Kun Yuan

Primary Cilia Are Decreased in Breast Cancer: Analysis of a Collection of Human Breast Cancer Cell Lines and Tissues

Primary cilia (PC) are solitary, sensory organelles that are critical for several signaling pathways. PC were detected by immunofluorescence of cultured cells and breast tissues. After growth for 7 days in vitro, PC were detected in ∼70% of breast fibroblasts and in 7–19% of epithelial cells derived from benign breast (184A1 and MCF10A). In 11 breast cancer cell lines, PC were present at a low frequency in four (from 0.3% to 4% of cells), but were absent in the remainder. The cancer cell lines with PC were all of the basal B subtype, which is analogous to the clinical triple-negative breast cancer subtype. Furthermore, the frequency of PC decreased with increasing degree of transformation/progression in the MCF10 and MDA-MB-435/LCC6 isogenic models of cancer progression. In histologically normal breast tissues, PC were frequent in fibroblasts and myoepithelial cells and less common in luminal epithelial cells. Of 26 breast cancers examined, rare PC were identified in cancer epithelial cells of only one cancer, which was of the triple-negative subtype. These data indicate a decrease or loss of PC in breast cancer and an association of PC with the basal B subtype. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

Gli1 enhances migration and invasion via up-regulation of MMP-11 and promotes metastasis in ERα negative breast cancer cell lines

Gli1 is an established oncogene and its expression in Estrogen Receptor (ER) α negative and triple negative breast cancers is predictive of a poor prognosis; however, the biological functions regulated by Gli1 in breast cancer have not been extensively evaluated. Herein, Gli1 was over-expressed or down-regulated (by RNA interference and by expression of the repressor form of Gli3) in the ERα negative, human breast cancer cell lines MDA-MB-231 and SUM1315. Reduced expression of Gli1 in these two cell lines resulted in a decrease in migration and invasion. Gli1 over-expression increased the migration and invasion of MDA-MB-231 cells with a corresponding increase in expression of MMP-11. Silencing MMP-11 in MDA-MB-231 cells that over-expressed Gli1 abrogated the Gli1-induced enhancement of migration and invasion. Sustained suppression of Gli1 expression decreased growth of MDA-MB-231 in vitro by increasing apoptosis and decreasing proliferation. In addition, silencing of Gli1 reduced the numbers and sizes of pulmonary metastases of MDA-MB-231 in an in vivo experimental metastasis assay. In summary, Gli1 promotes the growth, survival, migration, invasion and metastasis of ERα negative breast cancer. Additionally, MMP-11 is up-regulated by Gli1 and mediates the migration and invasion induced by Gli1 in MDA-MB-231.

Cell surface associated alpha-L-fucose moieties modulate human breast cancer neoplastic progression.

Glycosylation drives critical processes important for mammalian cell–cell and cell–matrix interactions. Alpha-l-fucose (α-l-f) is a key monosaccharide component of oligosaccharides that has been found to be overexpressed during tumor progression. Modification of cell surface fucosylation, we hypothesized, alters tumor cell phenotype and function at the end of the neoplastic progression cascade including tumor invasion. Alpha-l-fucosidase (α-l-fase) is a glycosidase that specifically removes (α-l-f) from oligosaccharide sites. We first verified the effectiveness of the α-l-fase to specifically decrease the level of α-l-f on the cell surface of several human breast cancer cell lines and also examined the recovery time for these cells to repopulate their surfaces. To investigate the potential effect of defucosylation on tumor functions, we studied the proliferation, and invasion in vitro of human breast cancer MDA-MB-231 cells as the representative cell model. We further examined several fucose-associated molecules previously shown to be involved in tumor progression, including CD44 and CD15 (Lewis X antigen). We found that α-l-fase pretreatment significantly decreased the invasive capability of breast cancer cells. Deoxyfuconojirimycin (DFJ), a specific α-l-fase inhibitor, reversed this effect. After fucosidase treatment, the level of both CD15 and CD44 were found to be reduced as measured by flow cytometry. α-l-fase treatment, further, did not affect tumor cell proliferation in vitro under identical experimental conditions. Gelatin zymography of conditioned media from tumor cells treated with α-l-fase demonstrated no change in MMP-2 activity while MMP-9 was significantly reduced. In summary, fucose containing glycans were found widely distributed on the cell surface of breast cancer cells and could be effectively removed by α-l-fase treatment. This decreased fucosylation, in turn, was seen to impair the interaction between tumor cells and extracellular matrices, and thus affected key cell functions modulating tumor invasion. Further elucidation of the molecular pathways involved in the inhibition of tumor cell invasion may suggest a rationale for the use of glycobiologic therapeutics to deter tumor progression.

