Bo Ri Seo

Implanted adipose progenitor cells as physicochemical regulators of breast cancer

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

Obesity leads to fibrotic remodeling of mammary adipose tissue, and the resulting increase in interstitial extracellular matrix stiffness promotes breast tumor malignancy. Fat fibrosis and breast cancer One of the many risk factors for cancer is obesity—but why? Seo et al. examined the cellular, structural, and molecular changes that happen in breast tissue in obese animals and people. They found that obesity induces fibrotic remodeling of the mammary fat pad, leading to changes in extracellular matrix (ECM) mechanical properties, via myofibroblasts and adipose stem cells (ASCs), regardless of ovary function. Through altered mechanotransduction, ECM from obese mice promoted human breast cancer cell growth, as well as the growth of premalignant breast cells (those that have yet to become cancerous). Tissues from obese patients revealed more severe fibrotic remodeling around tumors and higher levels of a key mechanosignaling component, YAP/TAZ, than their lean counterparts. The authors further demonstrated that caloric restriction in obese mice decreased fibrosis in mammary fat, suggesting a therapeutic angle for obesity-related cancers. By linking tumorigenesis to the behavior of fat cells and ECM mechanics, the authors point toward new drug targets for preventing cancer progression. However, a cautionary tale also exists in the use of adipose tissue and cells for patients after mastectomy, as ASCs from obese individuals may have the capacity to promote breast cancer recurrence. Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.

Parylene peel-off arrays to probe the role of cell–cell interactions in tumour angiogenesis

Microenvironmental conditions impact tumour angiogenesis, but the role of cell-cell interactions in modulating the angiogenic capability of tumour cells is not well understood. We have microfabricated a peel-off cell-culture array (PeelArray) chip to spatiotemporally control interactions between tumour cells in a large array format and to analyse angiogenic factor secretion in response to these conditions. The PeelArray chip consists of a polyethylene glycol (PEG) treated glass coverslip coated with a parylene-C template that can be easily peeled off to selectively micropattern biomolecules and cells. We have designed the PeelArray chip to reproducibly deposit large uniform arrays of isolated single cells or isolated cell clusters on fibronectin features of defined surface areas. We have utilised this microfabricated culture system to study the secretion of angiogenic factors by tumour cells, in the presence or absence of cell-cell contact as controlled by micropatterning. Our results indicate that cell-cell interactions play a synergistic role in regulating the expression of angiogenic factors (i.e., vascular endothelial growth factor [VEGF] and interleukin-8 [IL-8]) in various cancer cell lines, independent of other more complex microenvironmental cues (e.g. hypoxia). Our PeelArray chip is a simple and adaptable micropatterning method that enables quantitative profiling of protein secretions and hence, a better understanding of the mechanisms by which cell-cell interactions regulate tumour cell behaviour and angiogenesis.

Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells

Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell–matrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell – Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches.

In vitro models of tumor vessels and matrix: engineering approaches to investigate transport limitations and drug delivery in cancer.

Tumor-stroma interactions have emerged as critical determinants of drug efficacy. However, the underlying biological and physicochemical mechanisms by which the microenvironment regulates therapeutic response remain unclear, due in part to a lack of physiologically relevant in vitro platforms to accurately interrogate tissue-level phenomena. Tissue-engineered tumor models are beginning to address this shortcoming. By allowing selective incorporation of microenvironmental complexity, these platforms afford unique access to tumor-associated signaling and transport dynamics. This review will focus on engineering approaches to study drug delivery as a function of tumor-associated changes of the vasculature and extracellular matrix (ECM). First, we review current biological understanding of these components and discuss their impact on transport processes. Then, we evaluate existing microfluidic, tissue engineering, and materials science strategies to recapitulate vascular and ECM characteristics of tumors, and finish by outlining challenges and future directions of the field that may ultimately improve anti-cancer therapies.

