CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma

Abstract

Background Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. Methods T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. Results First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. Conclusions Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.

GSDMD-dependent pyroptotic induction by a multivalent CXCR4-targeted nanotoxin blocks colorectal cancer metastases

“…No differences in histology were observed between control buffer-treated mice and T22-PE24-H6-treated mice in these organs. This lack of toxicity was previously found in the same organs, including normal colon, kidney, liver, and spleen or bone marrow, in a lymphoma model (Falgàs et al., 2020 ) and in a head and neck cancer model (Rioja-Blanco et al., 2022 ) treated with the same T22-PE26-H6 dosage. Consequently, the glomeruli and surrounding renal tubules were clearly visible and presented no cytoplasmic vacuolation or eosinophilic protein accumulation.…”

A Novel CXCR4-Targeted Diphtheria Toxin Nanoparticle Inhibits Invasion and Metastatic Dissemination in a Head and Neck Squamous Cell Carcinoma Mouse Model

“…In this framework, the T22-DITOX-H6 nanotoxin represents a promising approach for HNSCC treatment, as it aims to deliver cytotoxic compounds exclusively to CXCR4 + cancer cells. Our previous work demonstrated the selective accumulation of nanoparticles in CXCR4-overexpressing tumor tissues [21], together with a CXCR4dependent cytotoxic effect and a potent antitumor effect in vivo [22]. Here, we demonstrate, for the first time, that the T22-DITOX-H6 nanotoxin induces potent anti-invasive and antimetastatic effects in vivo.…”

“…The detection of these CXCR4 + tumor cells in the invasive front of the primary tumors exhorted us to investigate the potential anti-invasive effect of the T22-DITOX-H6 nanotoxin. T22-DITOX-H6 includes the CXCR4 ligand T22, fused to the cytotoxic domain of the diphtheria toxin, which is able to selectively internalize and eliminate CXCR4 + HNSCC cancer cells [21,22]. In this context, we generated an orthotopic HNSCC mouse model through the inoculation of the 74B-Luci cells in the mouse tongues.…”

The current status of nanotechnological approaches to therapy and drug delivery in otolaryngology: A contemporary review

“…These technologies not only allow for the localization of NPs to tumor cells but also the release of chemotherapies and subsequent cellular destruction through PDT. There is additional work looking at the integration of nanotechnology with bacterial machinery using synthetic biology, 64 and, more recently, the incorporation of bacterial toxins into protein NPs to target HNSCCs 65 . Rioja‐Blanco et al 65 describe the incorporation of Pseudomonas aeruginosa exotoxin A and the diphtheria exotoxin from Corynebacterium diphtheriae into protein NPs to target HNSCCs.…”