Antitumor activity of Lactococcus lactis cell-free supernatant on human glioblastoma cell lines

Antitumor activity of Lactococcus lactis cell-free supernatant on human glioblastoma cell lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antitumor activity of Lactococcus lactis cell-free supernatant on human glioblastoma cell lines Ida De Chiara, Antonia Feola, Milena Della Gala, Rosangela Marasco, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5353727/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the last few years, probiotics have gained much attention within the medical, pharmaceutical, and food fields, given the health benefits provided by their consumption. They include several lactic acid bacteria (LAB) species, mostly belonging to the genera Lactobacillus , Lactococcus , and Streptococcus . Postbiotics are bioactive compounds (organic acids, short-chain fatty acids, enzymes, and neurotransmitters) produced by bacterial fermentation that exert different health effects. It is well known that probiotics, as health-promoting microorganisms, show different therapeutic properties, including anti-pathogenic, anti-inflammatory, and cholesterol-lowering activities. Recently, accumulating evidence has shown that certain commensal bacteria play protective roles against cancer; thus, anti-carcinogenic activity is one of the most interesting probiotics properties that is currently under investigation. Here, we studied the anticancer properties of postbiotics produced by three different Lactococcus lactis subsp lactis strains isolated from natural whey starter cultures on human glioblastoma cell lines. MTT and Trypan Blue exclusion assays revealed a significant reduction in cell proliferation, and flow cytometry analysis corroborated this data, demonstrating a cell cycle arrest in treated cells. Moreover, other cancer hallmarks, such as wound healing rate closure and migration, were markedly inhibited by postbiotics. On the other hand, primary astrocytes viability and the blood-brain barrier (BBB) integrity were not impaired, suggesting a selective effect of postbiotics on proliferating-undifferentiated cells. This preliminary study highlights, for the first time, the potential anticancer properties of postbiotics from some L. lactis strains on human glioblastoma cell lines. Biological sciences/Cancer/Cns cancer Biological sciences/Microbiology/Bacteria/Bacterial host response Probiotics Postbiotics Lactococcus lactis glioblastoma cancer therapy blood-brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Lactic acid bacteria (LAB) are generally recognized as safe, active, and functional ingredients for food production and preservation. LAB-derived fermented food has been an important component of the human diet for millennia 1 . They are important constituents of human microbiota and include many probiotic strains. Well-documented health-promoting effects of LAB include the improvement of gastrointestinal disorders and bacterial vaginosis, as well as treatment of allergies, obesity, and depression 2 – 4 . Recently, several evidence suggested that gut microbial balance may contribute to cancer prevention and help the effectiveness of anti-cancer therapies. Modulation of gastrointestinal microflora, enhancement of the host’s immune response, induction of apoptosis, antioxidative, and antiproliferative properties are among the mechanisms of action linked to probiotics to exert their anticancer effect 5 – 7 . Recently, health-promoting LAB strains have also been proposed for the production of postbiotics, namely, water-soluble products deriving from bacterial metabolism or being by-products from bacterial cells after their lysis. Postbiotics, produced by microbiota-beneficial bacteria, represent last-generation health-promoting molecules showing different advantages since they are more stable and safer than probiotics. Indeed, they should not be subjected to the same safety measures as products that include live microorganisms 8 , 9 . In addition to cell-free supernatants (CFSs) from bacterial fermentation, postbiotic components include also microbial molecules and metabolites such as short-chain fatty acids (SCFAs), enzymes, peptides, teichoic acids, peptidoglycan-derived muropeptides, endo- and exopolysaccharides (EPSP), cell surface proteins, vitamins, plasmalogens, and organic acids. For this reason, CFSs could be proposed as adjuvant in oncology 10 . Several preclinical studies demonstrated their capability to effectively arrest cancer cell growth, both in vitro and in vivo in several cancer types including oral, colon, cervical, and breast cancer. CFSs and EPSP from Lactobacillus and Bifidobacterium species were found to inhibit colon cancer progression and induce apoptotic cell death 11 . Previously, Liu and coworkers (2012) showed that EPSP from LAB had anti-oxidative properties and antiproliferative effects on hepatoma HepG2 cells 9 , 12 – 14 . Several studies demonstrated the therapeutic potential of SCFAs in protection against colorectal, pancreatic and gastric cancer 11 , 13 , 14 . Postbiotics from Lactobacillus plantarum strains showed an inhibitory effect on the proliferation of human breast cancer cells 5 . Although preclinical studies suggest that oral administration of postbiotics exhibits beneficial effects against various types of cancer, nowadays, nothing is known about the effect of postbiotics on brain cancers yet 11 , 13 , 14 . Among brain tumors, glioblastoma multiforme (GBM) represents the most common and most aggressive subtype of glioma, with an overall incidence of less than 10 per 100,000 people, representing approximately 15% of brain tumors. In recent years, the number of GBM cases in adulthood or young adulthood is continuously rising. Due to the early invasion of the brain parenchyma, its complete surgical removal is almost impossible. Currently, therapeutic approaches consist of surgical resection followed by radiotherapy and chemotherapy. Drugs used to treat GBM are temozolomide (TMZ) and cisplatin 15 , the first-choice cytotoxic agents for gliomas, that alkylate the DNA of both cancer and normal cells. However, tumors treated with TMZ soon develop chemo-resistance largely due to several mechanisms such as the activation of alternative DNA repair systems, epigenetic modifications (high methyl-guanine methyltransferase (MGMT) levels), the inactivation of mismatch repair enzymes MLH1 and MSH29, and over-expression of multidrug resistance proteins 16 . Moreover, one of the worst side effects of TMZ chemotherapy is represented by cognitive impairment such as loss of memory and learning, which is even more severe in the case of the childhood brain 15 . Although the underlying cellular and molecular mechanisms are largely unknown, these effects, often referred to as “chemo-brain”, are due to the alkylating property of these drugs which target DNA of both cancer and healthy cells. Therefore, identifying new more effective, and non-toxic compounds represents an urgent need in the therapy of GBM. The investigation of the gut microbiome's relationship with the brain is a rapidly evolving field, which may lead to potential therapeutic avenues for a disease with limited treatment efficacy. Here, we reported the biological effects of CFSs from three different Lactococcus lactis subsp lactis strains, previously isolated from natural whey starter culture 17 on different glioblastoma cell lines. In particular, we tested the ability of CFSs to inhibit proliferation, cell cycle, migration, and tumorosphere formation in GBM cell lines. We also show that the tested CFSs are non-toxic on healthy primary astrocytes and on an in vitro model of BBB, preserving its integrity. Results Lactococcus lactis cell-free supernatants (CFSs) impair cell viability of glioblastoma cell lines. CFSs derived from eight different L. lactis strains (A3, A5, B1, D1, D3, I1, I4, I7) were used to preliminarily test, by MTT assay, their effect on U87MG cell proliferation. A dose-dependent (50, 5, 0.5mg/ml) and time-course analysis (24, 48, 72 hours) was performed (Supplementary Figure S1 ). Based on previous results concerning probiotic properties 17 and the effect of CFSs on U87MG cell proliferation, D1, I4, and I7 L. lactis strains were selected for further analysis, performed also on U251 and T98 cell lines, at the lowest concentration tested (0.5mg/ml). As shown in Fig. 1 a, no differences were detected 24 hours after treatment as compared to untreated cells for none of the three CFSs on U87MG. A decrease in cell viability was observed 48 hours (42, 50, and 55% for D1, I4, and I7, respectively), and 72 (53, 83, and 45%) hours after CFSs administration as compared to the untreated cells (Fig. 1 a). Similar effects were also observed on the U251 cell line (Fig. 1 b). Contrariwise, CFSs of D1, I4, and I7 strains did not affect T98 cells, in which an increase in cell viability was observed (Fig. 1 c). This resistance in T98 cells is likely due to the upregulation of efflux transporters and enhanced DNA repair mechanisms, which could mitigate the cytostatic effects of the CFSs 18 . MTT data were further supported by the Trypan blue exclusion assay. Cells were stained, counted, and reported as number of live/dead cells. Untreated cells were used as a positive control. CFS of L. lactis D1, I4, and I7 decreased the cell viability of U87MG cells at the concentration of 0.5 mg/mL after 24 hours (Fig. 1 d). Contrariwise, no significant effects between live and dead cells were observed in the other two cell lines (Figs. 1 e, 1 f). Taken together, these data indicate a cytostatic effect of the three selected CFSs, rather than cytotoxic. CFSs from L. lactis D1-I4-I7 arrest proliferation in various cell cycle phases, but do not induce apoptosis To test the effects of the CFSs from D1, I4, and I7 strains on the cell cycle progression, U251, U87MG, and T98 cell lines were treated or not with CFSs for 12 and/or 24 h. Results demonstrate that all the treatments induce an arrest of glioblastoma cell cycle in different phases. Particularly, D1, I4, and I7 treatments on U251 induce a weak decrease of G0/G1 phase in 12 h compared to the control. On the other hand, more than 5.0% (27.8% ± 1.0 Vs 22.6% ± 0.8), 7.0% (29.0% ± 0.5 Vs 22.6 ± 0.8) and 8.0% (30.0% ± 0.5 Vs 22.6% ± 0.8) of cells, respectively, begin to accumulate in G2/M phase compared to the untreated control (Fig. 2 ). However, 24h after D1 and I4 treatments, U251 cells are arrested in G2/M, while 24 hours after I7 treatment (Fig. 2 a and Supplementary figure S2), the cells re-enter the S-phase, starting a new cell cycle. In U87MG cells, treatment with D1 and I7 CSFs for 12 hours, induce an accumulation in G0/G1 phase of about 12.0% (45.8% ± 0.4 Vs 34.2 ± 1.0) and 7.5% (41.7% ± 0.3 Vs 34.2 ± 1.0), respectively; and in G2/M phase of about 8.4% (32.9% ± 1,0 Vs 24.5% ± 0.8) and 5.9% (30.4% ± 0.5 Vs 24.5% ± 0.8), respectively. Consequently, a decrease of about 10% in the S-phase is observed. I4 treatment does not show statistically significant differences in the cell cycle phases compared to control conditions (Fig. 2 b). Concerning T98 cells, D1 and I4 CFSs show an increase of about 12% in G0/G1 phase (67.0% ± 0.5 Vs 55.6 ± 0.8) and 11.0% (66.5% ± 0.5 Vs 55.6% ± 0.8), respectively, compared to control (Fig. 2 b,c). Contrariwise, I7 treatment induces an increase of 11% of cells in the S-phase. To investigate the potential mechanism underlying the cell cycle arrest Annex V/ PI test was performed, revealing no apoptotic cells in all treatments (D1, I4, I7), neither on U251 nor on U87MG cell line at different time points (Supplementary Figure S3), supporting the hypotheses of a cytostatic rather than cytotoxic nature. Wound healing rate closure is inhibited by CFSs from L. lactis D1, I4, and I7 strains. To evaluate the effect of CFSs from L. lactis D1, I4, and I7 strains on cell migration and cell-cell interaction, the wound healing assay was performed. As shown in Figs. 3 a and 3 b, both U87MG and U251 cell lines, in the absence of treatment, were able to close the gap within 24 hours. In contrast, in the presence of 0.5 mg/mL of all CFSs used, the residual space was significantly higher compared to control conditions. Similar effects were also obtained for T98 cell line, except for D1 (Fig. 3 c). In particular, in the presence of 0.5 mg/mL of CFSs, the residual space was 85%, 65%, and 73% in treated U87MG (Fig. 3 d), 50%, 78%, and 90% in treated U251 (Fig. 3 e), and 63%, 92%, and 81% in treated T98 (Fig. 3 f), from L. lactis D1, I4, and I7 strains respectively. These data indicate inhibition of wound closure induced by the preincubation with the postbiotics produced by L. lactis D1, I4, and I7 strains, suggesting an inhibitory effect on proliferation, migration, or both. Cell migration of GBM cell lines is inhibited by CFS of L. lactis I4 and I7, but not D1. To further investigate the effects of CFSs on cell migration of U87MG, U251, and T98 cells, a chemotactic assay in Transwell Plate 8 µm Pore Size in the presence of 0.5mg/mL of CFSs was performed. Migration of untreated cells was used as positive control. No significant difference was observed in the migration of cells treated with 0.5 mg/mL of CSF from L. lactis D1 strain in all three cell lines, suggesting an inhibitory effect on proliferation, but not on migration. In contrast, treatment with 0.5 mg/mL of CFSs from L. lactis I4 and I7 strains reduced migration ability (Fig. 4 ). In particular, CFS from L. lactis I4 exhibits a stronger inhibitory effect on U87MG cell migration (Fig. 4 a and 4 d) compared to the other cell lines, whereas the CFS from L. lactis I7 predominantly inhibits the migration of T98 cells (Fig. 4 c and 4 f). In the case of U251 cells, both CFSs (I4 and I7) appear to have a comparable effect (Fig. 4 b and 4 e). This may suggest that the inhibition of migration by these extracts may occur in a specific and selective manner. CFSs from L. lactis D1, I4, and I7 strains inhibit spheroid growth over time. Recently, multicellular tumor spheroids have been used to track the evolution of GBM, elucidate resistance mechanisms, and propose novel therapy strategies 19 . Here, we induced spheroids formation of U87MG cells by self-aggregation of cells in the bottoms of non-adherent round bottom 96-well plates. Once formed, spheroids were treated with 0.5mg/mL of each CFSs under study and observed daily. Spheres diameter was measured from 24 hours to 6 days after treatments. As shown in Fig. 5 a, untreated spheroids appear tighter and denser over time. Meanwhile, after 6 days, the spheroid structure was totally disintegrated when treated with postbiotics, showing a loose and uneven cell aggregate. Furthermore, the treated samples showed a much larger cell distribution area than the untreated ones. Representative images of spheroids with and without postbiotics are shown in Fig. 5 a. Overall, postbiotics exposure inhibits spheroid growth over time, whereas the diameter of non-treated spheroids continues to grow (Fig. 5 b). CFSs from L. lactis strains do not affect the integrity of the Blood-Brain-Barrier nor astrocytes viability To better elucidate the effects of these CFSs on healthy non-proliferating cells and on the blood-brain barrier, we established an in vitro co-culture model as previously described in detail 20 . In this Transwell setup, primary porcine brain endothelial cells (pBECs) were seeded on semipermeable membranes pre-coated with proteins of the basement membrane (fibronectin and collagen IV), and astrocytes cultured in the bottom compartment as a non-contact co-culture (Fig. 6 a). The astrocytes are part of the blood-brain barrier and secrete stimulating factors that are essential for maturation and maintenance of the brain endothelial cells in order to be tight, polarised, and have a functional vesicular subcellular transport system 21 . Following barrier induction with differentiation factors, the barrier integrity was validated by immunofluorescence analysis of the adherens junction protein p120 catenin. Our results demonstrate no significant alteration in the expression and in the localization of the endothelial barrier, showing in vivo -like paracellular localization (Fig. 6 c). Integrity and permeability of the in vitro BBB model system were also evaluated by Trans Endothelial Electrical Resistance (TEER) 22 , measured over time (1, 6, and 24 hours) after the treatment with CFSs (Fig. 6 b). Replacement of medium in the in vitro BBB models is expected to introduce an immediate drop in TEER values and the observed drop after media change and inclusion (Fig. 6 b) is therefore expected. However, the fact that the TEER drop does not fall below 300–400 Ω·cm², coupled with the rapid increase observed in the following hours, suggests that tight junctions remain intact at a level that prevents paracellular transport of small molecules 20 . Imaging of tight junctions and TEER measurements confirm that none of the three selected CFSs impair the blood-brain barrier integrity. Moreover, to test the cytotoxicity and specificity of these CFSs, in a separate experiment, a Trypan blue exclusion test was performed on astrocytes. Untreated astrocytes were used as a positive control. CFSs of L. lactis D1, I4, and I7 did not affect the viability of astrocytes, demonstrating a selective effect only on proliferating undifferentiated cells (Fig. 7 ). Discussion This study represents the first investigation into the effects of cell-free supernatants (CFSs) from three distinct Lactococcus lactis subsp. lactis strains (D1, I4, and I7), isolated from natural whey culture, on in vitro glioblastoma models. We evaluated their impact on key cancer hallmarks, including proliferation, migration, and spheroid formation, using U87MG, U251, and T98 glioblastoma cell lines. Despite their common glioblastoma origin, these cell lines exhibit distinct biological features. Indeed, it is well known that U87MG cells proliferate and migrate faster than the others under the same culturing conditions, while U251 cell line has an up-regulated glycolysis pathway, resulting from the “Warburg effect” that is associated with several cancer cell properties such as adaptation to low nutrient conditions and resistance to oxidative stress and apoptotic stimuli 23 . Moreover, T98 cell lines overexpress MRPs (multidrug resistance proteins) and BCRP (breast cancer resistance protein) that pump out various anticancer agents, conferring them multidrug resistance 18 . All the experiments reported in this study were performed on the three cell lines, showing similar effects and suggesting that CFSs are able to affect different molecular mechanisms underlying all the cancer hallmarks that make GBM difficult to treat. A preliminary screening was performed to evaluate the effect of the CFSs from eight L. lactis different strains, on the proliferation of the U87MG cell line 17 . Results showed that all postbiotics tested can affect cell viability in a time- and dose-dependent manner. However, based on previous results concerning probiotic properties 17 , A3, A5, I4, and I7 L. lactis strains exhibited pronounced probiotic properties in terms of survival to simulated gastrointestinal stress and competitive exclusion with pathogens, compared to all other strains tested. Nevertheless, based on the MTT assay on U87MG cells, CFSs from A3 and A5 demonstrated a cytotoxic rather than a cytostatic effect. For these reasons, postbiotics from A3 and A5 were excluded, and CFS from D1 strain, together with I4 and I7, were selected for further analysis since they exhibited a less toxic effect at a concentration of 0.5mg/ml. These selected CFSs were further tested on cell viability of U251 and T98 cells as well. In particular, CFSs of L. lactis D1, I4, and I7 decreased cell viability of both U87MG and U251 after 48 and 72 hours, but no effects were detected after 24 hours. Contrariwise, CFSs of D1, I4, and I7 strains did not affect T98 cells, as they seemed to increase the cell viability. This resistance in T98 cells is likely due to the upregulation of efflux transporters 24 and enhanced DNA repair mechanisms 25 , which could mitigate the cytostatic effects of the CFSs. Future studies should investigate whether targeting these resistance pathways could enhance the efficacy of postbiotic treatments. Moreover, it is important to consider that MTT assay can be limiting, despite its continued use for assessing cell viability. This assay measures the activity of succinate dehydrogenase, a mitochondrial enzyme involved in the Krebs cycle and ATP production. However, in cells undergoing imminent death, this enzyme's activity can appear upregulated, as mitochondria may release more enzymes. As a result, the MTT assay, in some cases, may not accurately reflect true cell viability, since increased mitochondrial activity could indicate a cell’s attempt to sustain function prior to death, rather than actual cell health. This limitation can affect the interpretation of results, particularly in stressed or damaged cells 26 . Trypan blue exclusion assay supports the observation that these CFSs are able to impair cell viability. The anticarcinogenic effect of microbial metabolites may be due to different mechanisms, including induction of apoptosis, and/or inhibition of cell proliferation 27 , 28 . FACS flow cytometry was performed to investigate whether L. lactis D1, I4, and I7 CFSs may impair cell proliferation through cell cycle arrest. U87MG, U251, and T98 cells treated with these supernatants showed an accumulation in G1 or G2/M phase compared to the untreated control. The only exception was represented by I7 CFS, which induces an increase of T98 cells in S-phase. This accumulation may result from forced entry into the S-phase, causing cells to initiate DNA replication prematurely. Consequently, the replication fork encounters replication stress, leading to the arrest of T98 cells in S-phase, likely due to the activation of the intra-S-phase checkpoint. In addition, Annex V/ PI staining demonstrates that none of the selected postbiotics induced apoptosis, suggesting that CFSs inhibit proliferation in a cytostatic manner without killing tumour cells. Inhibition of proliferation of tumor cells without inducing cell death could potentially reduce cytotoxicity to normal, non-tumor host cells, particularly if those normal tissues are low- or non-proliferating, as in the case of central nervous system cells 29 . Indeed, data reported in this study show that none of the CFSs under investigation is able to affect the viability of primary astrocytes, the differentiated healthy counterpart of GBM cells. Tumour progression is dependent on the ability of cells to migrate and invade, infiltrating adjacent tissues 30 . The wound healing assay is a technique used to study cell migration and cell-cell interaction. It is specifically a 2D cell migration approach to semi-quantitatively measure cell migration of a sheet of cells. Our results reported that non-treated cells migrate towards the wound, thereby closing the wounded edges, whereas treatment with CFSs from the three probiotics under study significantly decreases wound healing rate closure in all the tested GBM cell lines. To further investigate the inhibition of cell migration properties of microbial metabolites, a Transwell assay was performed. Indeed, these two experimental approaches involve distinct assays: the wound healing assay is a 2D migration method that is useful for investigating cell-cell interactions, where cells adhere to a polystyrene surface. In this case, the inhibitor's effect may be more related to proliferation rather than migration. Unlike the wound healing assay, the transwell assay is used to study directional migration towards a chemoattractant and typically separates migration from proliferation, as the cells are not allowed to proliferate. FBS-directional cell migration was inhibited by I4 and I7, but not D1, suggesting that the latter influences proliferation but not migration. Moreover, CFSs from I4 and I7 appear to have different behaviours depending on the treated cell type. These findings suggest that CFSs exert differential effects based on strain-specific metabolic profiles. The inhibition of migration may be linked to alterations in cytoskeletal dynamics or extracellular matrix remodelling. Future studies should explore whether these postbiotics influence metalloproteases (MMPs) activity or integrin signalling, which are critical regulators of glioblastoma cell motility. 2D individual cell-tracking experiments and Transwell assays cover different phenotypes and hallmarks of cell motility and adhesion 31 , providing orthogonal information that can be used collectively to better describe the biological effects of CFSs on GBM cancer cells. However, the main drawback in anticancer therapy development is the use of 2D cultures in vitro models. On this issue, here we developed a 3D cell culture models that resemble tumour tissues by reproducing the in vivo complex architecture more faithfully. The phenotypic changes observed during exposure to postbiotics released by L. lactis strains can be used as a measure of their efficacy. Here, for the first time, we assessed the effect of L. lactis postbiotics on spheroid structures and growth, demonstrating that all the CFSs under study can inhibit spheroid growth and induce their disaggregation when added to the culture media. The establishment of tumour spheroids significantly increased the opportunity to investigate anti-cancer agents in vitro since spheroids represent the best mimics of solid tumours in vivo as they exhibit cellular heterogeneity, diffusion-limited distribution of oxygen and nutrients, cell-cell signalling, growth kinetics, cell-cell interactions, and therapeutic resistance to treatment modalities. Taken together, our results suggest that metabolites produced by L. lactis D1 are able to inhibit cell proliferation but not migration, whereas metabolites produced by L. lactis I4 and I7 are able to inhibit cell proliferation as well as cell migration. No one is able to induce cell death, but they all impair 3D cell spheroid growth. One possibility is that secreted bacterial metabolites interfere with intercellular adhesion molecules, which are important for migration and invasion, or hypoxia-related signalling, crucial for spheroid maintenance. One of the major challenges in cancer treatment is selectivity, namely, the ability of potential therapeutic agents to distinguish between malignant tumor cells and healthy ones represented by neurons, astrocytes, and endothelial cells of the blood-brain barrier (BBB) 32 . Indeed, some chemotherapy might lead to the development of central neurotoxicity, due to neuroinflammation, neuroendocrine changes, and alterations in the BBB that allow increased access of cytotoxic agents and pro-inflammatory cytokines to neurons and supportive glial (astrocytes and microglia) cells, as well as secondary activation of glial cells and myelin-producing (oligodendrocyte lineage) cell defects 33 . To better investigate the effects of CFSs, here we established an in vitro model of BBB and showed that CFSs do not affect endothelial cell viability nor BBB integrity, demonstrating once again that these compounds are safe for non-proliferating-differentiated healthy cells and their effects are exerted only on proliferating undifferentiated cancer cells. Moreover, the presence of the BBB reduce the effectiveness of anticancer therapy for human GBM. Endothelial cells of microvessels and capillaries of the brain, connected by tight contacts, limit the intercellular transport of hydrophilic drugs with a molecular weight > 500 Da 34 . The presence in CFSs of lipophilic compounds such as liposoluble vitamins, short-chain fatty acids, neurotransmitters, and other organic acids suggests that, at least partially, these substances could bypass the BBB hindrance and easily reach tumour cells 35 . Therefore, ongoing studies by HPLC and highly sensitive MS methods in our lab are aimed at evaluating the CFSs passage through the BBB and at individual screenings with purified fractions that will allow us to determine if the observed effects are caused by a single compound or by a synergistic effect. In conclusion, our study demonstrates for the first time that postbiotics from L. lactis strains D1, I4, and I7 possess promising anticancer properties by selectively inhibiting glioblastoma cell proliferation, migration, and spheroid growth without affecting healthy cells, such as astrocytes and endothelial cells, or BBB integrity. Future studies will focus on identifying specific bioactive molecules responsible for these effects and elucidating their mechanisms of action. Additionally, in vivo validation will be essential to assess therapeutic potential and optimize delivery strategies for glioblastoma treatment. These findings pave the way for exploring LAB-derived fermented foods as a natural source of bioactive compounds with potential applications in cancer therapy. Materials and Methods Bacterial Strains, Media, and Growth Conditions Lactococcus lactis subsp. lactis strains (A3, A5, B1, D1, D3, I1, I4, I7), used in the study, were previously isolated from natural whey starter cultures. Their different RAPD (Random Amplification of Polymorphic DNA) profiles and probiotic characteristics were described in 17,36 . Lactococcus strains were grown at 30°C in ESTY broth supplemented with lactose 1% (w/v). Medium was supplied from Condalab (Madrid, Spain). Postbiotic production L. lactis strains were cultured on ESTY broth overnight and then sub-cultured in 100mL of the same fresh medium. Absorbance was measured periodically at 600 nm until reached 1 OD. To separate supernatants from bacterial pellets, media were centrifuged at 3750 rpm, 30 min, 4°C. Supernatant samples were sterilized using a 0.22 μm pore size filter and neutralized (pH 7.00) with NaOH 1M. Cell-free supernatant (CFS) was stored at -80°C and then lyophilized (VirTis SP Scientific Wizard 2.0). Dried CFSs were resuspended in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Paisley, UK). Cell culture Human glioblastoma cell lines U87MG, U251, and T98 were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% fetal bovine serum (FBS, Life Technologies, Paisley, UK). 100 U/mL of penicillin/streptomycin was added to the culture media. Cells were incubated in a humidified atmosphere containing 37°C, 5% CO 2 , and 95% atmospheric air. Astrocytes purified from 1–2 days old Sprague–Dawley rats were used as a control of healthy cells and were grown in DMEM low glucose supplemented with 10% FBS and 100U/mL of penicillin/streptomycin for 3 weeks in a humidified incubator at 37°C, 5% of CO 2 , 95% atmospheric air. Cell viability assay Cell viability in response to treatment with CFSs, was assessed either by Trypan blue exclusion test or by thiazolyl blue tetrazolium bromide (MTT) assay as previously described with some modifications 6 . For the MTT assay, an initial screening was performed on U87MG cells, testing all the lyophilized CFSs of all the L. lactis strains, solved in DMEM media in desirable concentrations (50, 5, 0.5mg/mL). Subsequently, three CFSs ( L. lactis D1, I4, I7) were selected, and a single concentration (0.5 mg/mL) was chosen for further testing on U87MG, U251, and T98 cell lines. Briefly, 2.5 × 10 3 U87MG, 5 × 10 3 U251, and 5 × 10 3 T98 cells/well were grown in 96-well plates for 24 h and then treated with 0.5mg/mL of CFSs. Concomitantly, cells without treatments were used as a positive control. 10 µL of the 5 mg/mL MTT solution in PBS, (Invitrogen-Life Technologies, Eugene, OR, USA) were added to wells, and incubated for 3 h at 37 °C. After incubation, 100 µL of stop solution were added to each well. Then, the optical densities were measured at 595 nm using the Microplate Reader (GLO Max Discovery, Promega). The cell viability rate was assessed after 24, 48 and 72 hours and calculated as follows: cell metabolic activity rate (%) = (OD treated) / (OD control) x 100 For the Trypan blue exclusion test, 2.5 × 10 3 U87MG, U251, and T98 cells were grown and then treated with 0.5 mg/mL of three selected CFSs. Cells were then incubated at 37 °C, 5% CO 2 for 24 hours. Then, the cells were detached, stained with a 0.4% ( w / v ) Trypan blue solution, counted using a Burker chamber, and reported as the number of live/dead cells. Cell cycle analysis and apoptosis assay Cell cycle analysis was performed by Fluorescence-Activated Cell Sorting (FACS) as previously described 37 . Briefly, U87MG, U251, and T98 cells were treated with 5.0 mg/mL of D1 and I4, and 0.5 mg/mL of I7 CFS, after synchronization with 1% FBS for 12h, and incubated for 12 and/or 24 hours at 37°C. The cells were then washed two times with phosphate-buffered saline (PBS), fixed with EtOH 70%, permeabilized with Triton 0.1% and finally stained in 10 μg/mL of propidium iodide (PI) with 5 μg/mL RNase in the dark at 4°C overnight. Fluorescence was determined by using the FACSCalibur or Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). At least 15000 total events were acquired, of which 5000 events were PI positive and analyzed. Data were analysed with Floreada.io online software or Flowjo10.4 software Apoptotic cells were detected by annexin V-FITC/PI staining assay following the manufacturer instructions as previously described 38 (A432; Leinco Technologies, Inc.). Briefly, the cells were treated with 5 mg/mL of D1 and I4 and 0.5 mg/mL of I7 CFS, after synchronization with 1% FBS for 12h, and incubated for 24-48 hours, washed twice with annexin V-binding buffer and resuspended in 1ml of the same buffer (1x10 6 ). Fifteen minutes before flow cytometry analysis, 5ml of PI and 5ml di Annexin-V-FITC were added to 100 ml of each sample and then analyzed by Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). As a control of efficacy of materials and Annex V/ PI test reagent, NIH3T3 cell line were treated with Cisplatin 60mM, which induces cell death via an apoptotic pathway in a time-dependent manner (see Supplementary Figure S4). Wound healing assay The wound healing assay or scratch test was performed as previously described 39 . Briefly, 6 × 10 3 U87MG, U251, and T98 cells were seeded and grown to confluence in DMEM/10% FBS for 24 hours. Afterwards, the cell monolayers were wounded with a yellow tip and concomitantly treated with 0.5mg/mL of each CFS. Untreated cells were used as a positive control. Images were taken immediately after scratch formation (T0) and after 24 hours by using an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.), equipped with the AmScope 3.7 Software. To quantify the wound healing rate, arbitrary arrows pointing the wound edges were drawn on the T0 image and were further applied to the single photograms of each sample after 24 hours. The average margin distance was measured at five points and the results were expressed as a percentage of residual wound width at T24 compared to T0 of the same sample. Transwell migration assay The migration assay 40 was performed using Transwell Plate 8 µm Pore Size (Sarstedt, Darmstadt, Germany). Briefly, 7 × 10 5 U87MG, 4 × 10 4 U251, and 4 × 10 4 T98 cells treated and untreated with 0.5mg/mL of CFS, were plated on serum-free media and allowed to migrate for 4 hours at 37°C towards DMEM 5% FBS. At the end of the assay, cells in the upper chamber were removed using cotton swabs, while cells on the lower filter surface were fixed in cold MetOH 100% for 15 min, stained with 0.05% crystal violet for 30 min at room temperature, and then washed with tap water. Migrated cells were counted under an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.) in at least three randomly selected fields at 10X magnification. The number of migrating cells was calculated using ImageJ software. Tumour spheroid U87MG spheroids were formed by self-aggregation of cells in the bottoms of non-adherent round bottom 96-well plates (Bioflat, Sarstedt, Numbrecht, Germany), by following the method of Sivakumar, with some modifications 41 . Briefly, 100 to 500 cells in 100 μL of media were pipetted into individual wells and allowed to form cell-cell connections over the course of 24 hours. Once the sphere was formed, 0.5mg/mL of tested postbiotics were added. Pictures were taken after 0-24-48 hours and checked after 6 days of treatments by using an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.). Single sphere diameter was measured with the AmScope 3.7 Software. In vitro study on the Blood-Brain Barrier (BBB) Animals All animals used in the study have been treated according to relevant ethical guidelines. This includes that methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org ), on the ethical use of animals (European Communities Council Directive of 24 November 1986; 86/609/ECC), and Danish guidelines. Porcine brains were obtained as byproducts of the Danish food industry. Danish Slaughterhouses are under strict supervision and observation by the Danish Ministry of Environment and Food. According to the “Danish Crown” the pigs are anesthetized with 80-90 % CO 2 and then terminated by cutting their Carotid artery. Rats used for isolation of astrocytes were bred and group-housed in the local animal facility at an ambient temperature of 22 °C-23 °C and on a 12/12 h dark/light cycle under the inspection of the veterinarian and according to Danish regulations for lab animals. The rats were euthanized with the CO 2 method before they were sacrificed in accordance with international guidelines on the ethical use of animals (European Communities Council Directive of 24 November 1986; 86/609/EEC) and Danish guidelines. No in vivo experiments on animals or human material were used in these experiments. This research was approved Aarhus University Animal Facility veterinarian committee. Cell cultures and an in vitro BBB model establishment Astrocytes were purified from 1–2 days old Sprague–Dawley rats and brain microcapillaries were purified from 5–6 months old pigs. Selective cultures of porcine brain endothelial cells (pBECs) were established in a non-contact co-culture (NCC) in vitro BBB model as described 20 . Following primary cell purifications, astrocytes were cultured in poly-L-Lysine pre-coated 12-well plates in low glucose DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/ml) for three weeks before NCC establishment. Porcine brain microcapillaries were seeded on type IV collagen- (150 μg/mL) and fibronectin (50 μg/ mL) coated T75 flasks using DMEM-F12 supplemented with 10% plasma-derived serum (PDS) (First Link, Wolverhampton, United Kingdom, UK), penicillin (100 U/ mL), streptomycin (100 µg/ml), and heparin (15 U/mL). For the first four days in culture, pBEC were selected using puromycin (4 μg/mL). At 70% confuency, cells were passed with Trypsin/EDTA (2.5% trypsin, 0.1 nM EDTA in PBS) and seeded on type IV collagen (500 μg/mL) and fibronectin (100 μg/mL) coated Transwell inserts (12 mm, 0.4 μm pore polycarbonate membrane, cat. no: 3401, Corning, Kennebunk ME 04043, USA) at a density of 1.1x10 5 cells/insert. pBECs were co-cultured with astrocytes in the basal chamber in serum-free media. To further induce the barrier development, both chambers were supplemented with the differentiation factors hydrocortisone (550 nM), 8-(4-chlorophenylthio)-adenosine-3ʹ,5′-cyclic monophosphate (250 μM), and RO-201724 (17.5 μM) 1–2 days before experiments. The tightness of the model was validated by measurements of transendothelial electrical resistance (TEER) using an EndOhm-12 measurement device (World Precision Instruments), with values>1000 Ω cm2 accepted for experiments. Before all experiments, Transwell inserts with cultured pBECs were transferred to a new 12-well plate without astrocytes, washed two times with PBS, incubated with media without differentiation factors, and allowed to rest in the presence of 5% CO 2 for 2 hours at 37 °C, before further treatment. To investigate the effects of the L. lactis CFSs on the BBB, 0.5 mg/mL of each CFS was added in the apical compartment and incubated at 37 °C, in the presence of 5% CO2 up to 1-6-24 hours with a circular rotation of 100 rpm and an orbit of 3 mm. The tightness of the barrier model was validated before and after the addition of the CFSs by measurements of TEER. Subsequently, for each time point, media from the top and the bottom was collected and stored at -20°C for further analysis. Furthermore, the expression of adherens junction proteins was validated by immunocytochemistry according to the procedures described below. To test the cytotoxicity of these CFSs, Trypan blue exclusion test was performed on astrocytes as described above. Immunofluorescence staining BECs were fixed in 4% paraformaldehyde in cytoskeleton buffer (50 mM PIPES, 50 mM NaCl, 5% glycerol, 0.1% NP-40, 0.1% Triton X-100 and 0.1% Tween 20) at RT for 20 minutes, washed 3 times with PBS, and permeabilized with 0.1% Triton X-100 for 10 min and blocked in 2% BSA for 30 min. For immunostaining, 5 μg/mL of antibodies anti α-p120 catenin (610133, BD Transduction Laboratory) were applied for 1 hour at RT. Filters were then washed 3 times in PBS at RT before 30 min incubation in the dark with 1:200 anti-mouse antibodies Alexa Fluor 488 (A32731 Life technologies). For nuclear staining, cells were incubated with 0.125 μg/mL Hoechst stain solution (Sigma-Aldrich) for 10 min at RT. Finally, membranes were mounted on microscope glass slides #1.5, with ProLong Diamond Antifade Mounting medium (Invitrogen, Thermo Fisher Scientific). Confocal microscopy and image processing Confocal imaging was performed using an Olympus IX-83 fluorescent microscope with a confocal spinning disk unit (Yokogawa) and a 60x 1.2 NA objective. Pictures are presented as maximum-intensity z-stack projections unless other specific details are noted in figure legends. Image processing and spot segmentation analysis were performed using Fiji software 42 . Statical analysis All presented data are based on three independent experiments. The statistical analyses and graphs were prepared using Prism (8.0) (GraphPad Software). The bar plots in the graphs report mean values (±SD). The tests of significant difference were analyzed using the unpaired t -test with Welch’s correction, Oneway and 2way ANOVA with multiple comparisons. Declarations Acknowledgments We thank Annemette Boe Marnow and Donato Sardella for technical assistance Funding: This research was funded by the European Union and Italian MIUR, Project BIONUTRA—PON 2014–2020 (Development of Nutraceuticals from Natural Sources), Grant Number PON ARS01_01166. Author Contributions: Conception and design of the study, I.D.C., N.MS., MT.G., and L.M.; performing the research, I.D.C., F.A., and M.D.G; in vitro experiment on the Blood-Brain Barrier (BBB) I.D.C., and N.MS.; statistical analysis, I.D.C., A.P., and A.F.; analysis and/or interpretation of data, I.D.C, A.F., MT.G., R.M., and L.M.; drafting the manuscript, I.D.C, A.F., R.M., A.P., M.G., MT.G., and L.M. All authors provided critical feedback and helped to shape the manuscript. Competing interests: The authors declare no competing interests. All authors have no conflicts of interest relevant to this study to disclose. Approval for animal experiments : This research was approved Aarhus University Animal Facility veterinarian committee. Data Availability Statement: All the data that support the findings of this study are available in the paper and its Supplementary information published online. References Vera-Santander, V. E., Hernández-Figueroa, R. H., Jiménez-Munguía, M. T., Mani-López, E. & López-Malo, A. Health Benefits of Consuming Foods with Bacterial Probiotics, Postbiotics, and Their Metabolites: A Review. Molecules 28 , doi:10.3390/molecules28031230 (2023). Sebastián Domingo, J. J. Review of the role of probiotics in gastrointestinal diseases in adults. Gastroenterol Hepatol 40 , 417-429, doi:10.1016/j.gastrohep.2016.12.003 (2017). DE Leo, V. et al. Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota. Minerva Obstet Gynecol 75 , 432-439, doi:10.23736/S2724-606X.22.05087-4 (2023). Wu, Y. et al. A Review on Maternal and Infant Microbiota and Their Implications for the Prevention and Treatment of Allergic Diseases. Nutrients 15 , doi:10.3390/nu15112483 (2023). Chuah, L. O. et al. Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells. BMC Complement Altern Med 19 , 114, doi:10.1186/s12906-019-2528-2 (2019). Sharma, P., Kaur, S., Kaur, R. & Kaur, M. Proteinaceous Secretory Metabolites of Probiotic Human Commensal. Front Microbiol 9 , 948, doi:10.3389/fmicb.2018.00948 (2018). Abd Ellatif, S. A. et al. Assessment of probiotic efficacy and anticancer activities of Lactiplantibacillus plantarum ESSG1 (MZ683194.1) and Lactiplantibacillus pentosus ESSG2 (MZ683195.1) isolated from dairy products. Environ Sci Pollut Res Int 29 , 39684-39701, doi:10.1007/s11356-022-18537-z (2022). Nataraj, B. H., Ali, S. A., Behare, P. V. & Yadav, H. Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods. Microb Cell Fact 19 , 168, doi:10.1186/s12934-020-01426-w (2020). Scott, E., De Paepe, K. & Van de Wiele, T. Postbiotics and Their Health Modulatory Biomolecules. Biomolecules 12 , doi:10.3390/biom12111640 (2022). Banna, G. L. et al. GG: An Overview to Explore the Rationale of Its Use in Cancer. Front Pharmacol 8 , 603, doi:10.3389/fphar.2017.00603 (2017). Song, D., Wang, X., Ma, Y., Liu, N. N. & Wang, H. Beneficial insights into postbiotics against colorectal cancer. Front Nutr 10 , 1111872, doi:10.3389/fnut.2023.1111872 (2023). Liu, L. et al. A compatibility assay of ursolic acid and foodborne microbial exopolysaccharides by antioxidant power and anti-proliferative properties in hepatocarcinoma cells. Journal of Food, Agriculture and Environment 10 , 111-114 (2012). Elango, A., Nesam, V. D., Sukumar, P., Lawrence, I. & Radhakrishnan, A. Postbiotic butyrate: role and its effects for being a potential drug and biomarker to pancreatic cancer. Arch Microbiol 206 , 156, doi:10.1007/s00203-024-03914-8 (2024). Eladwy, R. A. et al. The postbiotic sodium butyrate synergizes the antiproliferative effects of dexamethasone against the AGS gastric adenocarcinoma cells. Front Nutr 11 , 1372982, doi:10.3389/fnut.2024.1372982 (2024). Zou, Y. et al. Brain Co-Delivery of Temozolomide and Cisplatin for Combinatorial Glioblastoma Chemotherapy. Adv Mater 34 , e2203958, doi:10.1002/adma.202203958 (2022). Singh, N., Miner, A., Hennis, L. & Mittal, S. Mechanisms of temozolomide resistance in glioblastoma - a comprehensive review. Cancer Drug Resist 4 , 17-43, doi:10.20517/cdr.2020.79 (2021). De Chiara, I. et al. Probiotic Properties of. Foods 13 , doi:10.3390/foods13060957 (2024). Nakatsuma, A. et al. Effects of kaempferol on the mechanisms of drug resistance in the human glioblastoma cell line T98G. Die Pharmazie 65 , 379-383, doi:10.1691/ph.2010.9807 (2010). Samara Rodrigues Alves and Italo Rodrigo Calori and Hong Bi and Antonio Claudio, T. Characterization of glioblastoma spheroid models for drug screening and phototherapy assays. OpenNano 9 , 100116, doi:https://doi.org/10.1016/j.onano.2022.100116 (2023). Nielsen, S. S. E. et al. Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells. J Vis Exp , doi:10.3791/56277 (2017). Toth, A. E. et al. The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro. Mol Neurobiol 55 , 8522-8537, doi:10.1007/s12035-018-0988-x (2018). Waithe, O. Y., Peng, X., Childs, E. W. & Tharakan, B. Measurement of Transendothelial Electrical Resistance in Blood-Brain Barrier Endothelial Cells. Methods Mol Biol 2711 , 199-203, doi:10.1007/978-1-0716-3429-5_16 (2024). Li, H. et al. Differences in Protein Expression between the U251 and U87 Cell Lines. Turk Neurosurg 27 , 894-903, doi:10.5137/1019-5149.JTN.17746-16.1 (2017). Bhatia, P. et al. Breast cancer resistance protein (BCRP/ABCG2) localises to the nucleus in glioblastoma multiforme cells. Xenobiotica 42 , 748-755, doi:10.3109/00498254.2012.662726 (2012). Erasimus, H., Gobin, M., Niclou, S. & Van Dyck, E. DNA repair mechanisms and their clinical impact in glioblastoma. Mutat Res Rev Mutat Res 769 , 19-35, doi:10.1016/j.mrrev.2016.05.005 (2016). Ghasemi, M., Turnbull, T., Sebastian, S. & Kempson, I. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. Int J Mol Sci 22 , doi:10.3390/ijms222312827 (2021). Kvakova, M., Kamlarova, A., Stofilova, J., Benetinova, V. & Bertkova, I. Probiotics and postbiotics in colorectal cancer: Prevention and complementary therapy. World J Gastroenterol 28 , 3370-3382, doi:10.3748/wjg.v28.i27.3370 (2022). Wu, J., Zhang, Y., Ye, L. & Wang, C. The anti-cancer effects and mechanisms of lactic acid bacteria exopolysaccharides in vitro: A review. Carbohydr Polym 253 , 117308, doi:10.1016/j.carbpol.2020.117308 (2021). Morley, K. L., Ferguson, P. J. & Koropatnick, J. Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells. Cancer Lett 251 , 168-178, doi:10.1016/j.canlet.2006.11.016 (2007). Fares, J., Fares, M. Y., Khachfe, H. H., Salhab, H. A. & Fares, Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther 5 , 28, doi:10.1038/s41392-020-0134-x (2020). Pijuan, J. et al. Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. Front Cell Dev Biol 7 , 107, doi:10.3389/fcell.2019.00107 (2019). Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R. & Begley, D. J. Structure and function of the blood-brain barrier. Neurobiol Dis 37 , 13-25, doi:10.1016/j.nbd.2009.07.030 (2010). Was, H. et al. Mechanisms of Chemotherapy-Induced Neurotoxicity. Front Pharmacol 13 , 750507, doi:10.3389/fphar.2022.750507 (2022). Pandit, R., Chen, L. & Götz, J. The blood-brain barrier: Physiology and strategies for drug delivery. Adv Drug Deliv Rev 165-166 , 1-14, doi:10.1016/j.addr.2019.11.009 (2020). Hoyles, L. et al. Microbiome-host systems interactions: protective effects of propionate upon the blood-brain barrier. Microbiome 6 , 55, doi:10.1186/s40168-018-0439-y (2018). Marasco, R., Gazzillo, M., Campolattano, N., Sacco, M. & Muscariello, L. Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk. Foods 11 , doi:10.3390/foods11020233 (2022). Di Zazzo, E. et al. The p85 regulatory subunit of PI3K mediates cAMP-PKA and insulin biological effects on MCF-7 cell growth and motility. ScientificWorldJournal 2014 , 565839, doi:10.1155/2014/565839 (2014). Boccellino, M. et al. H9c2 Cardiomyocytes under Hypoxic Stress: Biological Effects Mediated by Sentinel Downstream Targets. Oxid Med Cell Longev 2021 , 6874146, doi:10.1155/2021/6874146 (2021). Gentile, M. T., Pastorino, O., Bifulco, M. & Colucci-D'Amato, L. HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis. J Vis Exp , doi:10.3791/58591 (2019). Zhou, F. et al. MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA. Am J Transl Res 11 , 4584-4601 (2019). Sivakumar, H., Devarasetty, M., Kram, D. E., Strowd, R. E. & Skardal, A. Multi-Cell Type Glioblastoma Tumor Spheroids for Evaluating Sub-Population-Specific Drug Response. Front Bioeng Biotechnol 8 , 538663, doi:10.3389/fbioe.2020.538663 (2020). Dobens, A. C. & Dobens, L. L. FijiWings: an open source toolkit for semiautomated morphometric analysis of insect wings. G3 (Bethesda) 3 , 1443-1449, doi:10.1534/g3.113.006676 (2013). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5353727","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":448794285,"identity":"2c81eb83-ee2a-4bf8-a715-ee0656c83ebc","order_by":0,"name":"Ida De Chiara","email":"","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":false,"prefix":"","firstName":"Ida","middleName":"","lastName":"De Chiara","suffix":""},{"id":448794286,"identity":"b94d2444-c01a-41d1-83c1-89989a26773c","order_by":1,"name":"Antonia Feola","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"","lastName":"Feola","suffix":""},{"id":448794287,"identity":"f947ce3b-5046-4569-925b-d0eced0409f1","order_by":2,"name":"Milena Della Gala","email":"","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"Della","lastName":"Gala","suffix":""},{"id":448794288,"identity":"9ecd999b-bdd1-41bc-9848-cbc3f8325f9e","order_by":3,"name":"Rosangela Marasco","email":"","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":false,"prefix":"","firstName":"Rosangela","middleName":"","lastName":"Marasco","suffix":""},{"id":448794289,"identity":"e7ff68f9-6057-424a-8533-6d1cb25723a2","order_by":4,"name":"Morten Schallburg Nielsen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Morten","middleName":"Schallburg","lastName":"Nielsen","suffix":""},{"id":448794290,"identity":"bbd9569e-82f6-462b-a33e-b521c66c7d51","order_by":5,"name":"Antonio Porcellini","email":"","orcid":"","institution":"University of Naples “Federico II”","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Porcellini","suffix":""},{"id":448794291,"identity":"32f9a1e2-9a25-4a59-a93b-8a40fd7f0e95","order_by":6,"name":"Michele Grieco","email":"","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Grieco","suffix":""},{"id":448794292,"identity":"7997ac56-57f0-4cd5-99e4-30702a1e627f","order_by":7,"name":"Maria Teresa Gentile","email":"","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Teresa","lastName":"Gentile","suffix":""},{"id":448794293,"identity":"f7eeea7d-b034-4c7d-9c72-19b8808cf2f4","order_by":8,"name":"Lidia Muscariello","email":"data:image/png;base64,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","orcid":"","institution":"University of Campania Luigi Vanvitelli","correspondingAuthor":true,"prefix":"","firstName":"Lidia","middleName":"","lastName":"Muscariello","suffix":""}],"badges":[],"createdAt":"2024-10-29 11:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5353727/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5353727/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81623885,"identity":"41debbe4-9d17-4edc-892c-dd55480b3594","added_by":"auto","created_at":"2025-04-29 09:53:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCFSs from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. lactis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e D1-I4-I7 impair GBM cell viability.\u003c/strong\u003e \u003cstrong\u003e(a-b-c) \u003c/strong\u003eMTT assay on U87MG (\u003cstrong\u003ea\u003c/strong\u003e) U251 (\u003cstrong\u003eb\u003c/strong\u003e) and T98 (\u003cstrong\u003ec\u003c/strong\u003e) cell lines. Untreated cells (NT) were used as positive control. Error bars are representative of the SD of three independent experiments performed in triplicates. Significance of the data obtained was tested by 2way Anova with Sidak’s multiple comparisons (**** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, *\u003cem\u003ep\u003c/em\u003e= 0.02 compared to untreated cells; ns: not significant). \u003cstrong\u003e(d-e-f) \u003c/strong\u003eTrypan blue exclusion assay on U87MG (\u003cstrong\u003ed\u003c/strong\u003e), U251 (\u003cstrong\u003ee\u003c/strong\u003e) and T98 (\u003cstrong\u003ef\u003c/strong\u003e) cell lines. Untreated cells (NT) were used as positive control. Error bars are representative of the SD of three independent experiments performed in triplicates. Significance of the data obtained was tested by Welch’s test (*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 compared to untreated cells; ns: not significant).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/011184a877df3bf7ae63dbf8.png"},{"id":81623842,"identity":"05e0d452-6c25-4ff4-982c-63f829d112e8","added_by":"auto","created_at":"2025-04-29 09:53:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":465757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFACS flow cytometry cell cycle analysis of human glioblastoma cell lines. (a) \u003c/strong\u003eRepresentative Flow cytometry cell cycle analysis of U251 human glioblastoma cell line treated with CFSs from \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, I7 for the indicated time points. In each panel, boxes show the percentage mean of at least three independent experiments performed in triplicate SD of cells in the different phases of the cell cycle. \u003cstrong\u003e(b) \u003c/strong\u003eFlow cytometry histogram shows the percentage of cell cycle accumulation during treatments with CFS from \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 after 12 hours in U87MG, U251, and T98 cell lines. Error bars are representative of SD of the three independent experiments performed in triplicate. Significance of data obtained was tested by the 2way ANOVA with Dunnett’s multiple comparisons (**** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0,005, ns: non significant compared to untreated cells, NT). \u0026nbsp;\u003cstrong\u003e(c) \u003c/strong\u003ePercentage of U87MG, U251, and T98 treated with CFS of \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 for 12 hours in the different cell cycle phases.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/3ea2a1e37f47c4bdb73ecd9e.png"},{"id":81623896,"identity":"6b23d426-5a41-46f0-a563-1690057392e7","added_by":"auto","created_at":"2025-04-29 09:53:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4188465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWound healing assay in the presence of 0.5mg/mL CFSs from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. lactis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eD1, I4, and I7.\u003c/strong\u003e \u003cstrong\u003e(a-b-c)\u003c/strong\u003e Representative high-power microphotographs of U87MG (\u003cstrong\u003ea\u003c/strong\u003e), U251 (\u003cstrong\u003eb\u003c/strong\u003e), and T98 (\u003cstrong\u003ec\u003c/strong\u003e) cells treated with 0.5mg/mL of CFSs. Images were taken at time 0 (T0) and 24 h (T24) after CFSs treatment (10X total magnification, scale bar: 2,5 µm). T0 represents the wound width at the moment of the scratch. Arbitrary arrows pointing the wound edges were drawn on the T0 image and applied to each sample's single photograms after 24 hours. The average margin distance was measured at five points and the results were expressed as a percentage of residual wound width at T24 compared to T0 of the same sample. \u003cstrong\u003e(c-e)\u003c/strong\u003e Quantitative analysis of the migration rate tested on U87MG \u003cstrong\u003e(d)\u003c/strong\u003e, U251 \u003cstrong\u003e(e)\u003c/strong\u003e, and T98 \u003cstrong\u003e(f)\u003c/strong\u003e. Error bars are representative of the SD of three independent experiments. The significance of data obtained was tested by the Ordinary One-way ANOVA with Sidak’s multiple comparisons (**** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001, compared to non-treated cells, NT; ns: not significant; ND: not detected).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/b07999a9b724b084becdcebf.png"},{"id":81623834,"identity":"92c6921d-6b1d-4560-84ed-4b570f0e333c","added_by":"auto","created_at":"2025-04-29 09:53:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4375633,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of U87MG, U251, and T98 cellular migration upon treatment with CFSs from \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 strains. Cells were plated in serum-free media and allowed to migrate for 4 hours at 37°C towards DMEM 5% FBS. At the end of the assay, the cells on the lower filter surface were fixed, stained, and counted as described in Materials and Methods. \u003cstrong\u003e(a-b-c)\u003c/strong\u003e Representative high-power microphotographs of U87MG (\u003cstrong\u003ea\u003c/strong\u003e), U251 (\u003cstrong\u003eb\u003c/strong\u003e), and T98 (\u003cstrong\u003ec\u003c/strong\u003e) cells treated with 0.5mg/mL of CFSs. Cells were observed microscopically under an inverted microscope (4X total magnification) and counted (at 10X total magnification) in at least three randomly selected visual fields. Number of migrated cells was calculated using ImageJ software. \u003cstrong\u003e(d-e-f)\u003c/strong\u003e Number of migrated cells of U87MG (\u003cstrong\u003ed\u003c/strong\u003e), U251 (\u003cstrong\u003ee\u003c/strong\u003e), and T98 (\u003cstrong\u003ef\u003c/strong\u003e) cells. Untreated (NT) cells were taken as positive control. Error bars are representative of SD of the three independent experiments. Significance of the data obtained was tested by Welch’s test (**** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001, * \u003cem\u003ep \u003c/em\u003e=0.02, compared to untreated cells; ns: not significant).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/fa1a2670760f6e0a8d5f0118.png"},{"id":81623843,"identity":"6b28ef60-eb7a-4610-8539-2e5c7943f7ea","added_by":"auto","created_at":"2025-04-29 09:53:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3101187,"visible":true,"origin":"","legend":"\u003cp\u003eU87MG spheroid. Cells were plated and allowed to form spheroids. Following spheres formation, 0.5mg/mL of the tested postbiotics were added. (\u003cstrong\u003ea\u003c/strong\u003e) Representative photographs of spheroids with and without postbiotics. Pictures were taken from 24 hours up to 6 days after treatments (10X total magnification, scale bar: 2,5 µm). (\u003cstrong\u003eb\u003c/strong\u003e) Quantifications of spheroid diameter in the absence (NT) and presence of postbiotics (D1, I4, I7). Error bars are representative of SD of the three independent experiments performed in triplicate. Significance of data obtained was tested by the 2way Anova with Sidak’s multiple comparisons (**** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001, compared to spheroid diameter at time 0; ns: not significant).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/60086e55f3bd58b439005dbf.png"},{"id":81625429,"identity":"26e4a3f1-5a71-4925-a3ef-b2c215b78c54","added_by":"auto","created_at":"2025-04-29 10:09:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13708566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e assay of BBB model integrity. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic illustration of the applied non-contact co-culture (NCC) blood-brain barrier (BBB) model setup, including pBECs and astrocytes. (\u003cstrong\u003eb\u003c/strong\u003e) Validation of the barrier integrity by transendothelial electrical resistance (TEER) (Ω/cm2) presented as mean values (±SEM) and X axis marking the hours of barrier inducement (T= -1) and the hours of probiotics’ treatments. (\u003cstrong\u003ec\u003c/strong\u003e) Confocal microscopic imaging with 60X magnification of immunofluorescence staining visualizing the adherens junction (AJ) proteins p120 catenin (red), and Hoechst stain of nuclei (blue), with scale bar equal to 20 μm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/236153e55c8f850db1fa9a85.png"},{"id":81624569,"identity":"8581e221-642d-462a-b354-25659865a2c3","added_by":"auto","created_at":"2025-04-29 10:01:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":40420,"visible":true,"origin":"","legend":"\u003cp\u003eTrypan blue exclusion assay. Untreated cells (N.T.) were used as positive control. None of the three selected postbiotics alters astrocyte viability. Error bars are representative of the SD of three independent experiments performed in triplicate. Significance of the data obtained was tested by Welch’s test (*** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to untreated cells; ns: not significant).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/8a3bee3d59d054a44ac69474.png"},{"id":83653088,"identity":"92a40a90-e83d-40ba-b5b7-10e621d0ad59","added_by":"auto","created_at":"2025-05-30 07:47:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27411543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/2b1faa23-a652-4267-9e54-970bfa7b586d.pdf"},{"id":81623831,"identity":"4101c683-9d3d-4710-8582-d675b891e1ca","added_by":"auto","created_at":"2025-04-29 09:53:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2279095,"visible":true,"origin":"","legend":"","description":"","filename":"DeChiaraetalSupplementaryRevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-5353727/v1/80fff74964d715efe4b887d1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antitumor activity of Lactococcus lactis cell-free supernatant on human glioblastoma cell lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLactic acid bacteria (LAB) are generally recognized as safe, active, and functional ingredients for food production and preservation. LAB-derived fermented food has been an important component of the human diet for millennia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. They are important constituents of human microbiota and include many probiotic strains. Well-documented health-promoting effects of LAB include the improvement of gastrointestinal disorders and bacterial vaginosis, as well as treatment of allergies, obesity, and depression\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recently, several evidence suggested that gut microbial balance may contribute to cancer prevention and help the effectiveness of anti-cancer therapies. Modulation of gastrointestinal microflora, enhancement of the host\u0026rsquo;s immune response, induction of apoptosis, antioxidative, and antiproliferative properties are among the mechanisms of action linked to probiotics to exert their anticancer effect\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Recently, health-promoting LAB strains have also been proposed for the production of postbiotics, namely, water-soluble products deriving from bacterial metabolism or being by-products from bacterial cells after their lysis. Postbiotics, produced by microbiota-beneficial bacteria, represent last-generation health-promoting molecules showing different advantages since they are more stable and safer than probiotics. Indeed, they should not be subjected to the same safety measures as products that include live microorganisms\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In addition to cell-free supernatants (CFSs) from bacterial fermentation, postbiotic components include also microbial molecules and metabolites such as short-chain fatty acids (SCFAs), enzymes, peptides, teichoic acids, peptidoglycan-derived muropeptides, endo- and exopolysaccharides (EPSP), cell surface proteins, vitamins, plasmalogens, and organic acids. For this reason, CFSs could be proposed as adjuvant in oncology\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Several preclinical studies demonstrated their capability to effectively arrest cancer cell growth, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e in several cancer types including oral, colon, cervical, and breast cancer. CFSs and EPSP from \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e species were found to inhibit colon cancer progression and induce apoptotic cell death\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Previously, Liu and coworkers (2012) showed that EPSP from LAB had anti-oxidative properties and antiproliferative effects on hepatoma HepG2 cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several studies demonstrated the therapeutic potential of SCFAs in protection against colorectal, pancreatic and gastric cancer\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Postbiotics from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e strains showed an inhibitory effect on the proliferation of human breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although preclinical studies suggest that oral administration of postbiotics exhibits beneficial effects against various types of cancer, nowadays, nothing is known about the effect of postbiotics on brain cancers yet\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Among brain tumors, glioblastoma multiforme (GBM) represents the most common and most aggressive subtype of glioma, with an overall incidence of less than 10 per 100,000 people, representing approximately 15% of brain tumors. In recent years, the number of GBM cases in adulthood or young adulthood is continuously rising. Due to the early invasion of the brain parenchyma, its complete surgical removal is almost impossible. Currently, therapeutic approaches consist of surgical resection followed by radiotherapy and chemotherapy. Drugs used to treat GBM are temozolomide (TMZ) and cisplatin\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, the first-choice cytotoxic agents for gliomas, that alkylate the DNA of both cancer and normal cells. However, tumors treated with TMZ soon develop chemo-resistance largely due to several mechanisms such as the activation of alternative DNA repair systems, epigenetic modifications (high methyl-guanine methyltransferase (MGMT) levels), the inactivation of mismatch repair enzymes MLH1 and MSH29, and over-expression of multidrug resistance proteins\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, one of the worst side effects of TMZ chemotherapy is represented by cognitive impairment such as loss of memory and learning, which is even more severe in the case of the childhood brain\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Although the underlying cellular and molecular mechanisms are largely unknown, these effects, often referred to as \u0026ldquo;chemo-brain\u0026rdquo;, are due to the alkylating property of these drugs which target DNA of both cancer and healthy cells. Therefore, identifying new more effective, and non-toxic compounds represents an urgent need in the therapy of GBM. The investigation of the gut microbiome's relationship with the brain is a rapidly evolving field, which may lead to potential therapeutic avenues for a disease with limited treatment efficacy. Here, we reported the biological effects of CFSs from three different \u003cem\u003eLactococcus lactis\u003c/em\u003e subsp \u003cem\u003elactis\u003c/em\u003e strains, previously isolated from natural whey starter culture\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e on different glioblastoma cell lines. In particular, we tested the ability of CFSs to inhibit proliferation, cell cycle, migration, and tumorosphere formation in GBM cell lines. We also show that the tested CFSs are non-toxic on healthy primary astrocytes and on an \u003cem\u003ein vitro\u003c/em\u003e model of BBB, preserving its integrity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLactococcus lactis\u003c/b\u003e \u003cb\u003ecell-free supernatants (CFSs) impair cell viability of glioblastoma cell lines.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCFSs derived from eight different \u003cem\u003eL. lactis\u003c/em\u003e strains (A3, A5, B1, D1, D3, I1, I4, I7) were used to preliminarily test, by MTT assay, their effect on U87MG cell proliferation. A dose-dependent (50, 5, 0.5mg/ml) and time-course analysis (24, 48, 72 hours) was performed (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Based on previous results concerning probiotic properties\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and the effect of CFSs on U87MG cell proliferation, D1, I4, and I7 \u003cem\u003eL. lactis\u003c/em\u003e strains were selected for further analysis, performed also on U251 and T98 cell lines, at the lowest concentration tested (0.5mg/ml). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, no differences were detected 24 hours after treatment as compared to untreated cells for none of the three CFSs on U87MG. A decrease in cell viability was observed 48 hours (42, 50, and 55% for D1, I4, and I7, respectively), and 72 (53, 83, and 45%) hours after CFSs administration as compared to the untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similar effects were also observed on the U251 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Contrariwise, CFSs of D1, I4, and I7 strains did not affect T98 cells, in which an increase in cell viability was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This resistance in T98 cells is likely due to the upregulation of efflux transporters and enhanced DNA repair mechanisms, which could mitigate the cytostatic effects of the CFSs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. MTT data were further supported by the Trypan blue exclusion assay. Cells were stained, counted, and reported as number of live/dead cells. Untreated cells were used as a positive control. CFS of \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 decreased the cell viability of U87MG cells at the concentration of 0.5 mg/mL after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Contrariwise, no significant effects between live and dead cells were observed in the other two cell lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Taken together, these data indicate a cytostatic effect of the three selected CFSs, rather than cytotoxic.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCFSs from\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003eD1-I4-I7 arrest proliferation in various cell cycle phases, but do not induce apoptosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test the effects of the CFSs from D1, I4, and I7 strains on the cell cycle progression, U251, U87MG, and T98 cell lines were treated or not with CFSs for 12 and/or 24 h. Results demonstrate that all the treatments induce an arrest of glioblastoma cell cycle in different phases. Particularly, D1, I4, and I7 treatments on U251 induce a weak decrease of G0/G1 phase in 12 h compared to the control. On the other hand, more than 5.0% (27.8% \u0026plusmn; 1.0 Vs 22.6% \u0026plusmn; 0.8), 7.0% (29.0% \u0026plusmn; 0.5 Vs 22.6 \u0026plusmn; 0.8) and 8.0% (30.0% \u0026plusmn; 0.5 Vs 22.6% \u0026plusmn; 0.8) of cells, respectively, begin to accumulate in G2/M phase compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, 24h after D1 and I4 treatments, U251 cells are arrested in G2/M, while 24 hours after I7 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary figure S2), the cells re-enter the S-phase, starting a new cell cycle.\u003c/p\u003e \u003cp\u003eIn U87MG cells, treatment with D1 and I7 CSFs for 12 hours, induce an accumulation in G0/G1 phase of about 12.0% (45.8% \u0026plusmn; 0.4 Vs 34.2 \u0026plusmn; 1.0) and 7.5% (41.7% \u0026plusmn; 0.3 Vs 34.2 \u0026plusmn; 1.0), respectively; and in G2/M phase of about 8.4% (32.9% \u0026plusmn; 1,0 Vs 24.5% \u0026plusmn; 0.8) and 5.9% (30.4% \u0026plusmn; 0.5 Vs 24.5% \u0026plusmn; 0.8), respectively. Consequently, a decrease of about 10% in the S-phase is observed. I4 treatment does not show statistically significant differences in the cell cycle phases compared to control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eConcerning T98 cells, D1 and I4 CFSs show an increase of about 12% in G0/G1 phase (67.0% \u0026plusmn; 0.5 Vs 55.6 \u0026plusmn; 0.8) and 11.0% (66.5% \u0026plusmn; 0.5 Vs 55.6% \u0026plusmn; 0.8), respectively, compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). Contrariwise, I7 treatment induces an increase of 11% of cells in the S-phase.\u003c/p\u003e \u003cp\u003eTo investigate the potential mechanism underlying the cell cycle arrest Annex V/ PI test was performed, revealing no apoptotic cells in all treatments (D1, I4, I7), neither on U251 nor on U87MG cell line at different time points (Supplementary Figure S3), supporting the hypotheses of a cytostatic rather than cytotoxic nature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWound healing rate closure is inhibited by CFSs from\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003eD1, I4, and I7 strains.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the effect of CFSs from \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 strains on cell migration and cell-cell interaction, the wound healing assay was performed. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, both U87MG and U251 cell lines, in the absence of treatment, were able to close the gap within 24 hours. In contrast, in the presence of 0.5 mg/mL of all CFSs used, the residual space was significantly higher compared to control conditions. Similar effects were also obtained for T98 cell line, except for D1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In particular, in the presence of 0.5 mg/mL of CFSs, the residual space was 85%, 65%, and 73% in treated U87MG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), 50%, 78%, and 90% in treated U251 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), and 63%, 92%, and 81% in treated T98 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), from \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 strains respectively. These data indicate inhibition of wound closure induced by the preincubation with the postbiotics produced by \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 strains, suggesting an inhibitory effect on proliferation, migration, or both.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCell migration of GBM cell lines is inhibited by CFS of\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003eI4 and I7, but not D1.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the effects of CFSs on cell migration of U87MG, U251, and T98 cells, a chemotactic assay in Transwell Plate 8 \u0026micro;m Pore Size in the presence of 0.5mg/mL of CFSs was performed. Migration of untreated cells was used as positive control. No significant difference was observed in the migration of cells treated with 0.5 mg/mL of CSF from \u003cem\u003eL. lactis\u003c/em\u003e D1 strain in all three cell lines, suggesting an inhibitory effect on proliferation, but not on migration. In contrast, treatment with 0.5 mg/mL of CFSs from \u003cem\u003eL. lactis\u003c/em\u003e I4 and I7 strains reduced migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In particular, CFS from \u003cem\u003eL. lactis\u003c/em\u003e I4 exhibits a stronger inhibitory effect on U87MG cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) compared to the other cell lines, whereas the CFS from \u003cem\u003eL. lactis\u003c/em\u003e I7 predominantly inhibits the migration of T98 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). In the case of U251 cells, both CFSs (I4 and I7) appear to have a comparable effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). This may suggest that the inhibition of migration by these extracts may occur in a specific and selective manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCFSs from\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003eD1, I4, and I7 strains inhibit spheroid growth over time.