How to detect and purify tenascin-C

Giblin, Sean Patrick; Murdamoothoo, Devadarssen; Deligne, Claire; Schwenzer, Anja; Orend, Gertraud; Midwood, Kim S.

doi:10.1016/bs.mcb.2017.08.022

Cited by 5 publications

(7 citation statements)

References 24 publications

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“…Recombinant his-tagged human TNC was purified as described (36,37). and used for incubation with cells.…”

Section: Tnc Cloning and Purificationmentioning

confidence: 99%

“…After 48 hours of transfection, the medium was replaced with 0.5 µg/ml containing DMEM/F12 medium with 10% FCS and the cells were grown to confluency. The protein was then purified from the supernatant by using the Streptactin matrix (IBA, Lifesciences, catalog number 2-1021-001) following the manufacturer's guidelines and was then dialyzed 3 times against PBS as previously described (37).…”

Section: Tnc Cloning and Purificationmentioning

confidence: 99%

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Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma

Spenlé

Loustau

Murdamoothoo

et al. 2020

Cancer Immunology Research

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Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood.Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin  and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune suppressive stromal properties, reducing tumor growth, progression and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors as high tenascin-C expression and an immune suppressive stroma correlate to poor patient survival.

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“…Recombinant his-tagged human TNC was purified as described (36,37). and used for incubation with cells.…”

Section: Tnc Cloning and Purificationmentioning

confidence: 99%

Section: Tnc Cloning and Purificationmentioning

confidence: 99%

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma

Spenlé

Loustau

Murdamoothoo

et al. 2020

Cancer Immunology Research

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“…His-tagged human TNC (hTNC) was purified according to published protocols [99,100] and an additional lipopolysaccharide (LPS) removal washing step using 0.1% Triton-X 114 during nickel column purification was included. Murine strep-tagged TNC (mTNC) was purified according to published protocols [43].…”

Section: Recombinant Proteinsmentioning

confidence: 99%

“…For the quality control of hTNC and mTNC, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and for hTNC, Western blotting was also performed, both as described in [99]. A NuPAGE TM 4-12% BisTris gel (Thermo Fisher) was used.…”

Section: Gel Electrophoresis Coomassie Blue Staining Western Blotmentioning

confidence: 99%

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2

Gschwandtner,

Gammage,

Deligne

et al. 2023

IJMS

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Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.

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“…Full-length TN-C and TN-W were purified as described by Giblin et al (2018) (37). Before purification, histidine-tagged full-length TN-C and TN-W were precipitated from conditioned medium by stirring 2h at 4°C in presence of 2.2 M ammonium sulfate (Sigma-Aldrich), pelleted by centrifugation at 12,000g, for 20 min at 4°C, resuspended in 50 mL of Phosphate-Buffered Saline (PBS, Euromedex) containing 0.01% Tween-20 (VWR) and dialyzed 3 times (twice for 2h and once overnight) at 4°C in PBS containing 0.01% Tween-20.…”

Section: Recombinant Protein Production Purification and Adsorptionmentioning

confidence: 99%

Latent TGF-β Activation Is a Hallmark of the Tenascin Family

et al. 2021

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Transforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

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How to detect and purify tenascin-C

Cited by 5 publications

References 24 publications

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2

Latent TGF-β Activation Is a Hallmark of the Tenascin Family

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