In a recent study published in Communications Biology, researchers evaluated the therapeutic efficacy of Navoximod, combined with herpes simplex 1 (HSV-1) virotherapy, against hepatocellular carcinoma (HCC).

Study: Navoximod modulates local HSV-1 replication to reshape tumor immune microenvironment for enhanced immunotherapy via an injectable hydrogel. Image Credit: PaeGAG/Shutterstock.com

Background

Cancer immunotherapy has attained enormous success in treating various cancers. Nonetheless, HCC threatens the efficacy of immunotherapeutics as a result of low response rates and antagonistic outcomes due to complicated and potently immunosuppressive tumour microenvironment (TME). Thus, novel strategies are required to enhance HCC outcomes.

Oncolytic virotherapy can destroy tumour cells and generate generalized anti-tumour immune responses. Nonetheless, it has limited efficacy amongst humans as a result of impaired viral replication and the lack to beat TME.

Anti-tumour immunological activation and antiviral immunological suppression have to be rigorously modulated and balanced to optimize virus-based therapies.

The indoleamine 2, 3-dioxygenase 1 (IDO1) protein is a critical immunosuppressive molecule that’s reportedly upregulated in multiple tumours. IDOI inhibits cytotoxic [natural killer (NK) and cluster of differentiation 8-expressing (CD8+)] T and lymphocytes, improves regulatory T cell (Treg) activation, and facilitates myeloid-derived suppressor cell (MDSC) recruitment by accelerating the tryptophan to kynurenine conversion. Studies have reported IDO1 inhibitors as effective options for immunotherapy.

In regards to the study

The current study investigated whether Navoximod, an indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitor, could boost HSV-1-based therapy and improve HCC outcomes.

The team loaded Navoximod and HSV-1 in an injectable and biocompatible hydrogel for local-level drug delivery and maximized viral distribution for HCC for virotherapy. They performed single-cell ribonucleic acid sequencing (scRNA-seq) to evaluate immunological profiles on the tumour site.

The study then explored interferon-stimulated genes (ISGs) that inhibit HSV-1 proliferation to optimize oncolytic immunotherapy based on HSV-1.

Green fluorescent protein (GFP) signals generated from GFP-tagged HSV-1 were also used to guage HSV-1 multiplication. As well as, quantitative polymerase chain response (qPCR) and western blot analyses were performed to evaluate IDO1 levels in Hepa1-6 cells following HSV-1 treatment.

The impact of Navoximod on HSV-1 multiplication was verified using SMMC-7721 (human hepatocellular carcinoma) cells, 4T1 (murine breast cancer) cells, and in vivo using the rabbit VX-2 liver cancer model.

Silk-hydrogels were generated from cocoons to deliver the oncolytic virus. Scanning electron microscopy (SEM) and confocal microscopy (CFM) were performed to guage hydrogel porosity and virion distribution.

The team measured HSV-1 deoxyribonucleic acid (DNA) levels within the nervous system, blood, and peripherally situated organs following subcutaneous hydrogel injections to evaluate their cytotoxicity.

The tumour sections were stained using hematoxylin and eosin (H&E), TUNEL, and Ki67 stains to evaluate cytolytic damage to tumoral tissues. Flow cytometry (FC) evaluation and immunohistochemistry (IHC) staining were performed to substantiate the systemic changes of TME.

Results

The RNA-seq findings indicated that IDO1 was overexpressed in HCC, based on the Cancer Genome Atlas (TCGA) dataset. Navoximod promoted HSV-1 multiplication and herpes simplex virus-1-induced oncolysis in tumour cells and, subsequently, might be an efficient therapeutic option when combined with herpes simplex virus 1-based viral oncolytic therapy.

With a single injection, the hydrogel developed a regional depot to maximise viral reproduction and dissemination on the tumour location.

Of note, the hydrogel prolonged the disease-free life period of mice with HCC, protected them against tumour reoccurrence, and was effective within the rabbit orthotopic hepatic tumour model. Mechanistically, HSV-1 virotherapy and IDO1 inhibition, when combined, entirely reprogrammed the tumour microenvironment via scRNA sequencing.

The hydrogel served as a reservoir for regional delivery, allowing HSV-1 to take care of its cytotoxic activity and spread on the tumour site while reducing off-target harm to peripheral organs. The strategy produced complete responses to therapy in HCC subcutaneous malignancies, but HSV-1 or HSV-1/Navoximod mixes without being incorporated into the hydrogels produced only slight responses.

The hydrogel could counteract the immunosuppressive tumour microenvironment by modifying the immune cell population predominantly comprising effector memory (EM) and NK T lymphocyte accumulation. IDO1 upregulation inhibited HSV-1 proliferation, as indicated by the HSV DNA and gD envelope protein levels.

The outcomes were consistently observed using 4T1 and SMMC-7721 cells. IDO1 was found to be overproduced within the tumoural tissues and further increased by herpes simplex virus-1 therapy, acting as a type of negative feedback to inhibit herpes simplex virus-1 proliferation in cancer cells.

Myeloid-derived cells (M1 macrophages, monocytes, and dendritic cells) and T/NK cells predominantly infiltrated the tumour site.

Reverse transcription PCR indicated that the hydrogel treatment upregulated CC chemokine ligand 2 (CCL-2) and C-X-C motif ligand (CXCL)-10 and-11 genes, following the gene ontology (GO) enrichment evaluation results. IDO1 suppressed anti-tumour immunity, implying that IDO1 have to be blocked during herpes simplex virus-1-based immunotherapy.

The hydrogel rapidly eradicated tumour reoccurrence by inducing a conversion from naïve and central memory (CM) T lymphocytes to effector memory (EM) T lymphocytes. Given its efficacy, biosafety, and low systemic toxicity, it demonstrated promising potential as an oncolytic virus delivery route.

Conclusion

The study findings showed that combining Navoximod with HSV-1 could boost viral replication and reshape TME for tumour eradication through the hydrogel reservoir.

Navoximod strengthened the oncolytic and replication effects of herpes simplex virus-1 in vitro and in vivo and have to be investigated further to develop novel strategies and reduce the worldwide cancer burden.