Single-cell RNA reveals a tumorigenic microenvironment in the interface zone of human breast tumors.

BACKGROUND: The interface zone, area around invasive carcinoma, can be thought of as the actual tissue of the tumor microenvironment with precedent alterations for tumor invasion. However, the heterogeneity and characteristics of the microenvironment in the interface area have not yet been thoroughly explored. METHODS: For in vitro studies, single-cell RNA sequencing (scRNA-seq) was used to characterize the cells from the tumor zone, the normal zone and the interface zone with 5-mm-wide belts between the tumor invasion front and the normal zone. Through scRNA-seq data analysis, we compared the cell types and their transcriptional characteristics in the different zones. Pseudotime, cell-cell communication and pathway analysis were performed to characterize the zone-specific microenvironment. Cell proliferation, wound healing and clone formation experiments explored the function of differentially expressed gene BMPR1B, which were confirmed by tumor models in vivo. RESULTS: After screening, 88,548 high-quality cells were obtained and identified. Regulatory T cells, M2 macrophages, angiogenesis-related mast cells, stem cells with weak DNA repair ability, endothelial cells with angiogenic activity, fibroblasts with collagen synthesis and epithelial cells with proliferative activity form a unique tumorigenic microenvironment in the interface zone. Cell-cell communication analysis revealed that there are special ligand-receptor pairs between different cell types in the interface zone, which protects endothelial cell apoptosis and promotes epithelial cell proliferation and migration, compared to the normal zone. Compared with the normal zone, the highly expressed BMPR1B gene promotes the tumorigenic ability of cancer cells in the interface zone. CONCLUSIONS: Our work identified a unique tumorigenic microenvironment of the interface zone and allowed for deeper insights into the tumor microenvironment of breast cancer that will serve as a helpful resource for advancing breast cancer diagnosis and therapy.

Omega-3 polyunsaturated fatty acids ameliorated inflammatory response of mammary epithelial cells and mammary gland induced by lipopolysaccharide.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), essential fatty acids for humans and animals, have been reported to play a beneficial role in a variety of inflammatory diseases. In this study, we investigated the inhibitory effects and potential molecular mechanisms of n-3 PUFAs on the inflammatory response in lipopolysaccharide (LPS)-stimulated mammary alveolar cell line (MAC-T). Results showed that n-3 PUFAs could abate LPS-induced secretions of tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß in MAC-T cells through the nuclear transcription factor kappa B (NF-κB) signal pathway. Meanwhile, n-3 PUFA intervention attenuated histopathologic changes of mammary glands, the white blood cell number decrease, and the alkaline phosphatase level decrease in the serum of mice challenged by LPS. Furthermore, n-3 PUFA intervention improved the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS in mice. Collectively, both in vitro and in vivo experiments revealed that n-3 PUFAs have a positive effect on LPS-induced inflammatory response, which was possibly mediated by the NF-κB signaling pathway and the intestinal microbiota.

The lncRNA FAL1 protects against hypoxia-reoxygenation- induced brain endothelial damages through regulating PAK1.

Dysregulation of cerebral microvascular endothelial cells plays an important role in the pathogenesis of stroke. However, the underlying mechanisms still need to be elucidated. In the current study, we found that the long non-coding RNA (lncRNA) FAL1 was significantly reduced in response to oxygen-glucose deprivation and reoxygenation (OGD/R) stimulation in human primary brain microvascular endothelial cells (HBMVECs). Interestingly, overexpression of FAL1 ameliorated OGD/R-induced oxidative stress by reducing the production of reactive oxygen species (ROS) and increasing the level of reduced glutathione (GSH). Also, overexpression of FAL1 suppressed OGD/R-induced secretions of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and high mobility group box-1 (HMGB-1). We then found that OGD/R-induced reduction of cell viability and release of lactate dehydrogenase (LDH) were prevented by overexpression of FAL1. Additionally, exposure to OGD/R significantly reduced the phosphorylated levels of PAK1 and AKT as well as the total level of proliferating cell nuclear antigen (PCNA), which was restored by overexpression of FAL1. Importantly, overexpression of FAL1 restored OGD/R-induced reduction in the expression of endothelial nitric oxide synthase (eNOS) and the subsequent release of nitric oxide (NO). Our results implicate that FAL1 might be involved in the process of brain endothelial cell damage.

