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INTRODUCTION: Stromal-epithelial interactions are fundamental for normal organ development and there is a multitude of evidence that the different components of the microenvironment are also necessary for the maintenance and promotion of the "tumor organ". Deregulated tumor associated extracellular matrix (tECM) is a hallmark of cancer, causing an alteration in the amount and composition of the different components (i.e. proteins, proteoglycans, glycoproteins and polysaccharids) of the ECM. As epithelial-stromal interactions are reciprocal, it is possible that tECM itself is able to initiate tumor development. We therefore established a mouse model to examine the influence of tECM of murine breast cancer on developing breast tissue in mice. MATERIALS AND METHODS: Breast cancer was established in 5 BALB/c mice by subcutaneous injection of 1×106 4T1 cells in 100μl PBS into the left mammary fat pad. The mammary fat pad including the primary tumor was excised after two weeks, decellularised and labelled as tumor extracellular matrix (tECM). Tumor ECM of 4T1 tumors was implanted into the 4th inguinal mammary fat pad of BALB/c mice (n = 5) aged 5 days. After 12 weeks the fourth mammary fat pad including the primary tumor was excised. Tissue was used for paraffin embedding and mouse breast cancer PCR array. Murine breast cancer tissue (BCT) and normal murine breast tissue (BT) served as control. RESULTS: Gene array analysis of 84 breast cancer-specific transcripts revealed that the mammary gland cells which were exposed to tumor ECM (tECM-BT) showed a similarly high overexpression for 22 genes as apparent for breast cancer tissue (BCT). The corresponding scatter plot showed a high agreement in the expression of the examined genes between the mammary gland cells which were exposed to tumor ECM and the breast cancer tissue. DISCUSSION: Our results clearly demonstrate that the tECM is able to shift the gene expression pattern of murine mammary epithelial cells towards that of carcinoma, indicating a role in breast cancer initiation. These data underlines that the acellular component of the tumor (ECM) can lead to a transformation of mammary gland tissue cells. These data show for the first time that the interaction of normal breast tissue cells with tumor ECM leads to an exchange of information and a consecutive overexpression of tumor-specific genes. [ABSTRACT FROM AUTHOR]

Abstract: Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D‐collagen gel equivalent (3D‐CGE) and 3D cell‐synthetized stromal equivalent (3D‐CSSE). The 3D‐CGEs were obtained by means of the traditional collagen‐based cell technique and the 3D‐CSSE were obtained by bottom‐up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo‐2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix‐associated proteins from ISEMFs cultured in 3D‐CSSE compared to 3D‐CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D‐CSSE drove the CaCo‐2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus‐secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D‐CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial–stromal interface establishment. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis. To identify potential effective therapeutic drugs for PDAC, we established a screening system based on spheroid formation using 170#3 mouse PDAC cells with or without fibroblasts. We found that indirubin 3'-oxime (Indox) and 5-methoxyindirubin 3'-oxime (5MeOIndox) inhibited PDAC cell proliferation. Furthermore, PDAC xenograft growth was also inhibited in BALB/c nu/nu mice after administration of Indox and 5MeOIndox. Both phosphorylated CDK1 and cyclin B1 levels in 170#3 cells were significantly reduced by treatment with Indox and 5MeOIndox in vitro and in vivo. Cell cycle analysis revealed that 5MeOIndox, but not Indox, induced G2/M arrest. Annexin V-propidium iodide double-staining analysis demonstrated that Indox induced abundant non-apoptotic cell death of 170#3 cells, while 5MeOIndox predominantly induced early apoptosis, indicating that the cytotoxicity of 5MeOIndox is lower than that of Indox. These results suggest that one mechanism of 5MeOIndox is to induce G2/M arrest of PDAC cells via inhibition of CDK1/cyclin B1 levels, thereby leading to apoptosis. Our findings suggest 5MeOIndox as a potential useful anticancer agent in PDAC. [ABSTRACT FROM AUTHOR]

Excessive matrix production by pancreatic stellate cells promotes local growth and metastasis of pancreatic ductal adenocarcinoma and provides a barrier for drug delivery. Collagen type V is a fibrillar, regulatory collagen up-regulated in the stroma of different malignant tumors. Here we show that collagen type V is expressed by pancreatic stellate cells in the stroma of pancreatic ductal adenocarcinoma and affects the malignant phenotype of various pancreatic cancer cell lines by promoting adhesion, migration and viability, also after treatment with chemotherapeutic drugs. Pharmacological and antibody-mediated inhibition of β1-integrin signaling abolishes collagen type V-induced effects on pancreatic cancer cells. Ablation of collagen type V secretion of pancreatic stellate cells by siRNA reduces invasion and proliferation of pancreatic cancer cells and tube formation of endothelial cells. Moreover, stable knock-down of collagen type V in pancreatic stellate cells reduces metastasis formation and angiogenesis in an orthotopic mouse model of ductal adenocarcinoma. In conclusion, paracrine loops involving cancer and stromal elements and mediated by collagen type V promote the malignant phenotype of pancreatic ductal adenocarcinoma and underline the relevance of epithelial–stromal interactions in the progression of this aggressive neoplasm. [ABSTRACT FROM AUTHOR]

