Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts

Stromal fibroblasts influence the behavior of breast epithelial cells. Fibroblasts derived from normal breast (NAF) inhibit epithelial growth, whereas fibroblasts from breast carcinomas (CAF) have less growth inhibitory capacity and can promote epithelial growth. We sought to identify molecules that are differentially expressed in NAF versus CAF and potentially responsible for their different growth regulatory abilities. To determine the contribution of soluble molecules to fibroblast–epithelial interactions, NAF were grown in 3D, transwell or direct contact co-cultures with MCF10AT epithelial cells. NAF suppressed proliferation of MCF10AT in both direct contact and transwell co-cultures, but this suppression was significantly greater in direct co-cultures, indicating involvement of both soluble and contact factors. Gene expression profiling of early passage fibroblast cultures identified 420 genes that were differentially expressed in NAF versus CAF. Of the eight genes selected for validation by real-time PCR, FIBULIN 1, was overexpressed in NAF, and DICKKOPF 1, NEUREGULIN 1, PLASMINOGEN ACTIVATOR INHIBITOR 2, and TISSUE PLASMINOGEN ACTIVATOR were overexpressed in CAF. A higher expression of FIBULIN 1 in normal- than cancer-associated fibroblastic stroma was confirmed by immunohistochemistry of breast tissues. Among breast cancers, stromal expression of Fibulin 1 protein was higher in estrogen receptor α-positive cancers and low stromal expression of Fibulin 1 correlated with a higher proliferation of cancer epithelial cells. In conclusion, expression profiling of NAF and CAF cultures identified many genes with potential relevance to fibroblast–epithelial interactions in breast cancer. Furthermore, these early passage fibroblast cultures can be representative of gene expression in stromal fibroblasts in vivo

The patient perspective on absence of disease activity in rheumatoid arthritis: A survey to identify key domains of patient-perceived remission

© 2017, BMJ Publishing Group. All rights reserved. Background: Guidelines suggest treatment in rheumatoid arthritis (RA) to target remission, in close consultation with the patient. Our recent qualitative study of the patients' perspective on remission in RA identified 26 domains. The current study aimed to identify a short list of the most important aspects to inform future research. Methods: Patients with RA from the Netherlands, the UK, Austria, Denmark, France and the USA completed a survey that contained all domains identified in our qualitative study. They rated domains for importance ('not important', 'important' or 'essential' to characterise a period of remission) and if important or essential, whether this domain needs to be 'less', 'almost gone' or 'gone' to reflect remission. Respondents were also asked to determine their personal top 3 most important/ essential domains. Frequency of specific domains in the top 3 was calculated, and domains were sorted on the percentage of patients that evaluated a particular domain as 'essential'. Results: Of 274 respondents, 75% were female, mean (SD) age 57(13) years, disease duration 12(9) years. The top 3 were as follows: pain (67%), fatigue (33%) and independence (19%); domains most frequently rated as 'essential' were as follows: pain (60%), being mobile (52%), physical function (51%), being independent (47%) and fatigue (41%). Pain needed to be less (13%), almost gone (42%) or gone (45%) to reflect remission. Similar patterns were seen for fatigue, independence, mobility and physical functioning. Conclusion: Patients identified pain, fatigue and independence as the most important domains of RA disease activity that need to be improved to reflect remission

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells <it>in vitro </it>and <it>in vivo</it>. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women.</p> <p>Methods</p> <p>The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested <it>in vitro </it>via soft agar and invasion assays, and <it>in vivo </it>via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry.</p> <p>Results</p> <p>Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER)^-, progesterone receptor (PR)/PR^-cells were significantly more aggressive when in contact with AA ECM, as were ER⁺/PR⁺cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER⁺/PR⁺cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of approximately 8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue.</p> <p>Conclusions</p> <p>This study details normal premenopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.</p

Highly variable response to cytotoxic chemotherapy in carcinoma-associated fibroblasts (CAFs) from lung and breast

