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Compared with conventional treatments, bacterial-derived compounds may provide improved specificity with fewer off-target effects. This study examines soluble bacterial toxins as candidates for squamous cell carcinoma (SCC) therapy. 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J. Phillips 4 and David L. Becker 1,2,5, *  \nCitation: Ong, Z.X.; Kannan, B.; Phillips, A.R.J.; Becker, D.L. Investigation of Staphylococcus aureus Biofilm-Associated Toxin as a Potential Squamous Cell Carcinoma Therapeutic. Microorganisms 2024, 12, 293. [https://doi.org/10.3390/](https://doi.org/10.3390/)[ ](https://doi.org/10.3390/)microorganisms12020293  \nAcademic Editor: Roberta Gaziano  \nReceived: 27 December 2023  \nRevised: 17 January 2024  \nAccepted: 17 January 2024  \nPublished: 30 January 2024  \nCopyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ([https://](https://)[ ](https://)[creativecommons.org/licenses/by/](creativecommons.org/licenses/by/)[ ](creativecommons.org/licenses/by/)[4.0/](4.0/)) .  \n1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore  \n2 Skin Research Institute Singapore, Singapore 308232, Singapore  \n3 Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate Programme, Nanyang Technological University, Singapore 639798, Singapore  \n4 School of Biological Sciences, Auckland University, Auckland 1030, New Zealand; [a.phillips@auckland.ac.nz](a.phillips@auckland.ac.nz)  \n5 National Skin Centre, Singapore 308205, Singapore  \n* Correspondence: [david.becker@ntu.edu.sg](david.becker@ntu.edu.sg)  \nAbstract: Cancer therapies developed using bacteria and their components have been around since the 19th century. Compared to traditional cancer treatments, the use of bacteria-derived compounds as cancer therapeutics could offer a higher degree of specificity, with minimal off-target effects. Here, we explored the use of soluble bacteria-derived toxins as a potential squamous cell carcinoma (SCC) therapeutic. We optimized a protocol to generate Staphylococcus aureus biofilm-conditioned media (BCM), where soluble bacterial products enriched in the development of biofilms were isolated from a bacterial culture and applied to SCC cell lines. Bioactive components of S. aureus ATCC 29213 (SA29213) BCM display selective toxicity towards cancerous human skin SCC-12 at low doses, while non-cancerous human keratinocyte HaCaT and fibroblast BJ-5ta are minimally affected. SA29213 BCM treatment causes DNA damage to SCC-12 and initiates Caspase 3-dependent-regulated cell death. The use of the novel SA29213 bursa aurealis transposon mutant library led to the identification of S. aureus alpha hemolysin as the main bioactive compound responsible for the observed SCC- 12-specific toxicity. The antibody neutralisation of Hla eradicates the cytotoxicity of SA29213 BCM towards SCC-12 . Hla displays high SCC-12-specific toxicity, which is exerted primarily through Hla-ADAM10 interaction, Hla oligomerisation, and pore formation. The high target specificity and potential to cause cell death in a controlled manner highlight SA29213 Hla as a good candidate as an alternative SCC therapeutic.  \nKeywords: squamous cell carcinoma; Staphylococcus aureus; biofilm-conditioned media; transposon mutant library; selective toxin; hemolysin  \n1. Introduction  \nSquamous cell carcinoma (SCC) is the cancer of squamous epidermal keratinocytesand adnexal structures and usually develops on sun-exposed skin, such as the head and neck. Cutaneous SCC accounts for approximately 20% of all skin cancers [1] and reportedly affects approximately 2.2 million people at any one time [2] . Although SCC is second to basal cell carcinoma (BCC) in prevalence amongst non-melanoma skin cancers (NMSCs) [3,4], it is more likely to metastasize and cause mortality [5] . One of the primary causes of DNA damage in SCC is ultraviolet B (UVB) radiation [6,7] . Persons of li","cbCaisv3VpLmudHb","https://ap.wps.com/l/cbCaisv3VpLmudHb","pdf",3556365,21,"English","# Abstract\n# Introduction\n## SCC background and risk factors\n## Current treatment options and limitations\n## Rationale for bacterial-derived compounds","[{\"question\":\"What is the document’s main therapeutic focus for squamous cell carcinoma (SCC)?\",\"answer\":\"It investigates soluble Staphylococcus aureus biofilm-associated toxins as a potential SCC therapeutic candidate, aiming for selective cancer cell killing.\"},{\"question\":\"How were the Staphylococcus aureus biofilm-conditioned media (BCM) prepared and used?\",\"answer\":\"A protocol was optimized to generate S. aureus biofilm-conditioned media, isolate soluble biofilm-enriched bacterial products from culture, and apply them to SCC cell lines.\"},{\"question\":\"Why are bacterial-derived products considered as an alternative to traditional SCC treatments?\",\"answer\":\"They may offer higher target specificity compared with conventional therapies and avoid the infection risk associated with using live or attenuated bacteria.\"}]","Investigation of Staphylococcus aureus Biofilm-Associated Toxin as a Potential Squamous Cell Carcinoma Therapeutic | PDF",1789995575,53]