Oncolytic HSV-1 armed with CXCR4-antagonist interferes with glioblastoma stem-like cells properties and disrupt pro-tumoral microenvironment

Glioblastoma is the most frequent and aggressive primary brain cancer in adults, with a
median overall survival of approximately 15 months. Despite extensive research e􀆯orts,
no major therapeutic breakthrough has significantly improved patient prognosis over the
past 25 years.
CXCR4/CXCL12 pathway plays a major role in glioblastoma aggressiveness, resistance to
therapy, and recurrence. It promotes glioblastoma stem-like cells (GSCs) stemness and
self-renewal, tumour invasiveness, angiogenesis, and the establishment of an
immunosuppressive tumour microenvironment.
Virotherapy is a promising therapeutic strategy within the field of immunotherapy, which
has shown success in several cancers but limited e􀆯icacy in glioblastoma. Oncolytic
herpes simplex viruses (oHSVs) can selectively kill tumour cells while sparing healthy
tissue thereby triggering a local immune response. Importantly, these viruses can also be
“armed” with transgenes.
We engineered an oHSV expressing the CXCL12 antagonist CXCL12-P2G (oHSV/P2G) to
simultaneously inhibit multiple pathways involved in tumour progression while
stimulating anti-tumour immunity. In vitro, we confirmed P2G expression by oHSV/P2G
infected cells, demonstrated inhibition of CXCR4 downstream signalling, and reduced
CXCR4-dependent GSC stemness. Inhibition of invasiveness by oHSV/P2G was assessed
both in vitro and in immunodeficient (athymic) orthotopic xenograft NUDE mouse models
engrafted with human glioblastoma patient derived cell lines (GB138, T033).
In immunocompetent orthotopic syngeneic C57Bl6 mouse models engrafted with murine
glioblastoma cell lines (GL261N4, 005-GSCs), oHSV/P2G triggered a strong immune
response while impairing tumour angiogenesis. CXCR4⁺ tumour-associated
macrophages and microglia were significantly polarised to a pro-inflammatory
phenotype, CXCR4⁺ regulatory T cells were reduced, and other recruited lymphocytes (T
and B cells) were increased. This resulted in a myeloid-to-lymphoid transition of the
tumour microenvironment, associated with good prognosis. Notably, an indirect e􀆯ect
also promoted Th1 and cytotoxic profiles in CD4⁺ and CD8⁺ T cells, respectively, even
though these populations exhibit low CXCR4 expression. Importantly, in poorly
immunogenic mouse models (005-GSCs), oHSV alone did not improve median survival
compared with controls, whereas oHSV/P2G significantly prolonged survival.
Future studies should investigate combination strategies with immune checkpoint
inhibitors. Other immune populations, including dendritic cells, neutrophils, and natural
killer cells, should also be examined, along with the specificity and memory development
of the adaptive immune response. Finally, since virotherapy is currently considered a
second-line treatment and the CXCR4/CXCL12 pathway contributes to tumour
recurrence, relevant models should also be explored.