**3. Results**

#### *3.1. Deflamin Exhibits Anti-Tumor Activity in Zebrafish CRC Xenotransplanted Tumors*

In previous studies, we found that deflamin had anti-MMP-2 and -MMP-9 activities in CRC cell line HT-29, as well as anti-inflammatory role in mouse models of inducedintestinal colitis [21]. Therefore, we sought to investigate deflamin's role in cancer development using the zebrafish larvae CRC xenograft model. This is a well-established tumorigenesis model that displays several advantages: it is a quick assay with cellular resolution and allows the evaluation of crucial hallmarks of cancer, such as tumor proliferation, and metastatic and angiogenic potentials [27]. Therefore, CRC cell line HCT116 was labeled with a lipophilic dye (DiI) and injected into the periviteline space (PVS) of 48 hpf zebrafish embryos (Figure 1A). The day after injection (1 dpi), xenotransplanted zebrafish larvae were either treated with 100 μg/mL deflamin (the highest tolerable dose) or left untreated. Deflamin was extracted, isolated, purified, and concentrated from dry Lupinus albus seeds as previously described [20]. E3 medium, containing or not containing deflamin was refreshed every day for a total of 3 days of treatment. At 4 dpi, animals were fixed and stained, and analyses of tumor size, apoptosis (activated caspase3), and proliferation (mitotic figures) were performed by confocal microscopy (Figure 1A). Results showed that deflamin treated tumors were on average four times smaller than untreated tumors (Figure 1B,C, \*\*\* *p* < 0.001). Importantly, analysis of cell death suggests that deflamin induces approximately a four-fold increase in apoptosis when compared to untreated tumors (Figure 1B,D, \*\*\*\* *p* < 0.0001). Tumor proliferation was not significantly affected by deflamin treatment (Figure 1B,E).

Moreover, this zebrafish xenograft model provides the opportunity to analyze metastasis formation given that at 4 dpi human fluorescently labeled tumor cells can be found in distant sites such as the brain, optic cup, gills, and caudal hematopoietic tissue (CHT). To assess the value of deflamin in metastasis formation, the number of xenografted zebrafish with micrometastasis was assessed. Results revealed that under deflamin treatment, HCT116 tumors had a reduced capacity to colonize secondary tissues (about 40% reduction in micrometastasis formation, Figure 1F,G).

Taken together, these results indicate that deflamin has an anti-tumor and antimetastatic role in CRC development, suggesting important applications for cancer therapy.

#### *3.2. Deflamin Does Not Play a Direct Role in Cancer Cell Proliferation or Apoptosis*

To further investigate the mechanism of action of deflamin, we explored the direct effect of this molecule on cancer cell proliferation and apoptosis in vitro. To accomplish this, we treated HCT116, HT-29, and SW480 CRC cell lines with increased concentrations of deflamin (25 μg/mL, 50 μg/mL, and 75 μg/mL) for 5 days. The results showed no effect of this oligomeric protein on cancer cell proliferation rates, suggesting that deflamin does not play a direct role in cell cycle regulation (Figure 2A). Moreover, deflamin did not induce apoptosis of the HCT116, HT-29, and SW480 cells when caspase 3/7 activity was measured in vitro (Figure 2B), indicating that it does not hold a direct cytotoxic effect on cancer cells either. To further validate the absence of toxicity of deflamin in live organisms, we treated 72 hpf zebrafish larvae for three days with increased concentrations of deflamin (50 μg/mL and 100 μg/mL). Figure S1 shows no mortality associated with zebrafish treatments under the conditions tested.

**Figure 1.** Zebrafish xenotransplant model of HCT116 cells exposed to deflamin: (**A**) Human cancer cell line HCT116 was fluorescently labeled with DiI (red) and injected into the perivitelline space (PVS) 2 days post-fertilization (dpf) nacre/casper zebrafish larvae. Zebrafish xenografts were treated in vivo with deflamin for 72 h and compared with untreated controls regarding tumor size, cell death, cell proliferation, and metastasis formation; (**B**) At 4 days post-injection (dpi), zebrafish xenografts were imaged on PVS by confocal microscopy; (**C**) Analysis of tumor size (\*\*\*, *p* ≤ 0.001); (**D**) Analysis of activated caspase 3 (apoptosis, \*\*\*\*, *p* ≤ 0.0001); (**E**) Analysis of mitotic figures (proliferative cells,

(ns); **F**) Zebrafish xenografts were also imaged over the entire body by confocal microscopy. Representative images of HCT116 micrometastasis in gills and caudal hematopoietic tissue (CHT); (**G**) % of zebrafish exhibiting metastasis. The number of xenografts analyzed is indicated in the representative images. In the graphs, each dot represents one zebrafish xenograft. Statistical analysis was performed as described in the Statistical Analysis section (\*, *p* ≤ 0.05, \*\*\*, *p* ≤ 0.001, \*\*\*\*, *p* ≤ 0.0001). Scale bars represent 50 μm. All images are anterior to the left, posterior to the right, dorsal up, and ventral down.

