To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-of-origin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource.Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key  Lung cancer remains the leading cause of cancer death in the United States , 2 and iin vitro or in vivo , squamous cell carcinoma (LUSQ), small cell lung cancer (SCLC), and large cell carcinoma (LULCC) –42, the https://seer.cancer.gov/) program, a large United States-based cancer registry that at present includes over 331 million subjects from 17 regions, to assess lung cancer mortality for different races/ethnicities for the main histological types . Current recommendations are to test cell lines for mycoplasma and authenticity when they first reach a new laboratory, before publication, and every two months while in culture.Two important considerations when using cell lines for research are cell line quality and authenticity. The presence of contaminating microorganisms, particularly mycoplasma, and the cross-contamination with other cell lines can invalidate performed research , 50. MycWe investigated the availability of lung cancer cell lines representing different races/ethnicities using the resources listed in We identified 390 cell lines from Asian lung cancer patients , H/LWe found almost 300 cell lines for which race/ethnicity is unknown –73.We noted that the number of cell lines developed from men was over 2-fold higher than cell lines developed from women, and this excess was most prominent for the cell lines developed from Asian individuals   were established using SV40LgT antigen  and small airway epithelial cells (SAECs)  , 86. BEAThe availability of methods to establish immortalized alveolar and airway cells allows progress to be made in deriving additional cell lines from racially and ethnically diverse subjects. However, there is one important cell type for which no immortalized human cell lines have yet been established: pulmonary neuroendocrine (PNE) cells, the main cell-of-origin of SCLC . ImmortaDerivation of cell line types from induced pluripotent cells has also been used to obtain bronchial epithelial cells  and alvein vitro cultures can be useful for the study of cancer driver and tumor suppressor genes, epigenetic changes in cancer cells, the effects of environmental exposures, and the investigation of lung cancer risk SNPs, among other topics. Cell lines provide a relatively pure population of cells compared to heterogeneous tumor or tissue samples that can contain variable amounts of contaminating blood cells and stroma. This simplification can greatly facilitate analyses and provides one powerful strategy to leverage cell-based models. However, it lacks the complexity arising from growth in three-dimensional space or from the interactions with other cell types, such as fibroblasts and blood vessels. Growth of pure cell lines with a defined medium in three dimensions can provide the next level of complexity, while the addition of fibroblasts, endothelial, and blood cells can further simulate in vivo characteristics. Even further advanced are three-dimensional models, so-called “organs-on-a-chip”, which may incorporate an air-liquid interface and/or the movement associated with breathing  mice or humanized mice need to be used. Such models, known as xenografts, can be made using human cancer cell lines, primary patient tumors, or even circulating tumor cells , the number of sequenced lung cancer samples from White patients outnumbers that of Black patients by almost 9:1. Thus, data on driver mutations in underrepresented patients must also be expanded. Clearly, much work remains to be done. The first step is to highlight current shortcomings in knowledge and resources, and to disseminate information to lung cancer patients of all races and ethnicities about the need for cell lines representing lung cancer in their communities. Explaining how lung cancer cell lines and cell-of-origin lines can be used to improve research and develop new therapies for people of the patients’ own racial/ethnic backgrounds can help patients make an informed decision about whether to participate. In addition, it would be beneficial if the donations of tissues/cells were discussed with patients by researchers and/or clinicians from their own racial/ethnic group, supporting mutual trust and a better understanding of research goals  , Black non-Hispanic, White non-Hispanic (IAO), and Asian .


To investigate the mechanism of exosomes' role in the transformation of MDS to AML.Exosomes in culture supernatants of MDS and AML cell lines, were extracted by ultrafiltration and identified in three ways: morphology, size, and exosome protein surface markers. Exosomes from AML cell lines were then co-cultured with MDS cell lines and their impacts on MDS cell microenvironment, proliferation, differentiation, cell cycle, and apoptosis were analyzed by CCK-8 assay and flow cytometry. Furthermore, exosomes from MSC were extracted for further authentication.The transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and flow cytometry methods all verify that ultrafiltration is a reliable method to extract exosomes in the culture medium. Exosomes from AML cell lines inhibit the proliferation of MDS cell lines, block cell cycle progression, and promote apoptosis and cell differentiation. It also leads to increased secretion of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in MDS cell lines. In addition, MSC-derived exosomes were found to inhibit the proliferation of MDS cell lines, arrest cell cycle progression, promote apoptosis, and inhibit differentiation.Ultrafiltration is a proper methodology in extracting exosomes. The exosomes of AML origin and MSC origin may play a role in MDS leukemia transformation via targeting TNF-α/ROS-Caspase3 pathway. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders that are characterized by inefficient hematopoiesis, hemocytopenia, and an increase in blast cells, with nearly one-third of patients at risk of progression into acute myeloid leukemia(AML) , 2. In tExosomes are extracellular vesicles 30–150 nm in diameter formed by cells by way of endocytosis . ExosomeApoptosis is a programmed cell death caused by the death receptor pathway or the mitochondrial pathway . Tumor nAt present, there is still no effective therapeutic methodology in treating MDS. The low and intermediate risk MDS is mainly managed via hypomethylating agents and supportive care, such as transfusion therapy. Cytotoxic chemotherapy, targeted therapy, and immunomodulatory therapy are the common managements for high-risk MDS, Allogeneic hematopoietic stem cell transplantation (HSPC) is the only treatment possible to cure MDS, but the treatment-related mortality risk and the source of stem cells are still the biggest obstacles , 23.It is now generally accepted that tumor cell-derived exosomes can promote disease progression and metastasis . In this2.The human myelodysplastic syndrome cell lines SKM-1 and MUTZ-1 were purchased from Otwo Biotech , and the human myeloid leukemia cell lines THP-1, K562, and HL-60 are routinely maintained in our laboratory. SKM-1 was cultured in IMDM with 10% FBS and 1% penicillin, MUTZ-1, THP-1, K562 and HL-60 were cultured in RPMI-1640 with 10% FBS and 1% penicillin, and all cell lines were cultured at 37 °C and 5% COWhen density reached 60–70%, cells were collected by centrifugation at 300 g, 4 °C, 10 min, the supernatant was collected after 48 h of further incubation in a serum-free medium. The supernatant was centrifuged at 300 g, 4 °C, 10 min to remove cellular components; 2000 g, 4 °C, 10 min to remove dead cells; then 10000 g,4 °C,30 min to remove cell fragments. The supernatant was filtrated via a 0.22 μm filter to remove apoptotic vesicles. The exosomes were collected by ultrafiltration using a 10 KD ultrafiltration tube . Finally, the ultrafiltrate was resuspended in PBS to elute the medium components.Transmission electron microscope (TEM) and Nanoparticle Tracking Analysis (NTA) were sent to Beijing Ovison Gene Technology Company for analysis. Exosome proteins were lysed using RIPA lysate . Rough quantification was performed using a BCA protein concentration assay kit . Equal amounts of exosome protein (60 μg) were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring the protein onto 0.45 μm PVDF membranes . Using 5% skimmed milk powder for closure, incubated with primary antibody CD63, TSG101, Calnexin for 1.5 h and HRP-labelled goat anti-rabbit secondary antibody for 2 h at room temperature in a shaker. The results were finally developed using the ECL  chemiluminescence method. Exosomes were identified by flow cytometry method using CD9 -FITC, CD63- PE, CD81- FITC, CD33- APC, and CD14- APC antibodies as the surface markers.PKH26  binds to the lipid region of the cell membrane, showing as a sign of red fluorescence. The labeled exosomes were co-cultured with the MDS cell line for 48 h and then observed under a fluorescent microscope and photographed for documentation.5 cells were taken in a flow tube, 5 μl FITC, and 5 μl PI were added, mixed, and incubated for 15 min at room temperature and protected from light and then tested on the machine. Cell differentiation and proliferation indicators were detected using CD11b-PE and CD117- PC5 antibodies. The Seven-item Cytokine Assay Kit  was used to detect cytokines in the culture supernatant. Detection of intracellular reactive oxygen levels were used the Reactive Oxygen Species Assay Kit , Briefly, the cells were resuspended in DCFH-D diluted with serum-free medium, incubated in a 37 °C cell incubator for 20 min, washed with serum-free medium and then assayed on the machine.AML cell line-derived exosomes were co-cultured with MDS cell lines for 48 h before flow cytometric analyses of cell cycle, apoptosis, differentiation, cytokines, and ROS. Cells were fixed in pre-cooled 70% ethanol at 4 °C, stained using propidium iodide (PI) according to the Cell Cycle Assay Kit , and DNA content was determined by flow cytometry analysis, and the percentage of cells in G0/G1, S, and G2/M phases were counted for comparison. Apoptosis staining was performed using the Apoptosis Assay Kit . 