Melanoma is the most lethal form of skin cancer, with a worldwide increase in incidence. Despite the increased overall survival of metastatic melanoma patients given recent advances in targeted and immunotherapy, it still has a poor prognosis and available treatment options carry diverse severe side effects.
Carbohydrate Polymers 250 (2020) 116869 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Green does not always mean go: A sulfated galactan from Codium isthmocladum green seaweed reduces melanoma metastasis through direct regulation of malignancy features T D.L Bellana, S.M.P Biscaiaa, G.R Rossia, A.M Cristala, J.P Gonỗalvesa, C.C Oliveiraa, F.F Simasa, D.A Sabryb, H.A.O Rochab, C.R.C Francoa, R Chammasc, R.J Gilliesd, E.S Trindadea,* a Cell Biology Department, Universidade Federal Paraná, Curitiba, Paraná, Brazil Biochemistry Department, Universidade Federal Rio Grande Norte, Natal, Rio Grande Norte, Brazil Center for Translational Research in Oncology, Instituto Câncer Estado de São Paulo, São Paulo, São Paulo, Brazil d Cancer Physiology Department, Moffitt Cancer Center, Tampa, FL, USA b c A R T I C LE I N FO A B S T R A C T Keywords: Cancer Melanoma Galactan Seaweed Polysaccharide Melanoma is the most lethal form of skin cancer, with a worldwide increase in incidence Despite the increased overall survival of metastatic melanoma patients given recent advances in targeted and immunotherapy, it still has a poor prognosis and available treatment options carry diverse severe side effects Polysaccharides from seaweed have been shown to exert antitumor activities Here we show in vitro and in vivo antitumor activities of a sulfated homogalactan (named 3G4S) from Codium isthmocladum seaweed in the B16-F10 murine melanoma cell line 3G4S did not induce cytotoxicity or proliferation changes; however, it was able to reduce solid tumor growth and metastasis, while not inducing side effects in mice B16-F10 cells traits related to the metastatic cascade were also impaired by 3G4S, reducing cell invasion, colony-forming capacity and membrane glycoconjugates Therefore, 3G4S shows promising antitumor activities without the commonly associated drawbacks of cancer treatments and can be further explored Introduction Cancer, comprising at least 200 distinct diseases, is one of the leading causes of mortality worldwide with more than 9.6 million deaths in 2018 (Bray et al., 2018) The shadows of a terminal illness are cast when malignant cells from the primary tumor are able to complete the multi-step process known as metastasis, which is responsible for more than 90% of cancer related deaths (Lambert, Pattabiraman, & Weinberg, 2016) Because of its high metastatic capacity, melanoma is the most lethal form of skin cancer when diagnosed at later stages, being one of the few types of cancer with an increasing incidence rate over the last decades (Ward & Farma, 2017) Melanoma’s high metastatic capacity arises from its high mutation rate and epigenetic alterations, resulting in protein expression and glycosylation patterns associated with migratory and invasive traits, rapidly enabling the metastatic process over its progression (Moran, Silva, Perry, & Gallagher, 2017) Recent advances in metastatic melanoma treatment increased patient’s overall survival, ⁎ thanks to the development of targeted therapies such as BRAF-mutant inhibitors (vemurafenib and drabafenib) and immunotherapies such as the immune checkpoints inhibitors anti-CTLA-4 (ipilimumab) and antiPD-1 (nivolumab), overcoming the limited benefits of chemotherapies such as dacarbazine (Domingues, Lopes, Soares, & Populo, 2018) Despite the improvement in treatment, metastatic melanoma still poses as a major clinical challenge Targeted and immunotherapies are still limited in their efficacy and application to different patients given the heterogeneity of the tumor genetic landscape and the development of resistance, while patient’s quality of life is severely compromised by side effects induced by the available treatments (Kroschinsky et al., 2017; Melis, Rogiers, Bechter, & van den Oord, 2017) These obstacles engender the search for anti-tumor and specifically anti-metastatic compounds that can provide new treatment strategies with reduced toxicity Polysaccharides have shown to induce selective cytotoxicity besides promoting cell cycle arrest, affect cell migration and invasion and reduce solid tumors and metastatic progression (Khan, Date, Chawda, & Corresponding author E-mail address: estrindade@ufpr.br (E.S Trindade) https://doi.org/10.1016/j.carbpol.2020.116869 Received May 2020; Received in revised form 10 July 2020; Accepted 30 July 2020 Available online 13 August 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Patel, 2019), reaching even clinical trials (Zhang et al., 2018) Seaweeds