Abstract: Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE2, we found enhanced GJIC with 1nM PGE2. This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE2 was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE2 secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE2 to the cells. Our findings show that PGE2 may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE2 secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells. [Copyright &y& Elsevier]

To understand the molecular pathogenesis of oral submucous fibrosis (OSF), which is a chronic inflammatory disease, gene expression profiling was performed in 10 OSF tissues against 8 pooled normal tissues using oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes ( P  ≤≤  0.05 and fold change  ≥≥  1.5). Among these, 2884 are upregulated and 2404 are downregulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed upregulation of transforming growth factor-ββ1 (TGF-ββ1), TGFBIp, THBS1, SPP1, and TIG1 and downregulation of bone morphogenic protein 7 (BMP7) in OSF tissues. Furthermore, activation of TGF-ββ pathway was evident in OSF as demonstrated by pSMAD2 strong immunoreactivity. Treatment of keratinocytes and oral fibroblasts by TGF-ββ confirmed the regulation of few genes identified in microarray including upregulation of connective tissue growth factor, TGM2, THBS1, and downregulation of BMP7, which is a known negative modulator of fibrosis. Taken together, these data suggest activation of TGF-ββ signaling and suppression of BMP7 expression in the manifestation of OSF. [ABSTRACT FROM AUTHOR]

Purpose: Interleukin (IL)-6 signaling through its soluble receptor (sIL-6R) (IL-6 trans-signaling) plays an important role in various inflammatory states. We investigated production of sIL-6R in the corneal epithelium and examined the role of IL-6 trans-signaling in the cornea. Methods: In-vitro experiments were performed using SV40-transformed human corneal epithelial cells (HCEC) and primary human corneal fibroblasts (HCF, keratocytes). Ectodomain shedding in HCEC was stimulated by adding phorbol myristate acetate (PMA, 3 μ M) both with and without ectodomain shedding inhibition using TNF-α processing inhibitor-1 (TAPI-1, 250 ng/mL), and the concentration of sIL-6R in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Expression of differential sIL-6R mRNA splicing (DS-sIL-6R) in HCEC was examined by using reverse transcription (RT)-PCR. The recombinant IL-6 or combination of recombinant IL-6/sIL-6R was added to HCF culture medium and phosphorylation of STAT3 was analyzed by Luminex assay. Tear fluid from patients with Sjögren syndrome was collected and analyzed by ELISA for sIL-6R concentration. Results: In HCEC culture medium, sIL-6R release was increased significantly ( P < 0.01) by adding PMA and this increased release of sIL-6R was inhibited significantly by adding TAPI-1, indicating the participation of ectodomain shedding in sIL-6R production. In RT-PCR, DS-sIL-6R expression was noted in HCEC. IL-6/sIL-6R-induced STAT3 phosphorylation was recognized in cultured HCF, suggesting IL-6 trans-signaling induced inflammatory cellular signaling in HCF. In the tear fluid of the patients with Sjögren syndrome, sIL-6R expression was up-regulated (Sjögren syndrome; 2.38 ± 0.98 ng/mL, normal control; 0.16 ± 0.34 ng/mL). Conclusions: Production of sIL-6R was induced by both ectodomain shedding and mRNA splicing in the corneal epithelium. IL-6 trans-signaling can induce an inflammatory response in corneal fibroblasts. The up-regulation of sIL-6R in inflamed ocular surfaces suggests a pivotal role of sIL-6R at the ocular surface. [ABSTRACT FROM AUTHOR]

Abstract: The ovarian surface epithelium (OSE) origin of ovarian cancers has been controversial because these cancers often exhibit Müllerian-like features. One hypothesis is that ovarian neoplasia involves the gain of growth advantages by OSE cells via activation of Müllerian programs. The homeobox gene HOXA10 controls formation of the uterus from the Müllerian ducts, and is not expressed in normal OSE. We previously found that HOXA10 is expressed in ovarian cancers with endometrial-like features, and induces transformed OSE cells to form glandular tumors in mice. In the current study, we found that induction of HOXA10 in OSE cells promotes homophilic cell adhesion and prevents anoikis. HOXA10 expression stimulated interactions of OSE cells with the extracellular matrix proteins vitronectin and fibronectin, and with mesothelial cells of the omentum which is a common attachment site for ovarian cancer cells. HOXA10 also stimulated interactions of OSE cells with omental fibroblasts, and these interactions promoted OSE cell growth. Our findings indicate that aberrant activation of a Müllerian program in OSE cells confers growth advantages by stimulating cellular interactions with the microenvironment. [Copyright &y& Elsevier]