103/well with 100 μ cell suspension per well, incubate at 37 °C, 5% CO2. The plate was removed at 0 d, 1 d, 2 d, 3 d, and 4 d, and 10 μl of CCK-8 reagent was added to each well and the culture was continued for 2 h. The absorbance (OD) values of the corresponding wells were measured at 450 nm and the cell proliferation curves were plotted.Cell proliferation was measured using the Cell Counting Kit (CCK-8) . AML cell line-derived exosomes were co-cultured with the MDS cell line for 48 h and then inoculated into 96-well plates at 5 × 10Proteins from MDS cell lines were extracted using RIPA lysate  after 48 h co-culturing The extracted proteins were quantified using the BCA protein concentration assay kit . Equal amounts of protein (60 μg) were subjected to SDS-PAGE and subsequently transferred onto 0.45 μm PVDF membranes . The primary antibody Caspase3 and β-actin were incubated for 1.5 h on a shaker at room temperature. Then the HRP-labelled secondary antibody was incubated for 2 h. The final development was performed by chemiluminescence using ECL .t-test. p < 0.05 was considered statistically significant.SPSS (version 17.0) and GraphPad (Prism 7.0) were used for statistical processing and plotting. All data were obtained from three independent experiments and the means of the two samples were compared using the Exosomes as described above were scanned by transmission electron microscopy, the exosomes were membrane vesicles less than 200 nm in diameter and resembling a teacup tray-like shape Fig. A, B. NanTo identify whether exosomes of AML cell line could be translocated into the MDS cell line, the exosomes labeled with PKH26 were cultured with the MDS cell line. The result showed that the MDS cell line can take up the PKH26-labelled exosomes after two days of incubation  and that K562-exo made SKM-1 cells to stop in the G0/G1 phase (p < 0.001)  and K562-exo resulted in MUTZ-1 cells being blocked in the S phase (p < 0.001)  and K562-exo (p < 0.001) co-culturing increased SKM-1 cell apoptosis   Fig. A. In conp < 0.05, p < 0.001)  and MUTZ-1 cells (P < 0.01) and suppressed the expression of the proliferation marker CD117 (P < 0.001) in SKM-1 cells compared to the control, which was consistent with the results of the CCK-8 assay. Similarly, CD117, a cell proliferation marker, was not detected in MUTZ-1 cells  after 48 h of co-culture  , while the rest of the cytokines did not differ significantly or even showed a decreasing trend  Fig. A, which One-third of MDS patients will transform into AML, and 24% of MDS patients have been reported to die as a result of leukemic transformation , HoweverWe first extracted AML cell line-derived exosomes by ultrafiltration and then characterized them by transmission electron microscopy, nanoparticle tracking analysis, protein blotting, and flow cytometry. Next, AML cell line-derived exosomes were co-cultured with MDS cell lines, and exosomes were also labeled with PKH26 to demonstrate that the exosomes could be taken up by MDS cell lines and function as intercellular communication. After co-culturing for 48 h and detection of cell proliferation by CCK-8, we found that exosomes of AML cell line origin inhibited the growth of MDS cell lines, which we further verified with CD117, a protein closely related to proliferation, also known as c-kit , THP-1-eTo further investigate how AML cell line-derived exosomes cause apoptosis in MDS cell lines, we again examined the alteration of seven cytokines and reactive oxygen species after 48 h co-culturing. We found that co-culturing resulted in more secretion of TNF-α and ROS from MDS cell lines. TNF-α is a key ligand in the death receptor signal pathway and ROS is an important signaling molecule in the mitochondrial pathway, both of which activate the Caspase protease family in a cascade reaction that ultimately activates Caspase3 protein to cause apoptosis . We alsoMesenchymal stem cells (MSC) are multipotent stem cells with the ability of self-renewal and multi-directional differentiation, which are widely distributed in many tissues of the human body, such as bone marrow, umbilical cord, and fat . Some stIn conclusion, our study shows that exosomes derived from AML cell lines can inhibit the proliferation of MDS cell lines, block cell cycle progression, promote apoptosis and differentiation, and lead to increased secretion of TNF-α and ROS in MDS cells. The data from this study suggests that AML-derived exosomes can affect the apoptosis of MDS cell lines via the TNF-α/ROS-Caspase3 pathway, thus affecting the transformation of MDS to AML Fig. . We also


Altered MM cytogenetics did not affect dinaciclib response ex vivo, alone or in combined treatment, suggesting that these combinations could be a suitable therapeutic option for patients bearing cytogenetic alterations and poor prognosis. This work also opens the possibility to explore cyclin‐dependent kinase 9 inhibition as a targeted therapy in MM patients overexpressing or with high dependence on MCL‐1.A better understanding of multiple myeloma (MM) biology has led to the development of novel therapies. However, MM is still an incurable disease and new pharmacological strategies are needed. Dinaciclib, a multiple cyclin‐dependent kinase (CDK) inhibitor, which inhibits CDK1, 2, 5 and 9, displays significant antimyeloma activity as found in phase II clinical trials. In this study, we have explored the mechanism of dinaciclib‐induced death and evaluated its enhancement by different BH3 mimetics in MM cell lines as well as in plasma cells from MM patients. Our results indicate a synergistic effect of dinaciclib‐based combinations with B‐cell lymphoma 2 or B‐cell lymphoma extra‐large inhibitors, especially in MM cell lines with partial dependence on myeloid cell leukemia sequence 1 (MCL‐1). Simultaneous treatment with dinaciclib and BH3 mimetics ABT‐199 or A‐1155463 additionally showed a synergistic effect in plasma cells from MM patients,  L notably reduce cell resistance to death.Multiple myeloma (MM) remains an incurable disease. As a general rule, MM cells overexpress or are highly dependent on MCL‐1 to survive. Therefore, dinaciclib (a multiple CDK inhibitor) decreases MM cells viability due to CDK9 inhibition and subsequent MCL‐1 downregulation. Rational combinations with corresponding BH3 mimetics in cases of codependence on BCL‐2 or BCL‐X MM is characterized by the appearance of multiple malignant plasma cell foci in the bone marrow (BM) causing bone destruction, thrombocytopenia and neutropenia . VirtualCyclin‐dependent kinases (CDKs) are serine/threonine kinases that regulate progression through cell cycle, by means of complexes with specific cell cycle regulatory proteins called cyclins . Aside f50) in the low nanomolar range . These proteins sequester proapoptotic members of the same family inhibiting mitochondrial outer membrane permeabilization (MOMP)  for 30 min on ice. Solubilized proteins were resolved by SDS–15% PAGE and transferred to nitrocellulose membranes. Afterward, membranes were blocked in TBS‐T  containing 5% skimmed milk and incubated with primary antibodies  diluted in TBS‐T containing 5% bovine serum albumin and 0.05% NaN3. Primary antibodies anti‐MCL‐1 (sc‐12756 and sc‐819) and anti‐BAK (sc‐832) were provided by Santa Cruz Biotechnology, Inc. . Primary anti‐BAX antibody (#2772) and anti‐BCL‐XL  were supplied by Cell Signaling Technology . Finally, membranes were washed with TBS‐T and incubated with the corresponding peroxidase‐labeled secondary antibodies (Sigma‐Aldrich) in TBS‐T 2.5% skimmed milk and revealed by Amersham™ Imager 600 . Control of protein loading was achieved by reprobing with anti‐α‐tubulin mAb . Band quantification was performed using imagej software .Changes in the amount of cytosolic or mitochondrial proteins were determined by western blot of cell extracts. After treatment with the corresponding drugs (3 × 102.5−/− and Bak−/−) were selected by culture in media containing puromycin (2–3 μg·mL−1) followed by limiting dilution. As a control, MM.1S cells were infected and puromycin selected with lentiviral particles containing empty LentiCRISPR v2 vector (MM.1S Ø). Accordingly, BAX−/−, BAK−/− and DKO were confirmed by western blot.MM.1S cell line was serially spin infected  with le2.64 cells (U266 and MM.1S) were treated in a flat bottom, 96‐well (200 μL per well) plate with dinaciclib in combination with BH3 mimetics  in complete medium mixed with GrowDex™ matrix 0.25%. After 24 h of incubation, supernatant was discarded and 20 μL per well of GrowDase™ , mixed with complete RPMI 1640 medium, was added. GrowDase™ was then incubated for 3 h at 37 °C. Finally, samples were filtered through Corning® cell strainers (40 μm pore size), cells stained with annexin V‐FITC/PI and analyzed by flow cytometry.U266 and MM.1S were cultured in 3D system by using GrowDex™  as a 3D matrix. For that purpose, 6 × 102.7m pH 7.4, NaCl 140 mm, CaCl2 2.5 mm and 0.5 μL of annexin V‐FITC per condition. Recombinant annexin V was purified and conjugated with FITC in our laboratory following Logue et al. procedure . In some cases, the Chou‐Talalay method [compusyn software, supplied by ComboSyn, Inc., Paramus, NJ, USA) to determine possible synergies. Additionally, receiver operating characteristics (ROC) test was used for validating cut‐off values between patients' samples cohorts.Statistical analysis was performed by using  authors , 40, 41,y method  was also33.1m) for 24 h. Analysis of cell proliferation showed that U266 and MM.1S cells were more resistant to dinaciclib than NCI‐H929, OPM‐2 and RPMI 8226   Table . Dinacic0) Table . After 40) Table .Mitochondria have a key role in cell death and survival. BAX and BAK are the main proapoptotic proteins of the BCL‐2 family, which turn from harmless monomers into deadly oligomers causing MOMP . As repoL , S63845 (a MCL‐1 inhibitor) or A‐1155463 (a BCL‐XL inhibitor). We adjusted single drug concentration for each line so that the amount of cell death was not greater than 30%. The difference between OBS and EXP specific apoptosis for each combination was calculated as indicated in Section L and indirect inhibition of MCL‐1 could overcome double combination resistance in this cell line.We next tried to improve the We also analyzed the effect of dinaciclib combinations with BH3 mimetics in 3D culture models generated with the cell lines more resistant to dinaciclib (U266 and MM.1S). Dinaciclib combinations with BH3 mimetics were quite efficient in promoting cell death in these 3D models, as illustrated in Fig. 3.3m palbociclib and 72 h incubation were needed to induce death in MM.1S and NCI‐H929 cells. Furthermore, palbociclib‐treated cells seemed to acquire a senescent‐like phenotype with bigger size and internal microvesiculation . As illustrated in Fig. + cells with 12 nm dinaciclib. This concentration was close to the LD50 of the majority of the MM cell lines tested (Table m) dinaciclib concentration than samples from SMM patients; however, the differences among them were not statistically significant. At a higher concentration (12 nm), samples from MGUS patients were significantly less sensitive than those of SMM, MM or PCL  and Din‐S those whose death rates were upper than the first quartile. Both cut‐off values were validated by ROC analysis  and dinaciclib‐resistant (Din‐R) according to their is Table . As illuex vivo sensitivity to dinaciclib. No significant differences were found between Din‐S and Din‐R subgroups regarding the previous lines of treatment , t, t, t, t, gain/amplification 1q (+1q), TP53 alterations and IGH‐FGFR3/IGH‐MAF gene fusions. In this regard, no significant differences were found between both groups, suggesting that sensitivity to dinaciclib is independent of the presence of these recurrent MM cytogenetic alterations ; second, samples showing moderate apoptotic rates with each single drug that were potentiated by drug combinations, and, finally, insensitive cells to combinations of dinaciclib with BH3 mimetics . The difference between OBS and EXP specific apoptosis was just calculated for samples classified in the second group. As shown in Fig. ex vivo, significantly increased when combined with ABT‐199 or A‐1155463. We could observe clear ex vivo synergy in 13/36 (36.1%) samples for ABT‐199 or A‐1155463 dinaciclib‐based combinations, and in 6/35 (17.1%) samples for S63845 combinations.Since combination of dinaciclib and BH3 mimetics showed synergism in MM cell lines, we next analyzed whether this was also the case in plasma cells from myeloma patients. As illustrated in Fig. ex vivo could correlate with the response to combinations. As in the previous analysis, we only considered samples that were not sensitive to single drugs but displayed a positive synergy to combinations between dinaciclib and BH3 mimetics. As illustrated in Fig. We would also like to analyze whether the resistance or sensitivity to dinaciclib