Fibronectin Mechanobiology Regulates Tumorigenesis

Fibronectin (Fn) is an essential extracellular matrix (ECM) glycoprotein involved in both physiological and pathological processes. The structure–function relationship of Fn has been and is still being studied, as changes in its molecular structure are integral in regulating (or dysregulating) its biological activities via its cell, matrix component, and growth factor binding sites. Fn comprises three types of repeating modules; among them, FnIII modules are mechanically unstable domains that may be extended/unfolded upon cell traction and either uncover cryptic binding sites or disrupt otherwise exposed binding sites. Cells assemble Fn into a fibrillar network; its conformational flexibility implicates Fn as a critical mechanoregulator of the ECM. Fn has been shown to contribute to altered stroma remodeling during tumorigenesis. This review will discuss (i) the significance of the structure–function relationship of Fn at both the molecular and the matrix scales, (ii) the role of Fn mechanobiology in the regulation of tumorigenesis, and (iii) Fn-related advances in cancer therapy development.

Influencing the Tumor Microenvironment: A Phase II Study of Copper Depletion Using Tetrathiomolybdate in Patients with Breast Cancer at High Risk for Recurrence and in Preclinical Models of Lung Metastases

Purpose: Bone marrow–derived progenitor cells, including VEGFR2+ endothelial progenitor cells (EPCs) and copper-dependent pathways, model the tumor microenvironment. We hypothesized that copper depletion using tetrathiomolybdate would reduce EPCs in high risk for patients with breast cancer who have relapsed. We investigated the effect of tetrathiomolybdate on the tumor microenvironment in preclinical models. Experimental Design: Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition. Results: Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P = 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II–III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 50% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P = 0.04), LOX activity (P = 0.03), and collagen crosslinking (P = 0.012). Conclusions: Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted. Clin Cancer Res; 23(3); 666–76. ©2016 AACR.

Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation

UNLABELLED Neovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches. STATEMENT OF SIGNIFICANCE Neovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Breast cancer cells alter the dynamics of stromal fibronectin-collagen interactions.

Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel their extracellular matrix (ECM) and promote invasive tumor growth. Two major ECM components, fibronectin (Fn) and collagen I (Col I), are known to interact with each other to regulate cellular behavior. In this study, we seek to understand how Fn and Col I interplay and promote a dysregulated signaling pathway to facilitate tumor progression. Specifically, we investigated the evolution of tumor-conditioned stromal ECM composition, structure, and relaxation. Furthermore, we assessed how evolving Fn-Col I interactions gradually affected pro-angiogenic signaling. Our data first indicate that CAFs initially assembled a strained, viscous, and unfolded Fn matrix. This early altered Fn matrix was later remodeled into a thick Col I-rich matrix that was characteristic of a dense tumor mass. Next, our results suggest that this ECM remodeling was primarily mediated by matrix metalloproteinases (MMPs). This MMP activity caused profound structural and mechanical changes in the developing ECM, which then modified vascular endothelial growth factor (VEGF) secretion by CAFs and matrix sequestration. Collectively, these findings enhance our understanding of the mechanisms by which Fn and Col I synergistically interplay in promoting a sustained altered signaling cascade to remodel the breast tumor stroma for invasive breast tumor growth.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis

Significance Hydroxyapatite (HA) nanocrystals are key constituents of the bone extracellular matrix and thus likely to influence the pathogenesis of breast cancer skeletal metastasis. However, there is currently an insufficient understanding of HA nanocrystal properties at sites prone to bone metastasis formation. Here we report a novel application of X-ray scattering and Raman imaging to characterize HA nanostructure in mouse models of breast cancer. Our results suggest that bone regions linked with the initiation of metastasis contain less-mature HA nanocrystals and that mammary tumors enhance HA nanocrystal immaturity in these regions even prior to secondary tumor formation. Insights from this work will significantly advance the development of mineralized culture models to investigate how the bone microenvironment regulates breast cancer metastasis. Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.