*2.2. CTX Does Not Interfere in the Spheroid Formation and Prevents Non-Spheroid from Forming Cells*

The human adenocarcinoma cell line A549 was mixed with MRC-5 cells in a ratio of 1:4 for spheroid formation by the hanging drop method. We observed regular roundshaped spheroids composed only of MRC-5 cells (Figure 2A); the presence of CTX did not affect the spheroid formation (Figure 2B). On the other hand, A549 cells were unable to aggregate to form a spherical structure due to weak intercellular interactions (data not shown). When A549 cells were mixed with MRC-5 cells, compact spheres were observed with a small group of tumor cells around them. These tumor cells were unable to integrate into the spheres (Figure 2C,D). Interestingly, there was a drastic reduction in tumor cells around the spheroids formed by the CTX treated MRC-5 cells (Figure 2E,F). On the other hand, MRC-5/Calu-3 spheroids were less compact compared to MRC-5/A549 spheroids (Figure S2A,B), while in the presence of CTX, fewer tumor cells were observed around the spheroid (Figure S2C,D). In addition, live/dead staining of MRC-5/A549 cells in spheroids confirmed that CTX did not affect cell viability (Figure 2E,F).

**Figure 2.** Spheroid formation by MRC-5 cells alone or in combination with A549 tumor cells. (**A**) MRC-5 single spheroid formation by the hanging drop method. (**B**) MRC-5 single spheroid constitutively formed in the presence of 12.5 nM of CTX. (**C**) MRC-5/A549 spheroid formation by the hanging drop method. (**D**) A small number of cancer cells did not incorporate into the cell aggregates (white arrow) (**E**) MRC-5/A549 spheroid constituted in the presence of 12.5 nM CTX—after 24 h, cell aggregates formed compact structures. (**F**) A small number of cancer cells did not incorporate into the cell aggregates (white arrow). Images obtained from inverted microscope 4X. (**E**) Live (green)/Dead (red) image of MRC-5/A549 spheroids. Scale bar = 100 μm. (**F**) Quantitative analysis from live/dead assay measured by relative fluorescence intensity. All the data presented here are from three independent experiments.

#### *2.3. Spheroid Cell Invasion of Collagen Gel*

To analyze the effect of CTX on the invasive phenotype, MRC-5/A549 spheroids were embedded into polymerized collagen type I gels for up to 48 h. Invading cells from MRC-5/A549 spheroids were elongated and spindle-shaped (Figure 3A). Spheroids constituted in the presence of CTX showed a 50% reduction in the invaded gel region (Figure 3B,C). On the other hand, the invasion distance of MRC-5/Calu-3 spheroids was shorter and preserved cell–cell interaction compared to MRC-5/A549 spheroids, while in the presence of CTX, the invasion area was reduced by invading cells (Figure S3).

**Figure 3.** Invasion area of spheroids in 3D collagen gels. Representative images are of invasion into

type I collagen gel (1.2 mg/mL) (**A**) by cells of MRC-5/A549 spheroids or (**B**) MRC-5/A549 constituted with 12.5 nM of CTX. Cell invasion was photographed under phase-contrast microscopy at 48 h. (**C**) Cell invasion area was measured and analyzed on ImageJ software. \* *p* < 0.05 compared to the control group. The data presented here are from three independent experiments (*n* = 5).

#### *2.4. Effect of CTX on the Expression of EMT Markers in Composite Spheroids*

To investigate the mechanism underlying the migration ability of MRC-5/A549 spheroids, we performed Western blot analysis for epithelial marker E-cadherin and mesenchymal markers—N-cadherin, vimentin, and α-SMA. Our results showed that after 3 days, MRC-5/A549 spheroids lost E-cadherin, but the expression of all EMT markers was elevated. MRC-5/A549 constitutively formed in the presence of CTX showed a significant reduction in mesenchymal markers, α-SMA (43%), N-cadherin (46%), and integrin αv (41%) (Figure 4). However, there was no marked difference in vimentin levels. On the other hand, at the same incubation period, MRC-5/Calu-3 spheroids presented high expression of E-cadherin and low expression of mesenchymal markers, which means that MRC-5/Calu-3 spheroids need a longer period of incubation to induce EMT. However, the presence of CTX on this spheroid inhibited only α-SMA expression compared to the control group, while there was no difference in integrin αv expression (Figure S4).

### *2.5. Effect of Crotoxin on MMP-9, MMP-13, and Cytokine Secretions in 3D Collagen Gel Matrix*

To determine the effect of CTX on spheroids in the 3D collagen gel, the release of matrix metalloproteinases, MMP-9 and MMP-13 (ECM-digesting enzymes), in the culture media were measured. Our results showed that a monolayer of MRC-5, grown in serum-free media, produced endogenous MMP-9; the enzyme was undetectable in media harboring A549 cells monolayer (Figure 5A). MMP-13 were produced endogenously by all cell lines. CTX did not interfere with MMP-9 release by MRC-5 cells, even though CTX repressed MMP-13 (69%) release by A549 cells (Figure 5B). Interestingly, MRC-5/A549 spheroids embedded in collagen gel promoted secretions of MMP-9 and MMP-13. However, composite spheroids treated with CTX inhibited MMP-9 and MMP-13 secretions (37% and 39%, respectively).

In addition, cytokines, chemokines, and growth factors released during the spheroid invasion of 3D collagen gel were assessed by a membrane-based cytokine array. Our results showed that the concentrations of 32 out of 80 cytokines had reduced by 1.2- to 5-fold in the culture media containing CTX-treated spheroid cells (Figure 5C). We found that CTX inhibits tumor-related cytokines, particularly IL-6, IL-8, HGF, TGF-β1, and IGFBP-1. CTX also inhibits chemokines that bind to CXCR1 and CXCR2 receptors such as CXCL5,

CXCL1/2/3, CXCL1a, CXCL6, and CXCL8 (IL-8) that are involved in cancer angiogenesis and metastasis (Figure 5C).

**Figure 5.** Effect of crotoxin on the production of MMP-9, MMP-13, cytokines, and chemokines in the 3D collagen gel. Monoculture of human lung fibroblasts MRC-5 and human lung adenocarcinoma cell line A549 incubated with 12.5 nM of CTX for three days; invasion of collagen gel by composite spheroids of MRC-5/A549 at 48 h. After this period, spent media were collected to quantify the volume of MMP-9 (**A**) and MMP-13 (**B**) by ELISA. \* *p* < 0.05 compared to the control group. # *p* < 0.05 compared to the control group. \*\* *p* < 0.01 compared to the control group. (*n* = 6). In (**C**), cytokine array analysis using composite spheroid invaded gel of at 48 h. The graph indicates the decrease in the growth factors by more than 1.2-folds. A pool of *n* = 4 was used.