treatment As previously mentioned, MM samples were classified into two groups attending to their cytogenetic status. Again, for this analysis, we only considered samples that did not show high sensitivity to single drugs. In this sense, no statistically significant differences between patients with FISH negative or altered cytogenetics were found in the response to combinations of dinaciclib with BH3 mimetics Fig. . Interes4Throughout the last decades, a great improvement of the myeloma therapeutic toolkit has occurred. The introduction of targeted drugs, such as proteasome inhibitors and cell therapies resulted in an increase of the median overall survival . HoweverL and increases apoptosis rates in some MM cell lines and plasma cells from MM patients, ex vivo. This has been previously found with other CDK inhibitors and BH3 mimetics. In particular, Zhou et al. [L in soft‐tissue sarcomas. The combination of dinaciclib with ABT‐199 has also been proposed against acute lymphocytic leukemia [Interestingly, we observed that dinaciclib synergizes with BH3 mimetics binding to BCL‐2 or BCL‐Xu et al.  found syu et al.  found a leukemia .ex vivo inhibition of MCL‐1 [L was involved in observed synergy [It is well‐known that CDK9 plays a main role in the control of basal gene transcription, thus assuring cell transcriptional homeostasis . Previouof MCL‐1  and MCL‐of MCL‐1 . When coof MCL‐1 , 27, 44. synergy . The maiMoreover, Din‐R group of plasma cells from MM patients exhibited low sensitivity to S63845, according to the proposed link between dependence on MCL‐1 and dinaciclib sensitivity. Additionally, partial dependence on BCL‐2 could also be related to the sensitivity to dinaciclib, as we found in our samples from patients. Within this context, we also observed that KMS‐12‐BM cell line, considered to be highly dependent on BCL‐2 , was sende novo patients and 70% of relapsed/refractory patients [m than those of SMM, MM or PCL. Our work is the first to evaluate the sensitivity to MCL‐1 indirect inhibitor depending on disease stage of MM and so it is not possible to compare with previous works. In particular, it has been reported that in MGUS the prevalence of +1q is lower than in SMM or MM patients, as determined by FISH and next‐generation sequencing techniques [Multiple myeloma genome instability not only facilitates clonal proliferation and spread of disease, but also creates cell vulnerabilities which can be used to develop new therapeutic strategies. In this study, we have evaluated if cytogenetic alterations were associated to dinaciclib response in MM cells. In this sense, we could not observe any significant correlation between genetic aberrations and response in MM cells, either in cell lines or in plasma cells from patients. However, plasma cells displaying +1q were highly sensitive to dinaciclib, alone or in combination with BH3 mimetics. 1q21 amplification is one of the most common cytogenetic alterations occurring in around 40% of patients . The prepatients , and it chniques , 72, 73.5ex vivo analysis of particular characteristics of malignant plasma cells. In this sense, dynamic BH3 profiling (DBP) has emerged as a useful tool to identify cellular dependence on individual antiapoptotic proteins with a simple FACS analysis or a microfluidic‐based DBP device [L respectively. In this regard, reversible thrombocytopenia caused by A‐1155463 [L inhibitors [The future of MM treatment relies on precision medicine, with decision making based on the P device , 75. As P device . This obP device . Combina‐1155463  may be phibitors . To achihibitors , 78, 79.hibitors  which wiThe authors declare no conflict of interest.MB‐V, JN and IM designed the study; MB‐V, NJ‐A, MC, AS‐DV and AB performed sample analysis; MB‐V, VP, AA, JN and IM interpreted and analyzed data; RD, GA and LP collected samples, reviewed patient records and collected data; MB‐V and IM wrote the manuscript. All authors had full access to all data, carefully reviewed the manuscript and approved the final version.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/1878‐0261.13522.The peer review history for this article is available at Fig. S1. Validation and functional characterization of engineered MM cell lines.Click here for additional data file.Fig. S2. Analysis of cell death induced by combinations of dinaciclib and BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S3. OBS and EXP specific apoptosis induced by dinaciclib‐based combinations with BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S4. Synergy validation between dinaciclib and BH3 mimetics in U266 and MM.1S cell lines by CI calculation through the Chou‐Talalay formula.Click here for additional data file.Fig. S5. OBS–EXP specific apoptosis for triple combinations of dinaciclib, ABT‐199 and A‐1155463 in OPM‐2 and RPMI 8226 cells.Click here for additional data file.Fig. S6. Cell death induced by dinaciclib and BH3 mimetics combinations in MM cell lines partially dependent on MCL‐1 in 3D culture models.Click here for additional data file.Fig. S7. Cell death induced by combinations of NVP‐2 (CDK9 inhibitor) and BH3 mimetics in MM cell lines.Click here for additional data file.Fig. S8. Effect of CDK4/6 inhibition on MM cell lines.Click here for additional data file.Fig. S9. Cell death induced by dinaciclib‐based combinations with BH3 mimetics in Din‐S and Din‐R subgroups of patients' samples.Click here for additional data file.Table S1.In vitro dinaciclib lethal dose (LD50) in established multiple myeloma (MM) cell lines used in this study.Click here for additional data file.Table S2. Validation of dinaciclib‐resistant (Din‐R) and dinaciclib‐sensitive (Din‐S) cut‐off values: ROC analysis.Click here for additional data file.Table S3. Summary of samples with high‐risk cytogenetic alterations used in this work. Dinaciclib resistance (Din‐R) or sensitivity (Din‐S) of each sample is also indicated.Click here for additional data file.


In 786-O/S cells, the high level of phosphorylation of MET and AXL was not affected by cabozantinib. Despite cabozantinib hampering the high constitutive phosphorylation of MET, the Caki-2 cells showed low sensitivity to cabozantinib, and this was independent of sunitinib pretreatment. In both sunitinib-resistant cell lines, cabozantinib increased Src-FAK activation and impeded mTOR expression. The modulation of ERK and AKT was cell-line-specific, mirroring the heterogeneity among the patients. Overall, the MET- and AXL-driven status did not affect cell responsiveness to cabozantinib in the second-line treatment. The activation of Src-FAK might counteract cabozantinib activity and contribute to tumor survival and may be considered an early indicator of therapy response.The tyrosine kinase inhibitor (TKI) cabozantinib might impede the growth of the sunitinib-resistant cell lines by targeting MET and AXL overexpression in metastatic renal cell carcinoma (mRCC). We studied the role of MET and AXL in the response to cabozantinib, particularly following long-term administration with sunitinib. Two sunitinib-resistant cell lines, 786-O/S and Caki-2/S, and the matching 786-O/WT and Caki-2/WT cells were exposed to cabozantinib. The drug response was cell-line-specific. The 786-O/S cells were less growth-inhibited by cabozantinib than 786-O/WT cells ( Renal cell carcinoma (RCC) is the seventh most common form of neoplasm in developed countries, accounting for approximately 3% of all diagnosed cancers worldwide ,2, with VHL) gene; chromosome remodeling by PBRM1, BAP1 and SETD2 genes; hyperactivated PI3K/AKT pathways; and mTOR alterations are the main molecular features [VHL elevates the stability of the hypoxia-inducible factor (HIF) transcription factor, which, in turn, tunes the expression of hypoxia-responsive genes, vascular endothelial growth factor (VEGF), platelet-derived growth factor beta (PDGF-β) and transforming growth factor (TGF-β) associated with both aberrant angiogenesis and tumor growth [VHL, along with hypoxia, can promote MET expression, which has also been associated with worse outcomes in RCC [At present, therapeutic approaches used to treat patients with metastatic RCC (mRCC) are based on specific molecular characteristics that play an essential role in disease development . In cleafeatures ,8,9,10. r growth ,12,13,14r growth ,16. Thisr growth ,18. Systr growth , which rr growth . Althougr growth ,22 and ir growth  for RCC r growth ,24. Hower growth  as a trar growth  that cour growth ,28,29 rar growth . In thiss in RCC ,31,32,33s in RCC , as wells in RCC . Whereass in RCC ,37, litts in RCC ,38. To ds in RCC  and whicIn the present study, we investigated the role and possible mechanism of MET and AXL activation in response to cabozantinib, particularly following the long-term administration of sunitinib to patients. Preclinical testing seldom considers second treatment with a TKI in tumor cell models that have experienced long-term exposure to first-line TKI, closely resembling the clinical setting. Therefore, we used two age-matched cell lines, i.e., the treatment-naïve (wild type) and corresponding sunitinib-treated cell lines, which were challenged with a single dose of sunitinib (3 µM) over four months (long-term treatment). We analyzed the effects of cabozantinib both alone and in sequence on cell survival and on the modulation of signaling transduction proteins that are responsible for cell proliferation, survival, and migration. This provides a better understanding of the molecular pathways related to drug responses that can be integrated with the data obtained from large clinical trials to improve the selection of patients for TKI-based sequence treatment.50) reached 80 and 95%, respectively, resulting in values significantly higher than those for the corresponding age-matched untreated 786-O/WT and Caki-2/WT cell lines . The drug reduced the cell viability of the 786-O/S cells to 50% (IC50) following exposure to 13 µM (±0.4), which is a greater reduction that that observed in 786-O/WT cells with an IC50 value of 10 µM (±0.6)   using a positive control that showed the maximum response to cabozantinib (786-O). We considered this value as a constant to constrain the baseline of the dose–response curve and determined the point on the curve where a 50% inhibition was observed. The absolute IC50 values were 14.5 µM (±1.50) and 13.6 µM (±1.05) for the Caki-2/WT and Caki-2/S cell lines, respectively   a. We als = 0.03) b. Therefki-2/WT) b. Overal50 values of cabozantinib in all cell lines, allowing us to treat the cells with the same drug concentration. Overall, the sunitinib-resistant 786-O/S cell lines displayed a four- and two-fold higher constitutive level of P-MET and P-AXL, respectively, as well as an enhanced phosphorylation rate (phosphorylate/pan forms) compared with the treatment-naïve 786-O/WT cell lines , in addition to inducing AXL phosphorylation without changing its total protein level. In contrast to the 786-O/WT cell line, the high constitutive phosphorylation level of MET and AXL was not induced further by cabozantinib treatment in the 786-O/S cells at the same time points b. ChangeFor the Caki-2 cell lines , the dosCabozantinib strongly deactivated the receptor after 4 h and throughout all the following time points of treatment in both cell lines. However, the molecular response to the drug did not correspond to the functional response (cell viability). Similar to the 786-O/S cell line, high expression and activation of the AXL receptor were related to sunitinib resistance occurring in Caki-2/S cell lines. Cabozantinib dephosphorylated P-AXL exclusively in sunitinib-resistant cells. Moreover, we detected a decrease in the total forms of MET and AXL in the sunitinib-resistant Caki-2/S cells, suggesting that cabozantinib inhibits the total expression of the two RTKs and not exclusively their activity. Based on these data, we inferred that only the modulation of P-AXL activity depended on the sunitinib > cabozantinib sequencing treatment. Following long-term sunitinib treatment, the specific signaling proteins P-FAK and BCL-2 presented seven- and five-fold higher protein levels, respectively, in the Caki-2/S cell line in comparison with the treatment-naïve Caki-2/WT cell line . The treThe aberrant activation of receptor tyrosine kinases MET and AXL, or MET amplification, is a major cause of the poor prognosis of RCC, as they are potent activators of cell proliferation and survival pathways and the development of bone metastases; most importantly, their activity helps cancer cells to bypass the growth-inhibitory effect of VEGFR-TKI sunitinib ,43,44,45VHL gene mutation was reported for both cell lines (https://www.depmap.org (release 22Q4 (accessed on 7 December 2022)) [The human RCC cell lines used in this study, 786-O/WT and Caki-2/WT, displayed a heterogeneous response to cabozantinib, with Caki-2 found to be less responsive to treatment. This result may have been caused by the different biological backgrounds of these cells, although a r 2022)) ,54. Cakir 2022)) ,56, whicr 2022)) . Based or 2022)) ,38,57, wr 2022))  as a resr 2022)) , as wellr 2022)) ,61. In tr 2022)) . These or 2022)) ,63 and rr 2022)) ,65. Recer 2022)) ,67, reprr 2022)) . Our datr 2022))  describir 2022)) ,71,72. Tr 2022)) , as wellr 2022)) . A recenr 2022)) . In addir 2022))  indicateOverall, the data presented in this study indicate that the high expression of the main target of cabozantinib, MET, does not reflect the drug-responsiveness property, as discussed in other studies. Considering that a viability assay, such as WST-1, evaluates the cell proliferation rate, cell size, metabolic rate and survival, it cannot be directly compared with the protein expression data. Nonetheless, we can argue that the cell-line-specific response to cabozantinib might be the result of the off-target effects of the drug. As a general observation, the variability occurring in cellular responses to cabozantinib between 786-O and Caki-2 cell lines suggests the preferential engagement of a specific downstream pathway. For instance, cabozantinib activated ERK and completely hampered AKT signaling at the T308 and S473 sites only in the Caki-2 cell line, and these events were independent of pretreatment with sunitinib. On the contrary, the cabozantinib treatment schedule  of the 786-O cell line may affect the AKT signaling process, which is one of the most important pathways involved in cell proliferation, survival and transformation occurring in RCC tumorigenesis ,76, as wOur results shed light on the in vitro effects of therapy sequencing for mRCCs. In summary, the upregulation of P-MET and P-AXL observed in our cell models did not suffice to sensitize cells to cabozantinib in the second-line treatment. The relationship between the cytotoxicity of cabozantinib, MET and AXL activities might depend on sunitinib pretreatment, drug incubation time and the cell phenotype. The data presented here also suggest a cell-line-dependent mode of action of cabozantinib, reflecting the heterogeneity among patients. Receptors MET and AXL might be activated by cabozantinib as a feedback mechanism only in 786-O. However, the sustained activation of the two receptors was preserved following prolonged drug exposure only in the sunitinib-resistant 786-O/S cell lines. On the contrary, the strong P-MET inhibition caused by cabozantinib was not affected by sunitinib pretreatment in the Caki-2 cell line, whereas P-AXL downregulation was affected by treatment with the sunitinib > cabozantinib sequence. In general, sunitinib pretreatment of both 786-O/S and Caki-2/S cells resulted in Src and FAK overexpression, ensuring a low sensitivity value for the second-line treatment.Sunitinib malate was purchased from Tocris Bioscience , and cabozantinib was purchased from TargetMol Chemicals Inc. . The compounds were dissolved in DMSO, stored as aliquots at −80 °C and thawed shortly before use. Sunitinib malate was used to prepare cell stocks of the sunitinib-resistant cell lines prior to the investigation of sequential TKI treatment.TP53 and PTEN and wild-type PBRM1) ) were cultivated in a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and RPMI-1640 , whereas the Caki-2 cells (mutant PBRM1 and wild-type PTEN and TP53)  were propagated in RPMI-1640, both containing 10% heat-inactivated fetal bovine serum (FBS). The cell growth conditions were set at 37 °C in a humid environment with 5% CO2. Exponentially growing cells were used for all experiments.For the current study, human clear cell RCC 786-O and Caki-2 cell lines were purchased from the American Type Culture Collection (ATCC) . These cell lines are both representative of subtype ccRCCs bearing a VHL mutation (the main feature of ccRCCs). The differences in the genetic backgrounds between the two cell lines were also reported, which helped us to include the patient heterogeneity effect in our study. The 786-O cell lines (mutant 50 (inhibitor concentration that causes 50% cell death) value of sunitinib was determined by a dose–response assay performed over 72 h and measured by a WST-1 cell viability test, as described below. To generate sunitinib-resistant cells lines, 786-O and Caki-2 cell lines were exposed to the selective pressure of sunitinib at their respective IC50 values (3 µM) for approximately four months and defined as 786-O/S and Caki-2/S cells, respectively. During the development stage, age-matched untreated wild-type (WT) 786-O/WT and Caki-2/WT cells were also subcultivated. The sunitinib tolerance of 786-O/S and Caki-2/S was routinely assessed and compared with their WT counterparts with a WST-1 test. In addition, cell stocks collected from both cell phenotypes were regularly cryopreserved. A stable high-sunitinib tolerance was achieved within 34 passages of the cell culture. The cell stocks corresponding to this passage were thawed for the experiments conducted in this study. Prior to performing sequential treatment with cabozantinib, the sunitinib-resistant cells were cultivated as a monolayer in medium without sunitinib for five days, along with the corresponding age-matched wild-type cell lines.To assess the activity of cabozantinib in the second-line setting following failed sunitinib treatment, we referred to a panel of sunitinib-resistant cell lines previously generated at our institution (data not published). Briefly, the IC3 or 2 × 103/well, respectively, and allowed to adhere overnight at 37 °C. The cells were exposed to either DMSO  or increasing concentrations of cabozantinib (0.1–25 µM) for 72 h. Subsequently, a 10 µL/well of WST-1 solution  was added, and the cells were incubated further at 37 °C for 60 min. The optical density was measured at 450 nm and a reference of 620 nm using a microplate reader .A WST-1 assay was performed to evaluate cell survival rates in response to cabozantinib exposure. Exponentially growing cells were used for the experiments, as well as age-matched untreated wild-type and sunitinib-resistant cells. Briefly, the untreated wild-type 786-O/WT and Caki-2/WT cell lines and their corresponding sunitinib-resistant cells, 786-O/S and Caki-2/S, were seeded in a 96-well plate at a density of 1 × 105 cells/mL in a 60 mm Petri dish and allowed to adhere overnight. On the following day, the cells were left untreated  or were exposed to 12 µM cabozantinib. This concentration roughly corresponds to the average IC50 value of cabozantinib in all cell lines that were analyzed, allowing us to treat the cells with the same drug concentrations. A previously conducted preclinical study showed a rapid inhibitory effect of cabozantinib on receptor MET phosphorylation within 8 h [TM BCA protein assay kit . Subsequently, 20 µg of cell lysates was separated by SDS-PAGE  and transferred onto a methanol-activated polyvinylidene difluoride (PVDF) membrane  with a tank transfer system . The membranes were blocked in 5% blocking solution for 1 h at room temperature (RT). They were incubated further overnight with primary antibodies against PAN-MET, phospho-METY1234/5, PAN-AXL, phospho-AXLY779, phospho-ERK1/2T202/Y204, PAN-AKT, phosphor-AktT308, phosho-AktS473, phosphoS6S235/6, phosho-FAKY397 , phosphor-Src family, GAPDH  and BCL-2  under agitation at 4 °C overnight. The membranes were then washed three times in Tris-buffered saline and Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  for one hour at room temperature (RT). Antibody detection was performed using an ECL-plus HRP substrate kit . We performed a stripping and reprobing membrane protocol to visualize different proteins on the same membrane. Briefly, the membranes were agitated for 30 min in 25 mM glycine-hydrochloric acid (HCL), 1% sodium dodecyl sulfate (SDS) and pH 2 buffer at 50 °C. Subsequently, the blots were washed in 1X phosphate-buffered saline (PBS), equilibrated in Tris-buffered saline and 0.1% Tween 20® detergent (TBST) and incubated with 5% blocking solution prior to reprobing with another antibody.For analysis of receptor tyrosine kinases and downstream intracellular signaling, the wild-type and age-matched sunitinib-resistant cells were seeded at a density of 2 × 10thin 8 h . Therefo50 values were analyzed with a nonlinear regression model using GraphPad Prism version 9.2.0 for Windows, GraphPad Software , and the drc packagev3.0-1 [50. We calculated the concentration that provoked a response halfway between the blank  and a positive control (cells with a full inhibited response) using Graph Pad Prism® software. Analysis of variance (ANOVA) and Student’s t-test were applied for comparative analysis with IBM® SPSS® Statistics for Windows (Version 27.0). p-values of ≤0.05 were considered statistically significant. The data obtained from all the experiments are presented as mean ± standard error (SE) of biological replicates, each derived from technical replicates. Graphs were generated with GraphPad Prism or in Excel. Composite images for publication were generated with Inkscape . ImageJ 1.52a image analysis software  [Dose–response assays and ICgev3.0-1 . If the er 2022)  was usedOur in vitro study suggests that cabozantinib can efficiently primarily target P-MET and, to a limited extent, P-AXL in RCC. Despite a dose-dependent reduction in cellular viability following cabozantinib treatment, inferior drug sensitivity was observed in Caki-2/WT cells and sunitinib-resistant Caki-2/S and 786-O/S cell lines. Cabozantinib can induce intracellular signals involved in cell proliferation and survival processes and can also be considered an early indicator of response to therapy. The effect of cabozantinib on MET and MET-dependent signaling was highly variable between the two cell lines, highlighting the often-observed heterogeneity of patient sensitivity and responses to targeted therapy, which might complicate treatment standardization for all patients. Based on our analysis, we determined that the sequence of sunitinib followed by cabozantinib is not superior to first-line therapy with cabozantinib. In the second-line treatment, the MET- and AXL-driven status did not improve cell responsiveness to cabozantinib.


Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications. Despite considerable efforts, we still have a very limited understanding of how the brain works when it is healthy or sick. Non-invasive imaging methods performed in humans lack the spatial and temporal resolution to probe its microscopic anatomy and function. Thus, a simplified and accessible model of the human brain is urgently needed.Although animal models have provided a significant boost to neuroscience research and are still widely used, their limits have been extensively demonstrated. In fact, besides the ethical issues, studies have shown that the results of animal experiments often fail to translate into human clinical trials . MoreoveHuman neural cells cultured in highly controllable and monitorable environments have been widely used for investigating neurotoxicity, neuroprotection, drug screening and therapeutic assessment for both brain tumors and neurodegenerative diseases, e.g., Parkinson’s and Alzheimer’s ,8,9. PosSince soma are unrealistically flattened and neuronal axonal and dendritic outgrowth cannot occur in all directions in traditional monolayers , advanceThree-dimensional constructs have also been generated from neural cell lines and may provide a strategy for developing standardized models with better physiological relevance compared with traditional 2D  cultures. In this context, after an overview of the neural cell lines commonly used in the literature, we describe recent approaches exploiting cell lines for generating 3D models of brain tissue. Given the legislative and public urge to reduce the use of animals in scientific experiments, we suggest their re-evaluation in humane and human-relevant research, particularly for regulatory applications where standardized and reproducible inter-laboratory outcomes are crucial.To identify articles dealing with three-dimensional in vitro human-relevant models of brain tissue involving cell lines, we first conducted an analysis of the existing scientific literature. Web of Science was used with the query: (((neur* OR brain OR cereb*) AND model*) AND (neurosphere* OR 3d OR three-dimension*) AND human AND “cell* line*”). Only studies published from 2000 to 2022 were selected, thus identifying 304 original articles and 37 review papers.Oncology (18%) and science technology (18%) are the research areas where cellular models of human brain tissue are mainly involved . ScientiAfter this general inspection, a more precise analysis was carried out. Each abstract was read and assessed for inclusion according to whether its focus was on 3D models of brain tissue or neural tissue generated from immortalized human cell lines. Thus, a consistent number of papers was discarded because they focused on non-human models , in vivo models, models developed with induced pluripotent stem cells and primary cells, or papers describing models of tissues different from the brain . Thus, a total of 96 papers  was selected.Most of the cell lines used in 3D in vitro models in neuroscience research derive from tumor tissues, in particular glioblastoma and neuroblastoma. In the following paragraphs, we describe the studies identified with the literature search in which 3D in vitro models of brain tissue have been developed using cell lines derived from tumors and from healthy tissues.The cancer cell lines most exploited in cellular neuroscience research derive from glioblastoma  and neuroblastoma (35%). However, 3D models of brain tissue have also been generated using cells from embryonal carcinoma and medulloblastoma.Glioblastoma cell lines are used in 3D in vitro constructs for modeling gliomas, in particular glioblastoma multiforme (GBM), the most common malignant brain tumor with the poorest prognosis and survival ,27. MostAs detailed in Neuroblastoma is the most common extracranial solid tumor observed in childhood (less than five years old) ,45, origAs regards the applications, even for neuroblastoma cell lines, most of the efforts have been directed towards characterizing the effects of the microenvironment on cell behavior, e.g., in terms of proliferation, cell invasiveness and differentiation. However, it is interesting to note that SH-SY5Y cells have been successfully exploited for neurodegenerative studies, and in particular Parkinson’s and Alzheimer’s diseases, accounting for 14% of the articles. Indeed, they are usually chosen for their catecholaminergic (though not strictly dopaminergic) neuronal properties , which tOnce differentiated, SH-SY5Y cells show the formation of neural processes and functional synapses, as well as the production of neuron-specific enzymes, neurotransmitters and neurotransmitter receptors ,75,76. IIn addition to glioblastoma and neuroblastoma cell lines, 3D human brain models have been generated using cells originating from embryonal carcinoma and medulloblastoma. Human pluripotent embryonal carcinoma NTera2 (NT2) cells are widely used for in vitro neurotoxicity studies thanks to their ability to differentiate into post-mitotic neurons after treatment with RA ,79. NT2-The UW228-3 cell line was established from human posterior fossa medulloblastoma . This liAlthough the majority of cell lines identified by the literature search had tumor origins, cell lines derived from healthy tissue can be used in 3D in vitro models of the human brain. Among these, the Lund human mesencephalic (LUHMES) line, which originates from the mesencephalon of a human subject, was established in 1998. The LHUMES neuron-like immortalized cells have been extensively characterized as a robust neuronal model suitable for neurodevelopmental studies, neurotoxicity and the modeling of brain diseases ,87. IndeNeural Progenitor Cells (NPCs) have a self-renewal capability and can give rise to healthy neural cell lineages. They can be obtained from iPSCs or derived directly from brain tissues ,89. In tOver the last few decades, numerous studies have highlighted the superiority of 3D cultures with respect to monolayers as they are able to better recapitulate the morphology and architecture of tissues and cells in their native environment, both in physiological and diseases conditions. 3D systems based on human neural cell lines exhibit specific cellular and molecular features which occur in vivo ,97 and sSuch 3D models can be realized using both scaffold-less and scaffold-based strategies ,84. The Several examples of alginate-based spheroids laden with the cell lines described in this review can be found in the literature, in combination with different cell adhesive materials such as gelatin and collagen ,64,70,93Although the results obtained with the generation of spheroids are promising, more systematic studies are still needed to exploit their versatility. For instance, 3D constructs may differ significantly in terms of mechanical and transport properties. This is mainly due to the high variability of the biomaterials used for fabricating them. Matrigel- and ECM-derived matrices are known to suffer from ‘batch syndrome’; on the other hand, alginate is more reproducible, but it is available in different molecular weights, and the protocols for obtaining spheroids involve different concentrations and crosslinking methods. Moreover, as alginate is a marine-plant-derived material, it lacks cell binding sites such as RGD motifs. For this reason, some attempts to combine alginate with cell adhesive materials, e.g., gelatin , can be The applications of the 3D in vitro models described in The development of 3D in vitro models mimicking the in vivo cell environment and behavior is also essential to obtain relevant results in toxicity testing ,110. A nA wide class of pathologies is included under the definition of neurodegenerative diseases, all of them characterized by the degeneration and death of neural cells ,112. TheThe ability to produce in vitro models with neural cells has been fundamental to advancing the understanding of the central nervous system’s (CNS) function at the microscale, as well as of the disease mechanisms underlying neuropathologies and neurotoxicity ,113. We From our literature search, the first thing that stands out is the heterogeneity of the studies reported and the fact that many of them do not carry on a systematic characterization of the cell lines or materials involved. Long-term culture often leads to the accumulation of mutations in such cells, resulting in outcomes which are difficult to reproduce in laboratories . We alsoThe regulatory testing of chemical substances to define their limits of safety for humans and the environment using in vitro methods requires a high degree of inter-lab reproducibility and throughput, with tightly defined experimental protocols. This is necessary for compliance with the Guidance on Good Cell Culture Practice (GCCP) standards . Risk anWe suggest that the 3D culture of neural cell lines should be improved to promote their use in safety and toxicity testing of chemicals at a regulatory level, leveraging the versatility of cells such as SH-SY5Y to exploit their full potential. Further investigations should be performed towards the standardization of protocols and towards the identification of supplements able to generate different classes of neurons in a controlled manner. Alternatively, since glial cells modulate neuron function and signaling, while neurons generate and propagate electrical and chemical signals , neurobl


Studies about radiation-induced human cataractogenesis are generally limited by (1) the poor number of epithelial lens cell lines available (likely because of the difficulties of cell sampling and amplification) and (2) the lack of reliable biomarkers of the radiation-induced aging process. We have developed a mechanistic model of the individual response to radiation based on the nucleoshuttling of the ATM protein (RIANS). Recently, in the frame of the RIANS model, we have shown that, to respond to permanent endo- and exogenous stress, the ATM protein progressively agglutinates around the nucleus attracted by overexpressed perinuclear ATM-substrate protein. As a result, perinuclear ATM crowns appear to be an interesting biomarker of aging. The radiobiological characterization of the two human epithelial lens cell lines available and the four porcine epithelial lens cell lines that we have established showed delayed RIANS. The BFSP2 protein, found specifically overexpressed around the lens cell nucleus and interacting with ATM, may be a specific ATM-substrate protein facilitating the formation of perinuclear ATM crowns in lens cells. The perinuclear ATM crowns were observed inasmuch as the number of culture passages is high. Interestingly, 2 Gy X-rays lead to the transient disappearance of the perinuclear ATM crowns. Altogether, our findings suggest a strong influence of the ATM protein in radiation-induced cataractogenesis. Only a few months after the discovery of X-rays, the first radiation-induced (RI) eye damage was observed in exposed laboratory animal models . Later, The lens is considered to be the most radiosensitive tissue among not only the ocular structures but also among all the organs ,5. HowevTo date, industrial and medical workers  rThere are two major limits in the quest for the basic mechanisms of cataractogenesis. First, the lack of availability of epithelial lens cell lines, notably human ones, reflects not only the difficulties of cell sampling to establish cell lines in humans but also the difficulties of amplifying this type of cell up to high cell culture passages to mimic aging. To date, two human epithelial cell lines are available: The untransformed HLEpiC cell line from ScienCell  and the transformed HLE-B3 cell line from ATCC . It is noteworthy that, as documented with skin fibroblasts, the transformation or immortalization of cell lines may