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecently, multicellular tumor spheroids have been used to track the evolution of GBM, elucidate resistance mechanisms, and propose novel therapy strategies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Here, we induced spheroids formation of U87MG cells by self-aggregation of cells in the bottoms of non-adherent round bottom 96-well plates. Once formed, spheroids were treated with 0.5mg/mL of each CFSs under study and observed daily. Spheres diameter was measured from 24 hours to 6 days after treatments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, untreated spheroids appear tighter and denser over time. Meanwhile, after 6 days, the spheroid structure was totally disintegrated when treated with postbiotics, showing a loose and uneven cell aggregate. Furthermore, the treated samples showed a much larger cell distribution area than the untreated ones. Representative images of spheroids with and without postbiotics are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. Overall, postbiotics exposure inhibits spheroid growth over time, whereas the diameter of non-treated spheroids continues to grow (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCFSs from\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003estrains do not affect the integrity of the Blood-Brain-Barrier nor astrocytes viability\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo better elucidate the effects of these CFSs on healthy non-proliferating cells and on the blood-brain barrier, we established an \u003cem\u003ein vitro\u003c/em\u003e co-culture model as previously described in detail\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In this Transwell setup, primary porcine brain endothelial cells (pBECs) were seeded on semipermeable membranes pre-coated with proteins of the basement membrane (fibronectin and collagen IV), and astrocytes cultured in the bottom compartment as a non-contact co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The astrocytes are part of the blood-brain barrier and secrete stimulating factors that are essential for maturation and maintenance of the brain endothelial cells in order to be tight, polarised, and have a functional vesicular subcellular transport system\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Following barrier induction with differentiation factors, the barrier integrity was validated by immunofluorescence analysis of the adherens junction protein p120 catenin. Our results demonstrate no significant alteration in the expression and in the localization of the endothelial barrier, showing \u003cem\u003ein vivo\u003c/em\u003e-like paracellular localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Integrity and permeability of the \u003cem\u003ein vitro\u003c/em\u003e BBB model system were also evaluated by Trans Endothelial Electrical Resistance (TEER)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, measured over time (1, 6, and 24 hours) after the treatment with CFSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Replacement of medium in the \u003cem\u003ein vitro\u003c/em\u003e BBB models is expected to introduce an immediate drop in TEER values and the observed drop after media change and inclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) is therefore expected. However, the fact that the TEER drop does not fall below 300\u0026ndash;400 Ω\u0026middot;cm\u0026sup2;, coupled with the rapid increase observed in the following hours, suggests that tight junctions remain intact at a level that prevents paracellular transport of small molecules\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Imaging of tight junctions and TEER measurements confirm that none of the three selected CFSs impair the blood-brain barrier integrity. Moreover, to test the cytotoxicity and specificity of these CFSs, in a separate experiment, a Trypan blue exclusion test was performed on astrocytes. Untreated astrocytes were used as a positive control. CFSs of \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 did not affect the viability of astrocytes, demonstrating a selective effect only on proliferating undifferentiated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents the first investigation into the effects of cell-free supernatants (CFSs) from three distinct \u003cem\u003eLactococcus lactis\u003c/em\u003e subsp. \u003cem\u003elactis\u003c/em\u003e strains (D1, I4, and I7), isolated from natural whey culture, on \u003cem\u003ein vitro\u003c/em\u003e glioblastoma models. We evaluated their impact on key cancer hallmarks, including proliferation, migration, and spheroid formation, using U87MG, U251, and T98 glioblastoma cell lines. Despite their common glioblastoma origin, these cell lines exhibit distinct biological features. Indeed, it is well known that U87MG cells proliferate and migrate faster than the others under the same culturing conditions, while U251 cell line has an up-regulated glycolysis pathway, resulting from the \u0026ldquo;Warburg effect\u0026rdquo; that is associated with several cancer cell properties such as adaptation to low nutrient conditions and resistance to oxidative stress and apoptotic stimuli\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Moreover, T98 cell lines overexpress MRPs (multidrug resistance proteins) and BCRP (breast cancer resistance protein) that pump out various anticancer agents, conferring them multidrug resistance\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. All the experiments reported in this study were performed on the three cell lines, showing similar effects and suggesting that CFSs are able to affect different molecular mechanisms underlying all the cancer hallmarks that make GBM difficult to treat. A preliminary screening was performed to evaluate the effect of the CFSs from eight \u003cem\u003eL. lactis\u003c/em\u003e different strains, on the proliferation of the U87MG cell line\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Results showed that all postbiotics tested can affect cell viability in a time- and dose-dependent manner. However, based on previous results concerning probiotic properties\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, A3, A5, I4, and I7 \u003cem\u003eL. lactis\u003c/em\u003e strains exhibited pronounced probiotic properties in terms of survival to simulated gastrointestinal stress and competitive exclusion with pathogens, compared to all other strains tested. Nevertheless, based on the MTT assay on U87MG cells, CFSs from A3 and A5 demonstrated a cytotoxic rather than a cytostatic effect. For these reasons, postbiotics from A3 and A5 were excluded, and CFS from D1 strain, together with I4 and I7, were selected for further analysis since they exhibited a less toxic effect at a concentration of 0.5mg/ml. These selected CFSs were further tested on cell viability of U251 and T98 cells as well. In particular, CFSs of \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 decreased cell viability of both U87MG and U251 after 48 and 72 hours, but no effects were detected after 24 hours. Contrariwise, CFSs of D1, I4, and I7 strains did not affect T98 cells, as they seemed to increase the cell viability. This resistance in T98 cells is likely due to the upregulation of efflux transporters\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and enhanced DNA repair mechanisms\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, which could mitigate the cytostatic effects of the CFSs. Future studies should investigate whether targeting these resistance pathways could enhance the efficacy of postbiotic treatments. Moreover, it is important to consider that MTT assay can be limiting, despite its continued use for assessing cell viability. This assay measures the activity of succinate dehydrogenase, a mitochondrial enzyme involved in the Krebs cycle and ATP production. However, in cells undergoing imminent death, this enzyme's activity can appear upregulated, as mitochondria may release more enzymes. As a result, the MTT assay, in some cases, may not accurately reflect true cell viability, since increased mitochondrial activity could indicate a cell\u0026rsquo;s attempt to sustain function prior to death, rather than actual cell health. This limitation can affect the interpretation of results, particularly in stressed or damaged cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Trypan blue exclusion assay supports the observation that these CFSs are able to impair cell viability.\u003c/p\u003e \u003cp\u003eThe anticarcinogenic effect of microbial metabolites may be due to different mechanisms, including induction of apoptosis, and/or inhibition of cell proliferation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. FACS flow cytometry was performed to investigate whether \u003cem\u003eL. lactis\u003c/em\u003e D1, I4, and I7 CFSs may impair cell proliferation through cell cycle arrest. U87MG, U251, and T98 cells treated with these supernatants showed an accumulation in G1 or G2/M phase compared to the untreated control. The only exception was represented by I7 CFS, which induces an increase of T98 cells in S-phase. This accumulation may result from forced entry into the S-phase, causing cells to initiate DNA replication prematurely. Consequently, the replication fork encounters replication stress, leading to the arrest of T98 cells in S-phase, likely due to the activation of the intra-S-phase checkpoint. In addition, Annex V/ PI staining demonstrates that none of the selected postbiotics induced apoptosis, suggesting that CFSs inhibit proliferation in a cytostatic manner without killing tumour cells. Inhibition of proliferation of tumor cells without inducing cell death could potentially reduce cytotoxicity to normal, non-tumor host cells, particularly if those normal tissues are low- or non-proliferating, as in the case of central nervous system cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Indeed, data reported in this study show that none of the CFSs under investigation is able to affect the viability of primary astrocytes, the differentiated healthy counterpart of GBM cells. Tumour progression is dependent on the ability of cells to migrate and invade, infiltrating adjacent tissues\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The wound healing assay is a technique used to study cell migration and cell-cell interaction. It is specifically a 2D cell migration approach to semi-quantitatively measure cell migration of a sheet of cells. Our results reported that non-treated cells migrate towards the wound, thereby closing the wounded edges, whereas treatment with CFSs from the three probiotics under study significantly decreases wound healing rate closure in all the tested GBM cell lines. To further investigate the inhibition of cell migration properties of microbial metabolites, a Transwell assay was performed. Indeed, these two experimental approaches involve distinct assays: the wound healing assay is a 2D migration method that is useful for investigating cell-cell interactions, where cells adhere to a polystyrene surface. In this case, the inhibitor's effect may be more related to proliferation rather than migration. Unlike the wound healing assay, the transwell assay is used to study directional migration towards a chemoattractant and typically separates migration from proliferation, as the cells are not allowed to proliferate. FBS-directional cell migration was inhibited by I4 and I7, but not D1, suggesting that the latter influences proliferation but not migration. Moreover, CFSs from I4 and I7 appear to have different behaviours depending on the treated cell type. These findings suggest that CFSs exert differential effects based on strain-specific metabolic profiles. The inhibition of migration may be linked to alterations in cytoskeletal dynamics or extracellular matrix remodelling. Future studies should explore whether these postbiotics influence metalloproteases (MMPs) activity or integrin signalling, which are critical regulators of glioblastoma cell motility.\u003c/p\u003e \u003cp\u003e2D individual cell-tracking experiments and Transwell assays cover different phenotypes and hallmarks of cell motility and adhesion\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, providing orthogonal information that can be used collectively to better describe the biological effects of CFSs on GBM cancer cells. However, the main drawback in anticancer therapy development is the use of 2D cultures \u003cem\u003ein vitro\u003c/em\u003e models. On this issue, here we developed a 3D cell culture models that resemble tumour tissues by reproducing the \u003cem\u003ein vivo\u003c/em\u003e complex architecture more faithfully. The phenotypic changes observed during exposure to postbiotics released by \u003cem\u003eL. lactis\u003c/em\u003e strains can be used as a measure of their efficacy. Here, for the first time, we assessed the effect of \u003cem\u003eL. lactis\u003c/em\u003e postbiotics on spheroid structures and growth, demonstrating that all the CFSs under study can inhibit spheroid growth and induce their disaggregation when added to the culture media. The establishment of tumour spheroids significantly increased the opportunity to investigate anti-cancer agents \u003cem\u003ein vitro\u003c/em\u003e since spheroids represent the best mimics of solid tumours \u003cem\u003ein vivo\u003c/em\u003e as they exhibit cellular heterogeneity, diffusion-limited distribution of oxygen and nutrients, cell-cell signalling, growth kinetics, cell-cell interactions, and therapeutic resistance to treatment modalities. Taken together, our results suggest that metabolites produced by \u003cem\u003eL. lactis\u003c/em\u003e D1 are able to inhibit cell proliferation but not migration, whereas metabolites produced by \u003cem\u003eL. lactis\u003c/em\u003e I4 and I7 are able to inhibit cell proliferation as well as cell migration. No one is able to induce cell death, but they all impair 3D cell spheroid growth. One possibility is that secreted bacterial metabolites interfere with intercellular adhesion molecules, which are important for migration and invasion, or hypoxia-related signalling, crucial for spheroid maintenance.\u003c/p\u003e \u003cp\u003eOne of the major challenges in cancer treatment is selectivity, namely, the ability of potential therapeutic agents to distinguish between malignant tumor cells and healthy ones represented by neurons, astrocytes, and endothelial cells of the blood-brain barrier (BBB)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Indeed, some chemotherapy might lead to the development of central neurotoxicity, due to neuroinflammation, neuroendocrine changes, and alterations in the BBB that allow increased access of cytotoxic agents and pro-inflammatory cytokines to neurons and supportive glial (astrocytes and microglia) cells, as well as secondary activation of glial cells and myelin-producing (oligodendrocyte lineage) cell defects\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To better investigate the effects of CFSs, here we established an \u003cem\u003ein vitro\u003c/em\u003e model of BBB and showed that CFSs do not affect endothelial cell viability nor BBB integrity, demonstrating once again that these compounds are safe for non-proliferating-differentiated healthy cells and their effects are exerted only on proliferating undifferentiated cancer cells. Moreover, the presence of the BBB reduce the effectiveness of anticancer therapy for human GBM. Endothelial cells of microvessels and capillaries of the brain, connected by tight contacts, limit the intercellular transport of hydrophilic drugs with a molecular weight\u0026thinsp;\u0026gt;\u0026thinsp;500 Da\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The presence in CFSs of lipophilic compounds such as liposoluble vitamins, short-chain fatty acids, neurotransmitters, and other organic acids suggests that, at least partially, these substances could bypass the BBB hindrance and easily reach tumour cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, ongoing studies by HPLC and highly sensitive MS methods in our lab are aimed at evaluating the CFSs passage through the BBB and at individual screenings with purified fractions that will allow us to determine if the observed effects are caused by a single compound or by a synergistic effect.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates for the first time that postbiotics from \u003cem\u003eL. lactis\u003c/em\u003e strains D1, I4, and I7 possess promising anticancer properties by selectively inhibiting glioblastoma cell proliferation, migration, and spheroid growth without affecting healthy cells, such as astrocytes and endothelial cells, or BBB integrity. Future studies will focus on identifying specific bioactive molecules responsible for these effects and elucidating their mechanisms of action. Additionally, \u003cem\u003ein vivo\u003c/em\u003e validation will be essential to assess therapeutic potential and optimize delivery strategies for glioblastoma treatment. These findings pave the way for exploring LAB-derived fermented foods as a natural source of bioactive compounds with potential applications in cancer therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial Strains, Media, and Growth Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactococcus lactis\u0026nbsp;\u003c/em\u003esubsp. \u003cem\u003elactis\u0026nbsp;\u003c/em\u003estrains (A3, A5, B1, D1, D3, I1, I4, I7), used in the study, were previously isolated from natural whey starter cultures. Their different RAPD (Random Amplification of Polymorphic DNA) profiles and probiotic characteristics were described in\u003csup\u003e17,36\u003c/sup\u003e. \u003cem\u003eLactococcus\u003c/em\u003e strains were grown at 30\u0026deg;C in ESTY broth supplemented with lactose 1% (w/v). Medium was supplied from Condalab (Madrid, Spain).