The Klotho gene functions as an anti-aging gene. A previous klotho-knockout mice study indicated that neither male nor female gametocytes could accomplish the first meiotic division. It suggested that Klotho might regulate cell division. In this study, we determined the roles of Klotho in cytokinesis in cultural human cells (HEK293 and HeLa) and in zebrafish embryos. Immunoprecipitation, mass spectrometry analysis, and a zebrafish model were used in this study. The results showed that Klotho is located in the midbody, which correlated with cytokinesis related kinases, Aurora kinase B and citron kinases, in the late stage of cytokinesis. There was a spatial correlation between the abscission site and the location of Klotho in the cytokinesis bridge. A three-dimensional structural reconstruction study demonstrated there was a spatial correlation among Klotho, Aurora kinase B, and citron kinases in the midbody. In addition, Klotho depletion inactivated Aurora kinases; it was also indicated that Klotho depletion caused aberrant cell cycle and delayed cytokinesis in a cell model. The study with zebrafish embryos suggested that klotho knockdown caused early embryo development abnormality due to dysregulated cytokinesis. In conclusion, Klotho might have a critical role in cytokinesis regulation by interacting with the cytokinesis related kinases. Klotho is a type-I membrane protein related to β-glucuronidases, which is encoded by the KL gene in humans . Klotho It is also known that Klotho has a critical role in the calcium–phosphate hemostasis. The bone-derived growth factor, FGF23, was associated with parathyroid hormone and vitamin D, and mobilizes the sodium–phosphate cotransporters that control renal phosphate transport in proximal tubular epithelial cells. In the presence of Klotho, FGF23 activated its receptor, which influences the homeostasis of phosphate. As a co-receptor of FGF, Klotho converted canonical FGFR1 into a specific receptor for FGF23 ,6,7. FurIn addition to aging and calcium–phosphate hemostasis, accumulated evidence suggests that Klotho is a multifunctional protein. Recent studies have indicated that Klotho might have critical roles in inflammation, oxidative stress, glucose metabolism, cell proliferation, and apoptosis ,11,12,13Cytokinesis is the final stage of the cell division process during which the cytoplasm of a single eukaryotic cell divides into two daughter cells. Actin filaments and myosin-II motors, as well as many other proteins, are reported to function in the cytokinesis. Several kinases, such as Aurora kinases and polo kinase, have important regulatory roles in cytokinesis ,15. Prev2, 5% CO2) at 37 °C. For the cell cycle synchronization study, the cultured cells were treated with serum free medium or nocodazole  for 16 h [The human embryonic kidney (HEK) 293 cell lines and Hela cells were purchased from American Type Culture Collection . All of the cell lines were maintained in a DMEM medium  containing 10% FBS , and were supplemented with antibiotics and cultured in a humidified atmosphere (95% O5) were seeded in each well and cultured for 24 h at 37 °C. The cells were then co-incubated with 10 µL Lipofectamine® 2000  and 4 µg plasmids at 37 °C for 6 h. Subsequently, the cells were treated with fresh medium, followed by an additional 24 h of incubation at 37 °C. Following the transfection procedure, the cells with a GFP expression were selected with flow cytometry  and cultured for the further studies.The plasmids Klotho-sh-green fluorescent protein , membrane Klotho-V5, and secreted Klotho-V5  were used for the Klotho knock down and overexpression. HEK293 and HeLa cells (1 × 10Each immunoprecipitation (IP) was performed in triplicate. HEK293 cells were grown on 10 cm plates until 90% confluent, and were then harvested. The harvested cells were lysed with 500 μL of 0.5% NP40 lysis buffer, 50 mM HEPES pH 7.6, 150 mM KCl, 2 mM EDTA, 0.5% NP40, and 0.5 mM DTT, and protease/phosphatase  at 4 °C for 15 min. The lysates were cleared using IgG–Agarose . Then, 5 mg of the total protein was used for IP with 50 μg of either pre-cross-linked Klotho antibody  or non-specific IgG . The IP samples were then digested using 20 ng/mL of trypsin . Finally, the peptides were extracted, concentrated to dryness under vacuum, and stored at −20 °C until LC/MS/MS analysis. The type of search of mass spectrometer  was MS/MS ion search. The database search parameters were as follows: Database, SwissProt 2013_03 ; enzyme, Trypsin; fixed modification, carbamidomethyl (C); variable modifications, oxidation (M); taxonomy homo species, human; mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance: ±10 ppm; fragment mass tolerance, ±1 amu; max missed cleavages, 2; and instrument type, ESI-TRAP. Proteins with >two-folded changes (compared with the control group) and two peptides identified by mass spectrometer were considered significant. p value less than 0.05 were considered significant.The pathway analysis was done with Reactome V62 . All of The total protein was extracted using a commercial kit according to the manufacturer’s instructions . Thirty micrograms of protein from each sample was mixed with a sample loading buffer and loaded onto separate lanes on a 12% sodium dodecyl sulfate-polyacrylamide gel. The proteins were electro-transferred onto polyvinylidene fluoride membranes  and then immunoblotted with primary antibodies. The intensity of each band was quantified using NIH Image software , and the densitometric intensity corresponding to each band was normalized against the β-actin expression. The primary antibodies used for immunoblot were as follows: rabbit polyclonal β-actin antibody , rabbit polyclonal Klotho antibody , rabbit monoclonal cyclinB1 antibody , Aurora kinase A/-p antibody , Aurora kinase B/-p antibody , and mouse polyclonal citron kinase antibody .® Tubulin-RFP, BacMam 2.0). For the nucleus staining, cells were incubated with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) (abcam). The stained samples were observed under a confocal microscope . Imaris software  was used to generate a 3D model from the stacks that were acquired every 1 μm. Living cell images were observed using a Nikon Eclipse Ti-E  with TokaiHit WSKM chamber and were controlled by NIS-element AR. The objective lens used was 100× CFI Plan Apochromat Lambda 100× oil objectives (N.A.1.45). Time-lapse images were acquired at 3 s intervals for 2 min by Photometrics CoolSNAP MYO CCD.For immunofluorescence staining, cultured HeLa cells fixed by methanol were incubated with primary antibodies, followed by incubation with a fluorescent secondary antibody. The primary antibodies used for immunofluorescence staining were as follows: rabbit polyclonal Klotho antibody , mouse monoclonal Aurora kinase B antibody , and mouse polyclonal citron kinase antibody . The fluorescent staining for tubulin was done with a tubulin-RFP fusion protein construct  were maintained at 28 °C with a standard protocol . The embp-values of <0.05 were considered statistically significant.All of the data were expressed as mean ± standard error. One-way analysis of variance with Bonferroni corrections was performed for analyzing the data of the cell culture study. The data of the different animal groups were compared using the Wilcoxon–Mann–Whitney test. For exploring the potential function of Klotho, an IP/LC/MS/MS study was performed (using HEK293 cell lysates). There were 202 proteins identified by the above analysis . The reaFor determining the roles of Klotho in the cell cycle and mitosis, cell cycle markers, cyclin B1 and cyclin D1, were evaluated by Western blotting with nocodazole-arrested mitotic HEK293 cell extracts. In these cells, we found that the G2/M marker, cyclin B1, increased after the cells were released from nocodazole and declined after 6 h in the cells transfected with the control-GFP. In contrast, the cyclin B1 levels started to decrease soon after nocodazole release (0.5 h) in the cells with Klotho depletion A. FollowTo determine the subcellular localization of Klotho in mitotic cells (HeLa cells), endogenous Klotho were analyzed by immunofluorescent staining. During the mitosis, the expression of Klotho was unevenly distributed as a dot-like structure in the cytoplasm; meanwhile, membrane localized Klotho was noted A. DuringIt is known that two Klotho isoforms, membrane and secreted forms, are formed by RNA alternative splicing or protease cleavage . For defTo further investigate the association of cytokinetic abscission and Klotho localization, the spatial correlation between the abscission site and the localization of Klotho was analyzed by immunofluorescent staining. The results indicated that Klotho was localized at the body and outer edges of the midbody in the early to middle stage cytokinesis before bridge abscission starting A. At theTo explore the roles for Klotho, we monitored the cytokinesis abscission with HeLa cells with Klotho depletion. The study results indicated that the cytokinesis bridge structure is unusually elongated between divided cells. In addition, the abscission sites were not observed in the Klotho depleted cells in the late stage of cytokinesis D. The liThe reactome pathway analysis suggested that Klotho might have roles in Aurora kinase A activation. In addition, a potential physical interaction between Klotho and Aurora kinase B (protein ID: C7G535) was noted by the IP/LC/MS/MS study . Aurora For detailing the spatial localization of Klotho in the midbody and its correlation with cytokinesis related protein kinase, immunofluorescent staining for intrinsic Klotho and three dimensional (3D) reconstruction were performed. The results demonstrated that the midbody localization of Aurora kinase B happened in the early to middle stage of cytokinesis (upper panel of Cytokinesis is a critical process for controlling embryo cleavage and establishing embryonic cell polarity ,27. For Recent evidence indicates that Klotho has important roles in cell proliferation and differentiation ,28,29. IOur study further demonstrated that Klotho is co-localized with Aurora kinase B and citron kinases in the midbody during the cytokinesis, and is correlated with cytokinesis bridge abscission. The midbody is composed of several proteins localized to the preceding contractile ring and central spindle, which show a very precise and stereotypic distribution ,31. The Cytokinesis is the final stage of cell division. Cytokinesis failure in mammals is associated with carcinogenesis. Cell cleavage failure caused by impaired cytokinesis can lead to genetic instability and potentially to tumorigenesis . AccumulOur observation indicated that Klotho might have a critical role in cytokinesis regulation by interacting with the cytokinesis related kinases. Much remains to be done to characterize the roles of Klotho in cytokinesis. Of particular interest is the question of how Klotho is recruited during cytokinesis and regulates the abscission machinery.