<p>Abstract</p> <p>Background</p> <p>Carcinoma-associated fibroblasts (CAFs) can promote carcinogenesis and tumor progression. Only limited data on the response of CAFs to chemotherapy and their potential impact on therapy outcome are available. This study was undertaken to analyze the influence of chemotherapy on carcinoma-associated fibroblasts (CAFs) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>The <it>in vivo </it>response of stromal cells to chemotherapy was investigated in 22 neoadjuvant treated breast tumors on tissue sections before and after chemotherapy. Response to chemotherapy was analyzed <it>in vitro </it>in primary cultures of isolated CAFs from 28 human lung and 9 breast cancer tissues. The response was correlated to <it>Mdm2</it>, <it>ERCC1 </it>and <it>TP53 </it>polymorphisms and <it>TP53 </it>mutation status. Additionally, the cytotoxic effects were evaluated in an <it>ex vivo </it>experiment using cultured tissue slices from 16 lung and 17 breast cancer specimens.</p> <p>Results</p> <p>Nine of 22 tumors showed a therapy-dependent reduction of stromal activity. Pathological response of tumor or stroma cells did not correlate with clinical response. Isolated CAFs showed little sensitivity to paclitaxel. In contrast, sensitivity of CAFs to cisplatinum was highly variable with a GI50 ranging from 2.8 to 29.0 μM which is comparable to the range observed in tumor cell lines. No somatic <it>TP53 </it>mutation was detected in any of the 28 CAFs from lung cancer tissue. In addition, response to cisplatinum was not significantly associated with the genotype of <it>TP53 </it>nor <it>Mdm2 </it>and <it>ERCC1 </it>polymorphisms. However, we observed a non-significant trend towards decreased sensitivity in the presence of <it>TP53 </it>variant genotype. In contrast to the results obtained in isolated cell culture, in tissue slice culture breast cancer CAFs responded to paclitaxel within their microenvironment in the majority of cases (9/14). The opposite was observed in lung cancer tissues: only few CAFs were sensitive to cisplatinum within their microenvironment (2/15) whereas a higher proportion responded to cisplatinum in isolated culture.</p> <p>Conclusion</p> <p>Similar to cancer cells, CAF response to chemotherapy is highly variable. Beside significant individual/intrinsic differences the sensitivity of CAFs seems to depend also on the cancer type as well as the microenvironment.</p

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor

Evaluation of Candidate Stromal Epithelial Cross-Talk Genes Identifies Association between Risk of Serous Ovarian Cancer and TERT, a Cancer Susceptibility “Hot-Spot”

We hypothesized that variants in genes expressed as a consequence of interactions between ovarian cancer cells and the host micro-environment could contribute to cancer susceptibility. We therefore used a two-stage approach to evaluate common single nucleotide polymorphisms (SNPs) in 173 genes involved in stromal epithelial interactions in the Ovarian Cancer Association Consortium (OCAC). In the discovery stage, cases with epithelial ovarian cancer (n = 675) and controls (n = 1,162) were genotyped at 1,536 SNPs using an Illumina GoldenGate assay. Based on Positive Predictive Value estimates, three SNPs—PODXL rs1013368, ITGA6 rs13027811, and MMP3 rs522616—were selected for replication using TaqMan genotyping in up to 3,059 serous invasive cases and 8,905 controls from 16 OCAC case-control studies. An additional 18 SNPs with Pper-allele<0.05 in the discovery stage were selected for replication in a subset of five OCAC studies (n = 1,233 serous invasive cases; n = 3,364 controls). The discovery stage associations in PODXL, ITGA6, and MMP3 were attenuated in the larger replication set (adj. Pper-allele≥0.5). However genotypes at TERT rs7726159 were associated with ovarian cancer risk in the smaller, five-study replication study (Pper-allele = 0.03). Combined analysis of the discovery and replication sets for this TERT SNP showed an increased risk of serous ovarian cancer among non-Hispanic whites [adj. ORper-allele 1.14 (1.04–1.24) p = 0.003]. Our study adds to the growing evidence that, like the 8q24 locus, the telomerase reverse transcriptase locus at 5p15.33, is a general cancer susceptibility locus

Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo

Abstract Introduction Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process. Methods Primary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis. Results The combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion in vitro, as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior in vitro, as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone. Conclusions Herein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized in vivo model system. We confirmed our in vivo results with in vitro analyses, highlighting the value of this model to interchangeably substantiate in vitro and in vivo results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic in vivo model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis