*Article* **Crotoxin Modulates Events Involved in Epithelial–Mesenchymal Transition in 3D Spheroid Model**

**Ellen Emi Kato <sup>1</sup> and Sandra Coccuzzo Sampaio 1,2,\***


**Abstract:** Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the *Crotalus durissus terrificus* venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

**Keywords:** crotoxin; epithelial–mesenchymal transition; spheroid model; tumor stroma

**Key Contribution:** We demonstrated for the first time the modulatory effect of CTX on tumor– stroma interaction in a 3D model by showing its inhibitory capacity on crucial events of EMT and provide evidences that CTX is a potential modulator of the signaling cascade involved in the tumor progression.