BRCA genes are the leading cause of hereditary breast cancer. Current options to prevent cancer in these high-risk patients, such as anti-estrogen drugs and radical mastectomy, are limited by lack of efficacy, undesirable toxicities, or physical and emotional challenges. We have previously shown that PARP inhibitors can significantly delay tumor development in BRCA1-deficient mice. Here, we fabricated the PARP inhibitor talazoparib (TLZ) into spacer implants (InCeT-TLZ) for localized and sustained delivery. We hypothesized that this novel formulation will provide an effective chemopreventive strategy with minimal toxicity. TLZ was released gradually over 30 days as implants degraded. InCeT-TLZ significantly decreased proliferation and increased DNA damage in the mammary glands of BRCA1-deficient mice. Notably, the number of mice that developed hyperplasia in the mammary glands was significantly lower with InCeT-TLZ treatment compared to the control group. Meanwhile, InCeT-TLZ was also better tolerated than oral TLZ, without loss of body weight or anemia. This study provides proof of concept for a novel and safe chemopreventive strategy using localized delivery of a PARP inhibitor for high-risk individuals. Future studies will directly evaluate the effects of InCeT-TLZ for preventing tumor development.Mutations in  BRCA1) gene is an important tumor suppressor that protects against genomic instability1. BRCA1 regulates numerous cellular functions, including cell cycle, apoptosis and transcription3. Moreover, the BRCA1 protein plays a critical role in repairing double-stranded DNA breaks through the homologous recombination (HR) pathway. Mutations in BRCA genes are the leading cause of hereditary breast cancer. Women with BRCA mutations have up to an 80% lifetime risk of developing breast cancer4. The majority of BRCA1 mutated tumors are basal-like5, which is a subtype associated with a poor prognosis6. Developing effective cancer prevention strategies has become essential for these BRCA carriers.Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer deaths in women in the United States. In 2019, 268,600 women were expected to be diagnosed with breast cancer in the U.S. The breast cancer-associated  are successful examples of drugs that can prevent breast cancer. Tamoxifen and raloxifene have been approved by the FDA to prevent breast cancer in high-risk women17. Both BRCA and PARP (poly (ADP-ribose) polymerase) are essential for proper DNA repair. BRCA initiates DNA repair of double-strand DNA breaks though the homologous recombination repair pathway, while PARP mediates DNA repair of single-strand DNA damage. Inhibiting PARP by PARP inhibitors in BRCA-deficient cells results in genomic instability and induces cell death. Olaparib and talazoparib are two PARP inhibitors that have been approved by the FDA for the treatment of BRCA-mutated breast cancer18. Talazoparib is approximately 100 times more potent than olaparib because in addition to inhibiting the catalytic activity of PARP, talazoparib traps PARP at the site of DNA damage, i.e. “PARP trapping,” which further induces cell death19.PARP inhibitors were developed to treat BRCA-deficient cancers by inducing synthetic lethality20. Currently, PARP inhibitors are formulated for oral delivery. Low bioavailability with oral delivery of these drugs, poor drug accumulation in the target tissue and undesired toxicities because of high systemic exposure limit the use of these drugs for prevention.In addition to their efficacy for treating breast cancer, we have previously reported that PARP inhibitors are also effective for preventing the development of mammary gland tumors in BRCA1-deficient mice. When fed in diet, veliparib or olaparib (200 mg/kg diet or approximately 50 mg/kg body weight) delayed the average age of the first detectable tumor by 2.4 weeks and 6.5 weeks, respectively, compared to controls21. Here, InCeT-TLZ was first tested in vitro to validate its physicochemical properties, including stability, loading efficiency, release kinetics and biological functions. InCeT-TLZ was then tested in vivo using BrcaCo/Co;MMTV-Cre;p53+/− mice to evaluate its efficacy and toxicity profiles. To determine if InCeT-TLZ is effective for prevention, we first tested if these inserts can alter biomarkers before palpable tumors can be detected.In the present studies, we aim to improve local delivery of PARP inhibitors for development as effective chemopreventive agents with minimal toxicities. The PARP inhibitor talazoparib was fabricated into spacer implants (InCeT-TLZ), which can be injected directly into mammary glands using brachytherapy needles. This formulation using biodegradable polymers was designed to induce a sustained release of talazoparib for localized therapy. Poly (lactic-co-glycolic) acid (PLGA) has been approved by the FDA as a biodegradable and biocompatible copolymer for drug delivery. PLGA has flexible mechanical and degradation properties that have been widely used for developing controlled delivery systems for many different moleculesAll solvents used were HPLC grade and purchased from Sigma-Aldrich unless otherwise stated. Talazoparib , poly(lactic-co-glycolic) acid  , 1 mL tuberculin syringes  and SPX-50 silicone tubing  were purchased for implant fabrication.22. The implants were prepared with a composition of 5% (w/w) talazoparib embedded in poly(lactic-co-glycolic) acid matrix. Accordingly, talazoparib (9 mg) was dissolved in dimethyl sulfoxide, and PLGA (190 mg) was separately dissolved in chloroform. The polymer and drug solutions were mixed thoroughly by vortexing and sonication. The polymer/drug mixture was transferred to a 1 mL syringe and extruded into silicone tubing using an infusion pump (Harvard Apparatus) at a rate of 2.5 µL/min. After extrusion, the tubing was kept in an oven at 60°C overnight. The next day, the implants formed inside the tubing were ejected, cut to an appropriate size and stored in a closed vial at − 20°C. Blank PLGA implants without drug (empty spacers) were fabricated in the same method described above.Talazoparib implants were fabricated using a polymer extrusion method with modificationsA Hitachi S-4800  field emission scanning electron microscope (SEM) was used to observe cross-sections of the implants. To prepare the samples for SEM analysis, the implants were dipped in liquid nitrogen to flash freeze and a cross-section of the cylindrical implant was cut with a frozen razor. The cut implants were mounted on SEM stubs using conductive carbon adhesive and sputter-coated with 10–15 nm of platinum for 5 min using a Denton Vacuum DV-502 system . The implants were imaged using SEM at 5–15 kV.3PO4, with a gradient flow rate of 0.8 mL/min, and detected with ultraviolet absorbance at 230 nm. To determine the drug loading per unit length (mm) of implant, 4 mm long implants (n = 3) were first dissolved in dimethylformamide (0.1 mL), ethanol (0.4 mL) was added, and the extracted drug was quantified by HPLC analysis. A standard curve was fit with a linear regression curve and the curve was used to quantify the concentration of talazoparib in each implant. To determine the drug releasing behavior of the implants, 4 mm long TLZ implants (n = 3) were incubated with 1 mL of PBS (pH 6.0) in a microcentrifuge tube at 37 °C. At scheduled time intervals, the release medium was entirely withdrawn from the microcentrifuge tube and replaced with 1 mL of fresh PBS. The amount of talazoparib released in the withdrawn medium was quantified by HPLC using a standard curve. The drug released from the implants was plotted as a function of time.The drug loading and releasing behavior of InCeT-TLZ implants was determined by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II system and a reverse phase SUPELCOSIL LC-18 HPLC column. The samples were run by HPLC using a mobile phase of acetonitrile and water (1:1), both solvents containing 0.1% H23. Cells were cultured in DMEM media + 10% FBS + 1% Pen/Strep . W0069 and W780 cells were treated with empty spacer or InCeT-TLZ (2 mm or 4 mm) in 6-well plates for 48 h, and then cells were harvested and proteins were extracted for western blotting. In the cell viability assay, cells were seeded into 96 well plates and treated with empty spacer or InCeT-TLZ (1 mm) for 5 days. Cell viability was assessed by the MTT assay.W0069 and W780 cells were derived from mammary tumors of BRCA1-deficient mice and provided by Dr. Chuxia Deng at the NIH 24. W780 and W0069 cells treated with empty spacer or InCeT-TLZ were lysed in RIPA buffer  with protease inhibitors . The BCA assay was used to quantify the concentration of protein samples. 20 µg of protein were loaded and separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Primary antibodies were applied to detect the corresponding proteins, including γH2AX , cleaved-caspase 3 , PARP/cleaved-PARP , PCNA , and vinculin . Secondary antibodies (anti-rabbit or anti-mouse linked to HRP) were purchased from Cell Signaling Technology. ECL Western blotting substrate  was used to detect the signal. Images shown are representative of 3 independent experiments.Western blotting was performed as previously describedBrcaCo/Co;MMTV-Cre;p53+/−) were randomized into three groups (N = 33–35/group): InCeT-TLZ group , empty spacer group (2 mm of blank PLGA InCeT implant), and oral TLZ treatment group . All implants were inserted into the left abdominal mammary gland . Every effort was made to minimize suffering. Mice were euthanized by inhalation of carbon dioxide followed by cervical dislocation. Age-matched, and when possible littermate-matched, female BRCA1-deficient mice  were then harvested and sectioned for histopathology and immunohistochemistry. Citrate buffer  was used for antigen retrieval. Slides were incubated in 3% hydrogen peroxide for 10 min to quench the endogenous peroxidase activity. Sections were stained with PCNA  or γH2AX  antibodies for 1 h at room temperature or overnight at 4°C, respectively. Anti-mouse and anti-rabbit secondary antibodies conjugated to HRP were purchased from Cell Signaling. Signal was detected using a DAB kit . Sections were counterstained with hematoxylin (Vector). The percentage of positively stained cells was quantified using ImageJ. All the analysis was done blinded as to group identity to eliminate any potential bias.Immunohistochemistry was conducted as described previously24. A paired t-test was used to compare body weight before and after treatment. For the histopathology of mammary glands, a Chi-Square test was used to compare proportions24. p < 0.05 was considered statistically significant.The in vitro experiments were repeated at least three times. Results were expressed as mean ± SE. For the in vivo experiments, results were analyzed using one-way ANOVA followed by a Tukey test if the data fit a normal distribution; the Kruskal–Wallis one-way ANOVA on ranks was used followed by the Dunn test for multiple comparisons if the data did not fit a normal distribution (Prism 6)InCeT-TLZ implants were fabricated using a solvent-based polymer extrusion method. The fabrication procedure is simple, reproducible and cost-effective. The implants produced are solid cylindrical rods Fig. A, which To determine the consistency of drug loading in the implants, the amount of drugs in the implants produced in two small-scale batches were compared by HPLC. The