Abstract: Objectives: Expression of uPA mRNA is massively up-regulated in the stroma of poorly differentiated ovarian tumors. We hypothesized that this expression was induced by paracrine signals from the epithelial tumor cells, and established an in vitro model of ovarian cancer microenvironment to study intercellular cross-talk. Methods: ES-2 clear cell carcinoma cells were grown in tissue culture inserts in a double-chamber system with fibroblastic stromal LEP cells embedded in Matrigel. Binding-site directed antibodies were used to neutralize soluble cytokines in ES-2 conditioned medium (CM) before incubation with LEP cells. Real time PCR measured uPA mRNA in LEP cells, as well as mRNA for cytokines in both cell types. Results: Co-culture with ES-2 cells as well as incubation with ES-2 CM induced uPA mRNA in LEP cells about two-fold. In short time (12 h) incubation of LEP cells with CM, antibodies to EGF and bFGF reduced induction of uPA mRNA, suggesting that these cytokines function as paracrine signals. EGF mRNA and bFGF mRNA were also found in ES-2 cells. At longer incubation (24 h) antibodies to bFGF, HB-EGF, HGF, IGF-1, and IL-1α reduced uPA mRNA induction, suggesting an autocrine function for these cytokines in LEP cells. In fact, expression of the same five cytokines was up-regulated in LEP cells exposed to CM. Conclusion: We identified two cytokines as paracrine signals, and five cytokines as autocrine signals in ovarian cancer cell induced up-regulation of uPA mRNA in stromal fibroblastic cells. It is crucial to understand intra-tumoral cross-talk, since it can offer new therapeutic approaches. [Copyright &y& Elsevier]

Cancer metastasis involves complex cell behavior and interaction with the extracellular matrix by metabolically active cells. To observe invasion and metastasis with sub-cellular resolution in vivo, multiphoton microscopy (MPM) allows imaging more deeply into tissues with less toxicity, compared with other optical imaging methods. MPM can be combined with second harmonic generation (SHG), fluorescent lifetime imaging microscopy (FLIM), and spectral-lifetime imaging microscopy (SLIM). SHG facilitates imaging of stromal collagen and tumor–stroma interactions, including the architecture and remodeling of the tumor microenvironment. FLIM allows characterization of exogenous and endogenous fluorophores, such as the metabolites FAD and NADH to score for metabolic state and provide optical biomarkers. SLIM permits additional identification and separation of endogenous and exogenous fluorophores by simultaneously collecting their spectra and lifetime, producing an optical molecular “fingerprint”. Both FLIM and SLIM also serve as an improved method for the assessment of Förster (or fluorescence) resonance energy transfer (FRET). Hence, the use and further development of these approaches strongly enhances the visualization and quantification of tumor progression, invasion, and metastasis. Herein, we review recent developments of multiphoton FLIM and SLIM to study 2D and 3D cell migration, invasion into the tumor microenvironment, and metastasis. [ABSTRACT FROM AUTHOR]

Abstract: Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R =0.3967, p =0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors. [Copyright &y& Elsevier]

Background. Protein tyrosine phosphatase γ (PTPγ) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicate that estradiol-17β (E2)-induced suppression of PTPγ may play a role in mammary tumorigenesis. Zeranol (Z), a nonsteroidal growth promoter with estrogenic activity that is used by the US meat industry, induces estrogenic responses in primary cultured breast cells and breast cancer cell lines. Methods. PTPγ mRNA expression in human breast tissues and cells isolated from surgical specimens of mammoplasty and breast cancer patients were detected and quantified by RT-PCR. Immunohistochemical staining was used to localize PTPγ in human breast tissues. Breast epithelial and stromal cells were isolated and co-cultured to determine the involvement of cell–cell interaction in the regulation of PTPγ mRNA expression by E2 and Z. Results. PTPγ mRNA expression was lower in cancerous than in normal breast tissues. Both E2 and Z suppressed PTPγ mRNA levels in cultured normal breast tissues by ∼80%, but had a lesser effect in cultured epithelial cells isolated from normal breast tissues. In the co-culture system, both E2 and Z suppressed PTPγ mRNA to a greater degree in epithelial cells than in stromal cells. In whole breast tissues, PTPγ was immunolocalized to the epithelium. Treatment with E2 or Z diminished PTPγ staining indicating reductions in PTPγ at the protein level. Conclusions. The results indicate that both E2 and Z regulate PTPγ expression in human breast and that epithelial–stromal cells interaction is important in the regulation of PTPγ expression by estrogenically active agents. [ABSTRACT FROM AUTHOR]

