Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

Antitumor and immunomodulatory activity of fucoidan from the brown alga

Lessonia trabeculata (Lessoniaceae) on breast cancer spheroids

Rosa Condori-Macuri1; https://orcid.org/0000-0002-2177-3462

Libertad Alzamora-Gonzales1*; https://orcid.org/0000-0002-7425-7453

Raisa Cruz-Riquelme1; https://orcid.org/0000-0003-3128-087X

Erasmo Colona-Vallejos1; https://orcid.org/0000-0001-9759-288X

Nadia Chauca-Torres1; https://orcid.org/0000-0003-1356-1344

1. Research Group on Immunomodulators and Antitumor of Natural and Synthetic Origin, Immunology Laboratory,

Universidad Nacional Mayor de San Marcos, Lima 110058, Perú; rosa.condori@unmsm.edu.pe, lalzamorag@unmsm.

edu.pe (*Correspondence), raisat13cruzr@gmail.com, ecolonav@unmsm.edu.pe, nadia.torres.hsj@ssss.gouv.qc.ca

Received 24-IV-2023. Corrected 28-VIII-2023. Accepted 30-X-2023.

ABSTRACT

Introduction: The therapeutic benefits of the brown algae fucoidan in the treatment of breast cancer have

attracted considerable interest in recent years. However, research using spheroids which provide relevant results

in trials for antitumor and immunomodulatory products because they adequately simulate the tumor microen-

vironment, is limited.

Objective: To evaluate the antitumor and immunomodulatory activity of Lessonia trabeculata fucoidan (LtF),

native to the Peruvian Sea, on two types of multicellular tumor spheroids.

Methods: The study was conducted from January to December 2021. Two types of spheroides were elabo-

rated: from 4T1 tumor cells (MTS), and from 4T1 tumor cells+mouse splenocytes (MTSs). The antitumor

activity of LtF was evaluated in MTS by quantifying cell viability with MTT. Immunomodulatory activity was

determined in MTSs using the IC50 for two types of treatment: simple, fucoidan alone (LtF) and combined,

fucoidan+doxorubicin (LtF+Dox). Pro-inflammatory (TNF-α, IL-6) and anti-inflammatory (IL-10, TGF-β)

cytokine production was quantified by sandwich ELISA 72 h after treatment. Dox was used as positive control

in all assays.

Results: LtF exerted antitumor activity as evidenced by increased necrotic zone and cell debris formation com-

pared to the untreated control. Antitumor activity was concentration dependent between 100 and 6 000 μg/ml.

In MTSs, simple treatment increased IL-6 and decreased IL-10 and TGF-β production. The combined treatment

significantly reduced TGF-β production. In both treatments and Dox, there was an increase in IL-6 compared

to the untreated control. The highest production of IL-10 and TGF-β was observed in the untreated control,

compatible with a highly immunosuppressive tumor microenvironment.

Conclusions: LtF is a good candidate for the treatment of breast cancer and can immunomodulate the tumor

microenvironment alone or in combination with Dox.

Key words: anti-inflammatory cytokines; antineoplastic; fucoidan; pro-inflammatory cytokines; tumor spheroids.

https://doi.org/10.15517/rev.biol.trop..v71i1.54918

BIOMEDICINA

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

INTRODUCTION

Seaweed is considered an excellent source

of bioactive natural products with anti-inflam-

matory, antioxidant, immunomodulatory, and

antitumor effects, among others (Mayer et

al., 2019). The advantage of using seaweed is

its high availability and low processing costs

compared to terrestrial plants (Lee et al., 2022).

Recently, the use of seaweed as a source of

bioactive principles for the treatment of vari-

ous cancers, including breast cancer (BC), has

been explored. In the United States, BC is the

second leading cause of cancer death among

women after lung cancer, and the leading cause

of cancer death among black and Hispanic

women (Giaquinto et al., 2022). Conventional

cancer therapy causes negative side effects that

debilitate patients. Therefore, there is a need to

search for products that complement the classi-

cal management of the disease and help mitigate

the side effects of treatment (Abudabbus et al.,

2017). Proper management of the immune

response is important in the fight against can-

cer. Monitoring the production of pro-inflam-

matory cytokines is key to the detection of an

efficient anti-tumor immune response, whereas

the predominance of anti-inflammatory cyto-

kines is an indication that the tumor is gaining

ground in the individual and has successfully

reversed the immune defense (Lan et al., 2021).