MicroRNA-27b Modulates Inflammatory Response and Apoptosis during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis poses a significant global health threat. MicroRNAs play an important role in regulating host anti-mycobacterial defense; however, their role in apoptosis-mediated mycobacterial elimination and inflammatory response remains unclear. In this study, we explored the role of microRNA-27b (miR-27b) in murine macrophage responses to M. tuberculosis infection. We uncovered that the TLR-2/MyD88/NF-κB signaling pathway induced the expression of miR-27b and miR-27b suppressed the production of proinflammatory factors and the activity of NF-κB, thereby avoiding an excessive inflammation during M. tuberculosis infection. Luciferase reporter assay and Western blotting showed that miR-27b directly targeted Bcl-2-associated athanogene 2 (Bag2) in macrophages. Overexpression of Bag2 reversed miR-27b-mediated inhibition of the production of proinflammatory factors. In addition, miR-27b increased p53-dependent cell apoptosis and the production of reactive oxygen species and decreased the bacterial burden. We also showed that Bag2 interacts with p53 and negatively regulates its activity, thereby controlling cell apoptosis and facilitating bacterial survival. In summary, we revealed a novel role of the miR-27b/Bag2 axis in the regulation of inflammatory response and apoptosis and provide a potential molecular host defense mechanism against mycobacteria.

Aplysin Retards Pancreatic Necrosis and Inflammatory Responses in NOD Mice by Stabilizing Intestinal Barriers and Regulating Gut Microbial Composition.

Aplysin is a brominated sesquiterpene with an isoprene skeleton and has biological activities. The purpose of this study is to investigate the inhibitory effect of aplysin on spontaneous pancreatic necrosis in nonobese diabetic (NOD) mice and its potential mechanisms. Results showed that NOD mice at 12 weeks of age showed obvious spontaneous pancreatic necrosis, damaged tight junctions of intestinal epithelia, and widened gaps in tight and adherens junctions. Aplysin intervention was able to alleviate spontaneous pancreatic necrosis in NOD mice, accompanied with decreased serum endotoxin levels and downregulated expressions of Toll-like receptor 4 and its related molecules MyD88, TRAF-6, NF-κB p65, TRIF, TRAM, and IRF-3, as well as protein levels of interleukin-1ß and interferon-ß in pancreatic tissues. In addition, we observed obvious improvements of intestinal mucosal barrier function and changes of gut microbiota in the relative abundance at the phylum level and the genus level in aplysin-treated mice compared with control mice. Together, these data suggested that aplysin could retard spontaneous pancreatic necrosis and inflammatory responses in NOD mice through the stabilization of intestinal barriers and regulation of gut microbial composition.

RNA-seq analysis of different inflammatory reactions induced by lipopolysaccharide and lipoteichoic acid in bovine mammary epithelial cells.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are key virulence factors of Escherichia coli and Staphylococcus aureus respectively, and both of them could cause inflammatory reaction in bovine mammary glands. In this study, we used bovine mammary epithelial cells (BMECs) as pattern recognition receptors and stimulated them with LPS or LTA to investigate the global transcriptional response variations of BMECs to these two different virulent factors through RNA-Seq analysis. We found 100 differentially expressed genes (DEGs) with 95 up-regulated and 5 down-regulated genes in LPS-treated group, whereas 24 DEGs with 12 up-regulated and 12 down-regulated genes in LTA-treated group compared to control. Although the number and expression changes of DEGs are significantly different between LPS vs Control and LTA vs Control, KEGG pathway enrichment analysis showed the majorities of DEGs in each pair were enriched on cytokine-cytokine receptor interaction, NF-κB signaling pathway, and NOD-like receptor signaling pathway, especially cytokines and chemokines. These results provided a comprehensive analysis of gene expression profiles elicited by LPS and LTA in BMECs, contributing to the understanding of early "pathogen-host" interactions during intramammary infections.

