**1. Introduction**

The key role of glycosaminoglycans (GAGs), natural polysaccharides found in mammalian tissue, has been recognized in the regulation of cell properties and functions. They are now considered as pharmacological targets to treat several diseases and notably metastatic cancers. GAGs play a central role in the organization of the extracellular matrix (ECM) through their binding to various molecules (ECM components, cell-surface receptors,

**Citation:** Muñoz-Garcia, J.; Mazza, M.; Alliot, C.; Sinquin, C.; Colliec-Jouault, S.; Heymann, D.; Huclier-Markai, S. Antiproliferative Properties of Scandium Exopolysaccharide Complexes on Several Cancer Cell Lines. *Mar. Drugs* **2021**, *19*, 174. https://doi.org/ 10.3390/md19030174

Academic Editors: Irina M. Yermak and Viktoria Davydova

Received: 27 February 2021 Accepted: 18 March 2021 Published: 23 March 2021

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growth factors and cytokines). Nowadays, several therapeutic strategies are developed with the aim of restoring cell function due to a degradation of endogenous GAGs either in protecting endogenous GAGs or in using exogenous GAGs or GAG-mimetics [1–4].

Unfractionated heparin and its low-molecular-weight (LMW) derivatives are widely used to prevent or treat venous thromboembolism, which is a common complication in cancer patients. Clinical trials have shown that cancer patients treated with heparins have improved, with a decrease in cancer progression and a longer survival time [5,6], which may be explained by heparin's functional interference with critical biological steps of metastasis spread [7]. Despite their therapeutic potential, side-effects induced by their anticoagulant properties (e.g., hemorrhagic complications and heparin-induced thrombocythemia) may occur, limiting thus a long-term treatment. Heparin could present also a contamination with prion proteins or oversulfated chondroitin sulfate [8]. Heparin's use in therapy is sometimes limited due to this hazard profile. As a result, the number of studies on heparin mimetic molecules has increased in recent years. Polysaccharides may be a new source of heparin-like molecules among the possible drug candidates. Traditional algal or modern bacterial polysaccharides may be used as a substitute for mammalian GAGs [9]. Furthermore, preliminary animal studies have shown that polysaccharides extracted from eukaryotes and prokaryotes living in the marine environment could reveal a better benefit/risk ratio [9]. Algal polysaccharides like fucoidans–especially low-molecular-weight fucoidan preparations–have previously been shown to have heparin-like properties with low hemorrhagic risks [10]. As soluble molecules or polymers, marine polysaccharides derived from bacteria have a lot of promise in cell therapy and tissue engineering. When compared to other polysaccharides from eukaryotes, they can be produced in bioreactors under completely regulated conditions [11].

Among these marine polysaccharides, an exopolysaccharide (EPS) of interest was isolated from the culture broth of a hydrothermal bacterium called *Alteromonas infernus*. A monosulfated nonasaccharide composed of six neutral hexoses and three hexuronic acids, including one galacturonic acid unit bearing one sulfate group, makes up the repeating unit of this EPS. It presents a high molecular weight (>10<sup>6</sup> g mol−1) and a low sulfate content (<10%) [12–14]. When compared to heparin, this native EPS had a very poor anticoagulant effect. To improve its biological activities and provide a GAG mimetic compound, native EPS has been modified first by radical depolymerization, then by oversulfation to create EPS derivatives [15,16]. In the following, EPS-DR denotes native EPS that has undergone radical depolymerization, whereas EPS-DRS denotes EPS that has been further over-sulfated. These two chemical modifications allow for a decrease in molecular weight while increasing sulfate content [17]. The anticoagulant efficacy of this LMW highly sulfated EPS derivative is increased over that of its native precursor, but it is still 10 and 5 times less potent than unfractionated and LMW heparin, respectively. Following the oversulfation stage, an oversulfated EPS structure (EPS-DRS) has been suggested, with three additional sulfate groups on both glucose and galactose constitutive of the repeating unit (unpublished data). It is made up of three hexose units that have been oversulfated and have been reported elsewhere [15–17].

In vitro studies on bone remodeling have displayed different levels of bone resorption regulation by EPS-DRS, most of them leading to pro-resorptive effects [15]. These results have brought to consider those compounds to be tested on osteosarcoma, characterized by high potency to induce lung metastasis [18]. EPS-DRS of different sizes have been tested against two osteosarcoma cell lines (mouse POS-1 and human HOS) revealing that an EPS derivative of 15 kDa could effectively inhibit both migration and invasiveness of osteosarcoma cells in vitro, while it could be very efficient at inhibiting the establishment of lung metastases in vivo [19].

Since their natural tropism in targeting malignant tumor and their metastasis, EPS have been recently considered as vector to be combined with theranostic radionuclide pairs. Through the possibility of doing therapy and diagnostic at the same time, theranostic compounds represent new innovative clinical tools to be employed in a perspective of "personalized/precision" medicine [20]. More precisely, a theranostic pair 44Sc/47Sc could be both employed for Positron Emission Tomography (PET) imaging to detect primary and secondary tumors, as well as for Targeted RadioTherapy in order to bombard tumor tissues with gamma radiations [21]. Complexation between EPS and scandium has already been assessed and quantified in a previous work [22]. The EPS derivatives selected for this study are a highly sulfated one, called DRS (M w = 27 kDa) and a lower-sulfated one, called DR (with M w = 16 kDa). The complexation properties of these two EPS with scandium were higher than the ones on glucuronic and galacturonic acids, even though Sc-EPS complexes appear to be less stable ( *K*ScEPS-DR = 9.14; *K*ScEPS-DRS = 7.69) than scandium complexes with 1,4,7,10- Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA), classically used in radiopharmaceutical drugs.

For further use as a therapeutic vector, it is important to ensure that EPS may keep the ability of targeting cancer cells even when complexed with scandium. For this reason, in vitro studies concerning both cell proliferation and cell viability of various tumor cell lines were realized here to evaluate if there is a synergetic effect of Sc-EPS complexes on different cancer cell lines, compared to the EPS alone and scandium alone. To this aim, the cell index of human osteosarcoma (MNNG/HOS), human melanoma (A375), human lung cancer (A549), human glioblastoma (U251), and human breast cancer (MDA231) were monitored over a week using XCELLigence® technology. XCELLigence measures the electric impedance of cell assessed. This impedance is directly related to the cell adhesion process and, consequently, to cell proliferation and/or cell death. Moreover, the effect of the metal-to-ligand ration of the EPS-Sc complexes was assessed and, a heparin was used as a reference system for these EPS.