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePostbiotic production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL. lactis\u003c/em\u003e strains were cultured on ESTY broth overnight and then sub-cultured in 100mL of the same fresh medium. Absorbance was measured periodically at 600 nm until reached 1 OD. To separate supernatants from bacterial pellets, media were centrifuged at 3750 rpm, 30 min, 4\u0026deg;C. Supernatant samples were sterilized using a 0.22 \u0026mu;m pore size filter and neutralized (pH 7.00) with NaOH 1M. Cell-free supernatant (CFS) was stored at -80\u0026deg;C and then lyophilized (VirTis SP Scientific Wizard 2.0). Dried CFSs were resuspended in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Life Technologies, Paisley, UK).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman glioblastoma cell lines U87MG, U251, and T98 were cultured in DMEM (Dulbecco\u0026rsquo;s Modified Eagle Medium) containing 10% fetal bovine serum (FBS,\u0026nbsp;Life Technologies, Paisley, UK). 100 U/mL of penicillin/streptomycin was added to the culture media. Cells were incubated in a humidified atmosphere containing 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% atmospheric air. Astrocytes purified from 1\u0026ndash;2\u0026nbsp;days old Sprague\u0026ndash;Dawley rats were used as a control of healthy cells and were grown in DMEM low glucose supplemented with 10% FBS and 100U/mL of penicillin/streptomycin for 3 weeks in a humidified incubator at 37\u0026deg;C, 5% of CO\u003csub\u003e2\u003c/sub\u003e, 95% atmospheric air.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability in response to treatment with CFSs, was assessed either by Trypan blue exclusion test or by thiazolyl blue tetrazolium bromide (MTT) assay as previously described with some modifications\u003csup\u003e6\u003c/sup\u003e. For the MTT assay, an initial screening was performed on U87MG cells, testing all the lyophilized CFSs of all the \u003cem\u003eL. lactis\u003c/em\u003e strains, solved in DMEM media in desirable concentrations (50, 5, 0.5mg/mL). Subsequently, three CFSs (\u003cem\u003eL. lactis\u003c/em\u003e D1, I4, I7) were selected, and a single concentration (0.5 mg/mL) was chosen for further testing on U87MG, U251, and T98 cell lines. Briefly, 2.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e U87MG, 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e U251, and 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e T98 cells/well were grown in 96-well plates for 24 h and then treated with 0.5mg/mL of CFSs. Concomitantly, cells without treatments were used as a positive control. 10 \u0026micro;L of the 5 mg/mL MTT solution in PBS, (Invitrogen-Life Technologies, Eugene, OR, USA) were added to wells, and incubated for 3 h at 37 \u0026deg;C. After incubation, 100 \u0026micro;L of stop solution were added to each well. Then, the optical densities were measured at 595 nm using the Microplate Reader (GLO Max Discovery, Promega). The cell viability rate was assessed after 24, 48 and 72 hours and calculated as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecell metabolic activity rate (%) = (OD treated) / (OD control) x 100\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the Trypan blue exclusion test, 2.5 \u0026times; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eU87MG, U251, and T98 cells were grown and then treated with 0.5 mg/mL of three selected CFSs. Cells were then incubated at 37 \u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. Then, the cells were detached, stained with a 0.4% (\u003cem\u003ew\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) Trypan blue solution, counted using a Burker chamber, and reported as the number of live/dead cells. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle analysis and apoptosis assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle analysis was performed by Fluorescence-Activated Cell Sorting (FACS) as previously described\u003csup\u003e37\u003c/sup\u003e. Briefly, U87MG, U251, and T98 cells were treated with 5.0 mg/mL of D1 and I4, and 0.5 mg/mL of I7 CFS, after synchronization with 1% FBS for 12h, and incubated for 12 and/or 24 hours at 37\u0026deg;C. The cells were then washed two times with phosphate-buffered saline (PBS), fixed with EtOH 70%, permeabilized with Triton 0.1% and finally stained in 10 \u0026mu;g/mL of propidium iodide (PI) with 5 \u0026mu;g/mL RNase in the dark at 4\u0026deg;C overnight. Fluorescence was determined by using the FACSCalibur or Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). At least 15000 total events were acquired, of which 5000 events were PI positive and analyzed. Data were analysed with Floreada.io online software or Flowjo10.4 software\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApoptotic cells were detected by annexin V-FITC/PI staining assay following the manufacturer instructions as previously described\u003csup\u003e38\u003c/sup\u003e (A432; Leinco Technologies, Inc.). Briefly, the cells were treated with 5 mg/mL of D1 and I4 and 0.5 mg/mL of I7 CFS, after synchronization with 1% FBS for 12h, and incubated for 24-48 hours, washed twice with annexin V-binding buffer and resuspended in 1ml of the same buffer (1x10\u003csup\u003e6\u003c/sup\u003e). Fifteen minutes before flow cytometry analysis, 5ml of PI and 5ml di Annexin-V-FITC were added to 100 ml of each sample and then analyzed by Accuri C6 Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). As a control of efficacy of materials and Annex V/ PI test reagent, NIH3T3 cell line were treated with Cisplatin 60mM, which induces cell death via an apoptotic pathway in a time-dependent manner (see Supplementary Figure S4). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wound healing assay or scratch test was performed as previously described\u003csup\u003e39\u003c/sup\u003e. Briefly, 6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e U87MG, U251, and T98 cells were seeded and grown to confluence in DMEM/10% FBS for 24 hours. Afterwards, the cell monolayers were wounded with a yellow tip and concomitantly treated with 0.5mg/mL of each CFS. Untreated cells were used as a positive control. Images were taken immediately after scratch formation (T0) and after 24 hours by using an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.), equipped with the AmScope 3.7 Software. To quantify the wound healing rate, arbitrary arrows pointing the wound edges were drawn on the T0 image and were further applied to the single photograms of each sample after 24 hours. The average margin distance was measured at five points and the results were expressed as a percentage of residual wound width at T24 compared to T0 of the same sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Transwell migration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe migration assay\u003csup\u003e40\u003c/sup\u003e was performed using Transwell Plate 8 \u0026micro;m Pore Size (Sarstedt,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDarmstadt, Germany). Briefly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e7 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e U87MG, 4 \u0026times; 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eU251, and 4 \u0026times; 10\u003csup\u003e4 \u0026nbsp;\u003c/sup\u003eT98 cells treated and untreated with 0.5mg/mL of CFS, were plated on serum-free media and allowed to migrate for 4 hours at 37\u0026deg;C towards DMEM 5% FBS. \u0026nbsp;At the end of the assay, cells in the upper chamber were removed using cotton swabs, while cells on the lower filter surface were fixed in cold MetOH 100% for 15 min, stained with 0.05% crystal violet for 30 min at room temperature, and then washed with tap water. Migrated cells were counted under an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.) \u0026nbsp;in at least three randomly selected fields at 10X magnification. The number of migrating cells was calculated using ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumour spheroid\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eU87MG spheroids were formed by self-aggregation of cells in the bottoms of non-adherent round bottom 96-well plates (Bioflat, Sarstedt, Numbrecht, Germany), by following the method of Sivakumar, with some modifications\u003csup\u003e41\u003c/sup\u003e. Briefly, 100 to 500 cells in 100 \u0026mu;L of media were pipetted into individual wells and allowed to form cell-cell connections over the course of 24 hours. Once the sphere was formed, 0.5mg/mL of tested postbiotics were added. Pictures were taken after 0-24-48 hours and checked after 6 days of treatments by using an inverted microscope (Nikon Eclipse TE300, NIKON INSTRUMENTS INC.). Single sphere diameter was measured with the AmScope 3.7 Software. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003estudy on the Blood-Brain Barrier (BBB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals used in the study have been treated according to relevant ethical guidelines. This includes that methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org ), on the ethical use of animals (European Communities Council Directive of 24 November 1986; 86/609/ECC), and Danish guidelines.\u003c/p\u003e\n\u003cp\u003ePorcine brains were obtained as byproducts of the Danish food industry. Danish Slaughterhouses are under strict supervision and observation by the Danish Ministry of Environment and Food. According to the \u0026ldquo;Danish Crown\u0026rdquo; the pigs are anesthetized with 80-90 % CO\u003csub\u003e2\u003c/sub\u003e and then terminated by cutting their \u0026nbsp;Carotid artery.\u003c/p\u003e\n\u003cp\u003eRats used for isolation of astrocytes were bred and group-housed in the local animal facility at an ambient temperature of 22 \u0026deg;C-23 \u0026deg;C and on a 12/12 h dark/light cycle under the inspection of the veterinarian and according to Danish regulations for lab animals. The rats were euthanized with the CO\u003csub\u003e2\u003c/sub\u003e method before they were sacrificed in accordance with international guidelines on the ethical use of animals (European Communities Council Directive of 24 November 1986; 86/609/EEC) and Danish guidelines. No \u003cem\u003ein vivo\u003c/em\u003e experiments on animals or human material were used in these experiments.\u003c/p\u003e\n\u003cp\u003eThis research was approved Aarhus University Animal Facility veterinarian committee. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eCell cultures and an\u0026nbsp;\u003c/em\u003ein\u0026nbsp;vitro\u003cem\u003e\u0026nbsp;BBB model establishment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Astrocytes were purified from 1\u0026ndash;2 days old Sprague\u0026ndash;Dawley rats and brain microcapillaries were purified from 5\u0026ndash;6 months old pigs. Selective cultures of porcine brain endothelial cells (pBECs) were established in a non-contact co-culture (NCC) in vitro BBB model as described\u003csup\u003e20\u003c/sup\u003e. Following primary cell purifications, astrocytes were cultured in poly-L-Lysine pre-coated 12-well plates in low glucose DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100\u0026nbsp;U/mL), and streptomycin (100\u0026nbsp;\u0026micro;g/ml) for three weeks before NCC establishment. Porcine brain microcapillaries were seeded on type IV collagen- (150\u0026nbsp;\u0026mu;g/mL) and fibronectin (50\u0026nbsp;\u0026mu;g/ mL) coated T75 flasks using DMEM-F12 supplemented with 10% plasma-derived serum (PDS) (First Link, Wolverhampton, United Kingdom, UK), penicillin (100\u0026nbsp;U/ mL), streptomycin (100\u0026nbsp;\u0026micro;g/ml), and heparin (15\u0026nbsp;U/mL). For the first four days in culture, pBEC were selected using puromycin (4\u0026nbsp;\u0026mu;g/mL). At 70% confuency, cells were passed with Trypsin/EDTA (2.5% trypsin, 0.1\u0026nbsp;nM EDTA in PBS) and seeded on type IV collagen (500 \u0026mu;g/mL) and fibronectin (100 \u0026mu;g/mL) coated Transwell inserts (12\u0026nbsp;mm, 0.4\u0026nbsp;\u0026mu;m pore polycarbonate membrane, cat. no: 3401, Corning, Kennebunk ME 04043, USA) at a density of 1.1x10\u003csup\u003e5\u003c/sup\u003e cells/insert. pBECs were co-cultured with astrocytes in the basal chamber in serum-free media. To further induce the barrier development, both chambers were supplemented with the differentiation factors hydrocortisone (550 nM), 8-(4-chlorophenylthio)-adenosine-3ʹ,5\u0026prime;-cyclic monophosphate (250 \u0026mu;M), and RO-201724 (17.5 \u0026mu;M) 1\u0026ndash;2 days before experiments. The tightness of the model was validated by measurements of transendothelial electrical resistance (TEER) using an EndOhm-12 measurement device (World Precision Instruments), with values\u0026gt;1000 \u0026Omega; cm2 accepted for experiments. Before all experiments, Transwell inserts with cultured pBECs were transferred to a new 12-well plate without astrocytes, washed two times with PBS, incubated with media without differentiation factors, and allowed to rest in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 hours at 37 \u0026deg;C, before further treatment. To investigate the effects of the \u003cem\u003eL. lactis\u003c/em\u003e CFSs on the BBB, 0.5 mg/mL of each CFS was added in the apical compartment and incubated at 37 \u0026deg;C, in the presence of 5% CO2 up to 1-6-24 hours with a circular rotation of 100 rpm and an orbit of 3 mm. The tightness of the barrier model was validated before and after the addition of the CFSs by measurements of TEER. Subsequently, for each time point, media from the top and the bottom was collected and stored at -20\u0026deg;C for further analysis. Furthermore, the expression of adherens junction proteins was validated by immunocytochemistry according to the procedures described below. To test the cytotoxicity of these CFSs, Trypan blue exclusion test was performed on astrocytes as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Immunofluorescence staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBECs were fixed in 4% paraformaldehyde in cytoskeleton buffer (50 mM PIPES, 50 mM NaCl, 5% glycerol, 0.1% NP-40, 0.1% Triton X-100 and 0.1% Tween 20) at RT for 20 minutes, washed 3 times with PBS, and permeabilized with 0.1% Triton X-100 for 10 min and blocked in 2% BSA for 30 min. For immunostaining, 5 \u0026mu;g/mL of antibodies anti \u0026alpha;-p120 catenin (610133, BD Transduction Laboratory) were applied for 1 hour at RT. Filters were then washed 3 times in PBS at RT before 30 min incubation in the dark with 1:200 anti-mouse antibodies Alexa Fluor 488 (A32731 Life technologies). For nuclear staining, cells were incubated with 0.125 \u0026mu;g/mL Hoechst stain solution (Sigma-Aldrich) for 10 min at RT. Finally, membranes were mounted on microscope glass slides #1.5, with ProLong Diamond Antifade Mounting medium (Invitrogen, Thermo Fisher Scientific).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003eConfocal microscopy and\u0026nbsp;image processing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal imaging was performed using an Olympus IX-83 fluorescent microscope with a confocal spinning disk unit (Yokogawa) and a 60x 1.2 NA objective. Pictures are presented as maximum-intensity z-stack projections unless other specific details are noted in figure legends. Image processing and spot segmentation analysis were performed using Fiji software \u003csup\u003e42\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eStatical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll presented data are based on three independent experiments. The statistical analyses and graphs were prepared using Prism (8.0) (GraphPad Software). The bar plots in the graphs report mean values (\u0026plusmn;SD). The tests of significant difference were analyzed using the unpaired \u003cem\u003et\u003c/em\u003e-test with Welch\u0026rsquo;s correction, Oneway and 2way ANOVA with multiple comparisons.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Annemette Boe Marnow and Donato Sardella for technical assistance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;\u003cbr\u003eThis research was funded by the European Union and Italian MIUR, Project BIONUTRA\u0026mdash;PON 2014\u0026ndash;2020 (Development of Nutraceuticals from Natural Sources), Grant Number PON ARS01_01166.