MCPIP1 mediates inflammatory responses induced by lipopolysaccharide and lipoteichoic acid in bovine mammary epithelial cells.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is a kind of zinc finger RNA binding protein, which exerts immune responses in a variety of cell types. However, the role of MCPIP1 in bovine mammary epithelial cells during mastitis has not been studied. In this study, we explored the functions of MCPIP1 in the inflammatory process induced by virulence factors of pathogens in bovine mammary alveolar cell-T (MAC-T) cell line. Our results showed that MCPIP1 was significantly highly expressed both in the mammary tissue of dairy cows with mastitis and in inflammatory MAC-T cells induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we found that overexpression of MCPIP1 in MAC-T cells abated the LPS-induced increase at the gene expression levels of inflammatory mediators tumor necrosis factor-α-α, interleukin (IL)-1ß, IL-6 and IL-8, enhanced the LPS- and LTA-induced inhibition of epithelial proliferation and promoted the LPS- and LTA-induced oxidative and DNA damage. These findings indicated that MCPIP1 has an enormous potential in regulating the inflammatory response of bovine mammary epithelial cells during infection and may provide an effective therapeutic target for bovine mastitis to reduce the damage caused by inflammatory reactions.

IL-1ß induces increased tight junction permeability in bovine mammary epithelial cells via the IL-1ß-ERK1/2-MLCK axis upon blood-milk barrier damage.

Bovine mastitis occurs frequently in dairy cows and is often caused by various aetiological organisms, for example, Escherichia coli. Lipopolysaccharide (LPS) is a key virulence factor of E. coli. In this study, we stimulated bovine mammary epithelial cells (BMECs) with LPS to investigate the global transcriptional response and identify specific proinflammatory factors that play important roles in blood-milk barrier damage during mastitis caused by E. coli. By performing RNA-seq, we identified a large number of significantly differentially expressed genes (DEGs) between the LPS-treated BMECs and the control cells. Among the DEGs, interleukin-1ß (IL-1ß) was selected because its messenger RNA expression was induced by LPS and its enrichment is involved in multiple inflammatory signal pathways, and its roles in blood-milk barrier damage during the process of mastitis were investigated. Exogenous IL-1ß treatment damaged the integrity of the blood-milk barrier, as indicated by the increased BMEC tight junction (TJ) permeability and confirmed by in vitro and in vivo experiments. Furthermore, the IL-1ß-induced increase in the BMEC TJ permeability was mediated by the IL-1ß-ERK1/2-MLCK axis pathway. Our data provide insights into the functions of IL-1ß in blood-milk barrier damage caused by mastitis in dairy cows.

CYP1A1 Relieves Lipopolysaccharide-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

The expression of cytochrome P4501A1 (CYP1A1) enzyme is changed in various organs during the host response to inflammation or infection, leading to alterations in the metabolism of endogenous and exogenous compounds. Results of this study showed that CYP1A1 expression was significantly downregulated in the mammary tissue of bovine with mastitis, in inflammatory epithelial cells (INEs) extracted from the tissue, and in lipopolysaccharide- (LPS-) induced INEs compared with their corresponding counterparts. Overexpression of CYP1A1 in bovine mammary epithelial cells alleviated the LPS-induced inhibition of epithelial proliferation, abated the LPS-induced increase of gene expression and protein secretion of inflammatory cytokine tumor necrosis factor-α and interleukin-6, and attenuated the LPS-induced activation of NF-κB signaling. These findings suggest that CYP1A1 has immense potential in the regulation of inflammatory responses in bovine mammary epithelial cells during mastitis and may serve as a useful therapeutic target in mitigating injuries caused by inflammatory overreaction.

TGF-ß1 Induces EMT in Bovine Mammary Epithelial Cells Through the TGFß1/Smad Signaling Pathway.

BACKGROUND/AIMS: Transforming growth factor-ß1 (TGF-ß1) plays a crucial role in chronic inflammation in various tissues, and is related to inflammation-caused organ fibrogenesis associated with the epithelial-mesenchymal transition (EMT) and the deposition of the extracellular matrix (ECM). However, the effect of TGF-ß1 on bovine mammary epithelial cells (BMECs) with mastitis, and its mechanism, remain unknown. METHODS: We analyzed the level of TGF-ß1 in inflamed mammary tissues and cells using western blotting. BMECs were treated with TGF-ß1, and EMT-related gene and protein expression changes were evaluated using quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunofluorescence. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor, and analyzed EMT-related protein expression by western blotting. In addition, we injected TGF-ß1 into mice mammary glands to investigate whether it can cause mammary fibrosis in vivo. RESULTS: The TGF-ß1 level was up-regulated in mammary tissues with mastitis and in inducible inflammatory BMECs. TGF-ß1 treatment activated the TGF/ Smad signaling pathway in BMECs during their transition to the EMT phenotype, as indicated by morphological changes from a cobblestone-like shape to a spindle-like one. TGF-ß1 treatment also up-regulated the expression of α-smooth muscle actin, vimentin, and collagen I, albumin, and down-regulated the expression of E-cadherin both in mRNA level and protein level. Furthermore, TGF-ß1 enhanced the gene expressions of MMP2, MMP7, and fibronectin in BMECs. TGF-ß1 injection induced mice mammary infection and fibrosis. CONCLUSION: These findings suggested that aberrant up-regulation of TGF-ß1 in bovine mastitic mammary glands might play an important role in bovine mammary fibrosis caused by unresolved inflammation.

