Retrograde tracing of breast cancer-associated sensory neurons.

Breast cancer is one of the leading causes of mortality among women. The tumour microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumour progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons, which innervate and interact with breast tumours, are unknown. To establish the tools for labelling and subtyping neurons that interact with breast cancer cells, we utilised two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro, we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalisation of the sensory neuron's soma and axons. In vivo, we utilised both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labelling CGRP+ neurons, CTB is superior in labelling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo. In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumour innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons. Lay description: Breast cancer is an aggressive disease that affects both women and men throughout the world. While it has been reported that the increasing size of nerves in breast cancer correlates to bad prognosis in patients, the role of nerves, especially sensory nerves, in breast cancer progression, has remained largely understudied. Sensory nerves are responsible for delivering signals such as pain, mechanical forces (pressure, tension, stretch, touch) and temperature to the brain. The human body is densely innervated, and nerves extending into peripheral organs can be as long as a few meters. Nerve classification and function can be very complex, as they contain bundles of extensions (axons) originating in different neuronal bodies (soma). Maintaining neurons and growing axons in cell culture conditions in order to mimic innervation is technically challenging, as it involves multiple organs of the human body. Here, we focus on tracing sensory axons from the breast tumours back to the neuronal soma, located in the dorsal root ganglia, inside the spine. To do so, we are using two different 'retrograde' tracers, WGA and CTB, which are proteins with a natural ability to enter axons and travel in a retrograde fashion, arriving at the soma, even if it means to travel distances longer than a meter. Both tracers are fluorescently labelled, making them visible using high-resolution fluorescent microscopy. We show that both WGA and CTB can label sensory neurons in tumours, or in cell culture conditions. The two tracers differ in efficiency of tracing different sensory neurons subpopulations: while WGA is more efficient in tracing small C-fibres (CGRP-positive), CTB is more efficient in tracing A-fibres (NF200+) of sensory neurons. In summary, we have successfully established retrograde tracing techniques for sensory neurons towards studying and targeting breast cancer innervation.

Retrograde tracing of breast cancer-associated sensory neurons.

Breast cancer is one of the leading causes of mortality among women. The tumor microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumor progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons which innervate and interact with breast tumors are unknown. To establish the tools for labeling and subtyping neurons that interact with breast cancer cells, we utilized two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro , we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalization of the sensory neuron's soma and axons. In vivo , we utilized both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labeling CGRP+ neurons, CTB is superior in labeling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo . In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumor innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons.

DACIT: Device for Axon - Cancer cell Interaction Testing in 2D and 3D.

In this protocol, we describe steps to design, fabricate and use the Device for Axon and Cancer cell Interaction Testing (DACIT) in 2D and in 3D. In the first section, we detail steps to generate the mask, the master and the smooth-on mold. Next, we describe the step-by-step protocol for fabricating the DACIT, loading sensory neurons and cancer cells in 2D or 3D. We compare axonogenesis using PC-12 cell line and primary embryonic or adult sensory neurons, demonstrating the superior neurite growth in primary cells. We demonstrate DACIT can be used to compartmentalize neuronal soma and axons and expose them to different conditions, or to form a temporary gradient of neurotransmitter. Finally, we show that DACIT can be used to measure spheroid invasion in 3D in the presence of axons.

Infiltrating CD8+ T cells and M2 macrophages are retained in tumor matrix tracks enriched in low tension fibronectin fibers.

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Tenascin-C immobilizes infiltrating T lymphocytes through CXCL12 promoting breast cancer progression.

Immune checkpoint therapy, where CD8 tumor infiltrating T lymphocytes (TIL) are reactivated, is a promising anti-cancer treatment approach, yet with low response rates. The extracellular matrix, in particular tenascin-C, may generate barriers for TIL. To investigate this possibility, we used a MMTV-NeuNT and syngeneic mammary gland grafting model derived thereof with engineered tenascin-C levels and observed accumulation of CD8 TIL in tenascin-C-rich stroma. Inhibition studies revealed that tenascin-C induced CXCL12 through TLR4. By binding CXCL12, tenascin-C retained CD8 TIL in the stroma. Blockade of CXCR4, the receptor of CXCL12, enhanced macrophage and CD8 TIL infiltration and reduced tumor growth and subsequent metastasis. Retention of CD8 TIL by tenascin-C/CXCL12 was also observed in human breast cancer by tissue staining. Moreover, whereas high CD8 TIL numbers correlated with longer metastasis-free survival, this was not the case when also tenascin-C and CXCL12 levels were high. Altogether, these results may be useful for improving tumor immunity as diagnostic tool and to formulate a future "TIL-matrix-release-and-reactivate" strategy.

Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. MATERIALS AND METHODS: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. RESULTS: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with ß1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. CONCLUSION: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Matrix-Targeting Immunotherapy Controls Tumor Growth and Spread by Switching Macrophage Phenotype.

