Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism.

<p>This file contains supplementary figures S1-S10 and supplementary materials and methods. Figure S1 shows experimental design of mesenchymal stem cells in this article. Figure S2 shows differentiation of transgenic mesenchymal stem cells. Figure S3 shows differentiation and gel contraction of mesenchymal stem cells. Figure S4 shows YAP activity in mesenchymal stem cells on a stiff matrix. Figure S5 shows expression and activation of YAP-related signaling molecules in mesenchymal stem cells. Figure S6 shows contribution of mesenchymal stem cells to tumor progression. Figure S7 shows prosaposin detection in mammary tumors. Figure S8 shows the role of prosaposin for ERK and caspase 3. Figure S9 shows contribution of prosaposin to late tumor growth and progression. Figure S10 shows the role of stiff matrix for mesenchymal stem cells from B6 mice.</p>

Supplementary Figure S1. Effects on haptotaxis are independent of effects on cell velocity.

Cell migration is an important process that is involved in the major developmental stages of all complex organisms and results in the arrangement of cells into a precise architecture, the organization of the nervous system, and the generation of specialized organs and tissues. For example, cell migration in the adult allows the immune cells to traffic through tissues and to arrive at wounds to facilitate the healing process. Cells migrate along extracellular matrix (ECM) proteins using cell-surface receptors composed of integrins and proteoglycans. In various pathological conditions, such as cancer metastasis, cells restructure the ECM and reacquire the ability to migrate, but to detrimental effect. For several years cell migration has been largely studied in the context of glass or plastic surfaces coated with ECM proteins, which has allowed an abundance of information about the events in cell migration and the signaling pathways involved. More recently, the field has moved toward the more complex problem of understanding cell migration in three-dimensional matrices, both in in vitro culture conditions and in whole tissues and organisms. This article presents recent findings on cell migration in the context of three dimensions and focuses on cancer cell invasion as an example allowing us to highlight aspects of cell migration in vivo.

Abstract Background Elevated mammographic breast density is a strong breast cancer risk factor with poorly understood etiology. Increased deposition of collagen, one of the main fibrous proteins present in breast stroma, has been associated with increased mammographic density. Collagen fiber architecture has been linked to poor outcomes in breast cancer. However, relationships of quantitative collagen fiber features assessed in diagnostic biopsies with mammographic density and lesion severity are not well-established. Methods Clinically indicated breast biopsies from 65 in situ or invasive breast cancer cases and 73 frequency matched-controls with a benign biopsy result were used to measure collagen fiber features (length, straightness, width, alignment, orientation and density (fibers/µm2)) using second harmonic generation microscopy in up to three regions of interest (ROIs) per biopsy: normal, benign breast disease, and cancer. Local and global mammographic density volumes were quantified in the ipsilateral breast in pre-biopsy full-field digital mammograms. Associations of fibrillar collagen features with mammographic density and severity of biopsy diagnosis were evaluated using generalized estimating equation models with an independent correlation structure to account for multiple ROIs within each biopsy section. Results Collagen fiber density was positively associated with the proportion of stroma on the biopsy slide (p < 0.001) and with local percent mammographic density volume at both the biopsy target (p = 0.035) and within a 2 mm perilesional ring (p = 0.02), but not with global mammographic density measures. As severity of the breast biopsy diagnosis increased at the ROI level, collagen fibers tended to be less dense, shorter, straighter, thinner, and more aligned with one another (p < 0.05). Conclusions Collagen fiber density was positively associated with local, but not global, mammographic density, suggesting that collagen microarchitecture may not translate into macroscopic mammographic features. However, collagen fiber features may be markers of cancer risk and/or progression among women referred for biopsy based on abnormal breast imaging.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Alt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Here we aim to review the association between mammographic density, collagen structure, and breast cancer risk. While mammographic density is a strong predictor of breast cancer risk in populations, studies by Boyd show that mammographic density does not predict breast cancer risk in individuals. Mammographic density is affected by age, parity, menopausal status, race/ethnicity, and body mass index (BMI). New studies normalize mammographic density to BMI and this may provide a more accurate way to compare mammographic density in women of diverse race and ethnicity. Preclinical and tissue-based studies have investigated the role collagen composition and structure in predicting breast cancer risk. There is emerging evidence that collagen structure may activate signaling pathways associated with aggressive breast cancer biology. Measurement of film mammographic density does not adequately capture the complex signaling that occurs in women with at-risk collagen. New ways to measure at-risk collagen potentially can provide a more accurate view of risk.