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis.

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5ß1 and αvß3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.

Generation of mastitis resistance in cows by targeting human lysozyme gene to ß-casein locus using zinc-finger nucleases.

Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine ß-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in ß-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

Effects of 3-hydroxyflavone on the cellular and molecular characteristics of bovine embryos produced by somatic-cell nuclear transfer.

This study aimed to investigate the effects of 3-hydroxyflavone, a natural antioxidant pigment enriched in vegetables, on the developmental cellular and molecular characteristics of bovine somatic-cell nuclear transfer (SCNT) embryos. There were no significant differences in the cleavage rate at 48 hr of culture or in the inner cell mass (ICM)-to-trophectoderm (TE) ratio between 3-hydroxyflavone addition and untreated (control) groups (P > 0.05). 3-hydroxyflavone (20 µM) did, however, increase the cleavage rate at 24 hr of culture and the blastocyst-formation rate on Days 6 and 7 (P < 0.05); decrease the levels of intracellular reactive oxygen species in two-, four-, and eight-cell stage embryos (P < 0.05); increase H3K9ac levels in two- and four-cell stages (P < 0.05); increase the total cell number; and decrease the apoptosis index in Day-7 blastocysts. Furthermore, the addition of 3-hydroxyflavone resulted in lower expression of the stress-related gene HSP70.1 and pro-apoptotic gene BAX, as well as higher expression of the anti-apoptotic gene BCL-xL and pluripotency-related genes OCT4 and SOX2 in Day-7 blastocysts produced by SCNT (P < 0.05). The addition of 3-hydroxyflavone during in vitro culture thus exerted beneficial effects on preimplantation development of bovine SCNT embryos both at the cellular and molecular levels.

PhiC31 integrase-mediated genomic integration and stable gene expression in the mouse mammary gland after gene electrotransfer.

BACKGROUND: PhiC31 integrase is capable of conferring long-term transgene expression in various transfected tissues in vivo. In the present study, we investigated the activity of phiC31 integrase in mouse mammary glands. METHODS: The normal mouse mammary epithelial cell line HC11 was transfected with FuGENE® HD Transfection Reagent (Roche Diagnostics, Shanghai, China). Transfection of the mouse mammary gland in vivo was performed by electrotransfer. Transgene expression was detected by western blotting and an enzyme-linked immunosorbent assay. Genomic integration and integration at mpsL1 was confirmed by a nested polymerase chain reaction. RESULTS: An optimal electrotransfer protocol for the lactating mouse mammary gland was attained through investigation of different voltages and pulse durations. PhiC31 integrase mediated site-specific transgene integration in HC11 cells and the mouse mammary gland. In addition, the site-specific integration occurred efficiently at the 'hot spot' mpsL1. Co-delivery of PhiC31 integrase enhanced and prolonged transgene expression in the mouse mammary gland. CONCLUSIONS: The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.

CD24⁺ ovary cancer cells exhibit an invasive mesenchymal phenotype.