**Figure 2.** Effect of Deflamin on viability and MMPs of CRC cell lines HCT116, HT-29, and SW480: (**A**) Viability of human CRC cell lines when exposed to deflamin; (**B**) Analysis of cell death by apoptosis of human CRC cell lines treated with deflamin; (**C**) Zymographic analysis of the antigelatinases activity of deflamin. Statistical analysis was performed as described in the Statistical Analysis section (\*, *p* ≤ 0.01; \*\*, *p* ≤ 0.01, \*\*\*, *p* ≤ 0.001).

Overall, deflamin appears as a safe polypeptide oligomer to be administered in vivo. Furthermore, these results suggest that the increased apoptosis seen in the zebrafish model, was not caused by a direct effect of deflamin on cancer cells, but rather an indirect role, possibly through MMPs inhibition.

#### *3.3. Deflamin Inhibits MMP-2 and MMP-9, Contributing to Impaired Cancer Cell Migration and Invasion*

Although deflamin was not found to have a direct effect on cancer cell viability, gelatinases MMP-2 and MMP-9 are known to be critical for the ability of cancer cells to migrate and invade since they act not only on the degradation of the ECM (contributing to the rearrangement of the matrix and release of growth factors) but also on the degradation of cell–cell and cell–matrix adhesion molecules [3]. Zymographic analysis of MMPs from HCT116, HT-29, and SW480 CRC cell lines showed that deflamin exerts an inhibitory role on the activity of both MMP-2 and MMP-9 in all cell lines tested, displaying the greatest effect

on SW480 cell line (a reduction of about 75% of MMPs activity at a deflamin concentration of 80 μg/mL, \*\*\* *p* < 0.001, Figure 2C). Therefore, we investigated the effect of deflamin on cancer cell migration using the wound healing in vitro assay (Figure 3A). Migration of cancer cells was shown to be impaired by the addition of deflamin to all cell lines tested, with the SW480 cell line showing the highest inhibition of cellular migration (about 77% reduction in migration at 80 μg/mL of deflamin concentration upon 72 h of treatment, \*\* *p* < 0.01, Figure 3A). We further tested the role of deflamin on cancer cell invasion using a 3D matrix of collagen (Figure 3B). MMP-2 and MMP-9 degrade collagen types III and I, respectively, being both able to cleave collagen types IV and V [28]. Therefore, analysis of 3D spheroids of the same cell lines showed a dose-dependent inhibition of invasion of 3D spheroids on a collagen matrix, being once more SW480 the cell line with the highest inhibition of invasive cells (reduction of about 40% invasiveness after 72 h of exposure to deflamin, \*\*\* *p* < 0.001, Figure 3B).

Overall, these results indicate that deflamin exerts an inhibitory effect on the activity of both MMP-2 and -9, as well as on cancer cell migration and invasion, with important implications for cancer development and progression.

#### *3.4. Deflamin Inhibits Collagen Degradation and Angiogenesis In Vivo*

For a tumor to continue to grow and start migrating/invading, two processes need to occur: (1) elimination of the physical barriers by ECM degradation; (2) generation of pro-angiogenic factors to allow the formation of new blood vessels. MMP-2 and MMP-9 are particularly important for both these processes since they increase the bioavailability of important factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor β (TGF-β) by degrading ECM components such as collagen type IV and perlecan [29]. Thus, we investigated the role of deflamin in both of these processes through analysis of the extracellular collagen degradation and tumor angiogenesis in the zebrafish xenotransplant model. For that, HCT116 cells were stably transduced with a fluorescent dtTomato expressing vector and xenotransplanted into the PVS of wild type AB and Tg(kdrl:EGFP) zebrafish larvae (Figure 4A). For the analysis of collagen degradation in vivo, we used the collagen hybridizing peptide (CHP) which is a 5-FAM conjugated synthetic peptide that specifically binds to denatured collagen strands through hydrogen bonding (Figure 4B). CHP is an extremely specific probe for unfolded collagen molecules, while showing negligible affinity for intact collagen molecules due to a lack of binding sites [30]. In order to investigate blood vessel formation in the tumor area, the zebrafish Tg(kdrl: EGFP) model, which has the vessels labeled with GFP, was used [31] (Figure 4C). Using these models, our results show an inhibitory role of deflamin on collagen degradation when compared to control tumors in vivo (about 85% reduction in collagen degraded area, \* *p* < 0.05, Figure 4D). Furthermore, analysis of tumor blood vessel formation showed about an 80% reduction in infiltrating vessels per tumor area (\*\*\* *p* < 0.001, Figure 4F).