There was an error in J. Cell Sci. (2016) 129, [1989-2002][1] ([doi:10.1242/jcs.180539][2]). The -YAP panels on stiff substrate in Fig. 8C were incorrectly duplicated in Fig. 6A. The journal has seen the original data for Fig. 6A and the corrected and original panels are shown below. This error

Increasing evidence demonstrates an important role for the extracellular matrix (ECM) in breast cancer progression. Collagen type I, a core constituent of the fibrous ECM, undergoes a significant set of changes that accompany tumor progression, termed Tumor Associated Collagen Signatures (TACS). Late stages of this progression are characterized by the presence of bundled, straight collagen (TACS-2) that become oriented perpendicular to the tumor-stromal boundary (TACS-3). Importantly, the presence of TACS-3 collagen is an independent predictor of poor patient outcome. At present, it remains unclear whether reorganization of the collagen matrix is the consequence of mechanical or compositional tissue remodeling. Here, we identify compositional changes in ECM correlating to collagen fiber reorganization from nineteen normal and invasive ductal carcinoma (IDC) patient biopsies using matrisome-targeted proteomics. Twenty-seven ECM proteins were significantly altered in IDC samples compared to normal tissue. Further, a set of nineteen matrisome proteins positively correlate and five proteins inversely correlate with IDC tissues containing straightened collagen fibers. Tenascin-C and thrombospondin-2 significantly co-localized with aligned collagen fibers in IDC tissues. This study highlights the compositional change in matrisome proteins accompanying collagen re-organization during breast cancer progression and provides candidate proteins for investigation into cellular and structural influences on collagen alignment.

Inflammation, and the organization of collagen in the breast tumor microenvironment, is an important mediator of breast tumor progression. However, a direct link between markers of inflammation, collagen organization, and patient outcome has yet to be established. A tumor microarray of 371 invasive breast carcinoma biopsy specimens was analyzed for expression of inflammatory markers, including cyclooxygenase 2 (COX-2), macrophages, and several collagen features in the tumor nest (TN) or the tumor-associated stroma (TS). The tumor microarray cohort included females, aged 18 to 80 years, with a median follow-up of 8.4 years. High expression of COX-2 (TN), CD68 (TS), and CD163 (TN and TS) predicted worse patient overall survival (OS). This notion was strengthened by the finding from the multivariate analysis that high numbers of CD163+ macrophages in the TS is an independent prognostic factor. Overall collagen deposition was associated with high stromal expression of COX-2 and CD163; however, total collagen deposition was not a predictor for OS. Conversely, local collagen density, alignment and perpendicular alignment to the tumor boundary (tumor-associated collagen signature-3) were predictors of OS. These results suggest that in invasive carcinoma, the localization of inflammatory cells and aligned collagen orientation predict poor patient survival. Additional clinical studies may help validate whether therapy with selective COX-2 inhibitors alters expression of CD68 and CD163 inflammatory markers.