The brown alga Lessonia trabeculata

Villouta & Santelices, known as aracanto, palo,

palo blanco (Peru) or huiro palo, huiro varilla

(Chile), is an endemic species of the eastern

Pacific coasts. It is distributed between the

meridians 14º (central coast of Peru) and 55º

(Chile) south latitude and is the dominant spe-

cies of the intertidal and shallow subtidal rocky

bottom (Santelices et al., 1980). Most studies

on this species have focused on ecological

RESUMEN

Actividad antitumoral e inmunomoduladora de fucoidan del alga parda Lessonia trabeculata

(Lessoniaceae) en esferoides de cáncer de mama

Introduccción: Los beneficios terapéuticos del fucoidan de algas pardas en el tratamiento del cáncer de mama

han despertado gran interés en los últimos años. Sin embargo, las investigaciones con esferoides son limitadas,

éstos proporcionan resultados relevantes en ensayos de productos antitumorales e inmunomoduladores porque

simulan adecuadamente el microambiente tumoral.

Objetivo: Evaluar la actividad antitumoral e inmunomoduladora del fucoidan de Lessonia trabeculata (LtF),

nativa del Mar Peruano, en dos tipos de esferoides tumorales multicelulares.

Métodos: El estudio se realizó de enero a diciembre de 2021. Se elaboraron dos tipos de esferoides: con células

tumorales 4T1 (MTS) y con células tumorales 4T1+esplenocitos de ratón (MTSs). La actividad antitumoral de

LtF se evaluó en MTS cuantificando la viabilidad celular con MTT. La inmunomodulación se determinó en MTSs

utilizando la IC50 para dos tipos de tratamiento: simple, fucoidan solo (LtF) y combinado, fucoidan+doxorubicina

(LtF+Dox). La producción de citoquinas proinflamatorias (TNF-α, IL-6) y antiinflamatorias (IL-10, TGF-β) se

cuantificó mediante ELISA sándwich 72 h post-tratamiento. En todos los ensayos se utilizó Dox como control

positivo.

Resultados: En los MTS, el LtF ejerció actividad antitumoral evidenciada por aumento de la zona necrótica y for-

mación de restos celulares respecto al control no tratado. La actividad antitumoral fue concentración-dependiente

entre 100 y 6 000 μg/ml. En los MTSs, con el tratamiento simple se incrementó IL-6 y disminuyeron IL-10 y

TGF-β. El tratamiento combinado redujo significativamente la producción de TGF-β. Los dos tratamientos y Dox

incrementaron IL-6 respecto al control no tratado. La mayor producción de IL-10 y TGF-β se observó en los no

tratados, compatible con un microambiente tumoral altamente inmunosupresor.

Conclusiones: El LtF es un buen candidato para tratar el cáncer de mama y puede inmunomodular el microam-

biente tumoral solo o en combinación con Dox.

Palabras clave: antineoplásico; citoquinas antiinflamatorias; citoquinas proinflamatorias; esferoides tumorales;

fucoidan.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

and reproductive aspects (Campos et al., 2021;

González et al., 2018; Tala et al., 2004).

The use of L. trabeculata is mainly arti-

sanal, and it is mainly used for the extraction of

alginates in the industrial sector (Gouraguine

et al., 2021). Some reports suggest alternative

uses, such as a bioabsorbent for Cd (II) and Hg

(III) in environments contaminated by met-

als (Boschi et al., 2011). Fucoidan is the least

studied of the bioactive compounds contained

in this bioresource. The structure and composi-

tion of fucoidan vary depending on the algal

species and abiotic factors of the marine envi-

ronment (Wang et al., 2020), so it is essential to

define the bioactive potential of fucoidan and

other compounds in each species.

Regarding its anticancer activity, fucoi-

dan has been shown to exert a cytotoxic effect

on tumor cells by modulating apoptosis and

the cell cycle (Jin et al., 2021). Fucoidan also

stimulates immune functions such as matura-

tion and proliferation of dendritic cells (Park

et al., 2020), activation of NK cells (Zhang et

al., 2021), macrophages (Ma et al., 2021), cyto-

toxic T lymphocytes (Kiselevskiy et al., 2022),

and modulates cytokine production (Colona,

2022; Takahashi et al., 2018). These properties

demonstrate that fucoidan is an enhancer of the

immune system’s anti-tumor activity.