introduce significant biases in the radiation response, and therefore, any radiobiological data from the transformed HLE-B3 cell lines may be interpreted with caution.Second, another difficulty in the cataractogenesis research area is the lack of reliable biomarkers for the RI aging process. Indeed, despite the considerable efforts provided by telomeres, telomerase activity, and residual DNA breaks, there is still no consensus about a molecular endpoint that would quantify a specific feature of aging .A model based on the RI nucleoshuttling of the ATM kinase protein (RIANS) has been proposed to quantify the risk linked to exposure to ionizing radiation ,24,25,26Two radioresistant skin fibroblast cell lines (1BR3 and MRC5) that originated from apparently healthy patients were used in this study as negative controls for radiosensitivity. These cell lines were purchased from the European collection of authenticated cell cultures  under the references, #90011801 and #05011802, respectively. Fibroblasts were routinely cultured as monolayers with Dulbecco’s Modified Eagle’s Minimum Medium (DMEM) , supplemented with 20% fetal calf serum, penicillin, and streptomycin.2+ and Mg2+, ScienCell), and then trypsinized with a trypsin/EDTA solution (ScienCell). The immortalized human lens epithelial cell line (HLE-B3) was purchased from the American Type Culture Collection  under the #CRL-11421 reference and was routinely cultured in the same conditions as indicated for human fibroblasts.The untransformed human lens epithelial cell line  and the untransformed human retinal pigment epithelial cell line  were purchased from ScienCell Research Laboratories . Note that Hamada’s group mentioned that ScienCell Research Laboratories proposed several lots of primary normal human lens epithelial cells derived from different donors  under the same name (HLEpiC) . Hence, One radioresistant porcine skin fibroblast cell line (PSF-1) that originated from a cutaneous biopsy from a butcher pig from a local slaughterhouse was used as a negative control for radiosensitivity. This biopsy was performed in the 1990s at Institut Gustave-Roussy  in agreement with the regulations of the French departments and the slaughterhouse veterinarians.2-independent serum . The eyes were cleaned for muscles and immersed for a few minutes in an antiseptic solution . The anterior segment of the bulb was removed, as well as the lens, the vitreous, and the retina. Lenses were collected in a saline solution at room temperature. Lenses were then placed on Petri dishes with their anterior side down. A small incision was made on the posterior capsule using sterile scalpels. The capsules were then opened and fixed on the Petri dishes using sterile forceps. The fibrous cellular mass was discarded, and only the capsules were kept  in agreement with the local regulatory department and the slaughterhouse veterinarians. This experimental step was performed with the assistance of T.M., and the procedure was described elsewhere . This prere kept .2+ and Mg2+), then trypsinized with TrypLE Express 1X solution (Gibco). All the experiments were performed with cells in the plateau phase of growth (95–99% in G0/G1). The cells used in the experiments were cultured at different passages (from passage 3 to passage 30).The capsules were incubated and cultured by following the same procedure as for fibroblasts, rinsed with PBS 1X (with Ca−1) [Irradiations were performed with a 6 MeV photon medical irradiator (SL 15 Philips) (dose rate: 6 Gy·min−1) .γH2AX, -pATM, and -MRE11 and secondary antibodies [BFSP2 antibody  and the polyclonal anti-rabbit anti-FYCO1 antibody  were used at 1:100. Nuclei were counterstained with 4′,6′-Diamidino-2-phenylindole  antibodies [The immunofluorescence protocol was described elsewhere, together with the references for the primary anti-tibodies ,31,32,33tibodies ,31,32,332 in which R was defined as the average nucleus radius  allows the visualization of endogenous protein–protein interactions at the single-molecule level . The PLAThe procedures of the cell extracts and immunoblots are described elsewhere ,31,32,33A two-way ANOVA was used to compare numerical values, and Spearman’s test was used to compare the kinetic data. The foci kinetic data were fitted to the so-called Bodgi’s formula . Statistp < 0.01) while it was similar to that of the control in the HRPEpiC   micronuclei and cellular radiosensitivity ,41. Twenp > 0.5) . p < 0.05) (p > 0.5). These last findings suggest that the repair rate of DSB recognized by NHEJ can be considered normal  A. As abu < 0.05) A. At 24 d normal A. pATM immunofluorescence was applied to the same cell lines with the same irradiation scenario. No spontaneous pATM foci were observed in the three cell lines tested. In the radioresistant skin fibroblasts, the number of pATM foci scored 10 min post-irradiation was 40 ± 4 per cell, in agreement with previous studies [p < 0.05), supporting a delay in the RIANS. The number of pATM foci decreased progressively to a value non-significantly different from zero at 24 h post-irradiation  [p < 0.05)  ,43. Convd cells) ,43. From < 0.05) C. p < 0.0001) (p < 0.05). At 24 h post-irradiation, the HLE-B3 cell line showed a number of γH2AX foci significantly higher than the HLEpiC cell line  (p < 0.01)  A. These  0.0001) B, sugges < 0.01) C. Howeve < 0.01) D.A first set of trials (Set 1) was carried out after adult cataract surgery. The trials consisted of obtaining lens epithelial cells from different conditions: One clear lens, one moderate cataractous lens, and one dense cataractous lens. Set 1 procedure was based on phacoemulsification, allowing the fragmentation of the lens and a UV probe to aspirate the cataract. This resulted in the destruction of some lens, and the challenge was to get a critical number of viable and clonogenic cells. However, this procedure failed to provide clones.A second set of trials (Set 2) consisted of obtaining whole lens from adult enucleation procedures. Set 2 procedure was based on the extraction of the whole lens, thus preserving the cells of interest. However, most of the donors were adults older than 50, and, again, this procedure failed to provide clones. Another critical step of the procedure was that only one layer of epithelial cells is present on the anterior side of the lens capsule, and breaking the capsule can cause the detachment of the cells from each other, therefore reducing the chances of cell proliferation.Faced with the lack of availability of human lens epithelial cells, the low proliferating ability of the HLEpiC cell line, and the biases associated with the transformed HLE-B3 cell line, we attempted to establish human lens epithelial cell lines from adult eyes, and various procedures were considered:The technical difficulty of establishing human lens epithelial cell lines prompted us to investigate the radiation response of lens cells in some animal models. There was an opportunity to work on the porcine lens, and we therefore established four cell lines to document their radiation response by applying the same experimental conditions and techniques as those described above and in Materials and Methods. In order to compare the radiobiological data from the lens, we used historical data obtained from a porcine skin fibroblast, PSF-1, established in the 1990s.p < 0.01). These results support significant RI genomic instability in the porcine lens epithelial cell lines tested (The number of micronuclei assessed 24 h after 2 Gy X-rays in the PSF-1 and in the four porcine lens (CP) cell lines was examined. The four porcine cell lines tested showed a significantly high number of micronuclei in comparison with the skin PSF-1 cell line . The number of γH2AX foci scored 10 min after 2 Gy in the radioresistant control PSF1 was 71 ± 3 per cell, while the corresponding numbers of γH2AX foci in the four porcine lens epithelial cells were significantly lower  (p > 0.5), suggesting a normal repair rate of DSB recognized by NHEJ  A, sugges by NHEJ A. Altogep < 0.05)  B. These  < 0.05) B.With regard to the MRE11 foci, the PSF-1 cells showed a maximum of 5 MRE11 foci per cell 1 h post-irradiation and 3 MRE11 foci per cell 4 h post-irradiation. Three of the four CP cell lines tested showed a curvilinear increase in the number of MRE11 foci per cell at 4 h post-irradiation  C. These Our findings showed that the lens cells tested systematically elicit delayed RIANS with fewer γH2AX and pATM foci than expected. As evoked in the Introduction, the delay of RIANS is generally due to proteins overexpressed in the cytoplasm that are substrates of ATM phosphorylation  . We, theAs a second step, we focused on the BFSP2 protein (3 SQ and 2 TQ). We first examined BFSP2 expression by immunofluorescence. BFSP2 appeared to be mainly cytoplasmic, with some nuclear forms. Interestingly, we observed some perinuclear crowns of BFSP2 staining before irradiation, disappearing up to 1 h after irradiation, and then reappearing 4 h after irradiation .We thereafter investigated the BFSP2 expression in cytoplasmic protein extracts of the fibroblast radioresistant control cell line (MRC5) and of the four porcine lens epithelial cell lines  by applying immunoblots . Cell exp < 0.001). The influence of the irradiation on the number of PLA foci reflecting the ATM–BFSP2 complexes was cell line dependent. Since the RIANS is delayed in the porcine lens epithelial cell lines and since the BFSP2 protein was found to be very abundant in the cytoplasm, we hypothesized that some BFSP2–ATM complexes may exist. The PLA assay was applied to the CP cell lines . The PLARecently, in this journal, we have pointed out that fibroblasts from patients suffering from Alzheimer’s disease (AD) specifically show spontaneous perinuclear pATM crowns . The per12 = 4096 cells if one passage corresponds to one doubling time and from 1 to 224 = 16,777,216 cells if one passage corresponds to two doubling times. Altogether, these findings are consistent with our model of accelerating aging with the agglutination of ATM around the nucleus. Further investigations are, however, needed to relate these findings to the formation of pATM crowns with the BFSP2 protein that would be phosphorylated by ATM in an inner layer of the crown, as recently observed in AD cells with the APOE protein [The percentage of cells with perinuclear pATM crowns was plotted against the number of cell passages, and some individual differences appeared. Mathematically, such differences cannot be due to errors in cell seeding or passage number calculation. The highest regression coefficients were obtained when the data were fitted to exponential and power laws A. By con protein .In the context of the RIANS-aging model, exposure to ionizing radiation may lead to a transient monomerization of ATM and therefore to a temporary disappearance of the perinuclear pATM crown . In ordeThis work aims to document the radiation response of human epithelial lens cells. However, the radiobiological characterization of this type of cell remains limited by the lack of availability of human epithelial lens cell lines, notably explained by the difficulties in amplifying them to high cell culture passages. By applying the RIANS biomarkers  to the HLEpiC cell line from ScienCell , a significant delay appeared in RIANS, suggesting that this lens cell line elicits moderate but significant radiosensitivity. Furthermore, the MRE11 foci kinetics found in HELpiC cells showed an analogy with those obtained from skin cells from aging syndromes, with a maximal value reached at 24 h post-irradiation. The radiobiological features of the transformed B3 lens cell line led to the same conclusions. Hence, while we are aware that too few human lens cell lines have been tested, our findings and literature data converge on the fact that (1) the human lens is a radiosensitive tissue  and (2) In addition, we identified a potential X-protein specific to the lens, i.e., an overexpressed cytoplasmic ATM substrate whose perinuclear sub-localization may facilitate the progressive agglutination of ATM around the nucleus and the formation of the perinuclear pATM crown. Lastly, in porcine lens cells, the number of perinuclear pATM crowns reached its maximal value at cell culture passage 12, which supports a limited lifespan in cell culture for this type of cell. The identification of a lens-specific X-protein (BFSP2), that is perinuclear, supports once again that the lens is a radiosensitive and radiodegenerative tissue.