are an important font of sulfated polysaccharides, being galactans one of them (Jiao, Yu, Zhang, & Ewart, 2011; Manlusoc et al., 2019) Sulfated galactans are mainly found in red seaweeds, being also present in minor amounts in green seaweed, especially in Codium sp (Pomin & Mourão, 2008) They are composed of α-L- and/or α-D- or βD-Galp units and usually have a molecular mass higher than 100 KDa (Pomin, 2010) Codium sp seaweeds have a wide global distribution and there are already described methods of their artificial cultivation (de OliveiraCarvalho, Oliveira, Pereira, & Verbruggen, 2012; Hwang, Baek, & Park, 2008) Interesting biological activities from polysaccharides obtained from Codium species are already reported, such as anticoagulant (Li et al., 2015), immunostimulant (Lee, Ohta, Hayashi, & Hayashi, 2010) and liver-kidney protection against induced obesity (Kolsi et al., 2017) The green seaweed Codium isthmocladum biosynthesize a highly sulfated homogalactan composed mainly of β-D-Galp 3-O-linked and 4O-sulfated units (named here as 3G4S, and SG1 in the original description paper), with Mw of 14 KDa (Farias et al., 2008) This galactanrich fraction has antioxidant and anticoagulant activity, as well as antiproliferative capacity when exposed to HeLa cell line (Costa et al., 2010), but none is known of its activity against melanoma Based on the relevance of sulfated polysaccharides with anti-cancer activities and the promising structure features and origin of 3G4S our hypothesis is that 3G4S acts directly on cancer cells modulating traits related to cancer progression Hence, the aim of this study was to test the anti-tumor and anti-metastatic activities of this compound against the highly metastatic B16-F10 cell line 12657029, Waltham, Massachusetts, USA) and sterilized in 0.22 μm membranes (Millipore, Cat SLGV033RS, Kenilworth, New Jersey, USA), and DMEM without FBS was used as control For in vivo experiments 3G4S was dissolved in phosphate buffer saline (PBS), and PBS alone was used as control Materials and methods 2.4 Cell morphology 2.1 Purification, characterization and preparation of C isthmocladum polysaccharide B16-F10 cells were treated with 100 μg/mL 3G4S for 72 h and then cell morphology was analyzed by confocal and scanning electronic microscopy (SEM) Cytoskeleton was labeled with ActinGreen ReadyProbes (Invitrogen, Cat.R37110, Waltham, Massachusetts, USA) and cells nuclei with DAPI (Invitrogen, Cat.D1306, Waltham, Massachusetts, USA), and imaged with A1R MP + confocal microscope (Nikon Instruments Inc, Tokyo, Japan) Cells were fixed in Karnovsky solution (glutaraldehyde %, paraformaldehyde %, CaCl2 mM in sodium cacodylate buffer 0.1 M), washed and post-fixed in % osmium tetroxide (in sodium cacodylate buffer 0.1 M) for h, and then dehydrated using increasing ethanol concentrations Samples were dried to critical point and metallized using gold Images were acquired by a JEOL JSM 6360 –LV (Tokyo, Japan) SEM microscope 2.2 Cell lines B16-F10 murine melanoma cell line, obtained from Banco de Células Rio de Janeiro (Rio de Janeiro, Brazil), was cultivated in DMEM, supplemented with 10 % FBS, 0.25 μg/mL of penicillin/streptomycin (Thermo Fisher, Cat 15140122, Waltham, Massachusetts, USA) and 1.57 g/L sodium bicarbonate (Merck, Cat 36486, Kenilworth, New Jersey, USA) Luciferase expressing B16-F10-luc-G5 murine melanoma cell line was purchased from Caliper LifeSciences (Hopkinton, USA) Cells were cultivated in DMEM/F-12 (Thermo Fisher, Cat 11320033, Waltham, Massachusetts, USA), supplemented with % FBS and 0.25 μg/mL of penicillin/streptomycin 2.3 Cytotoxicity and proliferation assays B16-F10 cells (500 cells/well) were exposed to 10, 100 or 1000 μg/ mL of 3G4S for 72 h Cytotoxicity and proliferation were measured using MTT (Mosmann, 1983) and Crystal Violet (Gillies, Didier, & Denton, 1986) assays, respectively Apoptosis and cell cycle analyses were performed after 72 h treatment with 100 μg/mL 3G4S, using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Cat 556447, Franklin Lakes, New Jersey, USA) and BD PI/RNAse kit (BD Biosciences, Cat 550825, San Jose, California, USA), respectively Specimens of C isthmocladum (Vickers) were collected from Pirambúzios beach, (Rio Grande Norte, Brazil - 5°59′01″S/ 35°07'20"W) with agreement of the Brazilian National System of Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN; protocols A8C31A3 and A72AD2B) The seaweed was identified according to its morphology (Wynne, 1986) and a voucher specimen was deposited in the Herbarium of the Biosciences Institute, Universidade Federal Rio Grande Norte (UFRN; registration code UFRN25933) 3G4S was isolated and purified as previously described (Farias et al., 2008) (Supplementary material) 3G4S molecular weight was estimated by reference