Tumor spheroids are three-dimensional

in vitro culture models that allow the study of

cell-extracellular matrix interactions, hypoxic

conditions, drug penetration and tumor physi-

ology. Because they more closely resemble the

tumor microenvironment, spheroids are a great

advantage for studying the use of fucoidan in

cancer treatment (Nii et al., 2020). The objec-

tive of our study was to evaluate the antitu-

mor and immunomodulatory activity of L.

trabeculata fucoidan (LtF) on two types of

multicellular tumor spheroids generated from

triple-negative BC cells. We demonstrated that

LtF has antitumor and immunomodulatory

activity on spheroids. The simple and com-

bined treatments modify the immunosuppres-

sive microenvironment, so with these results we

hope to promote the research and development

of fucoidan-based products from L. trabeculata

as a strategy for cancer prevention and treat-

ment, as well as contribute to the sustainable

use of Peru’s natural resources. This is the first

study to report the antitumor and immuno-

modulatory activity of LtF on 4T1 and adds to

the existing information on the utility of a 3D

model consisting of tumor and immune cells

(MTSs) for the evaluation of natural products.

MATERIALS AND METHODS

Chemical reagents and materials: Doxo-

rubicin (Dox, cat. D1515), Roswell Park Memo-

rial Institute medium (RPMI-1640, cat. R8005),

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-

tetrazolium Bromide (MTT, cat. M5655, 0.5

mg/ml) and dimethyl sulfoxide (DMSO, cat.

D2438) were purchased from Sigma-Aldrich

(Merck KGaA, USA); fetal bovine serum

(FBS, cat. S181H) was purchased from Biow-

est SAS (France); red blood cell lysis buffer

(cat. A10492) from Gibco (USA); agarose (cat.

16500500) from Invitrogen (USA); trypan blue

(cat. 1450013) from Bio-Rad (United King-

dom), and sandwich ELISA kits for TNF-α

(cat. 88-7324-88), IL-6 (cat. 88-7064-88), IL-10

(cat. 88-7105-88) and TGF-β (cat. 88-8350-88)

were purchased from Thermo Fisher Scientific

Inc. (USA). All chemicals and solvents were of

analytical grade.

0.22 μm Stericup-GP Sterile Vacuum Fil-

tration System (cat. S2GPU05RE) was pur-

chased from Millipore (USA). 25 cm2 flasks

(cat. 430639), 70 μm cell strainer (cat. 431751),

96-well round-bottom microplates (cat.

3799), and flat-bottom microplate (cat. 3599)

were purchased from Corning Inc. (USA).

And Immulon 4HBX flat-bottomed micro-

plates (cat. 3855) from Thermo Fisher Sci-

entific Inc. (USA).

Fucoidan: Lyophilized LtF (sugars = 59 %,

sulfates = 5.7 %, purity = 83.4 %) was pro-

vided by the company PSW SA (https://www.

pswsa.com, Lima, Peru), which collected the

samples from L. trabeculata in San Nicolas Bay

(15°15’39’’ S & 75º13’47’’ W), Marcona District,

Nasca Province, Ica Region, Peru.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

Preparation of fucoidan and doxorubicin

concentrations: A 10 mg/ml solution of LtF

was prepared, from which dilutions were made

to obtain concentrations of 1, 10, 100, 1 000,

2 000, 4 000, 6 000, 8 000, and 10 000 μg/ml.

In the case of Dox, a 10 μg/ml solution was

prepared from which dilutions were made to

obtain concentrations of 0.01, 0.1, 0.5, 1, 5, and

10 μg/ml. Dox is an antineoplastic drug used to

treat BC and was used as a positive control for

antitumor and immunomodulating assays. All

dilutions were prepared in RPMI-1640 supple-

mented with 10 % FBS (complete medium:

CM), filtered through 0.22 μm membranes, and

stored at 4 ºC.

Cell culture of 4T1: 4T1 is a mouse adeno-

carcinoma cell line (code BCRJ0022), meta-

static and triple negative breast cancer (TNBC),

was obtained from the Rio de Janeiro Cell Bank

(Brazil). Cells were cultured in 25 cm2 flasks in

complete medium under standard conditions

(37 ºC, 5 % CO2, and 95 % relative humidity)

to 80 % confluence. Cultures were maintained

in an Esco CelCulture® CO2 incubator (CCL-

170B-8, Singapore). Viable cell counts were

performed using a Neubauer chamber and

trypan blue.

Isolation of mouse splenocytes: Six nul-

liparous female BALB/c mice, 6 weeks of age,

were obtained from the National Institute of

Health (Lima, Peru). The use of these animals

in the study was reviewed and approved by the

Ethics Committee of the Faculty of Veterinary

Medicine of the Universidad Nacional Mayor

de San Marcos (CEBA code 2020-2). Spleno-

cytes were obtained according to the method-

ology of Nilofar et al. (2017). Each spleen was

fractionated into small pieces in CM, and the

resulting cell suspension was passed through

a 70 μm cell strainer. Cells were collected and

washed with CM at 1 500 rpm for 5 min. The

pellet was lysed with 2 ml red blood cell lysis

buffer at 4 ºC for 2 min. It was then washed a

second time with CM. Finally, the splenocyte

pellet was resuspended in 1 ml CM for viable

cell counting using a Neubauer chamber and

trypan blue.