Overall, our results indicate that, by inhibiting MMP-2 and -9 functions, deflamin impairs ECM remodeling through inhibition of collagen degradation and tumor angiogenesis, important processes for cancer progression, rather than having a direct action on cancer cell proliferation and apoptosis.

**Figure 3.** Migration and invasion of CRC cell lines HCT116, HT-29, and SW480 upon treatment with deflamin: (**A**) Analysis of migration by the wound healing assay of human CRC cell lines HCT116, HT-29 and SW480 treated with deflamin for 48 h and its corresponding quantification (%) (*n* = 3). (**B**) 3D spheroid invasion assay of human CRC cell lines HCT116, HT-29, and SW480 treated with deflamin and the corresponding quantification (number of absolute invasive cells). The number of spheroids analyzed is indicated in the graphs. Statistical analysis was performed as described in the Statistical Analysis section (\*, *p* ≤ 0.05; \*\*, *p* ≤ 0.01; \*\*\*, *p* ≤ 0.001).

**Figure 4.** Cancer microenvironment analysis of HCT116 zebrafish xenotransplants: (**A**) Human cancer cell line HCT116 was stably transduced with FUdtTW plasmid (tdTomato expression marker) and injected into the perivitelline space (PVS) of 2 days post-fertilization (dpf) nacre/casper zebrafish larvae. Zebrafish xenografts were treated in vivo with deflamin for 72 h and compared with untreated controls regarding collagen degradation and vessel density; (**B**,**C**) At 4 days post-injection (dpi), zebrafish xenografts were imaged on PVS by confocal microscopy; (**D**) Quantification of degraded collagen area by analysis of CHP staining (5-FAM stained area); (**E**) Total vessel density analysis by EGFP marker; (**F**) Tumor vessel infiltration analysis by EGFP marker. The number of

xenografts analyzed is indicated in the representative images. In the graphs, each dot represents one zebrafish xenograft. Statistical analysis was performed as described in the Statistical Analysis section (\*, *p* ≤ 0.05; \*\*\*, *p* ≤ 0.001). Scale bars represent 50 μm. All images are anterior to the left, posterior to the right, dorsal up, and ventral down.

#### **4. Discussion**

Over the past decade, cancer pharmaceutics has been facing the challenge of maximizing the effectiveness and specificity of treatments, as well as minimizing the toxicity and resistance of therapeutic regimens. The increase in MMPs activity detected in a wide range of cancers has been taken as evidence for their implication in the cancer invasive and metastatic potential, therefore marking MMPs as important targets for both diagnostic and therapeutic purposes [5]. This feature has been well demonstrated in several works, comprising selective inhibition and MMP-9-deficient mice, all of which pointed to MMP-9 as an important target of neoplastic diseases [32]. Indeed, in recent years a substantial amount of research has been made, attempting to develop synthetic, low-molecular-weight inhibitors of MMPs (MMPIs) for the potential treatment of diseases in which they play a major role. However, technical difficulties, side effects, and dose-dependent toxicity have greatly limited the success of these anti-MMP drugs [8–11]. Nevertheless, interesting results have been obtained with natural compounds with anti-inflammatory and anti-tumoral activity. Currently, studies on molecules of natural origin have shown promise in inhibiting MMPs, especially MMP-9, in inflammatory and oncogenic pathological processes [12–17]. Deflamin is a natural food component extracted from white lupine seeds (*Lupinus albus*) that shows anti-MMP-2 and MMP-9, as well as anti-inflammatory activities [20–23]. Importantly, deflamin has the advantage of being a water-soluble molecule easily extracted and isolated in vitro, that shows resistance to boiling and to digestive enzymatic reactions and has the potential to act locally in the intestinal system (without being absorbed into circulation), likely bypassing the problem of systemic toxicity associated with common MMPIs [20–23]. In this sense, this work aimed to explore deflamin therapeutic potential, using 3D cellular systems and zebrafish larvae models. Even though drug pharmacodynamics in zebrafish may differ from mammals, many compounds have been shown to block disease in a similar way in both organisms [33]. Therefore, this work corroborated that deflamin does not hold a cytotoxic effect on the CRC cell lines and zebrafish embryos, providing evidence for its safety as a potential therapeutic strategy. Moreover, deflamin showed inhibitory activity of the invasive process both in cellular systems and in zebrafish cancer models. Importantly, deflamin was effective in inhibiting MMP-2, MMP-9, and general ECM remodeling, favoring spatial constraints of tumor growth/progression and limited nutrient/oxygen supply, due to decreased angiogenesis. In agreement with this work, our previous studies have suggested that deflamin's mode of action involves direct inhibition of MMP-9 and -2, due to its biochemical features [21]. Hence, rather than inhibiting any of the regular pathways of MMP activation or expression, deflamin reduces gelatinase activity, without inducing direct alterations in the cell cycle or in gene expression. However, by limiting ECM remodeling, deflamin prevents the angiogenic process, as well as the degradation of the physical barriers necessary for tumor growth. In this context, physical constraint renders tumors unable to proliferate and metastasize, finishing by becoming apoptotic. Thus, by reducing gelatinase activities in situ, deflamin provides a simple manner to restrict the tumor and render it unable to progress.