In Vitro Matrices for Studying Tumor Cell Invasion

Metastasis is the major cause of death in patients with cancer and thus attempts have been made for more than a century to experimentally dissect its component parts. Multiple arbitrary divisions have been offered that often begin with immortalized and transformed cells demonstrating altered cell proliferation, cell cycle and apoptotic pathways initiating their journey of dissemination by detaching from the primary tumor mass, initiating angiogenesis, degrading and penetrating the basement membranes and the surrounding connective tissue boundaries, intravasating, circulating and extravasating through the blood or lymphatic circulation and after evading the immune system eventually reaching one or more distant metastatic sites where they must undergo a similar process in reverse order. Tumor cell invasion is one of the key steps in this complex process. As the metastatic potential of tumor cells is largely dependent on their ability to degrade and migrate through extracellular matrix (ECM) barriers, inhibition of its subroutines (proteolysis, ECM degradation, chemotaxis, haptotaxis etc.) become logical targets for experimental cancer therapy. To explore potential approaches to inhibit tumor invasion, various in vitro invasion assays have been devised and become widely-accepted surrogate endpoints to mimic the in vivo condition. Most invasion assay systems are based on measuring the ability of cells to invade across purified ECM components such as collagen or complex artificial or reconstituted ECMs. Each has certain strengths, quantitative abilities, ease of manipulation of treatments etc. and like the tumor cells themselves, each is counterbalanced by inherent weaknesses. These are reviewed in this chapter.

Primary Cilia in the Breast and Breast Cancer

Primary cilia are non-motile, microtubule-based appendages extending from the surfaces of most vertebrate cells. The function of primary cilia is cell-type dependent. However, they are known to serve as sensors of the extracellular environment by sensing mechanical forces and chemical constituents in several different cell types. They also function as critical platforms for several signaling pathways, including hedgehog and platelet derived growth factor (PDGF) signaling, and function in maintaining cell polarity. While present on many types of normal cells, including epithelial cells of the breast, they have been shown to be decreased or absent in many cancers. This review discusses the relative decrease in primary cilia in a variety of cancers and the known consequences of the presence or absence of primary cilia during tumorigenesis. This review also addresses possible etiologies for the loss of primary cilia in cancer, potential functions for primary cilia in human breast epithelium and subsequent consequences of the loss of primary cilia during breast carcinogenesis.

Abstract 331: A novel perfusion bioreactor system maintains long-term viability of a three dimensional in vitro breast carcinoma surrogate