Preparation of multicellular tumoral

spheroids formed with 4T1 (MTS): 96-well

round-bottom microplates were coated with 50

μl of 1.5 % agarose per well. Then 200 μl of 4T1

cells (1×104 cells/well) in CM were added. To

promote cell aggregation, the microplate was

shaken at 60 rpm for 20 min using an incuba-

tor-shaker (ZHWY-2102C, Zhicheng, China),

and then incubated for 96 h under standard

culture conditions. The formation of MTS was

verified using a Leica inverted microscope (DM

IL LED, Germany).

Preparation of multicellular tumor

spheroids composed of 4T1 cells and spleno-

cytes (MTSs): 100 μl of 9×103 4T1 cells were

combined with 100 μl of 1×103 splenocytes per

well and processed as described for the prepa-

ration of MTS. Several preliminary assays were

performed to obtain the appropriate cell ratios

for the formation of MTS and MTSs.

Antitumor activity on MTS: The cytotox-

icity of LtF was determined by a colorimetric

assay using MTT. 200 μl of the prepared con-

centrations of LtF or Dox were added to each

MTS and incubated for 72 h under standard

conditions. Each concentration was tested in

quadruplicate (N = 4). The MTS were then

independently transferred to a flat-bottom

microplate, 200 μl of MTT reagent dissolved

in CM was added and incubated for 4 h under

standard conditions. The medium from each

well was discarded, 100 μl of dimethyl sulfoxide

was added, and incubated for 30 min at 40 rpm

on the shaker, at 25 °C. The microplate should

be protected from light during the assay. The

amount of formazan was quantified at 570 nm

with a differential filter of 630 nm, on a spec-

trophotometer (EPOCH2, Biotek Instruments

Inc., USA). Dox-treated MTS were used as pos-

itive and untreated MTS as negative controls.

The percentage of viable cells was calcu-

lated using the formula: Viability (%) = (aver-

age absorbance value of cells treated with the

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

product or drug / average absorbance value of

negative control) × 100. Where the product is

LtF and the drug is Dox. At the end of the assay,

the MTS were observed under an inverted

microscope to analyze any changes in their

appearance. The degree of cytotoxicity of each

LtF or Dox concentration was quantified as the

percentage of cell viability using the absorbance

values obtained for each assay and the ISO

10993-2009 classification, 100-75 %: Non-cyto-

toxic, 74-50 %: Mildly cytotoxic, 49-25 %: Mod-

erately cytotoxic, 24-0 %: Extremely cytotoxic.

Determination of the half-maximal

inhibitory concentration (IC50): GraphPad

Prism software version 8.0.1 (GraphPad Soft-

ware Inc., San Diego, CA, USA) was used to

determine the IC50 for LtF and Dox, using the

following formula: Y = 100/1 + 10X-log (IC50)

where Y is the cell viability defined between

0 % and 100 %, and X is the logarithm of the

concentration of LtF or Dox.

Immunomodulatory activity on MTSs:

After 72 h of treatment, under standard con-

ditions, supernatants were collected from: (i)

IC50 LtF alone (simple treatment); and (ii)

IC50 [LtF+Dox] (combined treatment); posi-

tive control (IC50 Dox) and negative control

(untreated), centrifuged at 1 500 rpm for 5 min

to remove cell debris, and stored at -86 °C.

Quantification of cytokines: Concentra-

tions in the supernatants of MTSs cultures from

treatments (i) and (ii) were determined using

sandwich ELISA kits for the pro-inflamma-

tory cytokines: Tumor Necrosis Factor-Alpha

(TNF-α), Interleukin-6 (IL-6) and the anti-

inflammatory cytokines Interleukin-10 (IL-10),

Transforming Growth Factor-Beta (TGF-β) (N

= 6). 100 μl/well of capture antibody was added

to 4HBX flat bottom microplates correspond-

ing to the cytokine of interest diluted in coating

buffer. The microplate was sealed and incu-

bated overnight at 4 °C with shaking at 60 rpm

according to the manufacturer›s instructions.

The appropriate standard curve was gener-

ated to determine the cytokine concentrations.

Absorbance was measured in a UV/visible

spectrophotometer (EPOCH-2, Biotek Instru-

ments Inc., USA) at 450 nm with a differen-

tial filter at 570 nm. The assay results were

expressed as pg/ml.