Abstract Because we do not know who will develop breast cancer, or who will have a relapse of cancer following surgery, there is an urgent need for the development of biomarkers that can be detected specifically in normal, nondiseased tissue. For 30 years there has been a search for the genetic markers that will predict risk of developing breast cancer, but even while family history is a known risk factor, only 10% of breast cancer risk can be specifically linked to a genetic mechanism (BRCA-1, BRCA-2). Furthermore, individuals who are at high risk due to genetics or family history do not always develop breast cancer. Breast density is another indicator of future incidence, as high-density tissue is correlated with a 4-6 fold increased risk of developing cancer. We hypothesized that a population analysis of the properties of the individual collagen fibers that comprise the extracellular matrix (ECM) in nondiseased breast tissue is a biomarker for the onset of disease. We used multiphoton second-harmonic generation (SHG) imaging to generate high-contrast images of the collagen ECM that can be gathered from common histologic slides that require no additional staining. Our goal was to determine the range of heterogeneity in the properties of the collagen matrix in normal samples. We imaged samples from 141 normal patient donors to comprise 4 different cohorts that define this ground truth state of the matrix. The cohorts were from donors who had never been diagnosed with breast cancer; who had previously been diagnosed with breast cancer but now were disease free; who had never been diagnosed with breast cancer at the time of tissue donation but later went on to develop breast cancer; and those who had donated tissue while in remission from an initial breast cancer diagnosis but later went on to have a recurrence of breast cancer. The SHG images of each patient sample were then analyzed using the curvelet transform-based ctFIRE and CurveAlign software platforms to generate data (fiber length, width, straightness, density, angle with respect to a boundary of epithelial ducts/lobules, and the relative alignment of fibers to each other) on each individual collagen fiber. Advanced statistical measurements and principal component analysis were used to classify the nature of each of the cohorts. Based on our measurements of the structure and organization of collagen fibers, we found that while measurements within a cohort were consistent, there were unique attributes of the collagen matrix that defined each cohort. Furthermore, we performed regression analysis of our measurements against the standard clinical features (age, race, BMI, etc.). Because our various cohorts delineate the nondiseased, involuted, precancer, and prerecurrence normal matrix, respectively, we feel that these data serve as a highly useful, novel classifier, one that describes the clinical impact of the timbre of the matrix in nondiseased women. Citation Format: Ryan J. Gigstad, Tianjie Wang, Yifei Liu, Menggang Yu, Yuming Liu, Adib Keikhosravi, Kevin W. Eliceiri, Patricia J. Keely, Matthew W. Conklin. Classification of the collagen ECM in normal human tissue as a biomarker for future breast cancer incidence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3598.

Breast cancer is the most common cancer among women worldwide and ranks second in terms of overall cancer deaths. One of the difficulties associated with treating breast cancer is that it is a heterogeneous disease with variations in benign and pathologic tissue composition, which contributes to disease development, progression, and treatment response. Many of these phenotypes are uncharacterized and their presence is difficult to detect, in part due to the sparsity of methods to correlate information between the cellular microscale and the whole-breast macroscale. Quantitative multiscale imaging of the breast is an emerging field concerned with the development of imaging technology that can characterize anatomic, functional, and molecular information across different resolutions and fields of view. It involves a diverse collection of imaging modalities, which touch large sections of the breast imaging research community. Prospective studies have shown promising results, but there are several challenges, ranging from basic physics and engineering to data processing and quantification, that must be met to bring the field to maturity. This paper presents some of the challenges that investigators face, reviews currently used multiscale imaging methods for preclinical imaging, and discusses the potential of these methods for clinical breast imaging.

Abstract The tumor microenvironment contains cancer cells, non-cancerous cells, and extracellular components such as extracellular matrix (ECM). Previous studies have demonstrated that interactions between the cells and the microenvironment contribute to cancer progression via chemical stimuli, such as growth factors, and mechanical stimuli, such as stiffness of the ECM. Recently, it has been reported that mesenchymal stem cells (MSCs) differentiate into cancer associated fibroblasts (CAFs) in response to chemical stimuli from cancer cells and thereby promote cancer progression. However, the contribution of mechanical stimuli to MSCs in cancer is poorly understood. In this study, we revealed that MSCs showed CAF phenotypes and promote mammary cancer progression in response to mechanical stimuli. On a stiff substrate, MSCs treated with conditioned media from cancer cell culture expressed increased levels of alpha smooth muscle actin (alpha-SMA), a marker of CAFs, compared to the MSCs cultured on a soft substrate. MSCs grown on a stiff substrate displayed higher expression and activity of YAP and increased phosphorylation of myosin light chain (MLC) compared to MSCs grown on a soft substrate. In addition, knockdown of YAP by shRNA decreased the expression of alpha-SMA and phosphorylation of MLC in MSCs on a stiff substrate. Pharmacological inhibition of MLC phosphorylation by H1152 treatment also reduced expression of alpha-SMA and activity of YAP in MSCs. Cell-cell communication between MSCs and carcinoma cells was bi-directional, as conditioned medium from MSCs cultured on a stiff substrate, but not a soft substrate, increased growth of mammary carcinoma cells. The soluble factor prosaposin was highly secreted by the MSCs on a stiff substrate, and the addition of recombinant prosaposin increased proliferation and survival of mammary carcinoma cells. Furthermore, secretion of prosaposin was promoted in YAP-overexpressed MSCs on a soft substrate. Mammary carcinoma cells treated with prosaposin showed increased level of phosphorylation in Akt at T308. In addition, inhibition of phosphorylation of Akt at T308 prevented proliferation and survival in mammary carcinoma cells. These results suggest that increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSCs to CAFs via YAP and actomyosin contractility, secretion of prosaposion from MSCs, and as a result, trigger progression of mammary cancer via phosphorylation of Akt at T308. Citation Format: Seiichiro Ishihara, David R. Inman, Wan-Ju Li, Suzanne M. Ponik, Patricia J. Keely. Stiffness of extracellular matrix regulates breast cancer progression by stimulating mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5904. doi:10.1158/1538-7445.AM2017-5904