loading content of TLZ in two different batches was approximately 25 µg per unit length (mm) of implant. Moreover, HPLC chromatography confirmed the physical stability of the drug in the implants. HPLC data showed the retention time of a peak of talazoparib in the implant had the same peak as the standard talazoparib in the chromatogram.The release profile of talazoparib from the implants was also studied. To determine the release kinetics, InCeT-TLZ implants were cut to predetermined lengths and incubated in PBS medium (pH 6.0) at 37 °C. At each predetermined time point, the medium was completely removed and replaced with fresh medium. Each collected medium fraction was subjected to HPLC analysis to quantify the released drug. The release profile of talazoparib implants showed continuous drug release in vitro Fig. D. There 25. Talazoparib increased the expression of γH2AX, cleaved-caspase 3, and cleaved-PARP within 48 h24. Similarly, treatment with InCeT-TLZ increased the expression of γH2AX, cleaved-caspase 3 and cleaved-PARP in both cell lines  were treated with InCeT-TLZ at the indicated concentrations for 48 h. Biomarkers of DNA damage γH2AX) and cell death (cleaved-caspase 3 and cleaved-PARP) were detected. Both W780 and W0069 cells were derived from tumors that developed in BRCA1-deficient mice. Although W780 and W0069 share a similar genotype, W780 cells represent an adenocarcinoma phenotype and W0069 cells represent a fibroadenoma phenotype, which is representative of the heterogeneous histopathology found in these tumors in vivoAX and ceTo test the chemical stability and efficacy of drug in the implant, BRCA1-mutated breast tumor cells were incubated with the implants (1 mm) for 5 days. Cell viability was detected using a MTT assay. An empty spacer (1 mm) control was included in the assay in order to analyze the effects of the polymer matrix of the implant. After 5 days of incubation, the cell viability in the InCeT-TLZ implant and empty spacer treated groups were 7.22 ± 0.49% (p < 0.001 vs. DMSO control) and 88.6 ± 2.06%, respectively in W780 cells , before any tumors had developed. Age-matched female Brca1Co/Co;MMTV-Cre;p53+/− mice were randomized into three treatment groups. The mice in the InCeT-TLZ treatment group were implanted with a TLZ spacer into the left abdominal mammary gland  using 18G brachytherapy needles. The second group was implanted with an empty spacer (2 mm) as the blank control group. To compare to the oral delivery platform, an oral TLZ group was included (FreeTLZ) as the third treatment group. 50 μg free TLZ was divided into 13 injections and given by gavage three times a week  for 4 weeks (~ 3.84 μg per injection). All the mice were sacrificed 4 weeks after treatment and the left abdominal mammary glands were collected for analysis. Although cohorts of age-matched mice were started on treatment at different ages, the changes across the three treatment groups were consistent. Thus, the three different time points were pooled for data analysis.To evaluate the effects of InCeT-TLZ in vivo, we implanted the InCeT-TLZ spacers into the abdominal mammary glands of Brca1By the chosen end points , the majority of the mice had not developed palpable tumors. As expected, only mice from the 24-week-old cohort had developed tumors: two mice in the empty spacer group and one mouse each from the InCeT-TLZ and FreeTLZ groups. Despite the lack of malignant tumors, many of the mice displayed pre-malignant lesions, or hyperplasias, in the mammary gland Fig. A. In the24. By blocking DNA repair pathways, DNA damage and growth arrest are induced in the cells. We stained for the expression of PCNA and γH2AX in mammary gland tissues to evaluate cell proliferation and DNA damage, respectively. In the empty spacer control group, epithelial cells of the ducts were highly proliferative as shown by the dark brown PCNA+ staining  decreased body weight  decreased the red blood cell count compared to the control group Fig. B. In conIn this study, we developed a localized delivery platform for the PARP inhibitor talazoparib as a novel strategy for chemoprevention. We fabricated talazoparib into a spacer implant which can be inserted into the target tissue as a sustained drug release depot. InCeT-TLZ was stable at room temperature and released TLZ in a linear manner over 30 days. Drug loading was consistent across batches (25 μg/mm), and the dose can be adjusted by changing the length of the spacer. InCeT-TLZ did not alter the biological effects of TLZ as it induced DNA damage and cell death in BRCA1-deficient tumor cells. Moreover, InCeT-TLZ, at a low dose, significantly decreased the development of hyperplasia in BRCA-deficient mice and was more efficacious in inducing DNA damage, which is a biomarker for efficacy of PARP inhibitors, compared to empty spacer or oral FreeTLZ treatment. This localized delivery of TLZ was also well tolerated, as no significant weight loss or anemia was observed as in the systemic exposure group. Our results demonstrate that these TLZ spacer implants are effective chemopreventive agents with minimal toxicity in BRCA1-deficient mice. We anticipate the same potential benefits with this approach could be optimized for use in patients with BRCA deficiency, although translation into human patients will require optimization of the number of implants needed based on the diffusion of drug out of the spacer.BRCA mutations, many other people could potentially benefit from this chemopreventive strategy. PARP inhibitors are proposed to selectively target cells that undergo malignant transformation and acquire defects in DNA repair pathways. In addition to mutations in BRCA1/2 genes, other genetic alterations could also produce HR deficiency and make cells vulnerable to synthetic lethality28. This includes genetic alterations of RAD51, ATR, CHK1/2, ATM, FANCD2 and FANCA, which are all key players within the HR pathway29. It has already been shown that PARP inhibitors are effective in treating BRCA1/2 wild-type tumors30. Therefore, InCeT-TLZ could potentially impact more women beyond populations with BRCA mutations. Additional studies should test the InCeT-TLZ platform for preventing other subtypes of breast cancer.Besides the well-defined high-risk patients with known germline 7.Chemoprevention has been an underused approach to reduce cancer risk and mortality. The selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene remain the only approved drugs for breast cancer prevention over the last 20 years. Unfortunately, the ineffectiveness of SERMs in preventing BRCA-deficient breast cancer leaves bilateral prophylactic mastectomy (BPM) or “watchful waiting” the only options for BRCA carriers. Those not interested in BPM should be offered additional preventive therapies besides surveillance with annual mammograms or magnetic resonance imaging26, have shown efficacy in preventing or delaying breast cancer development in mouse models. Our study indicates TLZ, a more potent PARP inhibitor compared to olaparib and veliparib, could also serve as a potential chemopreventive agent for breast cancer. The synthetic triterpenoid CDDO-methyl ester has been tested in the BRCA-deficient mouse model and significantly delayed tumor development by an average of 5.2 weeks23. The synthetic retinoid fenretinide has shown promising results in clinical trials for breast cancer chemoprevention31. The bromodomain inhibitor I-BET 762 delays tumor onset in a mouse model of ER- breast cancer32. The histone deacetylase inhibitor vorinostat, alone or in combination with synthetic triterpenoids, also reduces tumorigenesis in the PyMT breast cancer mouse model33. Drugs that are approved and widely used for diseases other than cancer can also be explored for chemopreventive potential. Successful chemoprevention agents require comprehensive evaluation to balance between risk and benefits.A number of compounds, including PARP inhibitors (olaparib and veliparib)34. Here, we fabricated TLZ into PLGA spacers, which can be easily adapted using existing brachytherapy procedures. As a localized treatment, InCeT-TLZ could overcome some of the limitations of conventional oral delivery of PARP inhibitors, such as limited bioavailability, poor tissue accumulation, and early drug metabolism. By avoiding a first pass through the systemic circulation, local delivery also reduces the risk of systemic toxicities. Unlike other strategies, such as intermittent dosing, this implantable formulation bypasses the concerns of noncompliance in patients and further improves the therapeutic effects. The differences of effects between local delivery and intermittent therapy needs to be compared directly in future studies. Our proof-of-concept study provided a template that can be easily used to formulate other drugs for many other applications.A localized delivery platform is one way to lower risk and enhance efficacy. Sustained and local delivery of drugs becomes an option with the use of biodegradable materials. PLGA is a FDA-approved polymer with great biocompatibility and biodegradability. It has been broadly used to develop controlled delivery systems for drugs from small molecules, macromolecules or proteins35. By knowing the mechanism of action of a drug, predictive biomarkers can be used to evaluate drug efficacy and toxicity at early stages. Biomarkers are often evaluated as surrogate endpoints in chemopreventive clinical trials. For instance, histological modulation of intraepithelial neoplasias has been the primary phenotypic surrogate end point in the National Cancer Institute chemoprevention program36. Biomarkers of proliferation, differentiation, chromosomal damage, cell growth regulatory molecules, and biochemical activities are other potential surrogate end points36.Biomarker studies may serve as a model to screen new agents efficiently. Biomarkers are not only powerful tools for disease diagnosis and personalized medication, they are also playing increasing roles in drug discovery and development37. The decreased incidence of hyperplasia also suggests a promising chemopreventive effect of InCeT-TLZ. Although systemic administration of PARP inhibitors has shown efficacy in previous publications20, FreeTLZ failed to reduce the percentage of hyperplasia cases in our study. The main reason for the difference is that we used a much lower dose of talazoparib compared to previous studies. In the systemic administration (FreeTLZ) group, a total of 50 μg TLZ was divided into 13 injections and given 3 times a week over 4 weeks, which translates to only 3.85 μg per injection and approximately 0.13 mg/kg body weight. In previous prevention studies, up to 200 mg/kg diet olaparib (roughly 50 mg/kg body weight) was used. With a lower dose, lower efficacy was expected. Indeed, FreeTLZ significantly decreased the percentage of proliferating cells, which is predictive of delayed tumor development for prevention in this model. In future studies, we will directly evaluate the efficacy of InCeT-TLZ for preventing tumor development. Other drug candidates for chemoprevention and possible drug combinations will be explored using this localized delivery platform.In our study, we have confirmed the biological changes induced by the PARP inhibitor talazoparib. Induction of γH2AX, in particular, has been commonly used as an indicator of drug responseSupplementary Information.


