PV-10 triggers immunogenic cell death in head and neck squamous cell carcinoma via endoplasmic reticulum stress and apoptosis
Primary risk factors of head and neck squamous cell carcinoma (HNSCC) include human papillomavirus (HPV) infection and exposure to tobacco and excessive alcohol. Despite currently available treatments, patients with recurrent HNSCC still have poor survival, highlighting the need for innovative therapies. PV-10, also known as rose bengal sodium (RBS, 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein disodium), is a small molecule being developed as an intralesional (IL) therapeutic agent, exhibiting substantial anti-tumor activity across diverse cancer types, but current knowledge regarding the molecular mechanisms involved in response to PV-10 remains limited. We evaluated the cytotoxic effects of PV-10 in HNSCC and explored its molecular mechanisms. In vitro, we found that PV-10 induced cytotoxicity in mEER and MTE-RAS cell lines, primarily by increasing the production of reactive oxygen species and leading to apoptosis through a caspase-dependent mechanism. Additionally, we observed that PV-10 treatment increased the release of damage-associated molecular pattern molecules such as HMGB1 and ATP and enhanced expression of calreticulin, HSP-70, and HSP-90 indicating potent immunogenic cell death (ICD). In vivo, IL PV-10 injections led to significant tumor regression in both mEER and MTE-Ras models. Complete responses (CR) were observed in 7 of 21 (33%) mice in mEER although no CR was observed in MTE-Ras. Our data suggests that one of the possible mechanisms by which PV-10 triggers ICD is by inducing endoplasmic reticulum stress, autophagy, and apoptosis. Our findings contribute to further understanding of the underlying mechanisms of PV-10 induced cytotoxicity and to develop future clinical trials in locally recurrent HNSCC. Overall design: RNA-sequencing and data analysis For RNA-sequencing MTE-Ras cells were injected subcutaneously into the right flank of C57BL/6 mice. Total RNA was isolated from three mouse tumors each from PBS and PV-10 treated mice using the RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer's protocol, followed by DNase treatment using RNase-Free DNase Set (Qiagen, #79254). The RNA extracted from tumors was quantitated with the Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA) and screened for quality on the Agilent TapeStation 4200 (Agilent Technologies, Santa Clara, CA). The samples were then processed for RNA-sequencing using the NuGEN Universal RNA-Seq Library Preparation Kit with NuQuant (Tecan Genomics, Redwood City, CA). Briefly, 100 ng of RNA was used to generate cDNA and a strand-specific library following the manufacturer's protocol. Quality control steps were performed, including TapeStation size assessment and quantification using the Kapa Library Quantification Kit (Roche, Wilmington, MA). The final libraries were normalized, denatured, and sequenced on the Illumina NovaSeq 6000 sequencer with the SP-200 cycle reagent kit to generate 100-150 million 100-base read pairs per sample (Illumina, Inc., San Diego, CA). All work was performed by the Moffitt Cancer Center Molecular Genomics Core Facility (RRID:SCR_012333). Read adapters were detected using BBMerge (v37.02) (RRID:SCR_016970) and subsequently removed with cutadapt (v1.8.1) (RRID:SCR_011841). Processed raw reads were then aligned to mouse genome mm10 using STAR (v2.7.7a) (RRID:SCR_004463). Gene expression was evaluated as read count at the gene level using RSEM (v1.3.0) (RRID:SCR_000262) with the Gencode gene model vM21. Normalization and differential expression analysis between the experimental and control groups were performed using DESeq2 (RRID:SCR_015687) (27). Gene set enrichment analysis (GSEA) was performed on the ranked gene list, based on fold change between experimental and control groups, using the gseGO function from the cluster Profiler R package (v 4.6.2). The analysis focused on Gene Ontology (GO) terms.