to a calibration curve made by dextran sulfate standards (10, 40, 70, 147 and 500 KDa) (Sigma-Aldrich®, Cat 75027, St Louis, Missouri, USA) Monosaccharide composition was analyzed after total acid hydrolysis (4 M HCl, 100 °C, h) using a LaChrom Elite® HPLC system (VWRHitachi, Radnor, Pennsylvania, USA) coupled to a LichroCARTđ 250-4 column (250 mm ì 40 mm) (Merck, Cat MC1508330001, Kenilworth, New Jersey, USA) packed with Lichrospher® 100 NH2 (Merck, Kenilworth, New Jersey, USA) and equipped with a refractive index detector (L-2490) (VWR-Hitachi, Radnor, Pennsylvania, USA) A 2DNMR heteronuclear (1H–13C) HSQCed (Edited Heteronuclear Single Quantum Coherence) spectrum was obtained using Bruker Avance III Ascend 600 MHz (14.1 T) spectrometer (Billerica, Massachusetts, USA) equipped with a mm inverse probe The chemical shift of 1H and 13C were expressed in δ (ppm) relative to TMSP (trimethylilsilylpropionate) (Cambridge Isotope Laboratories, Tewksbury, Massachusetts, USA) as an internal standard (δ =0 ppm) For in vitro experiments 3G4S was dissolved in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Cat 12800-017, Waltham, Massachusetts, USA) without fetal bovine serum (FBS) (Gibco, Cat 2.5 Solid tumor and experimental metastasis mouse models C57BL/6 mice (8–12 week old) were maintained and treated in accordance with animal use ethical principles Procedures were previously approved by Ethics Committee on Animal Experimentation (Universidade Federal Paraná: certificate #1025; Faculdade de Medicina de São Paulo: process 049/17; Moffitt Cancer Center: IACUC #R IS00003462) B16-F10 cells (5 × 105 cells in 100 μL of PBS) were subcutaneously inoculated in the right flank of male mice After days, mice started receiving daily intraperitoneal (I.P.) doses of 3G4S (50 mg/kg) diluted in PBS or PBS alone (control group) for 10 days, an experimental design based on previous results from our group (Biscaia et al., 2017) Tumors were daily measured using a digital caliper, and tumor volume was calculated using the formula “V = dxdxDx0.52” (“d” = smaller tumor dimension, “D” = bigger dimension) 3G4S antimetastatic effect was analyzed by the experimental metastasis model A 24 h polysaccharide pre-treatment regimen was established in order to simulate an intervention to prevent and/or reduce metastasis progression after a diagnosed primary melanoma Male and Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig HSQC spectrum of 3G4S from C isthmocladum →3)-β-D-Galp4S-(1→ units and →2)-3,4-Pyruvylated-β-D-Galp-(1→ (amplified) Numbers refer to the position of each 1H/13C correlation paraformaldehyde, and incubated for 30 with ActinGreen™ ReadyProbes With a humidified cotton-swab, non-invasive cells were gently removed from Transwells top Inserts membranes were assembled into a glass microscope slide, using Fluoromount-G™ mounting medium with DAPI (Electron Microscopy Sciences, Cat 17984-24, Haltfield, Pennsylvania, USA) Slides were scanned in the VSlide Carl Zeiss and Metasystems, using 20x objective to capture the entire membrane The number of cells that invaded Matrigel was counted by detection of DAPI stained cells nuclei using ImageJ Fiji Software female mice were I.P pre-treated with 3G4S (50 mg/kg) diluted in PBS or PBS alone 24 h before B16-F10 cells (5 × 105 cells in 100 μL of PBS) intravenous inoculation Post-cell inoculation, treatment was carried out daily for days, following ex-vivo lungs imaging for metastasis foci counting, or for 20 days, following ex-vivo lungs imaging for analysis of colonized area in relation to total lung area, using ImageJ Fiji Software (Schindelin et al., 2012) and staining with hematoxylin and eosin (H& E) Entire lobe images were obtained on a histological slide scanner VSlide Carl Zeiss and Metasystems (Oberkochen, Germany) using a 20x objective Bioluminescence imaging of experimental metastasis progression was performed using In vivo Imaging System (IVIS) Male and female mice were pre-treated with 3G4S (50 mg/kg) I.P for 24 h, following B16-F10-luc-G5 cells intravenous inoculation (5 × 105 cells) Treatment was carried out daily for 15 days After h or days postcell inoculation, mice were I.P injected with 150 μg/mL XenoLight Dluciferin (PerkinElmer, Cat 122799, Walthman, Massachusetts, USA), and subsequently bioluminescence was captured in a Xenogen IVIS 200 (Xenogen Corporation, Hopkinton, USA) Ex vivo bioluminescence imaging of mice organs (lungs, kidneys, liver, pancreas and spleen) was performed 2.8 Glycoconjugates labeling B16-F10 cells (1.2 × 104 cells/well) were exposed to 100 μg/mL 3G4S for 72 h Cell labeling was performed with WGA lectin Alexa Fluor 488 conjugate (Invitrogen, Cat W11261, Waltham, Massachusetts) that specifically binds to N-acetylglucosanime and Nacetylneuraminic acid (sialic acid) Samples