The interplay between cancer cells and immune cells is a key determinant of tumor survival. Here, we uncovered how tumors exploit the immunomodulatory properties of the extracellular matrix to create a microenvironment that enables their escape from immune surveillance. Using orthotopic grafting of mammary tumor cells in immunocompetent mice and autochthonous models of breast cancer, we discovered how tenascin-C, a matrix molecule absent from most healthy adult tissues but expressed at high levels and associated with poor patient prognosis in many solid cancers, controls the immune status of the tumor microenvironment. We found that, although host-derived tenascin-C promoted immunity via recruitment of proinflammatory, antitumoral macrophages, tumor-derived tenascin-C subverted host defense by polarizing tumor-associated macrophages toward a pathogenic, immune-suppressive phenotype. Therapeutic monoclonal antibodies that blocked tenascin-C activation of Toll-like receptor 4 reversed this phenotypic switch in vitro and reduced tumor growth and lung metastasis in vivo, providing enhanced benefit in combination with anti-PD-L1 over either treatment alone. Combined tenascin-C:macrophage gene-expression signatures delineated a significant survival benefit in people with breast cancer. These data revealed a new approach to targeting tumor-specific macrophage polarization that may be effective in controlling the growth and spread of breast tumors.

Tenascin-C increases lung metastasis by impacting blood vessel invasions.

Metastasis is a major cause of death in cancer patients. The extracellular matrix molecule tenascin-C is a known promoter of metastasis, however the underlying mechanisms are not well understood. To further analyze the impact of tenascin-C on cancer progression we generated MMTV-NeuNT mice that develop spontaneous mammary tumors, on a tenascin-C knockout background. We also developed a syngeneic orthotopic model in which tumor cells derived from a MMTV-NeuNT tumor. Tumor cells were transfected with control shRNA or with shRNA to knockdown tenascin-C expression and, were grafted into the mammary gland of immune competent, wildtype or tenascin-C knockout mice. We show that stromal-derived tenascin-C increases metastasis by reducing apoptosis and inducing the cellular plasticity of cancer cells located in pulmonary blood vessels invasions (BVI), before extravasation. We characterized BVI as organized structures of tightly packed aggregates of proliferating tumor cells with epithelial characteristics, surrounded by Fsp1+ cells, internally located platelets and, a luminal monolayer of endothelial cells. We found extracellular matrix, in particular, tenascin-C, between the stromal cells and the tumor cell cluster. In mice lacking stromal-derived tenascin-C, the organization of pulmonary BVI was significantly affected, revealing novel functions of host-derived tenascin-C in supporting the integrity of the endothelial cell coat, increasing platelet abundance, tumor cell survival, epithelial plasticity, thereby promoting overall lung metastasis. Many effects of tenascin-C observed in BVI including enhancement of cellular plasticity, survival and migration, could be explained by activation of TGF-ß signaling. Finally, in several human cancers, we also observed BVI to be surrounded by an endothelial monolayer and to express tenascin-C. Expression of tenascin-C is specific to BVI and is not observed in lymphatic vascular invasions frequent in breast cancer, which lack an endothelial lining. Given that BVI have prognostic significance for many tumor types, such as shorter cancer patient survival, increased metastasis, vessel occlusion, and organ failure, our data revealing a novel mechanism by which stromal tenascin-C promotes metastasis in human cancer, may have potential for diagnosis and therapy.

Targeting Breast Cancer Stem Cells: A Methodological Perspective.

Cancer Stem Cells (CSCs) constitute a subpopulation at the top of the tumor cell hierarchy that contributes to tumor heterogeneity and is uniquely capable of seeding new tumors. Because of their biological properties, CSCs have been pointed out as therapeutic targets for the development of new therapies against breast cancer. The identification of drugs that selectively target breast CSCs requires a clear understanding of their biological functions and the experimental methods to evaluate such hallmarks. Herein, we review the methods to study breast CSCs properties and discuss their value in the preclinical evaluation of CSC-targeting drugs.

Tenascin-C Orchestrates Glioblastoma Angiogenesis by Modulation of Pro- and Anti-angiogenic Signaling.

High expression of the extracellular matrix component tenascin-C in the tumor microenvironment correlates with decreased patient survival. Tenascin-C promotes cancer progression and a disrupted tumor vasculature through an unclear mechanism. Here, we examine the angiomodulatory role of tenascin-C. We find that direct contact of endothelial cells with tenascin-C disrupts actin polymerization, resulting in cytoplasmic retention of the transcriptional coactivator YAP. Tenascin-C also downregulates YAP pro-angiogenic target genes, thus reducing endothelial cell survival, proliferation, and tubulogenesis. Glioblastoma cells exposed to tenascin-C secrete pro-angiogenic factors that promote endothelial cell survival and tubulogenesis. Proteomic analysis of their secretome reveals a signature, including ephrin-B2, that predicts decreased survival of glioma patients. We find that ephrin-B2 is an important pro-angiogenic tenascin-C effector. Thus, we demonstrate dual activities for tenascin-C in glioblastoma angiogenesis and uncover potential targeting and prediction opportunities.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibits breast tumor growth and metastasis.

Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.

One-oligonucleotide method for constructing vectors for RNA interference.

AIM: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. METHODS: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP, were cloned into plasmids driven by RNA polymerase III promoter H1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. RESULTS: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. CONCLUSION: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.