We recently reported that the subset of CD24(+) cells in ovarian cancer possesses various cancer stem cell properties. In this study, we further show that this subpopulation of ovarian cancer cells exhibits an epithelial-mesenchymal transition (EMT) phenotype, high invasive capacity, and CXCR4/SDF-1-mediated chemotactic migration. We evaluated CD24 expression in various ovarian cancer cell lines by flow cytometric analysis. CAOV3 and a primary ovarian cancer cell line Clone 4 were sorted into CD24(+) and CD24(-) subpopulations by FACS and Western blot, cell invasion, adhesion, and in vitro chemotaxis assays were performed with these two subpopulations. We also assessed the effects of shRNA depletion of CD24 in CAOV3 and Clone 4 cells by Western blot and cell invasion assays. CD24 expression in ovarian cancer cell lines correlated with aggressive histologic subtypes of epithelial ovarian cancer. The CD24(+) subpopulation was also more invasive than the CD24(-) subpopulation and showed higher CXCR4/SDF-1-mediated chemotactic migration. CD24(+) cells exhibited an EMT phenotype as characterized by loss of E-cadherin expression and gain of vimentin, Twist, and Snail1 expression. In addition, CD24(+) cells stimulated cell attachment to fibronectin through the activation of ß1 integrin. Depletion of CD24 expression by CD24 shRNA efficiently suppressed cell invasion and induced downregulation of CXCR4 as well as loss of the EMT phenotype. In conclusion, CD24 expression in ovarian cancer may be related to tumor aggressiveness, in particular cell invasion and chemotactic migration. Therefore, CD24 may be a good candidate for a therapeutic target for ovarian cancer.

Invasive breast cancer induces laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling.

INTRODUCTION: Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype. METHODS: Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3ß1 or α6ß4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin ß1, or integrin ß4. RESULTS: MDA-MB-231 cells induced laminin-332 upregulation and integrin ß4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3ß1. Integrin ß4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin ß4 expression suppressed fibroblast aggregation in conditions of anoikis. CONCLUSION: Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin ß4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.

Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro.

Fibroblasts were extracted from tissue in tumor burden zones, distal normal zones and interface zones between tumor and normal tissue of human breast carcinomas, and the corresponding fibroblasts were designated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). The crosstalk between three types of fibroblasts and breast cancer cells was evaluated using an in vitro direct co-culture model. We found that INFs grew faster and expressed higher levels of fibroblast activation protein than did NFs and CAFs. Compared with CAFs and NFs, INFs grown with breast cancer cells were significantly more effective in inducing an epithelial-mesenchymal transition (EMT) in cancer cells, as indicated by induction of vimentin and N-cadherin and downregulation of E-cadherin. This EMT process was also accompanied by activation of extracellular signal-regulated kinase (ERK) and modulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Additionally, INFs promoted breast cell migration to a larger extent compared with NFs and CAFs. Taken together, these findings indicate that INFs isolated from the tumor interface zone exhibited more robust biological modulatory activity than did NFs and CAFs isolated from normal and tumor zones of the same tumor tissue, suggesting that the interface zone of the tumor represents a dynamic region vital to tumor progression.

Targeting ILK and ß4 integrin abrogates the invasive potential of ovarian cancer.

Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of ß1 and ß4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of ß1 and ß4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of ß4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of ß4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting ß4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

Laminin-332-rich tumor microenvironment for tumor invasion in the interface zone of breast cancer.

Dense fibrosis, which is caused by desmoplastic reaction, is usually found in invasive ductal carcinoma and may represent the alteration of the tumor microenvironment preceding tumor invasion. Thus, the dense fibrotic zone around invasive ductal carcinoma can be considered to be the actual tissue site of tumor microenvironment, where the precedent alterations for tumor invasion occur. To characterize the dense fibrotic zone, we classified invasive ductal carcinoma tissue into a tumor zone, a normal zone, and the novel interface zone (IZ), which shows dense fibrosis. The postulated IZ is a 5-mm-wide belt that circles the tumor margin and overlaps with normal tissue. Of the extracellular matrix components, laminin-332 was specifically overexpressed in the IZ. Events that appear to be similar to the epithelial-mesenchymal transition, a novel source of myofibroblast formation from epithelial cells, were observed in the IZ, according to the following characteristics: overexpression of matrix metalloproteinase 3, membrane type 1-matrix metalloproteinase, snail, and zinc finger E-box-binding homeobox 1, and the gain of N-cadherin expression, as well as the down-regulation of miR200c. The myofibroblasts isolated from the IZ, which were designated interface zone-fibroblast, displayed laminin-332 and membrane type 1-matrix metalloproteinase overexpression, in contrast with both cancer-associated fibroblasts and normal breast fibroblasts. Taken together, our results suggest that the IZ, which shows dense fibrosis, may provide a specialized microenvironment for guiding tumor invasion: the fibrosis caused by laminin-332 overexpressing myofibroblast formation (interface zone-fibroblast) via epithelial-mesenchymal transition.