Three-dimensional cell cultures may better mimic avascular tumors. Yet, they still lack characterization and standardization. Therefore, this study aimed to (a) generate multicellular aggregates (MCAs) of four breast cell lines: MCF7, MDA-MB-231, and SKBR3 (tumoral) and MCF12A (non-tumoral) using ultra-low attachment (ULA) plates, (b) detail the methodology used for their formation and analysis, providing technical tips, and (c) characterize the MCAs using morphometry, qualitative cytology (at light and electron microscopy), and quantitative immunocytochemistry (ICC) analysis. Each cell line generated uniform MCAs with structural differences among cell lines: MCF7 and MDA-MB-231 MCAs showed an ellipsoid/discoid shape and compact structure, while MCF12A and SKBR3 MCAs were loose, more flattened, and presented bigger areas. MCF7 MCAs revealed glandular breast differentiation features. ICC showed a random distribution of the proliferating and apoptotic cells throughout the MCAs, not fitting in the traditional spheroid model. ICC for cytokeratin, vimentin, and E-cadherin showed different results according to the cell lines. Estrogen (ER) and progesterone (PR) receptors were positive only in MCF7 and human epidermal growth factor receptor 2 (HER-2) in SKBR3. The presented characterization of the MCAs in non-exposed conditions provided a good baseline to evaluate the cytotoxic effects of potential anticancer compounds.

Seaweed bioactive compounds have shown anticancer activities in in vitro and in vivo studies. However, tests remain limited, with conflicting results, and effects in combination with anticancer drugs are even scarcer. Here, the cytotoxic effects of five seaweed compounds (astaxanthin, fucoidan, fucosterol, laminarin, and phloroglucinol) were tested alone and in combination with anticancer drugs (cisplatin—Cis; and doxorubicin—Dox), in breast cell lines (three breast cancer (BC) subtypes and one non-tumoral). The combinations revealed situations where seaweed compounds presented potentiation or inhibition of the drugs’ cytotoxicity, without a specific pattern, varying according to the cell line, concentration used for the combination, and drug. Fucosterol was the most promising compound, since: (i) it alone had the highest cytotoxicity at low concentrations against the BC lines without affecting the non-tumoral line; and (ii) in combination (at non-cytotoxic concentration), it potentiated Dox cytotoxicity in the triple-negative BC cell line. Using a comparative approach, monolayer versus 3D cultures, further investigation assessed effects on cell viability and proliferation, morphology, and immunocytochemistry targets. The cytotoxic and antiproliferative effects in monolayer were not observed in 3D, corroborating that cells in 3D culture are more resistant to treatments, and reinforcing the use of more complex models for drug screening and a multi-approach that should include histological and ICC analysis.

Jaboticaba is a Brazilian native berry described as a rich source of phenolic compounds (PC) with health promoting effects. PC from jaboticaba peel powder (JPP) have low intestinal bio-accessibility and are catabolized by gut microbiota. However, the biological implication of PC-derived metabolites produced during JPP digestion remains unclear. This study aimed to evaluate the antiproliferative effects of colonic fermented JPP (FJPP) in a 3D model of colorectal cancer (CRC) composed by HT29 spheroids. JPP samples fermented with human feces during 0, 2, 8, 24 or 48 h were incubated (10,000 µg mL⁻¹) with spheroids, and cell viability was assessed after 72 h. Chemometric analyses (cluster and principal component analyses) were used to identify the main compounds responsible for the bioactive effect. The antiproliferative effect of FJPP in the CRC 3D model was increased between 8 h and 24 h of incubation, and this effect was associated with HHDP-digalloylglucose isomer and dihydroxyphenyl-γ-valerolactone. At 48 h of fermentation, the antiproliferative effect of FJPP was negligible, indicating that the presence of urolithins did not improve the bioactivity of JPP. These findings provide relevant knowledge on the role of colonic microbiota fermentation to generate active phenolic metabolites from JPP with positive impact on CRC.