were acquired using BD FacsVerse flow cytometer (Missouri, USA) 2.9 Colony formation assay 2.6 Treatment side effects assessment Anchorage-independent colony formation assay was performed using AlgiMatrix ™ 3D Culture System (ThermoFisher, Cat 12684023, Walthmam, Massachusetts, USA) 3G4S pre-treated B16-F10 cells (100 μg/mL of 3G4S for 72 h) were plated in the reconstituted AlgiMatrix hydrogel in new DMEM medium with 10 % FBS without the treatment DMEM was replaced after three days of incubation and kept for more three days Colonies-containing alginate matrix were fixed, stained with CV and counted using ImageJ Fiji Software Body weight differences from before and after treatment were recorded After animal anesthesia, cava vein blood was collected and stored in EDTA-containing tubes Blood cell count and biochemical parameters analyses were performed using a chemistry analyzer Mindray BS-200 (Shenzhen, China) Organs were harvested and weighed 2.7 Invasion assay 2.10 Statistical analysis B16-F10 cells (1.2 × 104 cells/well) were exposed to 100 μg/mL 3G4S for 72 h Cells were detached using a scraper and plated in DMEM (FBS free) on top of Matrigel™ (2.6 mg/mL, 35 μL/well) (BD Biosciences, Cat 356234, San Jose, California, USA) pre-coated Transwells (Millipore, Cat MCEP24 h48, Massachusetts, USA) DMEM with 10 % FBS at the wells bottom was used as chemoattractant, followed by 72 h incubation Transwells were fixed for h with % Significant differences between experimental and control groups were determined by Mann Whitney t-test and by Two-Way ANOVA for tumor volume over time, using GraphPad Prism software Data present as median ± interquartile range Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Table Chemical shift assignments of the HSQCspectrum of 3G4S from C isthmocladum Unit Structure A →2)-3,4-Pyruvylated-β-D-Galpb-(1→ A6S →2)-3,4-Pyruvylated-β-D-Galp6S-(1→b B →3)-β-D-Galp4S-(1→ a b Chemical shifts, δ (ppm)a H1 C1 H2 C2 H3 C3 H4 C4 H5 C5 H6 C6 4.73 103.5 4.73 103.5 4.56 103.0 3.57 73.4 3.82 74.3 3.72 70.3 3.84 82.4 4.24 78.6 3.80 70.7 4.30 70.8 4.12 74.9 4.86 77.4 3.72 75.0 4.06 73.0 3.93 73.1 3.80 61.1 4.34 67.1 3.80 61.1 Chemical shifts are referred to internal standard trimethylsilyl propionic acid (δ =0.00 ppm) Assignments based on (Farias et al., 2008) Signals at 1.62/23.3 ppm correspond to C2 and C3/H3 of pyruvic acid ketal linked to O-3 and O-4 of galactose units Results post-cell inoculation Ex vivo lungs imaging showed 77.03 % reduction in metastasis colonization after 3G4S treatment (Fig 5C-D) Besides, surface intratissue colonization was also reduced by 3G4S (Fig 5E) After 10 days of treatment, treated mice spleens were heavier than control mice spleens, relative to body weight After 21 days of treatment, control mice lost weight over the experiment course, while 3G4S mice gained corporal weight 3G4S treated mice spleens and livers were heavier than those from control mice, however biochemical parameters and blood cell count did not show any indication of hepatotoxicity nor nephrotoxicity (as verified by AST, ALT and urea measurements) (Table 2) Data represent the mean ± SD of at least animals per group for 10 days of treatment, and of at least animals per group for 21 days of treatment 3.1 Purification and characterization of 3G4S The characterization analysis performed (Supplementary data) confirmed that 3G4S was highly similar to SG1 previously described by Farias et al (2008) 3G4S was composed of galactose with 1.2 ° of sulfation (DS), and Mw of 14.1 KDa HSQC spectrum also confirms 3G4S identity (Fig 1) Two major spin systems are evident from the HSQC spectrum, named unit A and unit B, which have anomeric hydrogen and carbon signals at δ 4.73/ 103.5 and 4.56/103.0, respectively All units consist of β-D-Galp residues, and their respective chemical shifts are showed in Table 3.2 3G4S induces low cytotoxicity without inducing apoptosis, proliferation or morphology changes 3.5 3G4S alters metastatic melanoma dynamics 3G4S treatment of B16-F10 cell line reduced glycoconjugates present in cell membrane by 33.47 %, as shown by WGA labeling (Fig 6B) Anchorage-independent colony formation capacity in a tridimensional scaffold was reduced by 31.41 % (Fig 6E) B16-F10 invasion capacity was also reduced by 27.95 % (Fig 6G) MTT (Fig 2A) and CV (Fig 2B) assays did not show reduction in mitochondrial activity or in cell proliferation, respectively, in any tested concentration Based on previous results from our group (Bellan et al., 2020; Biscaia et al., 2017), as well as in recent literature (Khan et al., 2019) the concentration of 100 μg/mL was chosen to the subsequent in vitro assays 3G4S treatment did not induce apoptosis (Fig 2C) or cell cycle alterations (Fig 2D-E) Representative areas of confocal microscopy (Fig F