The development and progression of estrogen receptor alpha positive (ERα+) breast cancer has been linked epidemiologically to prolactin. However, activation of the canonical mediator of prolactin, STAT5, is associated with more differentiated cancers and better prognoses. We have reported that density/stiffness of the extracellular matrix potently modulates the repertoire of prolactin signals in human ERα + breast cancer cells in vitro: stiff matrices shift the balance from the Janus kinase (JAK)2/STAT5 cascade toward pro-tumor progressive extracellular regulated kinase (ERK)1/2 signals, driving invasion. However, the consequences for behavior of ERα + cancers in vivo are not known.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1 hi /DEC2 hi /p27 hi /TGFβ2 hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1 hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER − breast cancer cells, post-hypoxic ER + breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

Background: Collagen fibers surrounding breast ducts may influence breast cancer progression. Syndecan-1 interacts with constituents in the extracellular matrix, including collagen fibers, and may contribute to cancer cell migration. Thus, the orientation of collagen fibers surrounding ductal carcinoma in situ (DCIS) lesions and stromal syndecan-1 expression may predict recurrence. Methods: We evaluated collagen fiber alignment and syndecan-1 expression in 227 women diagnosed with DCIS in 1995 to 2006 followed through 2014 (median, 14.5 years; range, 0.7–17.6). Stromal collagen alignment was evaluated from diagnostic tissue slides using second harmonic generation microscopy and fiber analysis software. Univariate analysis was conducted using χ2 tests and ANOVA. The association between collagen alignment z-scores, syndecan-1 staining intensity, and time to recurrence was evaluated using HRs and 95% confidence intervals (CIs). Results: Greater fiber angles surrounding DCIS lesions, but not syndecan-1 staining intensity, were related to positive HER2 (P = 0.002) status, comedo necrosis (P = 0.03), and negative estrogen receptor (P = 0.002) and progesterone receptor (P = 0.02) status. Fiber angle distributions surrounding lesions included more angles closer to 90 degrees than normal ducts (P = 0.06). Collagen alignment z-scores for DCIS lesions were positively related to recurrence (HR = 1.25; 95% CI, 0.84–1.87 for an interquartile range increase in average fiber angles). Conclusions: Although collagen alignment and stromal syndecan-1 expression did not predict recurrence, collagen fibers perpendicular to the duct perimeter were more frequent in DCIS lesions with features typical of poor prognosis. Impact: Follow-up studies are warranted to examine whether additional features of the collagen matrix may more strongly predict patient outcomes. Cancer Epidemiol Biomarkers Prev; 27(2); 138–45. ©2017 AACR.

Abstract In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179–89. ©2017 AACR.

Abstract Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell–mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENAINV isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENAINV and FN levels were correlated in two breast cancer cohorts, and high levels of MENAINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell–intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration. Significance: Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell–intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516–31. ©2016 AACR. See related commentary by Santiago-Medina and Yang, p. 474. This article is highlighted in the In This Issue feature, p. 461

High levels of collagen deposition in human and mouse breast tumors are associated with poor outcome due to increased local invasion and distant metastases. Using a genetic approach, we show that, in mice, the action of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2) in both tumor and tumor-stromal cells is critical for breast cancer metastasis yet does not affect primary tumor growth. In tumor cells, DDR2 in basal epithelial cells regulates the collective invasion of tumor organoids. In stromal cancer-associated fibroblasts (CAFs), DDR2 is critical for extracellular matrix production and the organization of collagen fibers. The action of DDR2 in CAFs also enhances tumor cell collective invasion through a pathway distinct from the tumor-cellintrinsic function of DDR2. This work identifies DDR2 as a potential therapeutic target that controls breast cancer metastases through its action in both tumor cells and tumor-stromal cells at the primary tumor site.