Novel Human Tenascin-C Function-Blocking Camel Single Domain Nanobodies.

Sayda Dhaouadi,Sandrine Bichet,Thomas Loustau,Ruth Chiquet-Ehrismann,Zakaria Benlasfar,William Erne,Matthias Mörgelin,Balkiss Bouhaouala-Zahar,Ayoub Ksouri,Chérine Abou-Faycal,Gertraud Orend,Andreas Kungl,Rahma Ben Abderrazek,Sonia Ledrappier,Alain Jung,Ismaïl Hendaoui,Devadarssen Murdamoothoo

doi:10.3389/fimmu.2021.635166

Abstract

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

Highlights

Tenascin-C (TNC), discovered over three decades ago, is one of the extracellular matrix (ECM) molecules that is highly expressed in tumors such as breast, colorectal and gastric cancers [1,2,3,4]
Because of the presence of CH1 domain in conventional antibodies (IgG1) and different hinge size in nonconventional antibodies (HCAbs), three PCR products were observed by agarose gel electrophoresis: the 900, 790, and 720 bp fragments corresponding to the VH-CH1-Hinge-CH2 of the IgG1 and the VHH-Hinge-CH2 exons of the IgG2 and IgG3, respectively (Supplementary Figure 1A)
The matrix, a highly abundant component of tumors, could be considered as a good tumor biomarker as matrix is often more stable than e.g., antigens expressed by tumor cells [70, 71]

Summary

Introduction

Tenascin-C (TNC), discovered over three decades ago, is one of the ECM molecules that is highly expressed in tumors such as breast, colorectal and gastric cancers [1,2,3,4]. High TNC expression levels correlate with shortened lung metastasis-free survival in breast cancer and overall survival in glioma patients [5, 6]. In vitro studies demonstrated that various stimuli such as EGF, TGFβ, b-FGF, and TNF-α, can induce expression of TNC in breast cancer stroma [5, 24]. EGF induced TNC via its receptor EGFR which activated oncogenic Ras signaling. Mammary tumor cells produced transforming growth factor β1 (TGFβ1), which induced TNC expression in the surrounding stroma [25, 26]. An overview of factors regulating TNC expression is presented in Giblin et al [23]

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