{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T08:58:59Z","timestamp":1780909139941,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,4]]},"group-title":"Cancer Biology","reference-count":38,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T00:00:00Z","timestamp":1780531200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institute of Health","award":["R21NS130429"],"award-info":[{"award-number":["R21NS130429"]}]},{"name":"Alex Lemonade Stand Foundation Reach Grant","award":["ALEX23-27891"],"award-info":[{"award-number":["ALEX23-27891"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Glioblastoma (GBM) is a highly aggressive type of glioma that is resistant to immunotherapy and is associated with poor prognosis, largely due to its immunosuppressive tumor microenvironment. Bruton\u2019s tyrosine kinase (BTK) is a non-receptor kinase that not only plays an important role in oncogenic signaling, particularly in tumor growth, but also regulates the activity of tumor-infiltrating myeloid cells, including dendritic cells, macrophages, and microglia in brain tumors. High BTK expression is associated with poor survival in patients with glioma. Oncolytic herpes simplex virus type 1 (oHSV)-derived virotherapy, a novel treatment strategy, has demonstrated effectiveness against GBM; however, its efficacy is limited by the tumor microenvironment. In this study, we found that BTK is predominantly expressed in GBM-infiltrating myeloid cells. Intratumoral injection of oHSV not only promotes infiltration of myeloid cells and T cells but also activates BTK in these myeloid cells, thereby limiting oHSV infection and replication in tumor cells. Combination treatment with BTK inhibitor ibrutinib improves anti-tumor efficacy of oHSV in both human GBM12 xenograft and syngeneic murine GSC005 models. Mechanistically, BTK inhibition increases oHSV-mediated tumor cell death (cleaved caspase-3) and cytotoxic CD8\u207a T cell infiltration, while decreasing tumor cell proliferation (Ki-67). BTK inhibition not only suppresses oHSV clearance by tumor-infiltrating microglia and macrophages but also reduces their pro-invasive effects on tumor cells. Addition of IDO inhibitor, an immune modulator, further prolongs survival in tumor-bearing mice in a syngeneic GBM model. Single-cell mRNA sequencing (scRNA-seq) analysis indicates that combination treatment modifies key signaling pathways in both tumor-infiltrating myeloid cells (macrophages and microglia) and CD8\u207a T cells. Further analysis shows that BTK inhibition, with or without IDO inhibition, promotes the formation of tumor-infiltrating tertiary lymphoid structures (TLS) during intratumoral oHSV treatment, subsequently remodeling T cell, NKT cell, and monocyte\u2013macrophage populations. These results indicate that BTK inhibition exerts multifaceted effects in enhancing the anti-tumor efficacy of oHSV therapy.<\/jats:p>","DOI":"10.64898\/2026.06.01.729275","type":"posted-content","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T21:50:11Z","timestamp":1780609811000},"source":"Crossref","is-referenced-by-count":0,"title":["BTK inhibition enhances immunovirotherapy in glioblastoma via tertiary lymphoid structure modulation"],"prefix":"10.64898","author":[{"given":"Konstantina","family":"Kyritsi","sequence":"first","affiliation":[{"name":"Department of Pathology, Georgia Cancer Center at Augusta University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Cell Biology & Anatomy, Georgia Cancer Center at Augusta 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