a–d) and SEM (Fig 2F e–h) did not show cell morphology differences between control and 3G4S treated cells Discussion Currently melanoma treatments collateral effects are a significant drawback given their non-selective characteristics, so we first sought to determine 3G4S possible cytotoxic activity However, 3G4S, sulfated galactan from Codium isthmocladum did not induce cytotoxicity or proliferation changes (Figs and 2) Polysaccharides exerting direct cytotoxic and antiproliferative effects on cancer cells, inducing cell apoptosis and cell cycle arrest, are commonly found (Khan et al., 2019; Zong, Cao, & Wang, 2012) including sulfated polysaccharides from seaweeds (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer, 2011; Kim et al., 2007; Sae-Lao, Tohtong, Bates, & Wongprasert, 2017) Nonetheless, some non-cytotoxic polysaccharides already showed antimelanoma activities as a sulfated heterorhamnan from the green seaweed Gayralia brasiliensis (Bellan et al., 2020) and the partially 3-Omethylated mannogalactans from Pleurotus eryngii (Biscaia et al., 2017) Given the high number of collateral effects commonly associated with direct cytotoxic treatments, a non-cytotoxic compound still able to induce antitumor activities, as 3G4S, is of significant relevance Daily treatment with 50 mg/Kg 3G4S, a similar dose with satisfactory effects as used in other antitumoral polysaccharide studies (Jiang et al., 2014; Jin et al., 2007), significantly reduced tumor growth over time, final tumor volume as well as tumor weight (Fig 3), which is a desirable clinical effect in advanced stages of unresectable melanoma (Nixon et al., 2018; Perez et al., 2019) Based on the promising antitumor activity induced by 3G4S and the threat posed by metastatic melanoma, we sought to investigate its 3.3 Melanoma solid tumor progression is reduced by 3G4S Daily 3G4S treatment resulted in tumor volume reduction over time when compared to control group since 7th day of treatment (40.47 %; Fig 3B) Final tumor volume (Fig 3D) and tumor weight (Fig 3E) were also reduced (59.10 % and 37.79 % respectively) 3.4 3G4S shows antimetastatic effect Melanoma cells distribution in control and treated mice was observed h after cell inoculation by IVIS (Fig 4B and C) Nine days postcell inoculation, 3G4S treated group showed reduced metastatic cells presence (Fig 4B and C) IVIS ex vivo imaging of organs showed a reduction in metastatic colonization in all tissues analyzed for female and male mice (Fig 4D and E respectively) Female mice spleens and pancreas and male mice kidneys showed a statistically significant reduction in metastatic colonization (Fig 4F and G) Metastasis foci count days post-cell inoculation was also reduced in 3G4S treated mice, resulting in 74.79 % less foci (Fig 5A and B) The experimental metastasis model was also carried out for 20 days Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig 3G4S cell cytotoxicity, proliferation and morphology (A) MTT (B) Cell proliferation (C) Annexin V and AAD (D) Cell cycle (E) Cell cycle distribution histogram These results represent the set of at least three biologically independent experiments for A, C, D and E, and two for B Control represented as a dotted line for A and B Data normalized for A and B (F) Confocal microscopy (a,b – Control; c,d – 3G4S) (F) SEM (e,f – control; g,h – 3G4S) containing N-acetylglucosamine and sialic acid (Fig 6) Tumor progression and metastasis are accompanied by a series of glycosylation modifications, promoting sustained proliferative signals, resistance to cell death, immune evasion, migration and invasion amongst other tumor promoting effects (Peixoto, Relvas-Santos, Azevedo, Lara Santos, & Ferreira, 2019) One of the mechanisms responsible for increasing cancer cells migration and invasion through glycosylation modulation is sialylation, the addition of syalic acid in N-glycans, promoting cancer cell detachment through physical disruption of cell adhesion (Schultz, Swindall, & Bellis, 2012) Thus the observed reduction in glycans containing N-acetylglucosamine and sialic acid observed post 3G4S treatment corroborates with a less invasive phenotype, as shown in the invasion assay and in the experimental metastasis results Anchorage-independent colony formation capacity of B16-F10 cell line was reduced after 3G4S treatment (Fig 6F), in a similar manner antimetastatic potential IVIS bioluminescence capture and ex vivo images of lungs presented a reduction in 3G4S treated mice metastatic progression in all experiments endpoints (Fig 4) Histology images also show a visual reduction in tumors inside the lungs (Fig 5) In order to understand how 3G4S modulates metastasis progression, we analyzed its effects in cell malignancy related traits that corroborate to metastatic dissemination In vitro 