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConception and design of the study, I.D.C., N.MS., MT.G., and L.M.; performing the research, I.D.C., F.A., and M.D.G; \u003cem\u003ein vitro\u003c/em\u003e experiment on the Blood-Brain Barrier (BBB) I.D.C., and N.MS.; statistical analysis, I.D.C., A.P., and A.F.; analysis and/or interpretation of data, I.D.C, A.F., MT.G., R.M., and L.M.; drafting the manuscript, I.D.C, A.F., R.M., A.P., M.G., MT.G., and L.M. \u0026nbsp;All authors provided critical feedback and helped to shape the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAll authors have no conflicts of interest relevant to this study to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApproval for animal experiments\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was approved Aarhus University Animal Facility veterinarian committee. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the data that support the findings of this study are available in the paper and its Supplementary information published online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVera-Santander, V. E., Hern\u0026aacute;ndez-Figueroa, R. H., Jim\u0026eacute;nez-Mungu\u0026iacute;a, M. T., Mani-L\u0026oacute;pez, E. \u0026amp; L\u0026oacute;pez-Malo, A. Health Benefits of Consuming Foods with Bacterial Probiotics, Postbiotics, and Their Metabolites: A Review. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, doi:10.3390/molecules28031230 (2023).\u003c/li\u003e\n\u003cli\u003eSebasti\u0026aacute;n Domingo, J. J. Review of the role of probiotics in gastrointestinal diseases in adults. \u003cem\u003eGastroenterol Hepatol\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 417-429, doi:10.1016/j.gastrohep.2016.12.003 (2017).\u003c/li\u003e\n\u003cli\u003eDE Leo, V.\u003cem\u003e et al.\u003c/em\u003e Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota. \u003cem\u003eMinerva Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 432-439, doi:10.23736/S2724-606X.22.05087-4 (2023).\u003c/li\u003e\n\u003cli\u003eWu, Y.\u003cem\u003e et al.\u003c/em\u003e A Review on Maternal and Infant Microbiota and Their Implications for the Prevention and Treatment of Allergic Diseases. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, doi:10.3390/nu15112483 (2023).\u003c/li\u003e\n\u003cli\u003eChuah, L. O.\u003cem\u003e et al.\u003c/em\u003e Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells. \u003cem\u003eBMC Complement Altern Med\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 114, doi:10.1186/s12906-019-2528-2 (2019).\u003c/li\u003e\n\u003cli\u003eSharma, P., Kaur, S., Kaur, R. \u0026amp; Kaur, M. Proteinaceous Secretory Metabolites of Probiotic Human Commensal. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 948, doi:10.3389/fmicb.2018.00948 (2018).\u003c/li\u003e\n\u003cli\u003eAbd Ellatif, S. A.\u003cem\u003e et al.\u003c/em\u003e Assessment of probiotic efficacy and anticancer activities of Lactiplantibacillus plantarum ESSG1 (MZ683194.1) and Lactiplantibacillus pentosus ESSG2 (MZ683195.1) isolated from dairy products. \u003cem\u003eEnviron Sci Pollut Res Int\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 39684-39701, doi:10.1007/s11356-022-18537-z (2022).\u003c/li\u003e\n\u003cli\u003eNataraj, B. H., Ali, S. A., Behare, P. V. \u0026amp; Yadav, H. Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods. \u003cem\u003eMicrob Cell Fact\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 168, doi:10.1186/s12934-020-01426-w (2020).\u003c/li\u003e\n\u003cli\u003eScott, E., De Paepe, K. \u0026amp; Van de Wiele, T. Postbiotics and Their Health Modulatory Biomolecules. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, doi:10.3390/biom12111640 (2022).\u003c/li\u003e\n\u003cli\u003eBanna, G. L.\u003cem\u003e et al.\u003c/em\u003e GG: An Overview to Explore the Rationale of Its Use in Cancer. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 603, doi:10.3389/fphar.2017.00603 (2017).\u003c/li\u003e\n\u003cli\u003eSong, D., Wang, X., Ma, Y., Liu, N. N. \u0026amp; Wang, H. Beneficial insights into postbiotics against colorectal cancer. \u003cem\u003eFront Nutr\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1111872, doi:10.3389/fnut.2023.1111872 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, L.\u003cem\u003e et al.\u003c/em\u003e A compatibility assay of ursolic acid and foodborne microbial exopolysaccharides by antioxidant power and anti-proliferative properties in hepatocarcinoma cells. \u003cem\u003eJournal of Food, Agriculture and Environment\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 111-114 (2012).\u003c/li\u003e\n\u003cli\u003eElango, A., Nesam, V. D., Sukumar, P., Lawrence, I. \u0026amp; Radhakrishnan, A. Postbiotic butyrate: role and its effects for being a potential drug and biomarker to pancreatic cancer. \u003cem\u003eArch Microbiol\u003c/em\u003e \u003cstrong\u003e206\u003c/strong\u003e, 156, doi:10.1007/s00203-024-03914-8 (2024).\u003c/li\u003e\n\u003cli\u003eEladwy, R. A.\u003cem\u003e et al.\u003c/em\u003e The postbiotic sodium butyrate synergizes the antiproliferative effects of dexamethasone against the AGS gastric adenocarcinoma cells. \u003cem\u003eFront Nutr\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1372982, doi:10.3389/fnut.2024.1372982 (2024).\u003c/li\u003e\n\u003cli\u003eZou, Y.\u003cem\u003e et al.\u003c/em\u003e Brain Co-Delivery of Temozolomide and Cisplatin for Combinatorial Glioblastoma Chemotherapy. \u003cem\u003eAdv Mater\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, e2203958, doi:10.1002/adma.202203958 (2022).\u003c/li\u003e\n\u003cli\u003eSingh, N., Miner, A., Hennis, L. \u0026amp; Mittal, S. Mechanisms of temozolomide resistance in glioblastoma - a comprehensive review. \u003cem\u003eCancer Drug Resist\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 17-43, doi:10.20517/cdr.2020.79 (2021).\u003c/li\u003e\n\u003cli\u003eDe Chiara, I.\u003cem\u003e et al.\u003c/em\u003e Probiotic Properties of. \u003cem\u003eFoods\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, doi:10.3390/foods13060957 (2024).\u003c/li\u003e\n\u003cli\u003eNakatsuma, A.\u003cem\u003e et al.\u003c/em\u003e Effects of kaempferol on the mechanisms of drug resistance in the human glioblastoma cell line T98G. \u003cem\u003eDie Pharmazie\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 379-383, doi:10.1691/ph.2010.9807 (2010).\u003c/li\u003e\n\u003cli\u003eSamara Rodrigues Alves and Italo Rodrigo Calori and Hong Bi and Antonio Claudio, T. Characterization of glioblastoma spheroid models for drug screening and phototherapy assays. \u003cem\u003eOpenNano\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 100116, doi:https://doi.org/10.1016/j.onano.2022.100116 (2023).\u003c/li\u003e\n\u003cli\u003eNielsen, S. S. E.\u003cem\u003e et al.\u003c/em\u003e Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells. \u003cem\u003eJ Vis Exp\u003c/em\u003e, doi:10.3791/56277 (2017).\u003c/li\u003e\n\u003cli\u003eToth, A. E.\u003cem\u003e et al.\u003c/em\u003e The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro. \u003cem\u003eMol Neurobiol\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 8522-8537, doi:10.1007/s12035-018-0988-x (2018).\u003c/li\u003e\n\u003cli\u003eWaithe, O. Y., Peng, X., Childs, E. W. \u0026amp; Tharakan, B. Measurement of Transendothelial Electrical Resistance in Blood-Brain Barrier Endothelial Cells. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e2711\u003c/strong\u003e, 199-203, doi:10.1007/978-1-0716-3429-5_16 (2024).\u003c/li\u003e\n\u003cli\u003eLi, H.\u003cem\u003e et al.\u003c/em\u003e Differences in Protein Expression between the U251 and U87 Cell Lines. \u003cem\u003eTurk Neurosurg\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 894-903, doi:10.5137/1019-5149.JTN.17746-16.1 (2017).\u003c/li\u003e\n\u003cli\u003eBhatia, P.\u003cem\u003e et al.\u003c/em\u003e Breast cancer resistance protein (BCRP/ABCG2) localises to the nucleus in glioblastoma multiforme cells. \u003cem\u003eXenobiotica\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 748-755, doi:10.3109/00498254.2012.662726 (2012).\u003c/li\u003e\n\u003cli\u003eErasimus, H., Gobin, M., Niclou, S. \u0026amp; Van Dyck, E. DNA repair mechanisms and their clinical impact in glioblastoma. \u003cem\u003eMutat Res Rev Mutat Res\u003c/em\u003e \u003cstrong\u003e769\u003c/strong\u003e, 19-35, doi:10.1016/j.mrrev.2016.05.005 (2016).\u003c/li\u003e\n\u003cli\u003eGhasemi, M., Turnbull, T., Sebastian, S. \u0026amp; Kempson, I. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, doi:10.3390/ijms222312827 (2021).\u003c/li\u003e\n\u003cli\u003eKvakova, M., Kamlarova, A., Stofilova, J., Benetinova, V. \u0026amp; Bertkova, I. Probiotics and postbiotics in colorectal cancer: Prevention and complementary therapy. \u003cem\u003eWorld J Gastroenterol\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 3370-3382, doi:10.3748/wjg.v28.i27.3370 (2022).\u003c/li\u003e\n\u003cli\u003eWu, J., Zhang, Y., Ye, L. \u0026amp; Wang, C. The anti-cancer effects and mechanisms of lactic acid bacteria exopolysaccharides in vitro: A review. \u003cem\u003eCarbohydr Polym\u003c/em\u003e \u003cstrong\u003e253\u003c/strong\u003e, 117308, doi:10.1016/j.carbpol.2020.117308 (2021).\u003c/li\u003e\n\u003cli\u003eMorley, K. L., Ferguson, P. J. \u0026amp; Koropatnick, J. Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells. \u003cem\u003eCancer Lett\u003c/em\u003e \u003cstrong\u003e251\u003c/strong\u003e, 168-178, doi:10.1016/j.canlet.2006.11.016 (2007).\u003c/li\u003e\n\u003cli\u003eFares, J., Fares, M. Y., Khachfe, H. H., Salhab, H. A. \u0026amp; Fares, Y. Molecular principles of metastasis: a hallmark of cancer revisited. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 28, doi:10.1038/s41392-020-0134-x (2020).\u003c/li\u003e\n\u003cli\u003ePijuan, J.\u003cem\u003e et al.\u003c/em\u003e Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 107, doi:10.3389/fcell.2019.00107 (2019).\u003c/li\u003e\n\u003cli\u003eAbbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R. \u0026amp; Begley, D. J. Structure and function of the blood-brain barrier. \u003cem\u003eNeurobiol Dis\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 13-25, doi:10.1016/j.nbd.2009.07.030 (2010).\u003c/li\u003e\n\u003cli\u003eWas, H.\u003cem\u003e et al.\u003c/em\u003e Mechanisms of Chemotherapy-Induced Neurotoxicity. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 750507, doi:10.3389/fphar.2022.750507 (2022).\u003c/li\u003e\n\u003cli\u003ePandit, R., Chen, L. \u0026amp; G\u0026ouml;tz, J. The blood-brain barrier: Physiology and strategies for drug delivery. \u003cem\u003eAdv Drug Deliv Rev\u003c/em\u003e \u003cstrong\u003e165-166\u003c/strong\u003e, 1-14, doi:10.1016/j.addr.2019.11.009 (2020).\u003c/li\u003e\n\u003cli\u003eHoyles, L.\u003cem\u003e et al.\u003c/em\u003e Microbiome-host systems interactions: protective effects of propionate upon the blood-brain barrier. \u003cem\u003eMicrobiome\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 55, doi:10.1186/s40168-018-0439-y (2018).\u003c/li\u003e\n\u003cli\u003eMarasco, R., Gazzillo, M., Campolattano, N., Sacco, M. \u0026amp; Muscariello, L. Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk. \u003cem\u003eFoods\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, doi:10.3390/foods11020233 (2022).\u003c/li\u003e\n\u003cli\u003eDi Zazzo, E.\u003cem\u003e et al.\u003c/em\u003e The p85 regulatory subunit of PI3K mediates cAMP-PKA and insulin biological effects on MCF-7 cell growth and motility. \u003cem\u003eScientificWorldJournal\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, 565839, doi:10.1155/2014/565839 (2014).\u003c/li\u003e\n\u003cli\u003eBoccellino, M.\u003cem\u003e et al.\u003c/em\u003e H9c2 Cardiomyocytes under Hypoxic Stress: Biological Effects Mediated by Sentinel Downstream Targets. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, 6874146, doi:10.1155/2021/6874146 (2021).\u003c/li\u003e\n\u003cli\u003eGentile, M. T., Pastorino, O., Bifulco, M. \u0026amp; Colucci-D\u0026apos;Amato, L. HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis. \u003cem\u003eJ Vis Exp\u003c/em\u003e, doi:10.3791/58591 (2019).\u003c/li\u003e\n\u003cli\u003eZhou, F.\u003cem\u003e et al.\u003c/em\u003e MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/\u0026beta;-catenin pathway via direct targeting of Frizzled 7 mRNA. \u003cem\u003eAm J Transl Res\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 4584-4601 (2019).\u003c/li\u003e\n\u003cli\u003eSivakumar, H., Devarasetty, M., Kram, D. E., Strowd, R. E. \u0026amp; Skardal, A. Multi-Cell Type Glioblastoma Tumor Spheroids for Evaluating Sub-Population-Specific Drug Response. \u003cem\u003eFront Bioeng Biotechnol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 538663, doi:10.3389/fbioe.2020.538663 (2020).\u003c/li\u003e\n\u003cli\u003eDobens, A. C. \u0026amp; Dobens, L. L. FijiWings: an open source toolkit for semiautomated morphometric analysis of insect wings. \u003cem\u003eG3 (Bethesda)\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1443-1449, doi:10.1534/g3.113.006676 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotics, Postbiotics, Lactococcus lactis, glioblastoma, cancer therapy, blood-brain barrier","lastPublishedDoi":"10.21203/rs.3.rs-5353727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5353727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the last few years, probiotics have gained much attention within the medical, pharmaceutical, and food fields, given the health benefits provided by their consumption. They include several lactic acid bacteria (LAB) species, mostly belonging to the genera \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e. Postbiotics are bioactive compounds (organic acids, short-chain fatty acids, enzymes, and neurotransmitters) produced by bacterial fermentation that exert different health effects. It is well known that probiotics, as health-promoting microorganisms, show different therapeutic properties, including anti-pathogenic, anti-inflammatory, and cholesterol-lowering activities. Recently, accumulating evidence has shown that certain commensal bacteria play protective roles against cancer; thus, anti-carcinogenic activity is one of the most interesting probiotics properties that is currently under investigation. Here, we studied the anticancer properties of postbiotics produced by three different \u003cem\u003eLactococcus lactis\u003c/em\u003e subsp \u003cem\u003elactis\u003c/em\u003e strains isolated from natural whey starter cultures on human glioblastoma cell lines. MTT and Trypan Blue exclusion assays revealed a significant reduction in cell proliferation, and flow cytometry analysis corroborated this data, demonstrating a cell cycle arrest in treated cells. Moreover, other cancer hallmarks, such as wound healing rate closure and migration, were markedly inhibited by postbiotics. On the other hand, primary astrocytes viability and the blood-brain barrier (BBB) integrity were not impaired, suggesting a selective effect of postbiotics on proliferating-undifferentiated cells. This preliminary study highlights, for the first time, the potential anticancer properties of postbiotics from some \u003cem\u003eL. lactis\u003c/em\u003e strains on human glioblastoma cell lines.\u003c/p\u003e","manuscriptTitle":"Antitumor activity of Lactococcus lactis cell-free supernatant on human glioblastoma cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 09:53:14","doi":"10.21203/rs.3.rs-5353727/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c831cbea-29a9-4216-839d-f3376e6528d1","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47754069,"name":"Biological sciences/Cancer/Cns cancer"},{"id":47754070,"name":"Biological sciences/Microbiology/Bacteria/Bacterial host response"}],"tags":[],"updatedAt":"2025-05-30T07:38:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 09:53:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5353727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5353727","identity":"rs-5353727","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}