Malignant mesothelioma (MM) still represents a devastating disease that is often detected too late, while the current effect of therapies on patient outcomes remains unsatisfactory. Invasiveness biomarkers may contribute to improving early diagnosis, prognosis, and treatment for patients, a task that could benefit from the development of high-throughput proteomics. To limit potential sources of bias when identifying such biomarkers, we conducted cross-species proteomic analyzes on three different MM sources. Data were collected firstly from two human MM cell lines, secondly from rat MM tumors of increasing invasiveness grown in immunocompetent rats and human MM tumors grown in immunodeficient mice, and thirdly from paraffin-embedded sections of patient MM tumors of the epithelioid and sarcomatoid subtypes. Our investigations identified three major invasiveness biomarkers common to the three tumor sources, CAPG, FABP4, and LAMB2, and an additional set of 25 candidate biomarkers shared by rat and patient tumors. Comparing the data to proteomic analyzes of preneoplastic and neoplastic rat mesothelial cell lines revealed the additional role of SBP1 in the carcinogenic process. These observations could provide new opportunities to identify highly vulnerable MM patients with poor survival outcomes, thereby improving the success of current and future therapeutic strategies. The management of malignant mesothelioma (MM) remains a challenge today given its complex biology and aggressiveness, and the absence of specific early symptoms . The effProteomic analyzes of MM have already provided lists of putative cancer biomarkers, although significant differences are observed between primary and commercial MM cell lines , for exaRemembering the importance of potential sources of bias when identifying biomarkers with potential application in oncology ,13, to d6 cells i.p. into syngeneic rats also differs among the four models: five weeks for M5-T2, four weeks for F4-T2, and three and a half weeks for F5-T1 and M5-T1.The four rat MM tumor models shared a sarcomatoid morphology of tumor cells but differed in their infiltrative potential. The M5-T2 tumor is noninvasive, with tumor cell development restricted to the omentum without liver capsular breakthrough A, bottomscid, characterized by an absence of functional T cells and B cells) also differed markedly, with six weeks for MM34 versus three and a half weeks for MM163. These differences were also confirmed at microscopic level, as MM163 was characterized by tumor cells with heterogeneous nuclei in size and shape, prominent nucleoli, the presence of mitotic figures, and frequent atypia  were characterized by abundant intercellular collagen deposition, the presence of spindle-shaped tumor cells with oval nuclei, considerable heterogeneity in cell dimensions, and frequent atypia  relative to the non-invasive M5-T2 cell line (4.97)  polymerase 1 , involved in the blood coagulation cascade (anticoagulant), was the only one showing the same pattern of changes in human MM cell lines (MT-CO2), vesicle-trafficking protein SC22b (encoded by SEC22B), and fibronectin (encoded by FN1) (SELENBP1), an oxidoreductase also involved in protein transport, and synaptic vesicle membrane protein VAT-1 homolog (encoded by VAT1), which negatively regulates mitochondrial fusion  . These pl fusion . p > 0.090). By order of magnitude, proteins showing increased abundance with invasiveness included Ras-related protein Rab-31 (encoded by RAB31), Ragulator complex protein LAMTOR1 (encoded by LAMTOR1), isoform LCRMP-4 of dihydropyrimidinase-related protein 3 (encoded by DPYSL3), leucine-rich repeat-containing protein 59 (encoded by LRRC59), isoform 2 of tropomyosin alpha-3 chain (encoded by TPM3), endoplasmic reticulum resident protein 29 (encoded by ERP29), PRA1 family protein 3 (encoded by ARL6IP5), ferritin light chain (encoded by FTL), isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial (encoded by IDH3A), V-type proton ATPase subunit B, brain isoform (encoded by ATP6V1B2), and 40S ribosomal protein S18 (encoded by RPS18) (EHD2), septin-7 (encoded by SEPTIN7), serum albumin (encoded by ALB), and two subunits of hemoglobin (encoded by HBA1 and HBB) (The rest of the proteins listed in the 3 versus 1 comparison involved candidate biomarkers for which the difference in abundance between cells was insignificant (y RPS18) . Finallyand HBB) .PHB), and decrease in peroxiredoxin-6 (encoded by PRDX6) and ezrin (encoded by EZR) have previously been reported to be linked to the acquisition of invasive properties in rat MM models [GSN), profiling-1 (encoded by PFN1), glutathione-S-transferase P (encoded by GSTP1), keratin, type I cytoskeletal 10 (encoded by KRT10), and serpin H1 (encoded by SERPINH1) [Compared with the previous situation (3 versus 1), the numbers of common proteins found in conditions 2 versus 1, and 3 versus 2, were significantly reduced D. Among M models . MoreoveERPINH1) .Hif1a [We next investigated whether some of the 28 candidate biomarkers  common to the rat and human MM D exhibitSelenbp1) was the only one exhibiting a continuous decrease from the different subgroups of preneoplastic cell lines with epithelioid morphology to PNsarc1 and PNsarc2, including a final additional decrease in neoplastic cell lines  is 1 of 10 members of a family of proteins involved in intracellular fatty acid transport and lipid trafficking regulation in cells, which show different tissue-specific expression patterns . Its preThe first of an additional subset of common biomarkers of interest, which differed from the previous three by the absence of significant changes in xenografts (only a tendency), was represented by PARP1. The recent development of PARP1 inhibitors for the treatment of cancers presenting compromised HR repair has led to interesting findings on biomarkers associated with their clinical use against MM . MoreoveTwo other proteins caught our attention among the second additional subset of common biomarkers of interest, EHD2 and RAB31, characterized by highly significant changes in abundance in both rat and patient MM, but not in human cell lines or in xenografts. The level of the first protein, which belongs to the EHD family associated with plasma membrane, has been reported to be reduced in human esophageal squamous carcinoma in comparison with adjacent normal tissues, linked to increased motility of the tumor cells . SubsequHifa expression [Cdh1 and Il10, and parallel increase in the expression of Acta 2, Tgfb1 [Hifa [SELENBP1 expression, might be one factor in the development of hepatocellular carcinoma caused by HBV infection [0/G1 phase cell cycle arrest in bladder cancer [Both CAPG and RAB31 shared a similar pattern of changes during the course of rat mesothelial cell carcinogenesis. However, these changes were only observed in the first two subgroups of preneoplastic cell lines with sarcomatoid morphology, suggesting a link to increased pression . The pat2, Tgfb1 . Secondlb1 [Hifa  and EMT b1 [Hifa . Thirdlyb1 [Hifa . Interesb1 [Hifa . The mecb1 [Hifa . Althougb1 [Hifa , the comb1 [Hifa . Therefonfection . Wang etr cancer .The human studies were conducted according to the ethical guidelines of the Declaration of Helsinki. The paraffin-embedded human MM tumor pieces were prepared from samples of the Tumor Bank of the Reims University Hospital Biological Resource, Collection No. DC-2008-374, declared to the Ministry of Health according to French law, for the use of tissue samples for research. The two human cell lines MM34 (Meso 34) and MM163 (Meso 163) were established from pleural effusions of patients with suspected pleural MM , accordi2 atmosphere. Cells were collected from preconfluent 75 cm2 flasks and cell pellets of 2 × 106 cells were used for SWATH-MS proteomic analysis after washing in PBS buffer. The four rat neoplastic cell lines  were injected into syngeneic rats, and tumors collected and fixed as previously described [The 27 cell lines of the rat biocollection were grown in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin  at 37 °C in a 5% COescribed , and cul® HLB extraction cartridges , and the samples dried under SpeedVac. Peptide concentrations of the samples were determined using the Micro BCATM protein assay kit . The spectral libraries, DDA experiments, peptide identification, and peak extraction of the SWATH data were performed as previously described , using ep-value < 0.05 estimated by MarkerView were declared differentially expressed under different conditions.Five micrograms of each sample were analyzed with a SWATH-MS acquisition method. The method of acquisition, peak extraction of the SWATH data, calibration of the retention time of extracted peptide peaks and quantification followed the procedure already described in . For staThis study pointed to some proteins of interest that exhibited the same patterns of quantitative changes in different situations, and for which the relationship with tumor invasiveness has already been reported in the literature for other cancer types. Although this study was limited by the small number of samples, an interesting point was the similarity of observations made on malignant mesothelioma cells and tumors from different sources and from two different species. Extending these studies to a larger number of samples would be the logical next step, which may later contribute to improving current therapies for patients with the worst survival outcomes. Another interesting prospect is related to the questions raised by the additional involvement of the selenium-binding protein 1 in the carcinogenic process, a point that would present a good basis for further basic research in cancerology, and probably also for improving early MM diagnosis.