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

In this study, the novel exopolysaccharide (EPS) produced by the marine bacterium Alteromonas macleodii Mo 169 was used as a stabilizer and capping agent in the preparation of selenium nanoparticles (SeNPs). The synthesized nanoparticles were well dispersed and spherical with an average particle size of 32 nm. The cytotoxicity of the EPS and the EPS/SeNPs bio-nanocomposite was investigated on human keratinocyte (HaCaT) and fibroblast (CCD-1079Sk) cell lines. No cytotoxicity was found for the EPS alone for concentrations up to 1 g L⁻¹. A cytotoxic effect was only noticed for the bio-nanocomposite at the highest concentrations tested (0.5 and 1 g L⁻¹). In vitro experiments demonstrated that non-cytotoxic concentrations of the EPS/SeNPs bio-nanocomposite had a significant cellular antioxidant effect on the HaCaT cell line by reducing ROS levels up to 33.8%. These findings demonstrated that the A. macleodii Mo 169 EPS can be efficiently used as a stabilizer and surface coating to produce a SeNP-based bio-nanocomposite with improved antioxidant activity.

Breast cancer (BC) accounts for the most cancer-related deaths amongst women worldwide, implying an urgent need of finding new drugs more effective than those currently in use [1, 2]. Fucoxanthin (Fx) is a marine carotenoid derived from brown seaweed that has been showing antitumor effects on different cancer cell lines, mainly in 2D models [3]. However, 3D culture models have a better predictive capacity of in vivo cellular responses against cytotoxic compounds [4]. This study aimed to evaluate the potential anticancer effects of Fx versus Doxorubicin (Dox) (a conventional anticancer drug) in a panel of three BC cell lines, representative of different molecular subtypes (MCF-7, SKBR3 and MDA-MB-231), and in a non-tumoral BC cell line (MCF-12A), cultured under 2D and 3D conditions. Effects on cell viability, death, proliferation and DNA damage were targeted. Results from the 2D cultures showed that Dox and Fx caused cytotoxicity on all the cell lines. Cytotoxic activity of Dox involved mechanisms of cell death induction (in MCF-12A cells) and genotoxicity (in MDA-MB-231 cells), contrarily to Fx, where neither cell death nor genotoxic mechanisms were detected. Under 3D conditions, cells were less responsive to Dox and Fx, compared to 2D cultures. Antiproliferative effects were solely registered in SKBR3 cells exposed to Dox and Fx, and in MCF-12A cells exposed to Dox. The in vitro data revealed that the Fx may be a potential anticancer agent against BC cells, with differential effects according to the cell subtype. The data warrants further studies on the underlying anticancer mechanisms. Acknowledgments: We thank the fundamental financial support of the ICBAS of the UPorto, namely via its Laboratory of Histology and Embryology and Master Program in Environmental Contamination and Toxicology. This research was also partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by FCT and ERDF, in the framework of the program PT2020. [1] DeSantis, C.E., F. Bray, J. Ferlay, J. Lortet-Tieulent, B.O. Anderson, and A. Jemal, International Variation in Female Breast Cancer Incidence and Mortality Rates. Cancer Epidemiol Biomarkers Prev, 2015. 24(10): p. 1495-506. [2] Pádua, D., E. Rocha, D. Gargiulo, and A.A. Ramos, Bioactive compounds from brown seaweeds: Phloroglucinol, fucoxanthin and fucoidan as promising therapeutic agents against breast cancer. Phytochem Lett, 2015. 14: p. 91-98. [3] Kumar, S.R., M. Hosokawa, and K. Miyashita, Fucoxanthin: a marine carotenoid exerting anti-cancer effects by affecting multiple mechanisms. Mar Drugs, 2013. 11(12): p. 5130-47. [4] Santo, V.E., S.P. Rebelo, M.F. Estrada, P.M. Alves, E. Boghaert, and C. Brito, Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs. Biotechnol J, 2017. 12(1): p. 1600505.