-“radiosensitivity”: any RI clinical and cellular events attributable to cell death ;-“radiosusceptibility”: RI cancers or any RI event attributable to cell transformation;-“radiodegeneration”: any aspect of the response to ionizing radiation attributable to aging.The lens was considered one of the most radiosensitive tissues, such as the breast, thyroid, or skin ,47. HoweAs a consequence, the lens may be considered a radiosensitive and radiodegenerative but not radiosusceptible tissue (there is no report of cancer of the lens). Like with a number of genetic syndromes in our previous reports ,33,48,49Interestingly, by applying the same approach to the lens, we have identified at least one X-protein for the lens, the BSP2 protein, that shows perinuclear sublocalization and therefore may favor the formation of perinuclear pATM crowns in response to permanent stress. In the framework of the RIANS model, such proteins may be considered tissue-specific X-proteins, unlike the “syndromic” X-proteins defined in skin fibroblasts and specific to each human genetic disease. Hence, the lens appears to be a tissue whose radiosensitivity belongs to group II defined by the RIANS model  ,43 Figu.By combining the radiobiological characterizations accumulated with skin fibroblasts from genetic diseases associated with juvenile cataracts and those of the lens cells, the syndromic and lens-specific tissue X-proteins may contribute together to the sequestration of the ATM monomers in lens cells to explain both individual and tissue predispositions to aging. In other terms, the syndromic X-proteins may accelerate a process already “programmed” in the lens thanks to tissue X-proteins such as BFSP2 . Our finBy considering the perinuclear pATM crowns as specific biomarkers of aging , Figure 5 ATM monomers per Gy are produced in the cytoplasm [pATM antibodies, exposure to ionizing radiation may lead to the monomerization of the ATM dimers that compose the pATM crowns and therefore may cause them to disappear. However, exposure to ionizing radiation may not cause a definitive but a transient steric dispersion of the ATM monomers around the nucleus, and their local concentration may facilitate again the formation of ATM dimers after irradiation and therefore that of the pATM crowns. Hence, after irradiation and a “relaxation time” that would depend on the dose, the pATM crowns may reappear. In our conditions, the relaxation time deduced from our preliminary data was found to be between 24 h and 48 h. Exposure to ionizing radiation not only adds more DNA breaks in the nucleus but also triggers the monomerization of the ATM proteins that help recognize them. It has been suggested that, while about 40 DSB are induced per Gy per fibroblast, about 10ytoplasm . Since t5 ATM monomers. For the formation of the first monolayer, let us consider an average nucleus radius of lens cells of 5–10 μm, an average surface of the nuclear membrane of about 800 μm2, and an average number of nuclear pores of about 1000. The formation of a pATM crown monolayer may therefore require at least the agglutination of about 200 ATM monomers per nuclear pore and 250 ATM monomers per μm2. In order to relate our findings to RI cataractogenesis, further experiments are needed to link the number of cells with pATM crowns, the number of cells undergoing senescence, and the in vivo occurrence of cataracts. However, an interesting notion emerges from our findings: The predisposition to accelerated aging and to early cataracts that would be reflected by a non-negligible number of cells with pATM crowns at the lowest passages. Interestingly, Obviously, different scenarios of irradiation may fill such requirements  and are the subject of calculations in progress. Altogether, our findings support that all the epithelial lens cells tested, whether human or porcine, show a delay in the radiation-induced ATM nucleoshuttling, late MRE11 foci, and perinuclear ATM crowns, as already observed in cells from aging syndromes . In the


Gastric cancer (GC) ranked as the fifth most incident cancer in 2020 and the third leading cause of cancer mortality. Surgical prevention and radio/chemotherapy are the main approaches used in GC treatment, and there is an urgent need to explore and discover innovative and effective drugs to better treat this disease. A new strategy arises with the use of repurposed drugs. Drug repurposing coupled with drug combination schemes has been gaining interest in the scientific community. The main objective of this project was to evaluate the therapeutic effects of alternative drugs in GC. For that, three GC cell lines  were used and characterized. Cell viability assays were performed with the reference drug 5-fluororacil (5-FU) and three repurposed drugs: natamycin, nitazoxanide, and benztropine. Nitazoxanide displayed the best results, being active in all GC cells. Further, 5-FU and nitazoxanide in combination were tested in MKN28 GC cells, and the results obtained showed that nitazoxanide alone was the most promising drug for GC therapy. This work demonstrated that the repurposing of drugs as single agents has the ability to decrease GC cell viability in a concentration-dependent manner. Helicobacter pylori being the major risk factor for this disease  (MTT) assays were performed concerning the reference drug 5-FU and the three additional repurposed drugs, namely, NAT, NTZ, and BZT .Three human GC cell lines , originally obtained from Biobank-i3s , were used. All cell lines were originated from gastric carcinoma but harbor different genetics backgrounds.®, Grand Island, NY, USA) supplemented with 10% fetal bovine serum  and 1% (v/v) penicillin/streptomycin , maintained at a 37 °C and 95% humidified air with a 5% CO2 environment. Cells were cultured in monolayers in T25 cm2 flasks . Cell culture was observed daily, media was replaced every 3 days, and at 80% confluency cells were subcultured. In each passage, the culture media was removed, and the cells were carefully washed three times with phosphate-buffered saline solution  at room temperature. Then, the PBS was removed, and 0.25% trypsin-ethylenediaminetetraacetic acid , was added and incubated at 37 °C for 4–5 min. Cell detachment was confirmed visually in an inverted phase contrast microscope . Culture media was added to stop enzymatic detachment, and detached cells were transferred into a 15 mL falcon tube. The cell suspension was centrifuged at 1600 rpm for 10 min to promote the formation of a pellet, which was later resuspended in 0.850 mL of fresh culture medium to reach a final volume of 1 mL. Then cells were cryopreserved or re-plated after determining their number and cell viability using the trypan blue exclusion cell assay and an automated counter . All described procedures were performed under aseptic conditions in a flow chamber.Cells were cultured in RPMI-1640 medium (Gibcov/v)), and more medium to fill the vials, they were capped and transferred into a freezing container with isopropyl alcohol  for slow freezing (−1 °C/minute) at −80 °C for 3 days. For longtime storage, vials were transferred to liquid nitrogen storage containers (−196 °C) .For cell cryopreservation, an appropriate number of 2 mL cryovials was labeled . After automatic counting and determination of cell viability, the desired number of cells suspended in 1 mL of culture medium was transferred to each vial. After adding cryopreserving agent, dimethyl sulfoxide  at densities of 300 and 500 cells/well, in triplicate, for all cell lines. The plates were incubated for 14 days under standard conditions with daily monitorization to confirm the development of colonies with the exception of the MKN45 cell line that was incubated for 21 days. After 14 or 21 days, respectively, the medium was removed, and the cells were stained with 0.5% (v/v) crystal violet (CV) for 15 min at room temperature, and the number of colonies was determined. Only visible colonies were counted (>0.5 mm in diameter and without overlapping). The colony formation unit (CFU) assay evaluates the ability of the cell line to form colonies from a single cell. Cells at 80% confluency were passed and seeded into 6-well plates (Orange Scientific4 cells/well) and AGS cells (1 × 104 cells/well) were seeded onto 24-well plates, in triplicate for each cell line, to grow to full confluence in a monolayer for 72 h. Then, on the day before the scratch, the medium was changed to RPMI without FBS. A sterile 20–200 μL pipette tip was held vertically to make a scratch along the central area in each well. The detached cells and debris were removed by washing with 500 μL of PBS twice. Afterward, 500 μL of fresh medium was added and the plates incubated for 48 h to enable cells to migrate and invade the cell-free scratch. The scratch closure was monitored, and the progression registered at 0 h, 24 h, and 48 h at a magnification of 300×. Wound area closure was automatically quantified using ImageJ© software v.1.53t , measuring the gap size and tracking the gap width by drawing lines along the leading edges of each cell front. The results are presented as percentage of wound closure. To determine the invasive and migratory capacity of the cancer cells, cell mobility was assessed by an in vitro wound healing assay. MKN28 and MKN45 cells , for 30 min at 37 °C, and then the absorbance was read at 405 nm using a microplate photometer . The results are presented as optical density (OD), also referred to as absorbance.To perform the β-galactosidase assay, 6000 cells/cm®, Grand Island, NY, USA) for 2–4 h at 37 °C to arrest the cell cycle in metaphase. Cells were then harvested and suspended in a hypotonic solution . After centrifugation , cells were fixed (6:1 methanol to acetic acid), centrifuged, and resuspended in 3:1 methanol to acetic acid. Cell smears were prepared onto glass slides, air dried, and stained with Giemsa  according to a routine laboratory procedure. Karyotyping was performed according to the International System for Human Cytogenic Nomenclature [The cell karyotype of all cell lines was evaluated to determine chromosomal stability in terms of chromosome number and the occurrence of neoplastic changes. Cells were expanded, and once they were in the exponential growth phase and approaching 80% confluency, the cells were treated with 10 μg/mL colcemid solution  was prepared. Sections of 2 µm were cut, deparaffinized, dehydrated, and subjected to immunocytochemical analysis. The slides were placed for 10 min in xylene solution, and subsequently the slides were up and down 5 times in xylene solution, 100% alcohol, 96% alcohol, and 70% alcohol and washed in water for 5 min. Three antigen retrievals were used. In the retrieval solution/water bath, the slides were placed in 10% retrieval solution and incubated in a water bath at 100 °C for 30 min and cooled down for 10 min. In the microwave/Extran 0.5% Extran was boiled and the slides incubated in the microwave for 10 min. With the pepsin method, the slides were incubated 30 min/37 °C in a 4% pepsin solution, and then the solution was discarded; TBS was added and incubated 3 min in the