Neurotoxic effects of local anesthetics (LAs) on developing motor neurons have not been documented. We investigated the neurotoxic effects of LAs on developing motor neurons in terms of cell viability, cytotoxicity, reactive oxygen species (ROS), and apoptosis. Embryonic spinal cord motor neurons were isolated from Sprague-Dawley rat fetuses and exposed to one of the three LAs—lidocaine, bupivacaine, or ropivacaine—at concentrations of 1, 10, 100, or 1000 µM. The exposure duration was set to 1 or 24 h. The neurotoxic effects of LAs were determined by evaluating the following: cell viability, cytotoxicity, ROS production, and apoptosis. In the 1-h exposure group, the motor neurons exposed to lidocaine and bupivacaine had reduced cell viability and increased cytotoxicity, ROS, and apoptosis in a concentration-dependent manner. Lidocaine showed the highest toxicity, followed by bupivacaine. In the 24-h exposure group, all three LAs showed significant effects  on the motor neurons in a concentration-dependent manner. The neurotoxic effects of lidocaine were greater than those of bupivacaine and ropivacaine. Ropivacaine appeared to have the least effect on motor neurons. This study identified the neurotoxic effects of lidocaine and bupivacaine on developing spinal cord motor neurons. Local anesthetics (LAs) are widely used for pain control, and their roles in local and regional anesthesia have been established in clinical practice. However, studies have reported that LAs show neurotoxicity at high concentrations or after prolonged usage and that these neurotoxic effects may cause transient or persistent neurological dysfunction ,2,3. TheCloned cell lines—such as the mouse and human neuroblastoma cell lines—have been established and employed for investigating the neurotoxicity of LAs in vitro ,6,7,8; hExperimental procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (IRB number BA 111/063-01). Timed pregnant Sprague-Dawley rats were obtained. The primary rat spinal cord neurons were isolated from embryonic day 14–15 rat fetuses using a previously described method [Motor neurons were exposed to one of the following agents to examine the effects of LAs on developing motor neurons: (1) lidocaine, (2) bupivacaine, or (3) ropivacaine. The concentrations of each local anesthetic used were 1, 10, 100, and 1000 µM, and the exposure duration was either 1 or 24 h.2 incubator at 37 °C. The growth medium was then removed from each well; the wells were washed once with phosphate-buffered saline (PBS) and 100 mL of the LA was added to each well. After exposure, the LAs were removed by inspiration and all the wells were filled with 100 mL of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), a basal medium for the cell growth, and 10 μL of WST-8 solution. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt) is reduced by cellular dehydrogenase to a yellow-colored product called formazan whose amount is directly proportional to the number of viable cells. After incubation for 2 h at 37 °C, cell proliferation was measured by absorbance detection at 450 nm with a microplate reader.Cell viability was determined with a cell counting kit 8   at desigCytotoxicity was evaluated using a commercially available colorimetric assay kit that measured lactate dehydrogenase (LDH) leakage into the extracellular fluid. LDH is a stable cytoplasmic enzyme that is rapidly released into the cell culture supernatant when the cytoplasmic membrane is damaged . Thus, tCellular ROS was assessed using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay with a microplate reader . In thisCaspase activity was measured to quantify cellular apoptosis. Caspase-3 and caspase-7 play central roles in the process of cell death, and caspase 3/7 activity is widely used as an index of the proapoptotic response . The Cast-test after confirmation of normal distribution of the data. Statistical comparisons between groups were performed by one-way ANOVA, followed by Dunnett T3 post hoc comparisons. Data are expressed as mean (SD). P-values <0.05 were considered statistically significant.Each experimental condition was repeated 12 times using at least three different flasks of cells. All statistical analyses were performed using SPSS 19.0 software for Windows . Statistical analyses were performed by one-way analysis of variance followed by p < 0.001; Jonckheere-Terpstra test) after 1 h of exposure, and cell viabilities at all three lidocaine concentrations  were significantly lower than those in the control group. For the 24-h exposure, all three LAs caused a concentration-dependent decrease in cell viability . Compared with the control group, lidocaine markedly impaired cell viability at all concentrations (1–1000 µM), while bupivacaine decreased cell viability only at 100 and 1000 µM concentrations.Significant differences were observed in cell viability after 1-h exposure to the three LAs at the same concentration . In post hoc analysis, lidocaine had a significantly greater effect in decreasing cell viability than bupivacaine and ropivacaine at the same concentration. At an LA concentration of 1000 µM, cell viability was significantly lower in the bupivacaine group than in the ropivacaine group. For 24-h exposure, significant differences within the same concentration were found for all concentration levels (1–1000 µM). Post hoc analysis indicated that lidocaine markedly impaired cell viability at all concentrations (1–1000 µM), more so than the other LAs. Similarly, bupivacaine (10–1000 µM) significantly reduced cell viability when compared with corresponding concentrations of ropivacaine.For lidocaine, cell viability was significantly lower in the 24-h group than in the 1-h group at all concentrations. For bupivacaine, 24-h exposure caused a significant reduction in cell viability at 10, 100, and 1000 µM, but not at 1 µM.p < 0.001 and p < 0.001; Jonckheere-Terpstra test). Compared with that in the control group, the lidocaine (10–1000 µM) and bupivacaine (100 and 1000 µM) groups showed significantly higher cytotoxicity. With a 24-h exposure period, all three LAs showed concentration-dependent increases in cytotoxicity. Lidocaine and bupivacaine groups showed significant increases in cytotoxicity when compared to the control group at all concentrations; moreover, ropivacaine showed a more significant cytotoxic effect than the control group only at the highest concentration .At the same concentration (10–1000 µM), 1-h exposure to LAs caused significant differences in cytotoxicity among the three groups. In post hoc analysis, lidocaine had markedly greater cytotoxicity than bupivacaine and ropivacaine. In addition, at 100 and 1000 µM, the bupivacaine group had significantly higher cytotoxicity than the ropivacaine group. The 24-h exposure period was also associated with significant differences in cytotoxicity among the three groups at all concentration levels. In the post hoc analysis, lidocaine showed significantly greater cytotoxicity at all concentrations than bupivacaine and ropivacaine. Similarly, cytotoxicity was significantly higher with bupivacaine than ropivacaine at all concentrations (1–1000 µM).For lidocaine and bupivacaine, cytotoxicity was significantly greater with 24-h exposure than with 1-h exposure at all concentrations (1–1000 µM). However, for ropivacaine, significant differences in cytotoxicity between 1- and 24-h exposures were found only at the highest concentration (1000 µM).p < 0.001 and p < 0.001; Jonckheere-Terpstra test). Lidocaine (10–1000 µM) and bupivacaine (100–1000 µM) caused significantly higher oxidative stress than the control group. For the 24-h exposure, all three LAs induced concentration-dependent increases in ROS production . Lidocaine and bupivacaine significantly increased ROS production when compared to the control group at all concentration levels. In contrast, ropivacaine led to significantly greater oxidative stress than the control group only at the highest concentration .The effect of LAs on ROS indicated a similar pattern as that for cytotoxicity . For theExposure to LAs for 1 h resulted in significant differences in ROS production within the same concentration  among the three groups. In the post hoc analysis, ROS production in the lidocaine group was significantly higher than that in the other two LA groups. In addition, at 100 and 1000 µM concentrations, ROS production was significantly higher in the bupivacaine group than in the ropivacaine group. However, 24-h exposure to LAs induced significant increases in ROS production at all concentration levels among the three groups. Post hoc analysis showed that lidocaine induced significantly higher ROS production than the other two LAs at all concentrations (1–1000 µM). In addition, ROS production in the bupivacaine group was significantly higher than that in the ropivacaine group at all concentrations.p = 0.003).ROS production after 24-h exposure to lidocaine and bupivacaine was significantly greater than that after 1-h exposure at all concentrations. However, for ropivacaine, a significant increase in ROS production was noted after 24-h exposure when compared to after 1-h exposure only at 1000 µM . The lidocaine (10–1000 µM) and bupivacaine (1000 µM) group showed significantly higher apoptosis than the control group. After 24-h exposure, all three LAs showed concentration-dependent increases in apoptosis . Cellular apoptosis was significantly aggravated by lidocaine (1–1000 µM) and bupivacaine (10–1000 µM) when compared with the control group.For the 1-h exposure, there were significant differences in apoptosis at the same concentrations  among the three LA groups. Post hoc analysis revealed that lidocaine markedly aggravated cellular apoptosis when compared to bupivacaine and ropivacaine. For the 24-h exposure, all three groups showed significant differences in apoptosis at the same concentrations, at all concentrations. In the post hoc analysis, apoptosis was significantly higher in the lidocaine group than in the bupivacaine and ropivacaine groups at all concentrations. Comparisons between the bupivacaine and ropivacaine groups showed significant differences at 10, 100, and 1000 µM.Comparison between 1-h and 24-h exposure showed time-dependent increases in apoptosis at all lidocaine concentrations. However, significant time-dependent differences in apoptosis were found at bupivacaine concentrations of 10, 100, and 1000 µM and at a ropivacaine concentration of 1000 µM.In this study, we used developing motor neuron cells to investigate the neurotoxic effects of LAs. This is the first in vitro investigation on the neurotoxic effects of LAs in developing motor neurons. The major findings of the current study are that all LAs  had significantly different neurotoxicity profiles, as evidenced by the differences in cell viability, cytotoxicity, ROS production, and apoptosis. Among the three LAs, ropivacaine had the lowest toxicity, bupivacaine had medium toxicity, and lidocaine had the highest toxicity on developing motor neurons. These results were consistent with the potential neurotoxic clinical effects of lidocaine and bupivacaine, which may be produced by more than a single mechanism.In our study, cell viability was assessed using developing motor neurons, which are supposed to be susceptible to the neurotoxic effects of LAs. Lidocaine caused a concentration-dependent decrease in cell viability after a short-duration exposure (1 h). For the 24-h long-duration exposure, all three LAs caused a concentration-dependent decrease in cell viability. Ropivacaine had the least effect on the cell viability of developing motor neurons. This result is consistent with the findings of a previous study that investigated the neurotoxic effects of LAs administered intrathecally into the rabbit spinal cord . In thatOn the other hand, cytotoxic effects of LAs might exert a beneficial effect on cancer cells. In recent in vitro studies ,24,25,26A mechanism underlying the neurotoxicity of LAs is oxidative stress ; in thisAssessments of caspase activity were performed to evaluate the effect of LAs on cellular apoptosis, since both caspase-3 and caspase-7 play critical roles in cell death. Lidocaine and bupivacaine increased caspase activity with both increased exposure time and increased concentration, whereas ropivacaine showed no time-dependent effect on caspase activity, although it did show an effect at the highest concentration. Our findings are consistent with the results of previous studies in which lidocaine and bupivacaine caused neurotoxicity by inducing apoptosis in human SH-SY5Y neuroblastoma cells ,30. The In the current investigation, developing motor neurons were used since the sensitivity to LA neurotoxicity varies depending on the type and age of the cell. Motor neurons are more vulnerable to LA-induced neurotoxicity than sensory neurons , and groIn this study, we used equal concentrations of each LA. The relative potencies of the three different drugs should be considered while comparing the effects of LAs on developing motor neurons. A previous study comparing the neurotoxicity on the spinal cord between lidocaine, bupivacaine, and ropivacaine in a rabbit model used 2.5% lidocaine, 0.5% bupivacaine, and 0.5% ropivacaine . SimilarThere are a few limitations to be considered. First, this study used motor neurons with a specific development period (DIV7). The sensitivity of developing neurons to toxic stimuli depends on the age of the cell (development stage of the cell) , and morIn conclusion, among the commonly used LAs for pediatric patients , lidocaine and bupivacaine may show neurotoxicity on developing motor neurons of rats, since both these LAs induced cytotoxicity, increased ROS production, and induced apoptosis in a concentration- and time-dependent manner. The results also indicated that lidocaine showed the highest neurotoxicity, suggesting that the margin of safety regarding neurological complications of developing motor neurons may be the smallest with lidocaine. Further studies focusing on the toxicity of LAs at each stage of motor neuron development are needed to better understand these effects of LAs.