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Breast carcinomas are complex, three-dimensional (3D) tissues composed of breast cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Most in vitro models of carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. While more accurate 3D modeling allows for enhanced recapitulation of tumor biology and behavior, 3D culture is acknowledged to be challenging with cell viability decreasing dramatically overtime due to lack of available nutrients. Here-in, a novel perfusion bioreactor system supplies medium through 400 uM-diameter channels to maintain survival of a 3D breast cancer surrogate consisting of MDA-MB-231 (231) breast cancer epithelial cells, breast cancer fibroblasts (CAF) and ECM. For optimization of ECM in the breast cancer surrogates, collagen I concentration and species were varied and the effect on 3D morphology and cell viability was assessed. Methods: To assess the effect of collagen concentration on 3D morphology and cell viability, 231 cells and CAF (2:1 ratio) were incorporated into 1.9, 4, 6, or 8 mg/ml (bovine or rat tail) collagen I mixed with 10% basement membrane (BM, i.e. GFR Matrigel) and cultured for 7 days in 8-well chamber slides (non-perfused, solid 3D cultures). H&E stained histologic sections were prepared after fixation and paraffin embedding of the cultures. Cell aggregation, as a measure of 3D morphology, and viability were assessed on histologic sections by image analysis (ImageJ) and autofluorescence, respectively. To compare cell viability in solid 3D culture to surrogates in the perfusion bioreactor system, 231 cells and CAF (2:1 ratio) were incorporated into an ECM composed of 6 mg/ml bovine collagen I mixed with 10% BM and grown in solid 3D culture or in the bioreactor system. The conditions were compared at 7, 14, and 21 days. Results: Collagen I concentration and species had no significant effect on the extent of cell aggregation. However, cell viability was significantly greater in 6 and 8 mg/ml (69.6% and 67.0% alive, respectively) than 1.9 mg/ml (31.9% alive) bovine collagen (ANOVA, p≤0.05). A similar increase in viability with increasing concentration was not seen with rat-tail collagen. Therefore, 6 mg/ml bovine collagen I was used in cancer surrogates in the perfusion bioreactor system. Cell viability was increased in the perfused surrogate (87.9% alive) in comparison to solid cultures (69.6% alive, t-Test, p = 0.03) at 7 days. There was no significant decrease in viability at 14 and 21 days in perfused surrogates (93.8% and 76.7% alive, respectively). Conclusions: Bovine collagen I concentration affects viability of breast cancer cells in 3D. The perfusion bioreactor system promotes cell viability allowing for multi-week culture of breast carcinoma surrogates. Citation Format: Kayla F. Goliwas, Lauren E. Marshall, Kun Yuan, Joel L. Berry, Andra R. Frost. A novel perfusion bioreactor system maintains long-term viability of a three dimensional in vitro breast carcinoma surrogate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 331. doi:10.1158/1538-7445.AM2015-331

Abstract 2022: Importance of ECM and media permeation in 3D modeling of breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. 3D modeling of cancer is of particular importance in drug development where predicting in vivo effectiveness is challenging. Not only is the 3D structure important for proper modeling of cancer but the response from the surrounding microenvironment, including the extracellular matrix (ECM) and fibroblasts, is also necessary to accurately predict drug response. A major hindrance to 3D culture is loss of cell viability due to nutrient limitation. Herein, we demonstrate the ability of our novel bioreactor system to prolong viability of 3D cultures and the importance of ECM composition in breast cancer modeling. Methods: To gain further understanding of the effect of different ECM on the 3D arrangement of breast cancer cells and breast fibroblasts, three different variations of ECM were tested: 1) 100% basement membrane (BM, reduced growth factor Matrigel) diluted to 9-12 μg/ml, 2) an equal volume of BM and Collagen I (50% BM + 50% Collagen I), and 3) 10% BM in Collagen I. MDA-MB-231 (231) breast cancer cells were grown in each ECM in monoculture or co-culture with breast fibroblasts (ratio of 2:1) for 3 or 7 days. The formation of cell aggregates, as seen in most infiltrating carcinomas of the breast, was assessed by image analysis. To improve viability, 250 μM channels penetrated the 3D co-cultures (consisting of 231 cells and fibroblasts (2:1) mixed into 10% BM/Collagen I) in our perfusion bioreactor system. Proliferation, measured by Ki-67 immunostaining, was compared over time in solid co-cultures and perfused and non-perfused co-cultures after 3 or 7 days. Results: In 3D monocultures, significantly greater cell aggregation was seen with 100% BM compared to 50% and 10% BM at both 3 and 7 days (p<0.002, ANOVA). A similar result was seen in 3D co-cultures with fibroblasts (p<0.002, ANOVA). 3D cultures without channels (solid) demonstrated a reduced Ki-67 labeling index over time (65% at 1 day, 35% at 3 days, and 8.5% at 7 days). Whereas, 3D co-cultures with channels, both perfused and non-perfused, had a more constant Ki-67 labeling index over time (49.6% at 3 days and 37.3% at 7 days with perfusion and 37.4% at 3 days and 34.4% at 7 days without perfusion). Conclusions: Using 3D co-culture with fibroblasts and ECM to model breast cancer recapitulates in vivo tumor-stromal interactions in breast carcinomas better than monocultures in 2D. The formulation of ECM affected cell arrangement, with the presence of BM promoting cell aggregation. The use of our perfusion bioreactor system improved cell proliferation in comparison to solid 3D cultures, which did not sustain growth over time. We anticipate that further refinement of our 3D culture system will allow more accurate investigation of tumor-stromal interactions and drug testing in breast cancer. Citation Format: Kayla F. Goliwas, Lauren E. Marshall, Kun Yuan, Joel Berry, Andra R. Frost. Importance of ECM and media permeation in 3D modeling of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2022. doi:10.1158/1538-7445.AM2014-2022