Statistical analysis: For the analysis of anti-

tumor activity, the effect of LtF on cell viability

was considered, and values are expressed as the

mean of replicates ± standard error (SEM). For

the analysis of immunomodulatory activity,

cytokine production in treatments (i) and (ii)

was considered, and values are expressed as the

mean of replicates ± standard deviation (SD).

In both cases, differences between treatments

and controls were analyzed using a one-way

analysis of variance (ANOVA), followed by the

Tukey’s honestly significant difference (HSD)

test. P values: *P < 0.05, **P < 0.01 and ***P

< 0.001, compared with the untreated were

considered significant, P > 0.05 were not con-

sidered significant.

RESULTS

Characteristics of MTS and MTSs spher-

oids: The spheroids used were of the aggregated

type and could represent early-stage tumors

given the time spent in their generation and

testing (seven days total) with an average diam-

eter of 0.421 mm (Gallardo et al., 2006). At the

end of the trials, the untreated MTS and MTSs

controls remained compact to the touch, with

a dark central zone and a lighter peripheral

zone (proliferative zone). However, changes

such as cell detachment, increased cell debris,

morphologic deformation, and central zone

enlargement and darkening were induced by

LtF, LtF-Dox or Dox treatments (Fig. 1).

Antitumor effect of LtF on MTS: All LtF

concentrations showed a significant decrease in

cell viability compared to the untreated control

(CTRL-) (P < 0.001) (Fig. 2A). Cell viability

of MTS treated with 1 and 10 μg/ml. LtF and

10 μg/ml Dox was less than 20 %. LtF may be

considered extremely toxic on MTS at concen-

trations of 1, 10, 4 000, and 6 000 μg/ml. The

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

lowest percentage of viability was observed at

the 4 000 μg/ml (5.09 ± 1.52 %). A continuous

dose-dependent response observed in the 100

to 6 000 μg/ml range (Fig. 1). The IC50 of LtF

was 428 µg/ml.

Dox-treated spheroids showed a decrease

in cell viability of less than 12 % at 10 µg/ml

(Fig. 2B). The antitumor activity of Dox was

dose dependent and IC50 was 2 µg/ml. At a

concentration of 1 µg/ml, LtF showed a greater

cytotoxic effect than Dox (P < 0.001), and

at 10 µg/ml the effect was similar (P > 0.05);

however, the IC50 of Dox was much lower than

that of LtF.

Immunomodulatory activity on MTSs:

a) Pro-inflammatory cytokines. A significant

increase in IL-6 production was observed in

the combined and simple treatments compared

to the untreated (P ≤ 0.0001) (Fig. 3A). No

significant differences were observed between

LtF+Dox and Dox (P = 0.55) or between

LtF+Dox and LtF (P = 0.12). A significative dif-

ference was observed between LtF and Dox (P

= 0.0074) (Fig. 3A).

As for TNF-α, a significant increase in

production was observed only in LtF+Dox and

Dox treatments compared to LtF and untreated

(P = 0.001) (Fig. 3B). TNF-α production was

higher with LtF+Dox and Dox than with LtF

(P = 0.001, P < 0.001, respectively). There was a

similarity between LtF+Dox and Dox (P = 0.44)

(4.23 and 4.85 pg/ml, respectively). Although

the results for LtF treatment are not significant

Fig. 1. Morphology of MTSs. Arrow points to central necrotic zone. Abundant cellular debris and peripheral deformation

is observed in MTSs treated with LtF, LtF+Dox and Dox. Cell debris was reduced in the CTRL- ×100. IC50 was used for all

treatments (N = 4).

Fig. 2. Antitumor activity of LtF on MTS. A. LtF: 1, 10, 4 000 y 6 000 μg/ml demonstrated extreme cytotoxicity, B. Dox: 5

and 10 μg/ml were extremely cytotoxic. The IC50 value of LtF and Dox was 428 µg/ml and 2 µg/ml respectively. P values: *P

< 0.05 and ***P < 0.001, compared with untreated (CTRL-). P < 0.05 was considered significant. Data are expressed as mean

± SEM (N = 4).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

with respect to LtF+Dox and Dox; however,

it is interesting to note that the production of

this cytokine was four times higher than in the

untreated (1.03 and 0.25 pg/ml, respectively)

(P = 0.259). In addition, similar trends were

observed for both IL-6 and TNF-α, placing the

combined treatment at an intermediate point

(Fig. 3A, Fig. 3B).