3G4S treatment reduced B16-F10 cell line invasive activity (Fig 6) Polysaccharides are able to affect different cell dynamics associated with metastatic capacity, reducing MMPs production and activity as well as modulating cancer cell membrane surface receptors, reducing migratory and invasive capacities (Khan et al., 2019; Zong et al., 2013), modulations that can be associated with experimental metastasis reduction in vivo (Yu et al., 2018) 3G4S reduced glycoconjugates labeling, specifically glycans Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig Solid tumor progression is impaired with 3G4S (A) Experimental design (B) Tumor volume over time *p = 0.0141; ****p < 0.0001 (C) Solid tumors (a,b – Control; c,d – 3G4S) Representative tumors images from the same experiment based on median tumor weight (D) Final tumor volume (E) Tumor weight These results represent the set of four biologically independent experiments (Control N = 25; 3G4S N = 24; male mice) capacity to sustain proliferation, as well as making them more prone to cell death induced by immune system cells; and the B16-F10 3G4Streated diminished anchorage independent colonization capacity may be affecting the subsequent proliferation and lung colonization necessary for the metastatic progression Hence, the results described here point to the modulation of some of the most important cellular dynamics to the successful of the early stages of the metastasis cascade extravasation, tissue remodeling, immune evasion and colonization (Lambert et al., 2016) 3G4S treated mice gained corporal weight when compared to treatment start, while control mice lost weight (Table 2) This result could be an indicative of a protective effect from 3G4S against cancer cachexia, a disease adverse effect leading to skeleton muscle mass loss and progressive functional impairment (Fearon et al., 2011) Similar protective effects can be found exerted by other polysaccharides already described (Chen et al., 2018; Fitton, Stringer, Park, & Karpiniec, 2019) 3G4S treated mice also presented heavier spleens and liver when compared to control mice (Table 2) The increase in spleen weight should be further investigated as a possible collateral effect or an immune modulation activity, as shown by other polysaccharides in the form of spleenocyte proliferation and T cell activation (Ramberg, some polysaccharides are able to interfere in colony formation in the same concentration (100 μg/mL) and cell line (B16-F10) (Oliveira et al., 2019; Varghese, Joseph, Aravind, Unnikrishnan, & Sreelekha, 2017) After tissue extravasation the initial seeding of metastatic cells depends on its capacity to survive and colonize the new microenvironment, normally assisted by growth factors and inflammatory proteins released by the primary tumor, generating a premetastatic niche (Pachmayr, Treese, & Stein, 2017) To further generate micro and macrometastasis these cells need to enable sustained proliferative capacity, a trait simulated in the colony formation assay (Franken, Rodermond, Stap, Haveman, & van Bree, 2006) Cells treated with 3G4S presented a long lasting reduction in their colony formation capacity even after treatment removal, indicating a modulation in another metastatic cascade initial step (Fig 5D and E) Although the exact mechanism underlying 3G4S antimetastatic activity is still unclear, the modulation of metastatic related features demonstrated in vitro could be strongly correlated to the significant impairment of B16-F10 metastasis progression 3G4S may be affecting B16-F10 extravasation and lung’s tissue colonization through a reduction in cells invasive and tissue remodeling capacities; the reduction in glycoconjugates surrounding metastatic cells may be reducing their Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig Metastasis progression impairment by 3G4S observed by IVIS (A) Experimental design (B) Control and (C) 3G4S Bioluminescence capture h and days post-cell inoculation (Control N = 5, 3G4S N = 4; female mice) (D–E) Ex vivo organ bioluminescence (D- female mice; Control N = 5, 3G4S N = 4; E- male mice; Control N = 5, 3G4S N = 4) *p = 0.0286 (F–G) Incidence of metastasis in each mouse Presence of metastasis in the specified organ accounted for each mouse (F- female mice; Control N = 5, 3G4S N = 4; G- male mice; Control N = 5, 3G4S N = 4) The combination of structure features, namely molecular weight, sulfate content, monosaccharide composition and type of glycosidic linkage is closely related to the extent of polysaccharide activity However, the highly structural diversity of polysaccharides makes a direct relationship between structure and biological effects a complex subject (Jiao et al., 2011; Xu, Huang, & Cheong, 2017) Nonetheless, when comparing 3G4S structure and activities with some of the