freezer to stop the reaction. Slides were subjected to immunocytochemical analysis using a Novolink™ Max Polymer Detection Systems  kit according to the manufacturer’s instructions. Information on the primary antibodies and antigen retrieval methods used is presented in ®, Tokyo, Japan). For the different markers, the immunoexpression levels was scored according to the intensity of labelling . If distinct cytoplasmic and/or membrane staining was detected in at least 5% of the total cell populations, immunoreactivity was considered positive.The three cell lines were submitted to immunocytochemical (ICC) analysis to detect specific antigens. Cells were maintained in culture until they reached 70–80% confluence, and then enzymatic detachment was performed with 0.25% trypsin-EDTA solution, and a paraffin cytoblock (GmbH50) value was calculated. IC50 values > 100 μM were not considered.The potential of the antineoplastic drug 5-FU and three repurposing drugs  in the GC cell lines was analyzed. Cells were treated with 5-FU in concentrations ranging from 0.1 to 100 μM for 48 h, and cell survival was evaluated by MTT. Based on these results, a dose–response curve was obtained, and the half maximal inhibitory concentration  in different concentrations  and applied to the cells for 24 and 48 h. For each time point, the culture medium was removed, and, with the chamber light off, 100 μL of MTT was added to each well. Then, the cells were incubated for 2 h at 37 °C, protected from light. Finally, 100 μL/well of DMSO was added after MTT solution removal, and absorbance was measured at 570 nm using a microplate reader. To evaluate the chemotherapy pharmacodynamics, the three cell lines  were exposed to the following drugs: (1) 5-FU, (2) natamycin, (3) nitazoxanide, and (4) benztropine. Cell proliferation/viability was evaluated as mitochondrial metabolic activity performing an MTT reduction assay. To perform the MTT assay, cells in the logarithmic growth phase were seeded onto 96-well plates at a density of 5 × 1050 of 5-FU (12.41 μM) was combined with the IC50 of NTZ (6.713 μM) or increasing concentrations of the same drug . After 48 h, the MTT assay was performed as explained above, and the cell viability results were obtained.Afterward, 5-FU and NTZ were tested in combination on MKN28 cells. The ICt-test when comparing the control and treated cells from one cell line. Differences were considered statistically significant when p ≤ 0.05 for a confidence level of 95%. All the analyses were performed using the software GraphPad Prism version 8 . Significant results were presented using the symbol (*). * Statistically significant vs. control at p < 0.1. ** Statistically significant vs. control at p < 0.01. *** Statistically significant vs. control at p < 0.001. **** Statistically significant vs. control at p < 0.0001.The data presented are, at least, from three independent experiments. Data are expressed as mean ± standard deviation (M ± SD). The results were analyzed using one-way ANOVA, followed by a Student Almost all cell lines showed epithelial morphology when observed under light microscopy and after being cultured under the same conditions .The analysis revealed that all cells showed clear plastic adhesion. Additionally, they mainly presented polygonal or "epithelial-like" morphologies, demonstrating intercellular adhesion and often forming cell groups with variable sizes . Most ofTwo weeks after seeding , crystal violet-stained colonies were visualized in all cell lines, with the exception of MKN45, which took 21 days to grow. The ability to form colonies was lower for AGS and MKN28 cells when compared with the MKN45 cells at both densities. Indeed, regardless of the density, MKN28 cells presented the lowest percentage of clonogenicity. The results of the colony formation assays are shown in Each cell line displayed different wound closure dynamics . In the The AGS cell line was shown to have significant differences at 24 h and 48 h from the start of scratching. At 24 h, the MKN28 cell line displays fast wound closure; however, it seemed to decrease and stagnate at 48 h, not making much difference from that observed at 24 h, with no significant differences for any time point in this cell line.A cellular senescence assay was also performed, and the respective results are shown in In order to identify chromosomal abnormalities in GC, conventional karyotyping was performed in all cell lines. The frequency of normal and aneuploid cells detected in the different cell lines evaluation is depicted in The karyotype analysis showed that all the cell lines were aneuploid and were constituted by a very low percentage of cells with a normal karyotype .Regarding H&E, all the cell lines were constituted of round to oval cells with marked pleomorphism (cells with different sizes). The AGS neoplastic cells commonly exhibited cytoplasm vacuoles. The MKN45 neoplastic cells presented a predominant plasmacytoid morphology. The immunocytochemistry revealed cytoplasm immunoexpression of the epithelial markers AE1/AE3 and EpCam in all cell lines evaluated. However, the privation of E-cadherin expression was observed in the AGS cell line g. All ceAll the cell lines tested were also negative for the remaining antibodies  .After characterizing the cell lines, the evaluation of chemotherapy pharmacodynamics and the in vitro response of the different chemotherapeutic protocols in GC cell lines  were investigated. Cells treated with vehicle (culture medium with 0.1% of DMSO) were used as control. The results of the MTT assay for 5-FU in MKN28, AGS, and MKN45 cell lines are shown in The results of the MTT assay in the MKN28 cell line showed no significant differences at any time point (24 h and 48 h).The results of the MTT assay in the AGS cell line showed that at 24 h, only concentrations above 10 μM exhibited significant differences compared to the control, requiring 20 μM of the antineoplastic drug to kill 50% of the cells. At the 48h time point, from a concentration of 0.1 μM, significant differences were already observed, with less than 2 μM of 5-FU being necessary to reduce 50% of AGS viability.The results of the MTT assay in the MKN45 cell line showed that at 24 h, only two concentrations (10 μM and 100 μM) presented significant differences relative to the control. At 48 h, from a concentration of 10 μM, significant differences were already observed, with less than 2 μM of 5-FU being necessary to reduce 50% of MKN45 cells’ viability. The MTT assay for 5-FU treatment demonstrated a lack of efficiency after 24 h for MKN28 and MKN45 cell lines A,C, withThe potential antitumor effects of different repurposed drugs as single agents, namely, NAT, NTZ, and BZT in MKN28, AGS, and MKN45 GC cells were evaluated. The three cell lines were treated with increasing concentrations of each drug, from 1 to 100 μM, to investigate the viability of the cells 24 and 48 h after treatment.50 value of 4.42 μM and 6.71 μM, respectively  is an acute event in cancer metastasis, characterized by reciprocal loss of expression of epithelial cell-associated proteins, such as E-cadherin, and increased levels of mesenchymal markers, such as vimentin ,57.EPCam, an epithelial cell membrane molecule, was expressed in all cell lines. The increased levels of EPCam in GC and metastatic lesions indicate that this marker is a promising therapeutic target . All theH. pylori, one of the risk factors for the development of GC. In addition, like other anthelmintics, NTZ has shown good anti-tumor activity in other types of digestive tract cancers [In this investigation, 5-FU was chosen as the reference anti-neoplastic drug, highlighting its wide use in GC treatment . Based o cancers . Further cancers . Despite cancers .50 was determined for each drug and hour, and based on the best results, 5-FU and NTZ were selected for the drug combination. The results demonstrated that repurposing the drugs as single agents had the ability to decrease cell viability in all cell lines in a concentration-dependent manner. The cell viability of MKN28, AGS, and MKN45 cells treated with 5-FU resulted in concentration-dependent growth inhibition after 48 h. The drug NTZ, in the repurposing category, was the only active drug in all cell lines. The other repurposing drugs (NAT and BZT) demonstrated a lack of efficiency in reducing cell line viability after 24 h. After 48 h of treatment, these drugs showed some cytotoxic effects, namely, NAT on the MKN45 cell line and BZT on the AGS cell line.After evaluation with MTT, ICAs the cell lines have different histological origins, different results were expected for each cell line type. Based on the results, the drug NTZ seems to have a cytotoxic effect on both intestinal and diffuse GC. NAT was only shown to be cytotoxically active in the MKN45 cell line, which is derived from diffuse type GC. BZT was shown to have interesting results in that it was active in the AGS cell line, which is histologically derived from both GC types  but was not shown to have cytotoxicity in the remaining lines (MKN28 and MKN45), which are each derived from different GC types, namely, the intestinal and diffuse types.50 (NTZ) was selected and combined with the reference drug 5-FU for 48 h. This task aimed to evaluate if the combination of NTZ with the antineoplastic drug (5-FU) would enhance their cytotoxic effect on MKN28 cells. The results obtained proved that there is no synergetic effect of 5-FU and NTZ. In fact, NTZ displayed more cytotoxic effects than 5-FU alone. From literature research, studies that used an NTZ-based treatment for H. pylori identified and demonstrated a cure rate success higher than 80% [After determining the anticancer potential of each repurposing drug in MKN28, AGS, and MKN45 GC cells, the drug with the best ICthan 80% ,66,67. TWith the increasing resistance of various types of cancer, such as GC, to the reference drugs used for their treatment, it is important to find alternatives. The use of repurposing drugs as a treatment for GC has proven to be a promising area of research, having the potential to improve outcomes for patients with this type of cancer.In the present investigation it was demonstrated that the use of repurposing drugs as single agents has the ability to decrease cell viability in three GC cell lines in a concentration-dependent manner. NTZ was the only repurposed drug that significantly reduced cell viability in all cell lines. The remaining repurposing drugs, NAT and BZT, demonstrated a lack of efficiency in cell viability reduction at 24 h. The cytotoxic effects of these drugs were only significant in concentrations above 50 µM after 48 h of treatment. Then, MKN28 cell line was treated with a drug combination of NTZ with the antineoplastic drug 5-FU. In general, the use of NTZ alone was the most promising drug for GC therapy, since this drug displayed more cytotoxic effects than either drug by itself. However, thorough studies are strongly recommended to evaluate in more detail the anticancer properties of this drug combination and further evaluate its action in animal models and eventually in clinical trials. Taken together, these results demonstrate that there is a great potential in the use of repurposed drugs and in their inclusion in combination schemes in order to promote and develop novel therapeutic strategies for GC.Moreover, deeper mechanistic studies are strongly recommended to evaluate the anticancer properties of this drug combination and further confirmation on animal models and clinical trials. Taken together, these results demonstrate that there is great potential in the use of repurposed drugs for the treatment of GC. The inclusion of these drugs in combination schemes has also shown potential in order to promote and develop new therapeutic strategies for GC.