Immunomodulatory activity on MTSs:

b) Anti-inflammatory cytokines. Regarding

IL-10, a decrease in its production was found

in the simple and combined treatments, and

Dox respect to the untreated (P ≤ 0.0001). Dox

significantly reduced IL-10 levels compared to

LtF+Dox (P = 0.033); IL-10 production was

lower with LtF than with LtF+Dox (P = 0.01).

This cytokine was similarly inhibited by both

LtF and Dox (P = 0.945) (Fig. 4A).

Simple and combined treatments inhibited

TGF-β secretion compared to the untreated (P

< 0.001), LtF+Dox was highly significant (P ≤

0.0001) compared to the untreated. No signifi-

cant differences were found between LtF and

Dox (P = 0.804). LtF+Dox affected the produc-

tion of this cytokine with greater potency than

Fig. 3. Pro-inflammatory cytokines produced by MTSs. A. IL-6, B. TNF-α. MTSs were incubated alone or in combination

with LtF or LtF+Dox. LtF IC50 = 428 µg/ml, Dox IC50 = 2 µg/ml. Significance obtain by one-way ANOVA is indicated as ***P

< 0.001, and Tukey’s post-test significance is indicated as ###P < 0.001, ##P < 0.01; ns, statistically non-significant difference

using one-way ANOVA. Data are expressed as mean ± SEM (N = 6).

Fig. 4. Anti-inflammatory cytokines produced by MTSs. A. IL-10, B. TGF-β. MTSs were incubated alone or in combination

with LtF or LtF+Dox. LtF IC50 = 428 µg/ml, Dox IC50 = 2 µg/ml. Significance obtain by one-way ANOVA is indicated as ***P

< 0.001, and Tukey’s post-test significance is #P < 0.05, ##P < 0.01; ns, statistically non-significant difference using one-way

ANOVA. Data are expressed as the mean ± SEM (N = 6).

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

LtF and Dox (P = 0.008 and P = 0.0018, respec-

tively) (Fig. 4B).

It is interesting that in the simple treat-

ment, IL-6 and IL-10 are found in similar con-

centrations (~100 pg/ml), showing a pattern

that could be related to the efforts of immune

cells to restore homeostasis and highlighting

the immunomodulatory role of LtF.

It is also important to note that in untreat-

ed, pro-inflammatory cytokines were produced

at minimal levels, approximately 0.25 pg/ml

for TNF-α and 32.28 pg/ml for IL-6. It should

be noted that 4T1 cells also produce IL-6 and

TNF-α (Hsieh & Wang, 2018). In the positive

control (Dox), a pro-inflammatory environ-

ment was present, whereas in the untreated,

high levels of anti-inflammatory cytokines

indicate a strong immunosuppressive microen-

vironment that would promote tumor develop-

ment in vivo.

DISCUSSION

Breast cancer is the most common malig-

nancy in women, and its incidence is expected

to increase by more than 60 % over the next

20 years. Despite advances in detection and

treatment, the decline in mortality rates has

slowed since 2010, making it a key area of

research for the development of new therapeu-

tic alternatives (Lainetti et al., 2020). Standard

treatment with chemotherapy and radiotherapy

can cause various adverse side effects, such

as weakening of the immune system, making

the search for new treatments imperative. It is

important to note that the success of various

treatments depends on a proper assessment

that takes into account not only the neoplastic

cells, but also the tumor microenvironment

(Bożyk et al., 2022). In recent years, the use

of three-dimensional (3D) cultures or tumor

spheroids has gained popularity because they

more accurately simulate in vivo tumor char-

acteristics, including architecture that favors

cellular and physiological interactions in the

tumor microenvironment. This has reduced

the need for laboratory animals and improved

the effectiveness of in vitro assays in evaluating

new compounds and therapies (Nii et al., 2020;

Tevis et al., 2017).

Among the alternatives being explored

are products derived from algae, one of whose

polysaccharides is fucoidan, which has been

shown to have a cytotoxic effect on in vitro cul-

tures of mouse mammary adenocarcinoma 4T1

cells (Atashrazm et al., 2015; Hsu et al., 2013;

Xue et al., 2012; Xue et al., 2013) and an anti-

proliferative effect on the HeLa and U937 cell

lines due to selective apoptosis (Colona, 2022).