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  were tested alone and in combination with anticancer drugs , 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. Breast cancer (BC) is the most diagnosed cancer among women in high human development index countries and a leading cause of cancer death among females ,2. BC trTransversal to all cancer types is the problematic of drug resistance (innate or acquired)  and the Although it is almost unknown by the community in general, 50–60% of the drugs approved for cancer treatment are natural compounds and their derivatives ,20. The Screening for new anticancer drugs is often performed using in vitro studies, and typically with cancer cell lines cultivated in monolayer . NowadayWhen referring to the screening of effects of seaweed bioactive compounds in BC cell lines, there are no systematic studies using a panel of cancer cell lines with distinct biological characteristics while comparatively testing normal breast cell lines. Also, in what concerns combinations with drugs, it is poorly explored if the cell line characteristics can influence the type of response.In concord with the current state of the art, it is worth exploring the anticancer properties of selected seaweed compounds alone and in combination with reference drugs in a panel of breast cancer cell lines. For that, we selected three BC cell lines representative of the main BC subtypes: (i) MCF7 , corresponding to the most common BC type—Luminal A; (ii) SKBR3 , representing the HER-2 subtype; and (iii) MDA-MB-231, a triple-negative cell line , equivalent to TNBC ,53. We aFor this combinatory panel screening, we selected five brown seaweed bioactive compounds belonging to different chemical groups: (i) carotenoids: astaxanthin (Asta); (ii) polysaccharides: fucoidan (Fc) and laminarin (Lm); (iii) sterols: fucosterol (Fct); and (iv) phlorotannins: phloroglucinol (Phg).Carotenoids are fat-soluble organic pigments, naturally occurring in phototrophic organisms . Asta isSulphated polysaccharides are a major constituent of seaweeds’ cell walls that have attracted much attention as functional additives in the pharmaceutical, food and cosmetic industries . Fc is aLm is a water-soluble polysaccharide, corresponding to a storage glucan. Glucans are Food and Drug Administration approved compounds for lowering cholesterol levels  and theyPhytosterols represent a class of cholesterol-like molecules that integrate the cellular membranes of plants and algae, having a role in the regulation of membrane permeability . Fct hasPhg is a polyphenolic compound whose chemical structure includes an aromatic phenyl ring with three hydroxyl groups. Its biological activities include antioxidant and anti-inflammatory actions ,90. The As reference anticancer drugs, we chose two drugs used for treating many cancer types, including BC ,94: (i) In view of the above, this study aimed to evaluate the cytotoxic activity of the seaweed bioactive compounds Asta, Fc, Fct, Lm, and Phg, alone or combined with Cis and Dox, in three BC cell lines and one non-tumorous breast line, in monolayer culture (2D). The most promising combination of seaweed compound plus drug in monolayer was chosen to be investigated as to viability and proliferation, using a comparative approach with two in vitro systems (2D-monolayers versus 3D–MCAs).3 culture flasks  and maintained in the incubation chamber MCO 19AIC , with 5% CO2, at 37 °C. For cells’ growth and maintenance, the culture medium was replaced every two days. Cells were regularly observed using an inverted phase-contrast microscope CKX41 . The experiments were performed with cells under passage number 30. At 80% of cell confluence, monolayer cells were subcultured using trypsin/ethylenediamine tetraacetic acid (EDTA) (trypsin/EDTA) solution (0.25/0.02 in %).MCF7 was acquired from the European Collection of Authenticated Cell Cultures (ECACC). SKBR3 cell line was kindly provided by Professor Carmen Jerónimo . MCF12A and MDA-MB-231 cell lines were purchased from the American Tissue Culture Collection (ATCC). MCF7, MDA-MB-231, and SKBR3 were cultivated in high glucose Dulbecco’s modified Eagle’s medium (DMEM), without glutamine and phenol red, supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics solution penicillin/streptomycin (pen/strep) . MCF12A was cultivated in DMEM/Ham’s Nutrient Mixture F12 (DMEM/F12) medium without phenol red and supplemented with 20 ng/mL human epidermal growth factor (hEGF), 100 ng/mL cholera toxin, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 10% FBS, and 1% of the same antibiotic solution. All cell lines were cultivated in T75 cmDimethyl sulfoxide (DMSO) was purchased from VWR Chemicals . 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), astaxanthin , fucoidan ; fucosterol , laminarin ; phloroglucinol , cisplatin , doxorubicin ; insulin (I2643); cholera toxin (C8052), hydrocortisone (H088), and hEGF (E9644) were obtained from Sigma Aldrich . DMEM (F045), FBS, pen/strep, trypsin/EDTA, were acquired from Biochrom KG . DMEM/F12 was obtained from GE Healthcare . Resazurin  was purchased from Cayman . Cell Proliferation ELISA and BrdU (5′-bromo-2′-deoxyuridine) kit (colorimetric) were acquired from Roche . All other reagents and chemicals used were analytical grade.Stock solutions of astaxanthin (Asta), fucosterol (Fct), phloroglucinol (Pgh), and doxorubicin (Dox) were prepared in DMSO, while those of fucoidan (Fc) and laminarin (Lm) were prepared in supplemented cell culture medium. All those stock solutions were kept at −20 °C until used, except for the stock solutions of Fc and Lm that were prepared immediately before use. The stock solution of cisplatin (Cis) was prepared in a 0.9% NaCl solution and kept at 4 °C up to 1 month. Exposure solutions were prepared immediately before each experiment by diluting the stock solutions into the appropriate volume of respective fresh culture medium ensuring a final concentration of 0.1% DMSO.The study was performed in three phases according to a consecutive set of experiments . In Phas6 cells/mL, 100 µL/well, and incubated for 24 h for cell attachment. Then the culture medium was removed, and cells were exposed to the tested conditions for 72 h. At the end of the exposure period, respective cell viability or proliferation assays were performed, according to the phase of the study. Tested concentrations of seaweed bioactive compounds and reference drugs used in the screening of Phase 1 are detailed in Cells were seeded at the density of 0.05 × 10Prior to the exposures, the different solvent controls  were tested in the four cell lines, using MTT to evaluate the effects on cell viability. Four independent methodological experiments (with triplicates per each experiment) were performed and no significant differences among solvents were found (data not shown). For this reason, we opted for using the most common one for the set of compounds—medium with 0.1% DMSO— as the solvent control in all experiments.Regarding the selection of the concentrations to be tested in combination (Phase 2), we opted to use those that did not have a statistically significant effect on the cell viability of the tested cell lines or, in the case of the reference drugs, concentrations that did not reduce the cell viability below (in mean) 50%. These criteria were considered for each cell line. The selected combinations tested in Phase 2 are presented in 4 cells/mL, 200 µL/well. Plates were then centrifuged in a centrifuge Rotina 380 R  at 200× g for 10 min and placed in the incubator at 37 °C and 5% CO2 for 72 h to promote the MCAs formation. Then MCAs were incubated with the tested conditions for 96 h of exposure. After exposure, cell viability and proliferation assays as well as morphological analysis were performed.Cells were seeded in 96-well ultra-low attachment U-shaped spheroid plates  at a density of 40 × 102 at 37 °C. At the end of the incubation period, MCAs must be transferred from the 96-well ULA plates to flat-bottom 96-well plates with the help of a P1000 micropipette with a cut tip. Exposure medium was then aspirated, and formazan crystals were dissolved by adding 100 µL of DMSO:ethanol solution (1:1) (v/v) (monolayer) or 150 µL of DMSO (3D). Plates were left for 15 min under mild agitation to promote total formazan salt dissolution. Absorbance was measured at 570 nm in a microplate reader Multiskan GO . Results are expressed as percentage of cell viability and were calculated based on the absorbance ratio between treated conditions and the untreated control (cells incubated with culture medium with 0.1% (v/v) of DMSO). In both situations (tested condition and control), the absorbance of respective mediums without cells was subtracted in each situation to eliminate interferences related to the compounds or drugs.Cytotoxic effects of the tested conditions were assessed by MTT reduction assay. In short, 10 µL (monolayer) or 20 µL (3D) of MTT stock solution was added to each well, and incubated for 2 h (monolayer) and 4 h (3D), at 5% CO2 and at 37 °C. Similarly, to what was performed for the MTT assay, MCAs and respective mediums were transferred to flat-bottom 96-well plates. Fluorescence was then read using excitation wavelength at 560 nm and emission wavelength at 590 nm in the plate reader Synergy H1 . Results are expressed as a percentage of cell viability in relation to control and were calculated based on the fluorescence ratio between treated conditions and the untreated control (cells incubated with culture medium with 0.1% (v/v) of DMSO). In both situations (tested condition and control), the fluorescence of respective mediums without cells was subtracted in each situation to eliminate interferences related to the compounds or drugs.For resazurin assay, 1 µL (monolayer) or 2 µL (3D) of stock resazurin was added to each well. Plates were incubated for 3 h (monolayer) and 4 h (3D), with 5% CO2. Labeling medium was removed, and cells were stored at 4 °C overnight. Following the protocol, cells were fixed, and DNA denatured by the adding of 100 µL of FixDenant reagent (30 min). After removing the FixDenant Reagent, BrdU incorporation into cellular DNA was detected with mouse anti-BrdU conjugated with peroxidase working solution (diluted 1:100) (100 µL/well) for 90 min. Wells were rinsed with washing solution and 100 µL of substrate solution was added, to perform photometric