OX-40 Signaling Promotes Tumorigenesis in CTCL by Regulating ERK Activation

Evangelia Papadavid¹Karagianni Fani¹Eleni-Kyriaki Vetsika²Sara Valero-Diaz³Saire Edith Cordova-Hernandez³Christos Daniil¹Christina Piperi^1,4Berta Casar Martinez^3,5*

• ¹Center for Excellence on Rare Hematological Diseases of Children and Adults - Cutaneous Lymphoma University General Hospital "ATTIKON" , National and Kapodistrian University of Athens, Athens, Greece
• ²Centre of New Biotechnologies and Precision Medicine (CNBPM) School of Medicine, National and Kapodistrian University of Athens, Athens, Greece
• ³Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas (CSIC) -Universidad de Cantabria, Cantabria, Spain
• ⁴Department of Biological Chemistry, National and Kapodistrian University of Athens, Athens, Greece
• ⁵Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), Instituto de Salud Carlos III, Madrid, Madrid, Spain

Introduction: In Cutaneous T-cell Lymphoma (CTCL), T cells can be activated either by cytokines produced by malignant T cells or through immunological synapses, such as the interaction between OX-40 and OX-40L on dendritic cells. Both are co-expressed in tumor cells in Mycosis fungoides/Sézary syndrome and correlate with disease severity markers. Using a model of spontaneous metastasis in chick embryos, the present study aimed to determine the functional role of OX-40 in CTCL and assess its potential as a therapeutic target. Methods: OX-40 knockout MyLa and SeAx CTCL cells using CRISPR-Cas9 were engrafted onto the chorioallantoic membrane of chick embryos. We assessed tumor growth, dissemination, and TME modulation in the presence or absence of macrophages. Transwell-based transendothelial migration assays and co-culture experiments were performed to further explore the interactions between CTCL cells and macrophages. Angiogenesis and lymphangiogenesis have also been investigated. Results: OX-40 expression promoted intravasation, metastasis, and cytokine secretion, and increased M2 macrophages. Additionally, it restores transendothelial migration and dissemination in the presence of M2 macrophages, possibly through ERK activation. Co-culture experiments revealed that OX-40 promoted a Th2 cytokine profile in CTCL, correlating with M2 macrophages in xenografts. Although OX-40 did not affect angiogenesis in this model, it promoted lymphangiogenesis via VEGF-C expression. Discussion: Using the CTCL spontaneous metastasis model in chick embryos, we demonstrated that OX-40 regulates the TME to promote M2 increase, lymphangiogenesis, CAM intravasation, and metastasis. Therefore, the in vivo chick embryo metastasis model may serve as a valuable preclinical tool for identifying novel anti-tumor targets in CTCL. The OX-40 axis was identified as a key driver of CTCL progression, promoting tumor growth and metastasis through ERK activation while validating the chick embryo model as a preclinical tool for therapeutic testing.