The growth and morphogenesis of mammaryparenchyma varies substantially between species and isregulated by an array of systemic and local factors.Central to this regulation is the mammary fat pad, amatrix of adipose and connective tissue capable ofmediating hormone action and synthesizing an array ofgrowth regulatory molecules. In this article wehighlight differences between the morphologicaldevelopment of the mammary parenchyma in rodents, humans,and ruminant dairy animals, placing emphasis ondifferences in the cellular composition and structure ofthe mammary fat pad. While a great deal remains to be understood about the ability of stroma tolocally regulate mammary development, the significanceof its contribution is becoming increasingly apparent.The actions of several steroid and peptide hormones appear to be mediated by an array of growthfactors, proteases and extracellular matrix componentssynthesized by constituents of the mammary fat pad.Further, mammary adipose tissue represents a significant store of lipid which, by itself and through itsderivatives, could influence the growth of mammaryepithelium in diverse ways. This review describes theintegral role of the mammary fat pad duringmammogenesis, emphasizing the point that species differencesmust be addressed if local growth and morphogenicmechanisms within the mammary gland are to beresolved. [ABSTRACT FROM AUTHOR]

The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six-to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E2 or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated ( P<0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased ( P<0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted ( P<0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad ( P>0.05). Whereas hydrocortisone did not alter cell number, it attenuated ( P<0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens. [ABSTRACT FROM AUTHOR]

Tumor progression depends on the tumor-stroma interaction. In the breast, adipose tissue is the predominant stromal type. We have previously demonstrated that conditioned media (CMs) from explants of human adipose tissue of tumor breasts (hATT) increase proliferation and migration of breast cancer epithelial cells when compared to human adipose tissue from normal breasts (hATN). In this work, we aim to identify specific proteins and molecular/biological pathways associated with the secretion profile of hATT and hATN explants. hATT-CMs and hATN-CMs were separated by SDS-PAGE and analyzed by means of two-dimensional nano-liquid chromatography-mass spectrometry. The data was analyzed using ProteoIQ and FunRich software. In addition, 42 cytokines from hATT-CMs and hATN-CMs were assayed by a protein antibody assay. Compared to hATN-CMs, hATT-CMs showed greater protein diversity. We found that hATT-CMs presented a greater amount of proteins related to complement system activity, metabolism and immune system, as well as proteins involved in a variety of biological processes such as signal transduction and cell communication. Specifically, apolipoprotein AI and AII, complement component 3, and vimentin and desmin were significantly increased in hATT-CMs versus hATN-CMs. Moreover, a multivariate discriminant analysis of the cytokines detected by the array showed that IL-6, MCP-2 and GRO cytokines were sufficient and necessary to differentiate hATT-CMs from hATN-CMs. This analysis also showed that the levels of these three cytokines, taken together, correlated with stage and histological grade of the tumor in the hATT-CMs group, and with body mass index in the hATN-CMs group., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The epithelium of the pubertal mouse mammary gland grows and invades the mammary fat pad to form a primary ductal network. This outgrowth is tightly controlled by epithelial and stromal factors that are present in the environment around the terminal end buds (TEB) at the growth front and the newly formed ducts. Identifying the contribution that each cell type makes to this regulation is a major challenge. To identify the role that fibroblasts play during this process we have optimised a fibroblast isolation procedure, followed by cell cleanup, RNA extraction, and amplification from non-cultured, freshly isolated fibroblasts from around the TEB as well as the subtending ducts. This was facilitated by the use of mice that constitutively expressed EGFP, which allowed the visualization of the growth front of the pubertal mammary tree under UV light. The isolated RNA is of sufficiently high quality, giving reproducible qRT-PCR results, for transcriptome analysis after RNA amplification.

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that replicate in stratified squamous epithelia and cause a variety of malignancies. Current efforts in HPV biology are focused on understanding the virus-host interactions that enable HPV to persist for years or decades in the tissue. The importance of interactions between tumor cells and the stromal microenvironment has become increasingly apparent in recent years, but how stromal interactions impact the normal, benign life cycle of HPVs, or progression of lesions to cancer is less understood. Furthermore, how productively replicating HPV impacts cells in the stromal environment is also unclear. Here we bring together some of the relevant literature on keratinocyte-stromal interactions and their impacts on HPV biology, focusing on stromal fibroblasts, immune cells, and endothelial cells. We discuss how HPV oncogenes in infected cells manipulate other cells in their environment, and, conversely, how neighboring cells may impact the efficiency or course of HPV infection., (© 2016 Elsevier Inc. All rights reserved.)