detection. After 25 min, absorbances were immediately measured at 370 nm in a microplate reader Multiskan GO .Effects on cell proliferation were evaluated by BrdU assay . For monolayer, the assay was performed according to the manufacturer’s instructions. Briefly, cells were labeled with BrdU at a final concentration of 10 μM/well and incubated for 4 h at 37 °C, 5% CO2. Then, MCAs were transferred into a flat-bottom 96-well microplate and following the removal of the labelling medium, they were kept overnight at 4 °C. The following steps are identical to the monolayer protocol.For 3D culture some alterations were implemented. Briefly, after MCAs exposure, 100 µL of each well medium was removed, and then MCAs were incubated with 10 µL of BrdU labelling solution  for 5 h, at 37 °C, 5% COv/v) of DMSO).Results are expressed as a percentage of cell proliferation in relation to the control and were calculated based on the absorbance ratio between treated conditions and the untreated control .A total of 16 MCAs per tested condition/independent experiment were photographed at the end of the exposure time, using a stereomicroscope with darkfield SZX10 , equipped with a digital camera DP21 . The MCA areas were analyzed using the freeware AnaSP .At the end of exposure time, MCAs were collected to Eppendorf tubes with 10% buffered formalin  for fixation (24 h). For histological processing, MCAs were embedded in histogel , according to manufacturer’s instructions. The following processing protocol consisted in dehydration-1 h in a crescent series of ethanol ; clearing-1 h in each reagent: xylene: absolute ethanol (1:1); xylene (twice); and infiltrating in liquid paraffin (1 h twice). Paraffin blocks were obtained in an embedding station EG 1140H . Sections (3 µm) were performed in a microtome RM2255  and placed onto silane treated KP-frost slides . Slides were placed at 60 °C for 20 min, and then kept overnight at 37 °C.® Coverquick 2000 .Sections were deparaffinized in xylene and hydrated following the descendent sequence of ethanol , running tap water, 5 min each. Nuclei were stained with Mayer’s hematoxylin  for 3 min, and then slides were washed to remove dye excess. Following the protocol, sections were stained with 1% eosin Y for 5 min . Lastly, slides were dehydrated in ethanol , 5 min each, cleared in xylene (2 × 5 min), and mounted with the medium Q PathFor ICC, sections were deparaffinized and hydrated as described above. Heat antigen retrieval was made by sections immersion in citrate buffer 0.01 M, pH 6.0, using a pressure cooker (3 min after reaching maximum pressure). Slides were then allowed to cool, and then endogenous peroxidase blocking was performed with 3% hydrogen peroxide in methanol (10 min). After two washes in Tris saline buffer pH 7.6 (TBS) (5 min each), unspecific reactions were blocked using the appropriate reagent of the kit Novolink™ Polymer detection  (5 min), followed by two washes in TBST (TBS with 0.05% of Tween 20). Primary antibodies were diluted in phosphate buffer saline with 5% of bovine albumin serum and incubated 2 h at room temperature. For negative control, the primary antibody was substituted by the antibody diluent solvent only. Two primary antibodies were applied: rabbit monoclonal anti-Ki67, clone SP6  as cell proliferation marker ,103, dilhttps://folk.universitetetioslo.no/ohammer/past/) and GraphPad Prism 6.0 software . The normality and homogeneity of variance were confirmed by the Shapiro–Wilk and the Levene tests, respectively. The results are expressed as mean + standard deviation, except for the MCAs areas that were presented in median, maximum, minimum, and interquartile range (Q3–Q1), from at least five to six independent experiments (triplicate per each experiment). Significant differences (p < 0.05) were assessed by one-way ANOVA, followed by the post-hoc Holm–Šídák multiple comparison test. On selected cases, the significance of the difference between two groups of interest was tested with the Student’s t-test and using the sequential Holm–Bonferroni correction; the latter was implemented via a freeware spreadsheet calculator [Descriptive and inferential statistics were performed using Past3 (version 3.19) free software  alone did not show effects on cell viability, but when combined with Fct (1 and 5 µM), and Phg (10 and 50 µM) b,d. In lRegarding the SKBR3 line , Cis aloWith reference to the TNBC cell line MDA-MB-231 , Cis 10b,d,e.In relation to Dox, alone at 1 µM, it negatively affected cell viability. When combined with Fc (10 µg/mL) and Fct (1 µM), Dox at 1 µM seemed to have lost its action, while in combination with Fc (50 µg/mL) and Fct (5 µM) its effects were maintained b,c. The In relation to MCF12A cell line , Cis 1 .The results of the combinations in the panel of cell lines are summarized in For comparison purposes, we selected the most promising combination in which the seaweed compound and the drug alone did not have any effect on cell viability in relation to the control, but the combination potentiated the effect of the drug, that is, differing from the control and from the compound and drug alone. The selected combination was Fct 5 µM with Dox 0.1 µM in MDA-MB-231 cell line, as it revealed the most evident effect on cell viability, showing, on average, 46% less cellular viability than the drug alone. This selected combination was tested simultaneously in monolayer and in 3D culture, the latter providing multicellular aggregates (MCAs). Considering that 3D cultures are commonly more resistant to treatments ,108, we The results obtained in monolayer by the MTT assay a were veIn 3D culture b, the reThe resazurin assay performed in monolayer a reproduAlso, as in the MTT assay, cells in 3D culture b were moMDA-MB-231 cells cultivated in monolayer a showed The effects on cell proliferation in 3D culture b followeIn the stereo microscopic observation of the MCAs a, those After 96 h of exposure, MCAs were fixed, processed for paraffin embedding, and sectioned for hematoxylin-eosin (HE) staining and immunocytochemistry (ICC) analysis. By observing the MCAs stained with HE, the combinations in which morphological alterations were present are given in Antibodies against caspase-3 and ki67 were used for ICC. Here we show representative images of the control (C) and the combinations of Fct with Dox , as these were the conditions in which visual alterations of ICC staining existed in relation to the control . Also, iIn relation to the immunostainings for ki67, positive cells were also distributed all along the MCAs, with more predominance in their outer region . In Dox This study explored the cytotoxic effects of five brown seaweed compounds alone and combined with two reference drugs in a panel of breast cell lines, representing three BC subtypes and including a non-tumoral breast cell line. The study is justified considering that seaweed compounds, especially those from brown seaweeds, have been showing anticarcinogenic activities in many in vitro and in vivo studies related to many types of cancers, including BC ,109,110.50 values vary from study to study, from 2- to 10-fold of concentration within the same line [In this study, we started screening five seaweed compounds and two selected drugs in a panel of four breast cell lines, testing five concentrations of each, and then selected two of them for the combinations, according to pre-established criteria. Although there are some data related to the effects of the drugs in the used cell lines, the ICame line ,117,118.ame line .Regarding the cytotoxicity of the carotenoid Asta, alone (1–200 μM), it had no have effects on cell viability. This contrasts with previous studies that reported that Asta (50 μM) induced apoptosis in T-47D and MDA-MB-231 cells (both BC cell lines) , reducedIn relation to the polysaccharides, Fc and Lm alone caused significant cytotoxicity in MCF12A cell line at the highest tested concentration (1000 μg/mL). In contrast, other authors reported that Fc from 300 to 1000 μg/mL decreased cellular viability in a dose-dependent manner, induced G1 phase arrest, promoted ROS induction and triggered apoptosis through caspases-dependent pathway . AnotherIn combination, Fc modelled different effects according to the drug and the cell line tested. In SKBR3, Fc (10 μg/mL) in combination with Cis (10 μM) decreased Cis cytotoxicity, while the combination with Fc (50 μg/mL) statistically increased cell toxicity, differing from both the compound and the drug alone. A similar pattern of Cis enhancement effect with Fc was observed in MCF7, MDA-MB-231, and MCF12A cell lines, even if not with the same statistical significance, suggesting that Fc in higher concentrations may potentiate the effect of Cis in all cell lines. These findings are in line with previous studies where Fc significantly enhanced the cytotoxicity of Cis, Dox, and taxol in MCF7 cells . Fc at 4In some combinations of Fc with Dox—Fc at 10, 50 μg/mL with Dox at 0.1 μM, in MCF7 and MCF12A; Fc at 10 μg/mL with Dox at 1 μM, in MDA-MB-231—Fc seems to decrease Dox effect compared to Dox alone, since in the latter case Dox significantly differed from the control. In contrast, this effect was not verified in the referred combinations. Our results are in contradiction with a previous study describing that Fc enhanced Dox effects . In the The other tested polysaccharide, Lm, when tested alone, presented significant higher cellular viability than the control in MCF7. Although not statistically significant, the same tendency was observed in SKBR3 and MCF12A cells. Hypothetically, Lm can protect cells from oxidative stress produced by cellular metabolism. Treatment of mouse thymocytes with Lm suppressed apoptotic death around 2- to 3-fold and extended cell culture survival in about 20–30% . Our stuAs to the combinations with Cis, this drug lost its effects in some combinations with Lm . Conversely, in MDA-MB-231, Cis (20 μM) alone did not influence cell viability, but when in combination with Lm it presented lower cell viability, differing from the control. The literature related to the combination of Lm with Cis and Dox is very scarce, but it has been already described a protective effect against Cis-induced toxicity in auditory cells . When coConsidering the phlorotannins, Pgh alone had a cytotoxic effect in MCF7 from the concentration of 500 μM, and in MDA-MB-231 at 1000 μM. Our data corroborate a prior study