The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent ...metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.
We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.
When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.
Optical techniques, such as bioluminescence and fluorescence, are emerging as powerful new modalities for molecular imaging in disease and therapy. Combining innovative molecular biology and ...chemistry, researchers have developed optical methods for imaging a variety of cellular and molecular processes in vivo, including protein interactions, protein degradation, and protease activity. Whereas optical imaging has been used primarily for research in small-animal models, there are several areas in which optical molecular imaging will translate to clinical medicine. In this review, we summarize recent advances in optical techniques for molecular imaging and the potential impact for clinical medicine.
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Extracellular matrix regulates hallmark features of cancer through biochemical and mechanical signals, although mechanistic understanding of these processes remains limited by lack of ...models that recreate physiology of tumors. To tissue-engineer models that recapitulate three-dimensional architecture and signaling in tumors, there is a pressing need for new materials permitting flexible control of mechanical and biophysical features. We developed a hybrid hydrogel system composed of collagen and alginate to model tumor environments in breast cancer and other malignancies. Material properties of the hydrogel, including stiffness, microstructure and porosimetry, encompass parameters present in normal organs and tumors. The hydrogel possesses a well-organized, homogenous microstructure with adjustable mechanical stiffness and excellent permeability. Upon embedding multicellular tumor spheroids, we constructed a 3D tumor invasion model showing follow-the-leader migration with fibroblasts leading invasion of cancer cells similar to in vivo. We also demonstrated effects of CXCL12-CXCR4 signaling, a pathway implicated in tumor progression and metastasis, in a dual-tumor spheroid invasion model in 3D hydrogels. These studies establish a new hydrogel platform with material properties that can be tuned to investigate effects of environmental conditions on tumor progression, which will advance future studies of cancer cell invasion and response to therapy.
Our manuscript describes a novel design of hybrid hydrogel system composed of collagen and alginate modeling 3D tumor environments in breast cancer. The hydrogel possesses a well-organized, homogenous microstructure with adjustable mechanical stiffness. Upon embedding tumor spheroids, we successfully showed a 3D tumor invasion model showing follow-the-leader migration with fibroblasts leading invasion of cancer cells similar to in vivo. To the best of our knowledge, this is the first study showing two spheroids invade simultaneously and forming bridge-like connection and effects of chemical gradients in 3D hydrogel environment. This research provides a new model for tumor-stromal interactions in cancer cell migration and establishes a novel hydrogel system for analyzing physical and biochemical signals regulating cancer progression and response to therapy.
Chemokine CXCL12 gradients drive chemotaxis in a CXCR4-dependent mechanism and have been implicated in cancer metastasis. While CXCL12 gradients are typically studied in organized, defined ...environments, the tumor microenvironment is disorganized. In vivo, CXCL12 gradients depend on many factors: the number and arrangement of cells secreting and degrading CXCL12, isoform-dependent binding to the extracellular matrix, diffusion, and circadian fluctuations. We developed a computational model of the tumor microenvironment to simulate CXCL12 gradient dynamics in disorganized tissue. There are four major findings from the model. First, CXCL12-β and -γ form higher magnitude (steeper) gradients compared to CXCL12-α. Second, endothelial CXCR7+ cells regulate CXCL12 gradient direction by controlling concentrations near but not far from the vasculature. Third, the magnitude and direction of CXCL12 gradients are dependent on the local composition of secreting and scavenging cells within the tumor. We theorize that "micro-regions" of cellular heterogeneity within the tumor are responsible for forming strong gradients directed into the blood. Fourth, CXCL12 circadian fluctuations influence gradient magnitude but not direction. Our simulations provide predictions for future experiments in animal models. Understanding the generation of CXCL12 gradients is crucial to inhibiting cancer metastasis.
The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 were first identified in the context of trafficking and homeostasis of immune cells, such as T lymphocytes. Subsequently, it has been ...determined that CXCR4 regulates several key processes in a wide variety of cancers. Functions of CXCL12 and CXCR4 in cancer first were described in metastatic breast cancer, and more recent studies also have identified roles for this signaling pathway in primary breast tumors. This review focuses on functions of CXCR4 and CXCL12 in primary and metastatic breast cancer, including molecular mechanisms of action and relationships of this pathway to other key regulators of breast cancer progression. We also describe pre-clinical studies indicating the potential to exploit CXCR4 as a new molecular target for diagnosis and treatment of breast cancer in patients.
Mechanical properties of tumors differ substantially from normal cells and tissues. Changes in stiffness or elasticity regulate pro-metastatic behaviors of cancer cells, but effects have been ...documented predominantly in isolated cells or in vitro cell culture systems. To directly link relative stiffness of tumors to cancer progression, we combined a mouse model of metastatic breast cancer with ex vivo measurements of bulk moduli of freshly excised, intact tumors. We found a high, inverse correlation between bulk modulus of resected tumors and subsequent local recurrence and metastasis. More compliant tumors were associated with more frequent, larger local recurrences and more extensive metastases than mice with relatively stiff tumors. We found that collagen content of resected tumors correlated with bulk modulus values. These data establish that relative differences in tumor stiffness correspond with tumor progression and metastasis, supporting further testing and development of tumor compliance as a prognostic biomarker in breast cancer.
Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and ...fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC).
We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer.
Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer.
In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.
Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both ...of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the "competition" with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.
The chemokine receptor CXCR4 and its cognate ligand CXCL12 recently have been proposed to regulate the directional trafficking and invasion of breast cancer cells to sites of metastases. However, ...effects of CXCR4 on the growth of primary breast cancer tumors and established metastases and survival have not been determined. We used stable RNAi to reduce expression of CXCR4 in murine 4T1 cells, a highly metastatic mammary cancer cell line that is a model for stage IV human breast cancer. Using noninvasive bioluminescence and magnetic resonance imaging, we showed that knockdown of CXCR4 significantly limited the growth of orthotopically transplanted breast cancer cells. Mice in which parental 4T1 cells were implanted had progressively enlarging tumors that spontaneously metastasized, and these animals all died from metastatic disease. Remarkably, RNAi of CXCR4 prevented primary tumor formation in some mice, and all mice transplanted with CXCR RNAi cells survived without developing macroscopic metastases. To analyze effects of CXCR4 on metastases to the lung, an organ commonly affected by metastatic breast cancer, we injected tumor cells intravenously and monitored cell growth with bioluminescence imaging. Inhibiting CXCR4 with RNAi, or the specific antagonist AMD3100, substantially delayed the growth of 4T1 cells in the lung, although neither RNAi nor AMD3100 prolonged overall survival in mice with experimental lung metastases. These data indicate that CXCR4 is required to initiate proliferation and/or promote survival of breast cancer cells in vivo and suggest that CXCR4 inhibitors will improve treatment of patients with primary and metastatic breast cancer.
Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), ...recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.
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