**3. Discussion**

The present study aimed to investigate the possible modulatory effect of crotoxin, a toxin with PLA2 activity from the venom of the snake, *Crotalus durissus terrificus*, on EMT. Here, we used an in vitro spheroid model composed of human lung adenocarcinoma and human lung fibroblast cell lines. This model resembles early tumor–stroma interactions, mimicking an avascular tumor initiation step [13,34,35]. During tumor spheroid formation, fibroblasts become activated and acquire a myofibroblast-like phenotype referred to cancerassociated fibroblasts. These CAFs express contractile proteins (particularly α-smooth muscle actin), synthesize a large amount of ECM components, and secrete various matrix metalloproteinases [12,36].

In this study, we used a co-culture system to understand the reciprocal crosstalk between NSCLC tumor cell line, A549, and normal human lung fibroblast cell line, MRC-5. We found that tumor-conditioned media promoted the expression of α-SMA in MRC-5 cells, whereas CTX inhibited it. It has already been reported that the TGF-β pathway is the dominant mediator of crosstalk to initiate the process of CAF activation [37]. Previous studies have demonstrated the inhibitory effect of CTX on the functions of stromal cells such as macrophages [29] and endothelial cells [33] when co-cultivated with tumor-conditioned media. Thus, CTX impairs tumor progression. A recent study demonstrated that when human skin fibroblast cells were incubated with crude venom (CdtV) from *Crotalus durissus terrificus*, cells showed altered protrusions, formed highly polymerized actin filaments, and produced a high amount of fibronectin [38]. Based on these observations, we suggest that CTX plays varied roles in different microenvironments and may regulate the process of tissue repair.

Stadler and colleagues (2018) used different colon cancer cell lines for the spheroid formation and showed that some of the cells did not integrate into the spheroids. They hypothesized that the non-spheroid forming (NSF) cells are a subpopulation of tumor cells that had lost cell–cell adhesion properties and rendered them the ability to migrate [39]. Moreover, Sodek and colleagues (2009) correlated the ability of ovarian cancer cell lines to form compact spheroids with their migratory and invading capacity in 3D matrices. These cells exhibited myofibroblast-like features [40]. Our results showed that the presence of CTX in the composite spheroid prevented the loss of cell–cell adhesion properties of the cells and reduced the invasion area in a 3D collagen matrix.

To confirm our findings, the expression of a well-defined set of EMT-associated markers was analyzed by western blotting. Three-day-old MRC-5/A549 spheroids presented an upregulation of mesenchymal markers (such as N-cadherin, α-SMA, and integrin αv) and downregulation of E-cadherin in consensus with its rapid progression toward EMT. In contrast, as shown in the Supplementary Material, MRC-5/Calu-3 spheroids presented no alterations on EMT markers at the same experimental condition. These findings concur with a previous study that showed EMT progression in A549 and Calu-3 cells in vitro when exposed to TGF-β1 and pro-inflammatory cytokines. The authors suggest that differential cell plasticity and susceptibility to EMT may depend on tissue origin [41]. As fibroblasts become CAFs during spheroid formation, the interaction between tumor cells and CAFs leads to invasion strategies; CAFs turn into primary drivers to help tumor cells migrate by remodeling ECM and creating tracks [42]. Taken together, our data suggest that CTX significantly inhibits expression of N-cadherin, α-SMA, and integrin αv in MRC-5/A549 spheroids, which correlates with the reduced invasion area in the collagen gel. It also suggests an involvement of CTX with actin polymerization via integrin-dependent signaling pathway with subsequent impairment of migratory ability, a finding that was also observed in endothelial cells in the tumor microenvironment [33]. These findings are in agreement with a similar study conducted with PLA2 (BthTX-II) extracted from the venom of *Bothrops jararacussu*. BthTX-II displayed a weak catalytic activity and presented an inhibitory effect on the adhesion, proliferation, invasion, and migration of human breast cancer cells. It also inhibited EMT by modulating epithelial and mesenchymal markers [43]. Many studies have shown the inhibitory effect of other PLA2s on tumor cells, however the findings did not correlate the effect with the enzymatic activity of the molecule. CTX acts on focal adhesion kinases (FAK), a crucial component of integrin-mediated cell signaling in endothelial cells [33], and inhibits tyrosine phosphorylation, consequently inhibiting the activity of proteins involved in the intracellular signaling pathway of macrophages [44].

Our data suggest that CTX affected MMP-9 secretions, which significantly increased during tumor–CAF crosstalk in the spheroid model. Unsurprisingly, monocultures of A549 and MRC-5 cells released endogenous levels of MMP-9. It has been shown that high expression of MMP-9 is associated with the aggressiveness of malignant cells in solid tumors [45]. MMP-9 has also been reported to activate the bioactive form of TGF-β [46] and downregulate the expression of E-cadherin [47], thus initiating the process of EMT. Moreover, Eberlein and colleagues (2015) reported that the activation of normal fibroblast during tumor cell–fibroblast crosstalk occurs through the αvβ6/TGF-β signaling pathway [37]. We hypothesize that CTX regulates TGF-β activation since our findings showed reduced secretions of MMP-9 and αv integrin in the spheroid model. Moreover, human MMP-13 is expressed in skin fibroblasts and has a role in acute wound healing by remodeling fibrillar collagens [48]. In this study, we found an increased secretion of MMP-13 in MRC-5 monoculture incubated with CTX, suggesting its involvement in wound healing. The modulatory activity of CTX during the healing process in an inflammatory environment has already been shown in an earlier study [49]. Conversely, MMP-13 secretions were reduced in A549 monoculture in the presence of CTX as well as in the heterospheroid model. A study using NSCLC from patients showed that both MMP-9 and MMP-13 were associated with metastasis, invasion, and prognosis; MMP-13 mainly activates MMP-9 to participate in the invasion and metastasis of NSCLC [50]. This corroborates our finding of the microenvironment-dependent modulatory activity of CTX on the release of MMP-9 and MMP-13.

Cytokines released during MRC-5/A549 spheroids' invasion of collagen gel have shown that CTX drastically inhibits chemokines that bind to the receptor CXCR1 and CXCR2, such as CXCL5, CXCL-8 (IL-8), CXCL1/2/3, CXCL1a, and CXCL6. It is well established that the CXCL5/CXCR2 and IL-8/CXCR1/CXCR2 axes contribute to carcinogenesis by promoting tumor cell proliferation, migration, and invasion, and the EMT process of many tumor cells, including NSCLC [50–53], hepatocarcinoma cells [54], and papillary thyroid carcinoma cells [55]. Additionally, CTX inhibits primary growth factors involved with EMT such as HGF [56], TGF-β1 [37,57], and VEGF [53].
