Abstract B34: TGF-β-rich tumor-derived exosomes promote a proangiogenic phenotype in HNSCC
Authors: Ludwig Nils , Yerneni Saigopalakrishna S. , Azambuja Juliana H. , Razzo Beatrice M. , Hinck Cynthia S. , Pietrowska Monika , Hinck Andrew , Whiteside Theresa L. 
Published: September 2020

Abstract
TGF-β is a key regulator for tumor initiation and progression in head and neck squamous cell carcinoma (HNSCC). Tumor-derived exosomes (TEX) contain TGF-β and accumulate in the tumor microenvironment (TME). This study characterizes in vitro and in vivo the TGF-β content of HNSCC-derived exosomes and evaluates TGF-β signaling by exosomes that promotes angiogenesis. TEX were isolated from supernantants of 5 different HNSCC cell lines by mini size exclusion chromatography (mini-SEC) and characterized by electron microscopy, nanoparticle tracking analysis, and immunoblotting. TGF-β content in exosomes was evaluated by mass spectrometry (LC-MS/MS). Proliferation and migration of SVEC4-10 lymphendothelial cells as well as phosphorylation of Smad2 in response to TEX were investigated in vitro. These experiments were confirmed in vivo, using a matrigel plug model in mice. A novel trivalent TGF-β receptor trap (mRER) was used to inhibit TGF-β signaling in vitro and in vivo. The 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis mouse model was used to study TGF-β signaling in the TME during all phases of carcinogenesis. Exosomes isolated from plasma of these 4-NQO mice were quantified and the exosome TGF-β content was analyzed. Another cohort of 4-NQO mice received injections of TGF-β(+) TEX at early stages of carcinogenesis. In addition, TGF-β levels and activity were measured in exosomes isolated from plasma of 20 HNSCC patients. TEX carried high levels of TGF-β and were found to be potent inducers of angiogenesis in vitro and in vivo through functional reprogramming and phenotypic modulation of endothelial cells. Proliferation (p&lt;0.01) and migration (p&lt;0.01) by SVEC4-10 were stimulated by TEX and effects were inhibited by mRER treatment of SVEC4-10 (p&lt;0.05). TEX promoted formation of defined vascular structures in vivo and increased (p&lt;0.001) vascularization in matrigel plugs relative to controls. Those effects were inhibited by mRER treatment in a dose-dependent manner (p&lt;0.001). TGF-β expression increased in 4-NQO tumor tissue during carcinogenesis (p&lt;0.01) and correlated with increasing exosome numbers in plasma. TGF-β was found to be carried by plasma-derived exosomes throughout all stages of carcinogenesis. The injection of TEX into 4-NQO mice led to a systemic immunosuppression (p&lt;0.001), increased vascularization (p&lt;0.01), and enhanced the TGF-β levels in the tumor tissue (p&lt;0.05). Exosomes in plasma of HNSCC patients carried varying levels of TGF-β, and patients with nodal metastases had higher TGF-β levels (p&lt;0.01) relative to patients with no metastasis. The data show that TGF-β signaling by TEX in HNSCC promotes angiogenesis and drives tumor progression. Silencing of TGF-β in TEX promises to add new options to existing antiangiogenic therapies.
Citation Format: Nils Ludwig, Saigopalakrishna S. Yerneni, Juliana H. Azambuja, Beatrice M. Razzo, Cynthia S. Hinck, Monika Pietrowska, Andrew Hinck, Theresa L. Whiteside. TGF-β-rich tumor-derived exosomes promote a proangiogenic phenotype in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B34.