Comparative studies between 2D and 3D

cultures give different results for the same

product. This is the case for Fucus evanescens

fucoidan (100-800 µg/ml), which has cytotoxic

activity in SK-MEL-28 monolayers but loses

it when using SK-MEL-28 spheroids (Malya-

renko et al., 2021). In the present study, we

used MTS spheroids, so it can be said that the

results obtained are more like those that can

be obtained in vivo. Regarding the effect of the

treatments, the MTS or MTSs generally showed

a greater number of debris and partial defor-

mation of their appearance, which was more

pronounced in those treated with Dox. Baek et

al. (2016) observed similar effects on SH-SY5Y

(human metastatic neuroblastoma) spheroids,

in which the cytotoxic effects of Dox apparently

caused degradation of the extracellular matrix,

manifested by detachment of all cells from the

spheroid in an experiment monitored every 30

min for 5 days. In the present study, the cyto-

toxic effect was measured after 3 days, possibly

the longer the time, the complete deformation

of the spheroid would be observed.

Regarding the cytotoxic effect of fucoidan,

the reported concentrations for F. vesiculosus

on 4T1 monolayers range from 90 to 120 µg/

ml (Hsu et al., 2013) and from 50 to 200 µg/ml

(Xue et al., 2013). The concentrations used in

our study cover a wider range, from 1 to 10 000

µg/ml. It was shown that at a concentration of

1 µg/ml, LtF caused about 90 % cytotoxicity,

while at the same concentration Dox caused

about 20 %, suggesting that at low concentra-

tions, the fucoidan from this alga would have a

greater cytotoxic potency than Dox. At higher

concentrations of LtF (8 000 and 10 000 µg/

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

ml), cell viability is affected but not reduced to

the low levels of the previous concentrations.

This behavior may indicate a saturation of yet

unknown receptors on tumor cells through

which regulatory processes of the cell cycle and

apoptosis are stimulated as cytotoxic (antitu-

mor) mechanisms caused by fucoidan (Colona,

2022; Lin et al., 2020a). The presence of two

peaks in MTS viability could be explained by

the increased adaptation of tumor cells to these

LtF concentrations (Di Nicolantonio et al.,

2005). Results can be improved by using spher-

oids of uniform size, which can be obtained

using agarose supports and collagen I matrix

(Lin et al., 2020b) or at least two endpoint cell

staining methods when performing cytotoxic-

ity assessments (Holst & Oredsson, 2005).

The production of cytokines by immune

and tumor cells is related to the stage of the

cancer and therefore the treatment adminis-

tered, the potential outcome depends on the

timing of treatment administration. The shift

from anti-inflammatory to pro-inflammatory

cytokine production creates a favorable envi-

ronment for tumor progression (Paulsen et al.,

2017), which means that a modulating product

can be used, considering the tumor stage. Cyto-

kines may serve as markers of tumor stage. In

the early stages of cancer, pro-inflammatory

cytokines signal the proper functioning of the

cellular immune system, the main mechanism

for eliminating tumor cells (Berraondo et al.,

2019). In a study conducted by Takahashi et

al. (2018), fucoidan was administered orally to

patients with advanced cancer, which reduced

the levels of pro-inflammatory cytokines IL1-β,

IL-6, and TNF-α.

In our study IL-6 production was increased

after LtF treatment, like that reported for fucoi-

dan from Ascophyllum nodosum, F. evanescens,

F. vesiculosus, Sargassum fusiforme, Macrocystis

pyrifera, and Undaria pinnatifida in cultures of

splenic dendritic cells, bone marrow-derived

macrophages, activated splenic macrophages,

and human neutrophils (Hsu & Hwang, 2019).

Considering that splenocytes (T lymphocytes, B

lymphocytes, dendritic cells, macrophages and

monocytes) are found in MTSs, it is expected

that these cells will make direct contact with

4T1 tumor cells, become activated and produce

IL-6; however, IL-6 levels were lower in the

untreated than in the LtF, indicating that the

antigenic stimulus of 4T1 was not sufficient

and that there was a stimulatory effect caused

by LtF. This is a multifunctional cytokine whose

relationship to inflammation and BC is not easy

to establish because it has pro-inflammatory

and anti-inflammatory functions that depend

on the signaling pathway. Elevated levels of IL-6

secretion may accelerate tumor cell growth by

suppressing apoptosis and promoting angio-

genesis; however, the results are controversial.

In a study of patients with early-stage invasive

BC, high IL-6 expression was associated with

improved disease-free survival and specific

survival (Chen J., et al., 2022).

TNF-α controls immune and inflamma-

tory responses during the early stage of tumor

development (Lee et al., 2022). In the combined

treatment, there was a decrease with respect

to the Dox treatment, which could be related

to the modulation exerted by fucoidan, since

in the simple treatment the production of this

cytokine is not significant, although it is four

times higher than in the untreated. To clarify

the modulatory role of LtF in reducing TNF-α

production, it would be necessary to perform

assays taking into account the IC50 of Dox and

different concentrations of LtF to determine

the combination index (Yunita et al., 2020).