Abstract 118: Relationship of MMP11 to Gli1 and prognosis in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Gli1 is a transcription factor and oncogene that was first described in the context of Hedgehog signaling. We have previously shown that nuclear localization of Gli1 is predictive of a poor prognosis in estrogen receptor (ER)-negative (ER-), but not ER-positive (ER+) breast carcinomas (BrCa) [Xu et al. Breast Cancer ResTreat 2010:59]. In addition, we demonstrated that overexpression of Gli1 in ER- BrCa cell lines increases cell migration and invasion and, conversely, silencing of Gli1 decreases migration, invasion and metastasis [Kwon et al. Clinical Exp Metastasis 2011:437]. We also found that MMP11 is a down-stream target of Gli1 that is, at least in part, responsible for the pro-invasive/metastatic activity of Gli1. Methods: To extend these findings, we immunostained a microarray of 140 clinically-annotated BrCa for MMP11 and analyzed the results with regard to expression of Gli1 and prognosis. MMP11 was assessed in cancer epithelial cells and associated stromal cells. To study the function of Gli1 and MMP11 in metastasis, we derived a highly invasive variant of the ER- cell line SUM1315 by serial invasion-selection and compared expression of Gli1 and MMP11 in the parental cells and invasive variant. Results: There was no significant correlation between epithelial expression of MMP11 and nuclear localization of Gli1 (p=0.193, r=0.112) in cancer epithelium. However, stromal expression of MMP11 was positively correlated with nuclear localization of Gli1 in cancer epithelium (p=0.001, r=0.284), possibly due to increased secretion of MMP11 by Gli1-positive (i.e., those with nuclear Gli1) BrCa. In addition, stromal expression of MMP11 was higher in Gli1-positive than Gli1-negative BrCa (p=0.05). By Kaplan-Meier analysis, neither stromal nor epithelial expression of MMP11 alone was predictive of a poorer overall survival when considering all BrCa, ER+ and ER- BrCa. However, in ER- BrCa, high (> mean) epithelial expression of MMP11 and Gli1 nuclear localization identified a group with a particularly poor overall survival (p=0.035, Log-rank test), although Gli1 nuclear localization alone in ER- BrCa was also predictive of a poor overall survival (p=0.014, Log-rank test). By transwell invasion assay, the invasive variant of SUM1315 was approximately 3 times more invasive than parental cells. Expression of Gli1 by quantitative RT-PCR was similar in invasive variant and parental SUM1315, while expression of MMP-11 was 2-fold higher in the invasive variant. Conclusions: Stromal expression of MMP11 was higher in Gli1-positive than Gli-negative BrCa. Yet, expression of MMP11 alone was not a prognostic indicator nor did MMP11 expression improve the prognostic value of Gli1. Expression of MMP11 was modestly elevated in invasive variants of SUM1315. These findings suggest that MMP11 alone is not responsible for the increased invasion and poor prognosis resulting from Gli1 activity in BrCa. Funding provided by Susan G. Komen for the Cure Citation Format: Kun Yuan, Jenny L. Raines, Devanshu D. Kaushik, Andra R. Frost. Relationship of MMP11 to Gli1 and prognosis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 118. doi:10.1158/1538-7445.AM2013-118