Although the present work has some limitations as the zebrafish model is limited in terms of its similarity to mammal models, our results, paired with our previous findings on deflamin, bring a novel view on the use of MMP-2 and -9 inhibitors in cancer models. Indeed, despite MMP-9 being known for decades as an attractive target for anticancer therapies, the development of effective and safe MMP9 inhibitors as anticancer drugs has been shown to be extremely difficult. Recently, therapies have been aiming at more specificity through blocking antibodies that selectively inactivate MMP9, and these are currently in clinical trials [34]. It seems however that the feature of being able to reduce

gelatinolytic activity directly and in situ by a food component such as deflamin might be a similar and simpler approach for tackling cancer disease via MMP inhibition.

Overall, although MMP9 has been well established as an important target in anticancer treatments, there is still a need for selective, safe, and effective MMP-9 inhibitors. This is the first report on an effective food protein gelatinase inhibitor that reduces cancer development via a constriction of the tumor's 3D space distribution. Added to the fact that it is of food origin and easy to isolate, this work revealed the nutritional potential of deflamin as a co-adjuvant therapeutic agent in the treatment of CRC, as a nontoxic dietary supplement. Further work using more complex animal models of cancer and pre-clinical trials will, with no doubt, bring novel insights into the effectiveness of deflamin against CRC.

#### **5. Conclusions**

The work presented here demonstrated that deflamin was able to impair CRC angiogenesis and tumor microenvironment remodeling, via gelatinase inhibition, which led to a constriction and limitation of the tumor's spatial distribution and nutrient and oxygen supply. This type of mechanism seems to be a good depiction of what a specific MMP inhibitor should attain by reducing gelatinase activity, angiogenesis is efficiently impaired limiting tumor growth and inducing cancer cell apoptosis.

Our results suggest that being a natural compound, non-toxic, and resistant to digestion, deflamin holds the potential to be a novel nutraceutical, adjuvant or even a functional food to be used in treating and preventing CRC via a specific, in situ, gelatinase inhibition. Further studies of the use of this oligomeric protein are needed to assess its clinical and commercial value, but it seems plausible to infer that using this type of gelatinase inhibition could be a novel and effective approach to tackling gastrointestinal cancer disease.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers14246182/s1, Figure S1: Zebrafish lethality curves of the acute toxicity assay. At 72 hpf zebrafish embryos were exposed to increased concentrations of deflamin (n = 20) and mortality was evaluated during 72 h of treatment.

**Author Contributions:** S.S. was involved in in vitro and in vivo research models; B.B., J.M., A.L. were involved in deflamin purification and in vitro and in vivo research models; A.C. was involved in in vitro 3D models and supervision; R.C.-D. developed in vitro and in vivo research models and supervision; M.C. was involved in in vitro and in vivo models; L.C. (Lara Carvalhoand) zebrafish housing management, zebrafish model and supervision; L.C. (Luis Costa), R.F. and A.L. were involved in funding acquisition and supervision; R.F. project conceptualization, resources and supervision; M.M. project conceptualization, supervision, writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by national funds from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through the research unit UID/AGR/04129/2020 (LEAF), Project PTDC/BAA-AGR/28608/2017 and Project PTDC/OCE-ETA/4836/2021. Joana Mota was funded by Fundação para a Ciência e Tecnologia SFRH/BD/132832/2017. Raquel Cruz-Duarte was funded by SFRH/BD/ 139138/2018.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors acknowledge the support of IMM Fish facility and Bioimaging.

**Conflicts of Interest:** The authors declare no conflict of interest.