reporting that Pgh at 100 μM was not cytotoxic to MCF7 and SKBR3 cell lines . At the In combination with Cis, two different effects were observed: in MDA-MB-231, Pgh with Cis (20 μM) increased Cis cytotoxicity differing from the control, while in MCF12A, the combination with Cis (1 μM) negatively affected this drug action, rescuing cell viability to the control levels. Therefore, our results point to the possible protective and potentiating effects of Cis, depending on the cell line and the concentration used. The literature is also contradictory. On one hand, Pgh displayed a protective effect against Cis-induced cell death in normal human urothelial and bladder cancer cells . On the The combinations of Phg with Dox (0.01 μM) potentiated the effects of the drug in MCF7, where the combination differed from the control and both the compound and drug alone. A similar effect was observed with Dox (0.1 μM) in SKBR3, but did not differ from the compound and drug alone. However, in MCF12A, the opposite effect was observed. In the literature, we only found reports of the protective effects of Phg: cardioprotective agent against doxorubicin-induced cardiotoxicity . Some neDue to the conflicting information in the literature in relation to the combination of seaweed compounds with anticancer drugs, more studies are necessary to elucidate both methodological aspects and mechanisms beyond these contradictory results. The differences between our study and several others in relation to the cytotoxicity of seaweed compounds can be justified by many possible reasons. First, some concentrations largely differ, and, as we observed, different concentrations can trigger different responses within the same model. Secondly, there are differences in the sources of compounds. We used high purity commercial compounds, but most of the other studies used extracts from different seaweeds species, with diverse extraction processing and certainly different degrees of purity that could interfere with the results. Also, for some compounds, such as Fc, the molecular weight was considered a critical factor for its anticancer activity, and different fucoidans can present different molecular weights . SimilarFrom the five tested compounds, Fct had the most promising results. Fct alone at 10 μM induced cytotoxicity effects in the three BC cell lines, not affecting the non-tumoral cell line (MCF12A). Such pattern also existed in some other studies. Fct containing fractions of seaweeds were cytotoxic for colon cell lines and T47D BC cell line, without cytotoxic effects on the normal cell line . Fct fro50 ˃ 100 μM [Globally, and according to the present data and literature, Fct seemed to have less (or even no) impact on the non-tumoral cell lines than in cancer ones. This notion goes beyond breast cells. Indeed, besides colon cell lines , others ˃ 100 μM , therefo˃ 100 μM .For the Fct combination with drugs, we selected a non-cytotoxic concentration of the former. The combination of Fct with Cis did not differ from the effects of Cis alone. Contrary to our results, one study showed a synergistic anticancer effect of Fct in combination with Cis, related to the expression of apoptotic and angiogenic genes, in ovarian cancer cell lines .Differently, the combination of Fct with Dox enhanced cytotoxicity in MCF7 and MDA-MB-231 cells. In both situations, the lowest Dox concentration did not have cytotoxicity alone. Still, combined with Fct, the cytotoxic effects increased, differing from the control and the compound and drug alone. In MDA-MB-231, the potentiation of Dox effects was more evident, because Dox (0.1 μM) in combination with Fct (5 μM) differed from the control, from Fct alone and almost doubled the impact of the drug alone. In SKBR3 the same enhancement trend of Dox toxicity was observed, but without statistical significance. By the opposite, in MCF12A cell line, the combination of Fct with Dox increased cell viability suggesting a protective effect against Dox action.Notably, the most promising combination involving Fct was noted in the MDA-MB-231 cell line, representative of TNBC. From our data, Fct seems to be a promising drug adjuvant in this type of BC type. Owing to the lack of ER, PR, and HER2 receptors, which are nowadays the available BC target therapies, TNBC presents poor prognosis, being the systemic chemotherapy (often using Dox) the mainstream treatment ,143,144.The most promising combination determined here was further explored, moving to what we called Phase 3, where another set of experiments was performed, including different types of assays for assessing cell viability (MTT and resazurin assays). Additionally, BrdU assay was included to evaluate the effects on cell proliferation. The new set of experiments was made in monolayer and, simultaneously, in multicellular aggregates (MCAs), which are 3D cell cultures. It is expectable that the other combinations that had no effect in monolayer would also have no effect in 3D culture, however, we cannot ensure this is the case because we did not test it.In 3D culture models, cells are not attached to a plastic surface. Conversely, they form a three-dimensional cell arrangement. In the case of our study, the MCAs were self-assembled due to the use of low-attachment plates, and they were not grown in any matrix or scaffolds. These MCAs have cells in multilayers, which constitute a barrier to the penetration of chemicals to be tested , and theThe results obtained in this set of experiments (Phase 3) relative to monolayer cultures, replicated the results of Phase 2 in terms of cell viability, thus reinforcing the value of the obtained data. Both viability assays were concordant, because Fct alone did not present cytotoxicity while Dox (0.1 μM) plus Fct continued to cause effect, that significantly differed from the effects of both the compound and drug alone. Thus, Fct seemed to have potentiated Dox’s cytotoxicity. We consider these data as impressive in terms of future applications for cancer treatment, especially for TNBC: a low Dox concentration that alone did not have an effect on cell viability, when combined with a non-toxic compound, in this case, Fct significantly decreased cancer cells’ viability. This triad Fct/Dox/TNBC deserves further studies to explore the mechanisms involved in these interactions, namely using a pathway-focused gene expression analysis and additional cell-based assays. As to the latter, and because Fct may change the levels of reactive oxygen species (ROS) ,141, theInterestingly, the combination of Dox (0.1 μM) with Fct also had effects on cell proliferation, being more effective in inhibiting cell proliferation than the compounds in single exposure. Similar to what was seen in our study, Fct from two brown seaweeds induced antiproliferative effects on the MCF-7 cell line but using much higher concentrations than the ones applied here . As we sDespite the promising effects that we observed in monolayer cultures, they were not detected in 3D cultures. It was expectable that in 3D the same drug concentrations did not induce the same degree of cytotoxicity than in monolayer, as 3D cultures seem more resistant to drugs cytotoxicity ,152, incHowever, when MCAs were observed after processing for light microscopy analysis and ICC, the drug effects started to be noticed at 1 μM, with a discrete but noticeable increase of cells with a morphology that was compatible with apoptosis; presenting cell shrinkage, nuclear condensation, chromatin margination, karyorrhexis, and putative apoptotic bodies ,156. TheFrom the structural evaluation in 3D culture, we conclude that the qualitative histology complemented with ICC, is an important and useful tool for evaluating the cytotoxicity of the tested drugs, as the effects were observable in more detail, revealing damages that were neither detected by the stereomicroscopic images, and respective measurements of areas, nor by the viability assays. Cell-based assays represent a technically simpler and quick way of assessing drug effects, while histological and ICC analyses are more time-consumable, require more equipment and know-how, and can be more expensive, especially due to the reagents for ICC; yet, they can give information on the localization of the processes through the MCAs. Furthermore, by using paraffin sections a great number of proteins related to different outputs, such as stem cell, epithelial-mesenchymal transition, and senescence markers can be detected. Our data calls for using histological analysis as an output to be included in drug testing.This study tested the cytotoxic effects of five seaweed bioactive compounds  alone and in a range of combinations with the reference drugs , in a panel of breast cell lines. We described concentrations in which the seaweed compounds alone presented cytotoxic effects against the BC cell lines. Also, conditions existed where a non-toxic concentration of the seaweed compound revealed potentiation or inhibition of the drug’s cytotoxicity. The results did not unveil patterns, varying according to the cell line, compound concentration used for the combination, and the drug in the combination. The overall findings showed that seaweed compounds may have anticancer effects against BC cell lines. However, studying and establishing an effective combinatory therapy is complex, with variability between cell lines and the used compounds and concentrations.Among the tested compounds, Fct was the most promising compound concerning higher anticancer activity. Alone, Fct induced cytotoxicity at low concentrations against the three BC cell lines, without cytotoxic effects in the non-tumoral cell line. Also, in combination with Dox, it enhanced the drug’s effect under certain conditions. The data supported the importance of performing cytotoxicity screening in more complex culture models, as the effects found in monolayer were not reproducible in 3D, at least using the same bioassays. Our data stressed the importance of using other techniques, namely histological analysis, and ICC, for better understanding the cytotoxic effects and underlying mechanisms of seaweed bioactive compounds, alone and combined with drugs. Although there were no effects in the 3D model, the mixture of Dox with Fct, especially in TNBC needs further investigation, from increasing the concentration of Fct to recurring to other technologies for delivery of both types of chemicals.