Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy.

: 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. : 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.

[Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer.

Purpose: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer.

Materials And Methods: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation.

Unexpected softening of a fibrous matrix by contracting inclusions.

Cells respond to the stiffness of their surrounding environment, but quantifying the stiffness of a fibrous matrix at the scale of a cell is complicated, due to the effects of nonlinearity and complex force transmission pathways resulting from randomness in fiber density and connections. While it is known that forces produced by individual contractile cells can stiffen the matrix, it remains unclear how simultaneous contraction of multiple cells in a fibrous matrix alters the stiffness at the scale of a cell. Here, we used computational modeling and experiments to quantify the stiffness of a random fibrous matrix embedded with multiple contracting inclusions, which mimicked the contractile forces of a cell.

Estrogen receptor blockade and radiation therapy cooperate to enhance the response of immunologically cold ER+ breast cancer to immunotherapy.

Background: Most patients with estrogen receptor positive (ER+) breast cancer do not respond to immune checkpoint inhibition (ICI); the tumor microenvironment (TME) of these cancers is generally immunosuppressive and contains few tumor-infiltrating lymphocytes. Radiation therapy (RT) can increase tumor inflammation and infiltration by lymphocytes but does not improve responses to ICIs in these patients. This may result, in part, from additional effects of RT that suppress anti-tumor immunity, including increased tumor infiltration by myeloid-derived suppressor cells and regulatory T cells.

Model of collective detachment in high-grade serous ovarian cancer demonstrates that tumor spheroids produce ECM to support metastatic processes.

High-grade serous ovarian cancer (HGSOC) metastasizes through transcoelomic spread, with both single cells and spheroids of tumor cells observed in patient ascites. These spheroids may form through single cells that detach and aggregate (Sph-SC) or through collective detachment (Sph-CD). We developed an model to generate and separate Sph-SC from Sph-CD to enable study of Sph-CD in disease progression.

Nephronectin is required to maintain right lung lobar separation during embryonic development.

Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8β1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed allele were used to generate global knockouts (KOs).

Effect of hyaluronic acid on microscale deformations of collagen gels.

As fibrous collagen is the most abundant protein in mammalian tissues, gels of collagen fibers have been extensively used as an extracellular matrix scaffold to study how cells sense and respond to cues from their microenvironment. Other components of native tissues, such as glycosaminoglycans like hyaluronic acid, can affect cell behavior in part by changing the mechanical properties of the collagen gel. Prior studies have quantified the effects of hyaluronic acid on the mechanical properties of collagen gels in experiments of uniform shear or compression at the macroscale.

Multimodal imaging demonstrates enhanced tumor exposure of PEGylated FUD peptide in breast cancer.

In breast cancer, the extracellular matrix (ECM) undergoes remodeling and changes the tumor microenvironment to support tumor progression and metastasis. Fibronectin (FN) assembly is an important step in the regulation of the tumor microenvironment since the FN matrix precedes the deposition of various other ECM proteins, controls immune cell infiltration, and serves as a reservoir for cytokines and growth factors. Therefore, FN is an attractive target for breast cancer therapy and imaging.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature.

Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC.

Stromal Characteristics and Impact on New Therapies for Metastatic Triple-Negative Breast Cancer.

The heterogenous nature of triple-negative breast cancer (TNBC) is an underlying factor in therapy resistance, metastasis, and overall poor patient outcome. The lack of hormone and growth factor receptors lends to the use of chemotherapy as the first-line treatment for TNBC. However, the failure of chemotherapy demonstrates the need to develop novel immunotherapies, antibody-drug conjugates (ADCs), and other tumor- and stromal-targeted therapeutics for TNBC patients.

Primary head and neck tumour-derived fibroblasts promote lymphangiogenesis in a lymphatic organotypic co-culture model.

Background: In head and neck cancer, intratumour lymphatic density and tumour lymphangiogenesis have been correlated with lymphatic metastasis, making lymphangiogenesis a promising therapeutic target. However, inter-patient tumour heterogeneity makes it challenging to predict tumour progression and lymph node metastasis. Understanding the lymphangiogenic-promoting factors leading to metastasis (e.

Reduced synchroneity of intra-islet Ca oscillations in vivo in -deficient β cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes and are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca oscillations among β cells in vivo.

Effect of matrix heterogeneity on cell mechanosensing.

Cells sense mechanical signals within the extracellular matrix, the most familiar being stiffness, but matrix stiffness cannot be simply described by a single value. Randomness in matrix structure causes stiffness at the scale of a cell to vary by more than an order of magnitude. Additionally, the extracellular matrix contains ducts, blood vessels, and, in cancer or fibrosis, regions with abnormally high stiffness.

Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers.

It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined.

Hyperpolarized C Magnetic Resonance Spectroscopic Imaging of Pyruvate Metabolism in Murine Breast Cancer Models of Different Metastatic Potential.

This study uses dynamic hyperpolarized [1-C]pyruvate magnetic resonance spectroscopic imaging (MRSI) to estimate differences in glycolytic metabolism between highly metastatic (4T1, = 7) and metastatically dormant (4T07, = 7) murine breast cancer models. The apparent conversion rate of pyruvate-to-lactate (k) and lactate-to-pyruvate area-under-the-curve ratio (AUC) were estimated from the metabolite images and compared with biochemical metabolic measures and immunohistochemistry (IHC). A non-significant trend of increasing k ( = 0.

Elucidating cancer-vascular paracrine signaling using a human organotypic breast cancer cell extravasation model.

In cancer metastasis, extravasation refers to the process where tumor cells exit the bloodstream by crossing the endothelium and invade the surrounding tissue. Tumor cells engage in complex crosstalk with other active players such as the endothelium leading to changes in functional behavior that exert pro-extravasation effects. Most in vitro studies to date have only focused on the independent effects of molecular targets on the functional changes of cancer cell extravasation behavior.

Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

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.

Biomechanical Contributions to Macrophage Activation in the Tumor Microenvironment.

Alterations in extracellular matrix composition and organization are known to promote tumor growth and metastatic progression in breast cancer through interactions with tumor cells as well as stromal cell populations. Macrophages display a spectrum of behaviors from tumor-suppressive to tumor-promoting, and their function is spatially and temporally dependent upon integrated signals from the tumor microenvironment including, but not limited to, cytokines, metabolites, and hypoxia. Through years of investigation, the specific biochemical cues that recruit and activate tumor-promoting macrophage functions within the tumor microenvironment are becoming clear.

Breast Fibroblasts and ECM Components Modulate Breast Cancer Cell Migration Through the Secretion of MMPs in a 3D Microfluidic Co-Culture Model.

The extracellular matrix (ECM) composition greatly influences cancer progression, leading to differential invasion, migration, and metastatic potential. In breast cancer, ECM components, such as fibroblasts and ECM proteins, have the potential to alter cancer cell migration. However, the lack of in vitro migration models that can vary ECM composition limits our knowledge of how specific ECM components contribute to cancer progression.

A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells.

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells.

Matrix density drives 3D organotypic lymphatic vessel activation in a microfluidic model of the breast tumor microenvironment.

Lymphatic vessels (LVs) have been suggested as a preferential conduit for metastatic progression in breast cancer, where a correlation between the occurrence of lymph node metastasis and an increased extracellular matrix (ECM) density has been reported. However, the effect of ECM density on LV function is largely unknown. To better understand these effects, we used a microfluidic device to recreate tubular LVs in a collagen type I matrix.