Here, we used proliferation assay to characterize the plasma sensitivity of fourteen breast cancer cell lines. These assays showed that all tested cell lines were sensitive to NTP. In addition, a good correlation was found comparing cell sensitivity to NTP and radiation therapy (RT), where cells that were sensitive to RT were also sensitive to plasma. Moreover, in some breast cancer cell lines, NTP and RT have a synergistic effect. Adding a dose of PARP-inhibitor olaparib to NTP treatment always increases the efficacy of the treatment. Olaparib also exhibits a synergistic effect with NTP, especially in triple negative breast cancer cells. Results presented here help elucidate the position of plasma use as a potential breast cancer treatment.Cancer therapy has evolved to a more targeted approach and often involves drug combinations to achieve better response rates. Non-thermal plasma (NTP), a technology rapidly expanding its application in the medical field, is a near room temperature ionized gas capable of producing reactive species, and can induce cancer cell death both in vitro and  Each year, more than two million women are diagnosed with breast cancer, the most frequent cancer in women . DependiA new perspective in cancer treatment has come with the use of non-thermal plasma (NTP). Briefly, NTP consists of a partially ionized gas which allows to convert electrical energy into chemical and thermal energy. Usually obtained by applying an electric field to a flowing gas , NTP is BRCA1/2 status of the cell lines.As most studies on plasma oncology examined only a few cell lines to demonstrate the anticancer capacity of NTP, here we use a relatively high-throughput approach to characterize the sensitivity of fourteen human breast cancer cell lines to NTP. Hence, the aim of this work is twofold. First, to determine if the sensitivity of breast cancer cells to NTP is dependent on the molecular subtype. Second, to compare and evaluate the potential combination of NTP with RT and PARP (Poly (ADP-ribose) polymerase) inhibitor olaparib, two other therapies used in breast cancer. By comparing the plasma response of these cell lines to RT sensitivity, we observe a direct correlation between the efficacies of these two DNA damaging agents. Moreover, combining RT and plasma can result in a synergistic effect in a subset of cell lines. In addition, pretreatment with olaparib increased the efficacy of NTP in all tested cell lines. A synergistic effect was also measured for this combination, independent of the Precision oncology involves multidisciplinary approaches to define patient subgroups and adapt the use of various treatment modalities, either alone or in combinations, according to the disease’s sensitivities and patient’s needs. NTP can become a new member of the arsenal to treat patients affected by breast and other cancers. As NTP yield minor side effects ,22, locain vivo, plasma jets are most often used . Ho. Ho24]. γ modes in all cell lines. Cytotoxicity of the jet mode is similar to the cytotoxic effects of RT and in some cases greater than the response to 10 Gy . Additionally, the most radioresistant cell lines, HCC1428 and MDA-MB-175-VII, were sensitive to the jet mode using a sufficient dose of 120 s (p < 0.001).For comparison with plasma treatments, RT was used as a standardized reference for cytotoxic sensitivity of the different cell lines. A 4 Gy reference dose of RT tends to be more cytotoxic than both the Ω and 2 slightly increased the antiproliferative capacity of the treatment, while for other cell lines, the antiproliferative capacity remained unchanged. On the other hand, with the γ mode, addition of O2 to the treatment tends to improve the antiproliferative capacity of plasma for all cell lines. However, the sensitivity to the addition of O2 varies significantly between different cell lines. Comparison of the antiproliferative effect of O2 is illustrated in Another feature of the device is the possibility to inject a secondary gas directly into the plasma effluent or afterglow (via the hollow high-voltage electrode). γ modes is enhanced by the injection of O2 in the high-voltage electrode. However, from the use of GR values, it is possible to quantify the overall sensitivity of the different cell lines. As shown by 2 in the high-voltage electrode is almost inexistent in the Ω mode (p > 0.5 with t-test) but is significant in the γ mode (p < 0.001 with t-test). Even if the injection of O2 in our NTP device is performed downstream (in the effluent or the flowing afterglow region) rather than in the plasma itself, the antiproliferative capacity can benefit from the injection of O2. This feature is in agreement with other studies reporting on the anti-cancer effect of O2 addition to plasma [Results shown in o plasma ,29 but h50  and to RT is shown in 50 B. For ot < 0.005 B. This sWe then determined if sensitivity to plasma treatment can be correlated to a few different clinical aspects of breast cancer. We grouped the cell lines into their different receptor status subtypes . In It is known that NTP can produce RONS , which cγ mode with O2, but both displayed lower antiproliferative capacity than 4 Gy alone . Among the cell lines used, only HCC1569 and MDA-MB-361 contained a BRCA2 mutation. In those cell lines, only 40% (p < 0.001) and 20% (p > 0.05) of growth inhibition was observed, respectively, with olaparib alone. Indeed, the dose used was not enough to reach the synthetic lethality expected in BRCA mutants. Olaparib treatment alone resulted in growth inhibition ranging from 0% to 40% in other cell lines.As shown in In all cell lines, combination with olaparib tends to improve the cytotoxic effect of plasma. Interestingly, CI < 1 for seven out of eleven cell lines, demonstrating a synergistic effect of olaparib and plasma B. This wDNA damage can be visualized through the activation of DNA repair pathways. We used the detection of DNA-damage-associated foci phosphorylation of H2AX (γH2AX) to characterize the effect of our combination at the molecular level.p < 0.01 for HCC1428). In addition, pretreatment with olaparib further increased the number and intensity of foci following a treatment by jet mode and RT (p < 0.05). Interestingly, in some conditions, a strong pan-nuclear staining can be observed in a portion of the cells. This pan-nuclear