most common polysaccharides obtained from seaweeds, we can find interesting similarities and differences regarding structure and activity Nelson, & Sinnott, 2010) including in galactan-containing polysaccharides (Awadasseid et al., 2017; Zheng et al., 2016) Although the increase in 3G4S treated mice liver weight, biochemical analyses did not show hepatotoxicity (Table 2) Cancer treatment commonly induces severe patient’s quality life loss through a myriad of collateral effects, even reaching the point of treatment withdrawal by patients (Clarke, Johnston, Corrie, Kuhn, & Barclay, 2015), therefore 3G4S absence of toxicity results indicates another promising component of this polysaccharide Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig Metastasis progression impairment over time (A) Experimental metastasis end point days post cell inoculation (a–d) Control (e–h) 3G4S Lungs ventral view of animals (B) Metastasis foci count (Control N = 4; 3G4S N = 5; female mice) *p = 0.0317 (C) Experimental metastasis end point 20 days post cell inoculation (a–d) Control (e–h) 3G4S Ventral view of lungs (D) Total metastasis colonization area Total lung area / metastatic area (Control N = 12; 3G4S N = 8; female mice) ***p = 0.0002 (E) H &E from mice lungs Tumor colonies indicated by arrowheads (intratissue) and arrows (superficial) (a–d) Control (e–h) 3G4S Images corresponding to left lung lobe from the same lungs and in the same order as (C) activities of fucoidans, preponderantly inducing cell cycle arrest and selective cytotoxicity in cancer cells (Ale et al., 2011; Atashrazm, Lowenthal, Woods, Holloway, & Dickinson, 2015; Zhang, Teruya, Eto, & Shirahata, 2011) Differently from fucoidans, 3G4S presents Fucoidans are sulfated L-fucose-rich branched heteropolysaccharides obtained from brown seaweeds They present chains of α-(1→3)-L-Fuc and/or alternated α-(1→3)- and α-(1→4)-L-Fuc units (Li, Lu, Wei, & Zhao, 2008) A variety of studies present antitumor Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Table Analysis of physiological, biochemical and hematological parameters Parameter Treatment regimen: 10 days Corporal weight variation Lung weight/body weight Spleen weight/body weight Liver weight/body weight Kidney weight/body weight Biochemical parameters Alkaline phosphatase Creatinine Gamma GT Treatment regimen: 21 days Corporal weight variation Lung weight/body weight Spleen weight/body weight Liver weight/body weight Kidney weight/body weight Biochemical parameters Alanine transaminase Aspartate aminotransferase Creatinine Urea Total cholesterol Triglycerides VLDL Procalcitonin Complete blood count White blood cells Lymphocytes Monocytes Granulocytes Red blood cells parameters Red blood cells Mean corpuscular volume Mean corpuscular hemoglobin Red cell distribution width Platelets parameters Platelets Mean platelet volume Platelet distribution width Unit Control 3G4S P value % % % % % 8.05 ± 2.644 0.7799 ± 0.1211 0.4915 ± 0.04467 5.611 ± 0.4878 1.216 ± 0.02170 11.37 ± 2.538 0.7758 ± 0.1354 0.6153 ± 0.04842 ** 6.031 ± 0.4204 1.233 ± 0.1568 0.0952 0.8016 0.0079 0.3095 0.6667 U/L mg/dL U/L 95.88 ± 8.115 0.425 ± 0.05 4.85 ± 2.068 60.4 ± 5.662 0.35 ± 0.05774 5.833 ± 2.04 0.0286 0.2857 0.5714 % % % % % −1.298 ± 2.027 2.224 ± 1.135 0.421 ± 0.09374 5.215 ± 0.4865 1.527 ± 0.09001 + 1.375 ± 0.6455 *** 0.9443 ± 0.1482 ** 0.5601 ± 0.05745 ** 6.424 ± 0.4598 **** 1.512 ± 0.04374 0.0005 0.0011 0.0022 < 0.0001 0.7102 U/L U/L mg/dL mg/dL mg/dL mg/dL mg/dL % 45.84 ± 11.27 130.2 ± 53.05 0.375 ± 0.04629 59.88 ± 5.802 81.49 ± 4.041 100.9 ± 15.06 20.19 ± 2.998 0.2443 ± 0.0336 54.53 ± 7.751 179.6 ± 48.84 0.3667 ± 0.05164 51.95 ± 5.685 75.85 ± 6.95 90.53 ± 23.36 18.1 ± 4.704 0.2378 ± 0.03207 0.1419 0.1079 > 0.9999 0.0593 0.1512 0.3983 0.3983 0.7193 6.088 ± 1.454 4.475 ± 0.9498 0.125 ± 0.04629 1.488 ± 0.7019 7.783 ± 1.534 5.75 ± 1.343 0.15 ± 0.05477 1.883 ± 0.5636 0.1325 0.0593 0.5884 0.2278 x106 / μL fL pg % 8.237 ± 0.4923 41.3 ± 1.223 12.09 ± 0.2673 15.79 ± 1.145 7.775 ± 0.4598 40.67 ± 1.472 11.8 ± 0.3162 16.02 ± 0.5672 0.0734 0.5589 0.1282 0.3817 x103 / μL fL % 421.5 ± 53.71 5.8 ± 0.1309 14.74 ± 0.1847 395.5 ± 52.76 6.033 ± 0.3141 14.9 ± 0.2757 0.4715 0.1275 0.3124 x103 x103 x103 x103 / / / / μL μL μL μL Data represent the Mean ± SD of at least animals per group for 10 days of treatment, and of at least animals per group for 21 days of treatment modification associated with a higher degree of biological effects in seaweed polysaccharides (Patel, 2012) and that has also been linked to a higher biological and antitumor activity in polysaccharides in general (Xie et al., 2020) Antitumor and antimetastatic combined activities of 3G4S and its apparent absence of side effects described here represents a new step on melanoma treatment This promising compound