Also, assays on the production kinetics of this

cytokine should be included, considering times

shorter than the 72 h used in the present study.

In Dox-treated MTSs cultures, there was a sig-

nificant increase in TNF-α levels as reported

by Syukri et al. (2022), which was confirmed

in the present study. This cytokine favors the

activation, differentiation, survival or death of

cancer cells under certain conditions, an excess

could exacerbate the inflammatory process

and accelerate tumor development, so reduc-

ing its production in advanced stages of cancer

would be beneficial. Although the differences

are not significant, for both IL-6 and TNF-α

there is evidence of a trend towards a reduc-

tion of this cytokine with LtF+Dox. Under

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

highly inflammatory conditions, S. hemiphyl-

lum fucoidan reduced IL-6 and TNF-α levels

(Chen B., et al., 2022).

Overexpression of IL-10 is a poor prognos-

tic indicator associated with drug resistance,

metastatic cancer, and a high probability of

recurrence (Li et al., 2014). The decrease of this

cytokine observed in supernatants of MTSs as

a result of the treatments applied is significant

compared to the untreated. A better decrease

was observed with LtF and Dox than with

LtF+Dox, highlighting the immunomodulatory

capacity of LtF in the system used. Increased

production of this cytokine in LtF+Dox may be

beneficial in the treatment of advanced stage

BC, as it would attenuate the Dox-induced

inflammatory state.

Increased TGF-β production is associated

with poor survival in BC because it suppresses

the anti-tumor immune response and promotes

metastasis through increased levels of angio-

genic and connective tissue growth proteins

that promote epithelial-to-mesenchymal transi-

tion, particularly in BC. It also stimulates sev-

eral signaling networks involved in cell growth,

and differentiation (Lee et al., 2022). In our

study, the modulation exerted by LtF on Dox

was demonstrated by the remarkable decrease

of TGF-β in the combined treatment, as Dox

has been shown to stimulate TGF-β signaling

and consequently increase metastasis in BC

cells (Yunita et al., 2020). Likewise, TGF-β lev-

els decreased in all three treatments compared

to untreated. Similar results were obtained

by Xue et al. (2017) using fucoidan from F.

evanescens in a drug-induced rat mammary

carcinoma model. Chen L., et al. (2022) using

fucoidan combined with olaparib from Lami-

naria japonica in human monocytes, and Guo

et al. (2022) in mice with 4T1 adenocarcinoma,

successfully using micelles formed by fucoidan

and Dox to remodel the immunosuppressive

microenvironment and prevent metastasis.

In our study, the increase in IL-6 produc-

tion in the simple treatment and the decrease

in TGF-β levels in the combined treatment

indicate that LtF has a modulatory effect on the

tumor microenvironment. Due to the elevated

TGF-β concentration produced by untreated

MTSs, it is confirmed that this is a poor prog-

nostic marker for BC, even in early stage, these

results could be compared with serum samples

from individuals with early BC.

According to the literature review, the

present study is the first to demonstrate the

antitumor and immunomodulatory activity of

LtF from the Peruvian Sea on BC tumor spher-

oids, but assays with more complex spheroids

are needed to consolidate these results. In con-

clusion, the immunomodulatory activity of LtF,

the single and combined treatments induced a

significant production of IL-6. In the case of

TNF-α, a study of the kinetics of its production

in the model tested is required, since this cyto-

kine is the first to be secreted, it is suggested

to use shorter times than the one used. The

presence of anti-inflammatory cytokines in the

untreated controls confirms the strong immu-

nosuppression generated in the tumor micro-

environment. LtF is a good candidate for the

treatment of BC with the ability to immuno-

modulate the tumor microenvironment alone

or in combination with Dox, which should be

further investigated.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgement sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

This research was a part of the project

entitled “Estudio preclínico del potencial

inmunoadyuvante del fucoidan de Lessonia

trabeculata nativa (Alga Parda) en un modelo

experimental murino con tumor inducido 4T1,

para su utilización en el tratamiento de cáncer

de mama” funded by the Program PROCIEN-

CIA-Consejo Nacional de Ciencia, Tecnología

e Innovación Tecnológica, Peru (Contrato

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e54918, enero-diciembre 2023 (Publicado Nov. 02, 2023)

N° 133-2017-FONDECYT) and Universidad

Nacional Mayor de San Marcos PCONFIGI

2020, Code number: B20100241. This paper

is part of the undergraduate thesis of Rosa

Condori-Macuri. We also thank PSW-Peruvian

Seaweeds for providing the fucoidan used in

the study and its characteristics.

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