Deborah L Holliday

Choosing the right cell line for breast cancer research

Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research.

MiR‐26b is down‐regulated in carcinoma‐associated fibroblasts from ER‐positive breast cancers leading to enhanced cell migration and invasion

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs.

Clinical and functional significance of loss of caveolin‐1 expression in breast cancer‐associated fibroblasts

Loss of caveolin‐1 (Cav‐1) expression in breast cancer‐associated fibroblasts (CAFs) is predictive of poor prognosis in breast cancer, but its function has not been established. Our study tested the hypotheses that loss of Cav‐1 expression in breast fibroblasts was associated with poor prognosis in breast cancer, through promotion of breast cancer cell invasion. Cav‐1 stromal expression was immunohistochemically assessed in 358 breast cancers. Cav‐1 expression in primary breast fibroblasts was analysed by western blot. Modified Boyden chamber assays determined fibroblast ability to promote invasion of breast cancer cells. The impact of siRNA silencing of Cav‐1 in fibroblasts was evaluated using invasion assays and 3D co‐culture assays. Loss of Cav‐1 expression in breast stroma was significantly associated with decreased breast cancer‐specific and disease‐free survival (p = 0.01). Mean survival was 72 months (Cav‐1+ group) versus 29.5 months (Cav‐1− group). This was confirmed in multivariate analysis. Cav‐1 expression was significantly decreased in CAFs compared to normal fibroblasts (p = 0.01) and was associated with increased invasion‐promoting capacity. Cav‐1 siRNA‐treated fibroblasts promoted significantly increased invasion of MDA‐MB‐468 and T47D breast cancer cells from 27% (control) to 67% (p = 0.006) and from 37% to 56%, respectively (p = 0.01). 3D co‐cultures of MDA‐MB‐468 cells with myoepithelial cells led to the formation of organized cohesive structures when cultured with conditioned media from fibroblasts but resulted in a disorganized appearance in the presence of conditioned media from Cav‐1 siRNA‐treated fibroblasts, accompanied by loss of E‐cadherin expression in tumour cells. Our data confirm that loss of stromal Cav‐1 in breast cancer predicts poor outcome. At a functional level, Cav‐1‐deficient CAFs are capable of significantly increasing the invasive capacity of breast cancer cells. Copyright

The prognostic significance of tumour-stroma ratio in oestrogen receptor-positive breast cancer

Background:A high percentage of stroma predicts poor survival in triple-negative breast cancers but is diminished in studies of unselected cases. We determined the prognostic significance of tumour–stroma ratio (TSR) in oestrogen receptor (ER)-positive male and female breast carcinomas.Methods:TSR was measured in haematoxylin and eosin-stained tissue sections (118 female and 62 male). Relationship of TSR (cutoff 49%) to overall survival (OS) and relapse-free survival (RFS) was analysed.Results:Tumours with ⩾49% stroma were associated with better survival in female (OS P=0.008, HR=0.2–0.7; RFS P=0.006, HR=0.1–0.6) and male breast cancer (OS P=0.005, HR=0.05–0.6; RFS P=0.01, HR=0.87–5.6), confirmed in multivariate analysis.Conclusions:High stromal content was related to better survival in ER-positive breast cancers across both genders, contrasting data in triple-negative breast cancer and highlighting the importance of considering ER status when interpreting the prognostic value of TSR.

Phosphorylation of Estrogen Receptor β at Serine 105 Is Associated with Good Prognosis in Breast Cancer

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred score > or =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

The practicalities of using tissue slices as preclinical organotypic breast cancer models

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in breast cancer. Following ethical approval, 250 µm thick sections from surplus breast tumours (n=10) were prepared using a vibrating blade microtome. Triplicate tissue slices were placed in 6-well plates and cultured for up to 7 days±tamoxifen (1 nM) or doxorubicin (1 µM). Tissue slices were fixed and embedded before sectioning for morphological evaluation and immunohistochemistry. H&E showed good preservation of tissue morphology. Collagen production was evident. Biomarkers of proliferation and apoptosis could be evaluated using immunohistochemistry and used as surrogates to quantify drug effects. In summary, breast cancer tissue slices can be cultured in vitro as organotypic models. Nevertheless, although simple in concept, the delicacy of the model with regard to handling makes subsequent analytical processes challenging.

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

Background 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. Methodology/Principal Findings We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. Conclusions Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

Reponse to: comment on, 'Tumour-stroma ratio (TSR) in oestrogen-positive breast cancer patients'.

Reponse to: comment on, ‘Tumour-stroma ratio (TSR) in oestrogen-positive breast cancer patients’

Loss of CSMD1 disrupts mammary epithelial morphogenesis

CUB and Sushi multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene of unknown function. CSMD1 maps to chromosome 8p23, a region deleted in 50% of breast cancers (BC). We have examined the contribution of CSMD1 to the tumorigenic phenotype of mammary acini and evaluated its prognostic value in BC patients.

Role of miR-26b in carcinoma-associated fibroblasts and effect on migration and invasion of breast cancer epithelial cells

Abstract Background In recent years there has been an increasing awareness of the role of the microenvironment surrounding breast cancer epithelium, particularly the carcinoma-associated fibroblast (CAF), in modulating the behaviour of breast tumours. MicroRNAs, a family of small non-coding RNAs that are key in the post-transcriptional regulation of mRNAs, have a role in controlling the behaviour of cancers and have been studied extensively in breast cancer epithelial cells. We hypothesised that miRNAs have important roles in the behaviour of breast CAFs and, in turn, affect the behaviour of malignant breast epithelial cells. Methods We used laser capture microdissected normal fibroblast and CAFs from clinical samples, and a tissue co-culture model, to examine expression of miRNAs in breast CAFs. Deregulated miRNAs that were identified with this screening strategy were further assessed in a larger set of paired laser capture microdissected normal fibroblasts and CAFs. We assessed functional effects on fibroblasts by overexpressing, or knocking down miRNAs (or both) in immortalised fibroblast cell lines and using various growth, migration, and invasion assays. Functional effects of fibroblasts with reduced miRNA on breast cancer epithelial cells were examined in co-cultures. To identify potential miRNA targets and pathways downstream, we used mass spectrometry and in-silico analysis. The clinical relevance of these targets was examined by interrogating publicly available datasets. Findings Six miRNAs were consistently downregulated in CAFs compared with normal fibroblasts with a fold change of more that ten in both the tissue co-culture model and in patient samples. Of these miRNAs, miR-26b was downregulated in 12 of 14 cases of microdissected matched normal fibroblasts and CAFs from clinical formalin fixed paraffin embedded samples (Wilcoxon signed rank test, p=0·04), and consistently in a further four of four cases of matched primary cultures of normal fibroblasts and CAFs. Reduction of the level of miR-26b in immortalised breast fibroblasts showed a small decrease in proliferation but a very notable increase in migration (p COL12A1 was identified as a target of miR-26b. Increased stromal mRNA expression of COL12A1 was significantly associated with increased recurrence of breast cancer on multivariate analysis in publically available datasets (hazard ratio 17, 95% CI 1·9–159; p=0·01). Interpretation We have shown that downregulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and we have identified molecular changes that are downstream of miR-26b. These findings add to the growing body of evidence that epithelial-stromal interactions are important in modulating the behaviour of breast cancer. Funding UK Medical Research Council, Pathological Society of Great Britain and Ireland, Breast Cancer Campaign, National Institute of Health Research.