could be administered over a long period of time since the first diagnosis of a primary tumor, leading to tumor growth rate decrease and possible reduction and inhibition of metastatic progression antitumor activities without interfering in cell proliferation and viability This could be related, at least in part, to its differences in monosaccharide composition and glycosidic linkage types: 3G4S is a homogalactan mainly composed of β-(1→3)- linkage, whereas fucoidans are α-(1→3) and α-(1→4) linked and contain fucose units Interestingly, 3G4S has a Mw around 14 KDa, which is a value near of sulfated fucoidans molar mass that showed better antitumor activities (Choi & Kim, 2013; Kasai, Arafuka, Koshiba, Takahashi, & Toshima, 2015) Another example of abundant seaweed sulfated polysaccharides are carrageenans which are highly sulfated homogalactans They are composed of alternating units of D-Galp β-(1→3)-linked and D-Galp α(1→4)-linked, Galactose α-(1→4)-linked that can be replaced by 3,6anhydrogalactose units (Necas & Bartosikova, 2013) Many reports demonstrate carrageenans anticancer activities, especially through cell cycle arrest (Ling, 2012; Prasedya, Miyake, Kobayashi, & Hazama, 2016) and cell cytotoxicity, being those depolymerized carrageenans (with lower Mw) highly cytotoxic (Calvo et al., 2019; Z Jin, Han, & Han, 2013; Liu et al., 2019) Although monosaccharide composition and the relative low Mw of non-cytotoxic 3G4S approximate it to cytotoxic carrageenans, some structural features are different Carrageenans can be 2-O-, 4-O-, and 6-O-sulfated while 3G4S is preponderantly 4-O-sulfated (Campo et al., 2009) Moreover, 3G4S does not present alternated β-(1→3) and α-(1→4) glycosidic linkages nor 3,6-anhydrogalactose units, a structural moiety of carrageenans already linked to cell cytotoxic effects (Alves et al., 2012) Additionally, part of 3G4S antitumor activity could be associated to its high sulfate content, a structural characteristic and chemical Conclusion The polysaccharide 3G4S modulated in vitro malignancy features and reduced solid tumor and lung’s metastasis progression of melanoma without side effects This is the first report of a galactan from green seaweed with antitumor activities both in solid tumor model and experimental metastasis induced by the highly aggressive B16-F10 melanoma cell line, revealing it as a promising compound to further studies CRediT authorship contribution statement D.L Bellan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - review & editing S.M.P Biscaia: Data curation, Formal analysis, Investigation, Methodology G.R Rossi: Data curation, Formal analysis, Carbohydrate Polymers 250 (2020) 116869 D.L Bellan, et al Fig 3G4S modulates B16-F10 malignancy related traits (A) Glycoconjugate labeling representative histogram (B) Glycoconjugate labeling analysis ***p = 0.0007 (C–E) Colony formation anchorage-independent assay (C) Control (D) 3G4S (E) Colony formation analysis *p = 0.0286 (F) Invasion assay Representative image (a) Control (b) 3G4S (G) Invasion assay analysis **p = 0.0095 These results represent the set of at least three biologically independent experiments Data normalized Acknowledgements Investigation, Methodology A.M Cristal: Formal analysis, Investigation, Methodology J.P Gonỗalves: Data curation, Formal analysis, Investigation, Methodology C.C Oliveira: Conceptualization, Funding acquisition, Validation, Writing F.F Simas: Conceptualization, Funding acquisition, Validation, Writing D.A Sabry: Formal analysis, Investigation, Methodology H.A.O Rocha: Funding acquisition, Validation, Methodology C.R.C Franco: Funding acquisition, Validation, Supervision R Chammas: Conceptualization, Funding acquisition, Validation, Writing R.J Gillies: Conceptualization, Funding acquisition, Validation E.S Trindade: Conceptualization, Funding acquisition, Validation, Supervision, Writing, Project administration The authors would like to thank the Brazilian funding agencies CAPES (Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nớvel Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support (Grant numbers: CAPES- 001-CIMAR 1985/2014 and PROCAD 2965/2014; CNPq - 309260/2015-9) We 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antitumor activities (Choi & Kim, 2013; Kasai, Arafuka, Koshiba, Takahashi, & Toshima, 2015) Another example of abundant seaweed sulfated polysaccharides are carrageenans which are highly sulfated. .. Li, N., Mao, W., Yan, M., Liu, X., Xia, Z., Wang, S., Cao, S (2015) Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green alga Codium Divaricatum, 121,... units (named here as 3G4S, and SG1 in the original description paper), with Mw of 14 KDa (Farias et al., 2008) This galactanrich fraction has antioxidant and anticoagulant activity, as well as antiproliferative