BackgroundBreast cancer progression has been gradually recognized as a bidirectional interaction between cancer cells and tumor microenvironment including stroma cells, immune cells, and the dynamically altered ECM. However, there still lacks direct experimental evidences about how ECM properties modulate the activities of stroma and immune cells.MethodThe transcriptomic data and corresponding clinical information of breast cancer pawere obtained from TCGA. Patients were divided into ECM-high, ECM-median and ECM-low groups based on ssGSEA scores of C-ECM genes. The prognostic value of ECM was confirmed by univariate/multivariate Cox regression and survival analyses. GO and KEGG analyses were performed between ECM-high and -low groups. Then associations between ECM characteristics and clinical stages were verified by Masson’s trichrome and Sirius red/Fast Green staining of clinical breast cancer tissues. To evaluate the effects of ECM on CAF induction and T cell activation, the MRC-5, NIH/3T-3, primary T cells and Jurkat T cells were encapsulated in 3D collagen with different densities and organizations, and the expression levels of CAF biomarkers and secretion levels of IL-2 were assessed.ResultsECM scores showed broad variation across paracancerous and cancer samples as well as breast cancer molecular subtypes, and patients with different ECM groups showed distinct prognosis. Immunological activity and ECM associated biology processes were identified by GO and KEGG analyses across ECM-high and -low groups. According to MCP-counter algorithm, the infiltration of T cells was significantly lower in the ECM-high group, while CAF abundance was significantly higher. It is furtherly confirmed by clinical samples that collagen density and organization were associate with breast cancer progression. Finally, in vitro 3D-cultured fibroblasts and T cells validated that the density and organization of collagen showed significant effects on CAF induction and T cell activation.ConclusionOur study revealed a new mechanism of T cell immunosuppression and CAF induction, which could be of central importance for the breast cancer invasion and may constitute novel therapeutic targets to improve breast cancer outcomes.

Auxetic Structures for Tissue Engineering Scaffolds and Biomedical Devices

An auxetic structure utilizing a negative Poisson’s ratio, which can expand transversally when axially expanded under tensional force, has not yet been studied in the tissue engineering and biomedical area. However, the recent advent of new technologies, such as additive manufacturing or 3D printing, has showed prospective results aimed at producing three-dimensional structures. Auxetic structures are fabricated by additive manufacturing, soft lithography, machining technology, compressed foaming, and textile fabrication using various biomaterials, including poly(ethylene glycol diacrylate), polyurethane, poly(lactic-glycolic acid), chitosan, hydroxyapatite, and using a hard material such as a silicon wafer. After fabricating the scaffold with an auxetic effect, researchers have cultured fibroblasts, osteoblasts, chondrocytes, myoblasts, and various stem cells, including mesenchymal stem cells, bone marrow stem cells, and embryonic stem cells. Additionally, they have shown new possibilities as scaffolds through tissue engineering by cell proliferation, migration, alignment, differentiation, and target tissue regeneration. In addition, auxetic structures and their unique deformation characteristics have been explored in several biomedical devices, including implants, stents, and surgical screws. Although still in the early stages, the auxetic structure, which can create mechanical properties tailored to natural tissue by changing the internal architecture of the structure, is expected to show an improved tissue reconstruction ability. In addition, continuous research at the cellular level using the auxetic micro and nano-environment could provide a breakthrough for tissue reconstruction.

Atractylenolide-I Sensitizes Triple-Negative Breast Cancer Cells to Paclitaxel by Blocking CTGF Expression and Fibroblast Activation

This investigation was conducted to elucidate whether atractylenolide-I (ATL-1), which is the main component of Atractylodes macrocephala Koidz, can sensitize triple-negative breast cancer (TNBC) cells to paclitaxel and investigate the possible mechanism involved. We discovered that ATL-1 could inhibit tumor cell migration and increase the sensitivity of tumor cells to paclitaxel. ATL-1 downregulated the expression and secretion of CTGF in TNBC cells. Apart from inhibiting TNBC cell migration via CTGF, ATL-1 downregulated the expression of CTGF in fibroblasts and decreased the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblasts (CAFs), which in turn increased the sensitivity of TNBC cells to paclitaxel. In a mouse model, we found that ATL-1 treatments could enhance the chemotherapeutic effect of paclitaxel on tumors and reduce tumor metastasis to the lungs and liver. Primary cultured fibroblasts derived from inoculated tumors in mice treated with ATL-1 combined with paclitaxel expressed relatively low levels of CAF markers. Collectively, our data indicate that ATL-1 can sensitize TNBC cells to paclitaxel by blocking CTGF expression and fibroblast activation and could be helpful in future research to determine the value of ATL-1 in the clinical setting.

Abstract MP258: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

Background: A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results: The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage. Conclusions: Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle–mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie’s disease penile tunica albuginea

Background Peyronie’s disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)–mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV–mimetic NVs (PC–NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC–NVs in primary fibroblasts derived from human PD plaque. Methods Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)–mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. Results A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. Conclusion The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

Regeneration of Collagen Fibrils at the Papillary Dermis by Reconstructing Basement Membrane at the Dermal-Epidermal Junction

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibers in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

The J domain of sacsin disrupts intermediate filament assembly

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.

Monoacylglycerol disrupts Golgi structure and perilipin 2 association with lipid droplets

The two major products of intestinal triacylglycerol digestion and lipoprotein lipolysis are monoacylglycerols (MAG) and fatty acids. In the gut, these products are taken up by enterocytes and packaged into perilipin-coated cytosolic lipid droplets and then secreted as chylomicrons. We observed that fat feeding or intragastric administration of triacylglycerol oil caused the enterocyte Golgi to fragment into submicron puncta dispersed throughout the cytosol. Further, this apparent Golgi dispersion was also observed in cultured fibroblasts after treatment with fat (cream) and pancreatic lipase, but not when treated with deactivated lipase. We therefore hypothesized that a hydrolytic fat product, specifically monoacylglycerols, fatty acids or a combination of these molecules can trigger Golgi fragmentation. Disruption of coatomer function is known to cause Golgi to fuse with the ER, and blocks perilipin 2 delivery to lipid droplets. Thus, we assessed the effects of MAG on coatomer distribution, Golgi structure and perilipin 2 localization. We found that MAG, but not fatty acids, dispersed coatomer from the Golgi, fragmented the Golgi and caused perilipin 2 to accumulate on cellular membranes. Thus, our findings suggest that monoacylglycerol production during digestion disperses the Golgi, possibly by altering coatomer function, which may regulate metabolite transport between the ER and Golgi.