Intratumor (IT) inoculation of the rhino:poliovirus chimera, PVSRIPO, yielded objective radiographic responses with long-term survival in 20% of patients with recurrent glioblastoma (rGBM). PVSRIPO infects dendritic cells (DCs) and sets up non-cytopathogenic viral (v)RNA replication, which triggers sustained type-I IFN (IFN-I) signaling and antitumor T cell priming. Here we identify IFN-I signaling in glioma-draining cervical lymph nodes (cLN) as a mediator of polio virotherapy. Transient IFN-I signaling after IT therapy was rescued by cervical perilymphatic injection (CPLI) of PVSRIPO, targeting cLN directly. Dual-site (IT+CPLI) PVSRIPO induced profound inflammatory reprogramming of cLN, enhanced vRNA replication and IFN-I signaling in DCs and High Endothelial Venules (HEV), augmented anti-glioma efficacy in mice, and was associated with T cell activation in rGBM patients. A Ph2 clinical trial of IT+CPLI PVSRIPO is ongoing (NCT06177964). This work implicates the lymphatic system as a novel virotherapy target and demonstrates the CPLI concept to complement brain tumor immunotherapy.

G.P.C., M.G., M.C.B., A.D., D.D.B., and D.M.A own intellectual property related to PVSRIPO. All other authors declare they have no competing interests.

IND29534 IND29868 IND30411 NCT06177964

This work was supported by PHS grants R01 NS108773, R01 CA281320 (M.G.), and R00 CA263021 (M.B.). Microscopy analyses were supported by PHS grant 1S10-OD034340-01A1 to the Duke University School of Medicine Light Microscopy Core Facility.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Institutional Review Board (IRB) of Duke University School of Medicine gave ethical approval for this work (Pro00114031; Pro00113348; Pro00115266).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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All data produced in the present study are contained in the manuscript and transcriptomic raw data will be shared via a public repository.