**1. Introduction**

Events of metastasis begin with epithelial–mesenchymal transition (EMT), whereby epithelial cells lose their inherent characteristics, including apicobasal polarity, and acquire invasive and infiltrating mesenchymal properties [1–4]. The EMT program involves the loss of expression of E-cadherin. In contrast, the expression of N-cadherin is switched on along with upregulation of other mesenchymal markers, including vimentin, fibronectin, and metalloproteases [5–9]. Cancer-associated fibroblasts (CAFs), activated fibroblasts commonly found in the tumor microenvironment, are characterized by higher expression of myofibroblastic markers, including α-smooth muscle actin (α-SMA). CAFs are among the predominant cells within solid tumors and secrete several soluble growth factors, including transforming growth factor-β1 (TGF-β1), interleukin-6 (IL-6), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), stromal cell-derived factor-1 (SDF-1), and hepatocyte growth factor (HGF). Some of these growth mediators can induce EMT in carcinoma cells [10–12].

The majority of data that highlight the importance of EMT in carcinogenesis have been obtained using in vitro cell line models. Over the last decade, three-dimensional (3D) cancer cell culture systems in vitro have been developed to understand the interactions

**Citation:** Kato, E.E.; Sampaio, S.C. Crotoxin Modulates Events Involved in Epithelial–Mesenchymal Transition in 3D Spheroid Model. *Toxins* **2021**, *13*, 830. https://doi.org/10.3390/ toxins13110830

Received: 15 October 2021 Accepted: 19 November 2021 Published: 22 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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:// creativecommons.org/licenses/by/ 4.0/).

and crosstalk between cell–cell and cell–matrix that drives tumor progression [13–15]. The activation of EMT is mostly studied in various cancer spheroid models [16]. Therefore, the development of novel drugs that can inhibit the onset of EMT is one of the goals of cancer research. In this direction, several compounds derived from animal venoms are considered critical scientific tools. Firstly, animal venoms have practical therapeutic applications as they provide structural templates for the development of new drugs. Secondly, studies on animal venoms have contributed significantly to understanding the regulatory mechanisms guiding cell functions under normal and pathophysiological conditions, such as cancer [17].

Snake venoms are complex mixtures of bioactive molecules [17–19]. Phospholipase A2 enzymes (PLA2; EC 3.1.1.4) are among the most well-characterized components of known snake venoms. Among these, crotoxin (CTX), the major toxin from *Crotalus durissus terrificus* venom, is a heterodimer comprised of a basic subunit (CB), responsible for the phospholipase activity, and neurotoxic and myotoxic properties of the molecule. The CB subunit is associated non-covalently with crotapotin, an acidic, non-enzymatic peptide (CA). The CTX complex is a potent neurotoxin, while isolated subunits present low lethality [20–25]. Sixteen CTX isoforms were identified, resulting from a random combination of four CA isoforms (CA1, CA2, CA3, and CA4) and four CB isoforms (CBa2, CBb, CBc, and CBd) [22]. These combinations between the isoforms determine the formation of different complexes, responsible for the different pharmacological and biological properties reported for CTX [21]. Several studies have shown that CTX has in vivo and in vitro anti-inflammatory, immunomodulatory, and antitumoral properties [26–31]. Cura and colleagues (2002) suggested that CTX may have greater selectivity on solid tumors since CTX could inhibit in vivo growth of Lewis lung carcinoma and MX-I human mammary carcinoma. However, it has low antitumor activity against HL-60 leukemia cells [32]. Recent studies have demonstrated that CTX not only inhibits tumor growth but also modulates stromal cells in the tumor microenvironment, such as the reprogramming of endothelial cells and macrophages, thus exhibiting an antiangiogenic phenotype [26,28,29,33].

Based on the properties mentioned above of CTX on the tumor microenvironment we also present, herein, the ability of this toxin to modulate EMT. In this study, we show for the first time, the modulatory effect of CTX on EMT markers in the 3D-spheroid model composed of tumor cells and fibroblasts. CTX is a promising target for the future development of anti-metastasis therapeutics.