phosphorylation of H2AX signal may suggest cells potentially going through apoptosis following treatment [As expected, in reatment ,45. AccoThese results indicate that the effect of plasma on cancer cells can be improved by a combination with a DNA repair inhibitor such as olaparib. This reinforces the notion that the ability of NTP to induce cytotoxic effects occurs through DNA damage (whether SSB or DSB), similar to radiation.50 (treatment condition resulting in 50% relative viability) [50 is confounded by its slow cell division rate. The GR method is commonly used in a variety of open access databases compiling the drug sensitivity of different cell lines. The Library of Integrated Network-based Cellular Signatures (LINCS) [In this study, we compared the sensitivity to NTP with RT, a conventionally used modality in breast cancer treatment, on a large set of common breast cancer cell lines. A constraint to large-scale comparison of NTP sensitivity across cell lines is the heterogenous proliferation rates of different cell lines. Our method to quantify cell response to NTP was based on the normalized cell number 6 days post-treatment. Therefore, systematic variations in cell division time will affect relative response metrics such as ICability) . To over (LINCS)  and Phar (LINCS)  are two γ modes (in comparison to the jet mode) to reach a similar antiproliferative capacity turned out to be respected for all cell lines. As previously reported [2 to rare gas NTP increases the cytotoxicity of plasma. However, in the present case, O2 is not injected in the plasma per se, it is rather injected in the effluent region (in the Ω mode) or in the flowing afterglow (in the γ mode). As shown in 2 is significant in the γ mode. This suggests that injection of O2 in the flowing afterglow is sufficient to enhance the production of RONS, a fact in agreement with the enhancement of the O  atomic emission line in 2 in the Ω mode could be attributed to the low energy present in the plasma effluent. However, as 2, the mitigate effect of O2 could indicate that the 777.5 nm atomic line is not a good indicator of plasma antiproliferation capacity (at least with downstream O2 injection) or it could be simply due to the NTP dose that is too low to significantly impact cell proliferation. This will be the subject of further investigations.Beside the use of fourteen cell lines, three NTPs, described previously , have bereported ,29, addiWe previously reported that plasma can induce DNA damage in MDA-MB-231 cell line and drive the cells to undergo mitotic catastrophe . Here, i50 for the jet mode and RT, the doses required for combination could be better defined for each cell line in future experiments. Results from the current study supports the fact that the application of NTP in combination with RT may be complementary and a viable clinical strategy for further exploration.Using the same experimental method to investigate the sensitivity of cell lines to NTP and RT allowed to establish a strong correlation between both modalities’ antiproliferative capacity. This correlation highlights the similarity in the mechanisms of action of these modalities. In addition, using the same procedure, the combination of RT and NTP have also been tested. In agreement with previous work by Lin et al.  in otherOlaparib is approved by the US Food and Drug Administration (FDA) as a therapy for select breast, ovarian and prostate cancers, for which plasma treatment is also being evaluated in vitro ,33,54,55In our experiment, we chose to target PARP, which is mainly involved in SSB repair. Unresolved SSB will generally evolve in DSB, and direct induction of both types of DNA damage by RONS is probably the main mechanism of genotoxic stress by plasma treatment . In the In characterizing the relative sensitivity of a large set of publicly available breast cancer cell lines, this study built a platform to further explore the cellular mechanism of action of plasma. As these cell lines’ genomes have been sequenced ,61,62 an2 according to ATCC recommendations. Cells were used at a low passage number (lower than 25) upon reception from ATCC in order to maintain their parental phenotype and genotype. Genomic characterization of all investigated cell lines is publicly available [Breast cancer cell lines Panel 1  was purchased from the American Type Culture Collection . Cells were grown following ATCC recommendations , with or without insulin, with 1% penicillin-streptomycin (Pen-strep). Cells were incubated at 37 °C with or without 5% COvailable . This inγ mode or 4 min of Ω mode [The convertible plasma device used for the treatment of cells in suspension was described elsewhere . In brief Ω mode .−1 grating blazed at 300 nm with a resolution about 1.5 nm, Ocean Optics, Dunedin, FL, USA). The optical emission spectroscopy system was corrected by its complete response curve using an Intellical lamp  above 400 nm and a 900 W tungsten lamp  below.The optical emission of the discharge was collected by an optical fibre for emission spectroscopy . OpticalCell suspensions were prepared in DMEM containing pyruvate supplemented with 10% FBS and 1% Pen-strep. A fixed volume of 400 µL was dispensed in 1.5 mL microtubes for plasma or radiation treatment. Cell concentration was adjusted according to the different cell lines (ranging from 50 000 to 200 000 cells/mL). Plasma treatment was performed by positioning the nozzle of the device inside the tube at a constant distance of 5 mm from the surface of the media. Cells seeded in a 48-well plate were fixed at day 6 after treatment with a crystal violet solution . Fixed cells were then stained with DRAQ5 in PBS, washed and scanned with the Odyssey imaging system . A dilution curve of various cell numbers stained by DRAQ5 was prepared for each cell line in each experiment. DRAQ5 signal in each well was compared to the dilution curves to quantify the cell numbers in each well .x(c) the cell number of the treated sample at concentration c, x0 the cell number at the time t = 0 s , ctlx the cell number of the control sample at the same time as x(c), according to the following equationDose-response curves were calculated using the GR inhibition metrics . This mex0 value were inferred from the number of cells plated, fixed at 18 h and stained with DRAQ5. GR50 for jet and RT were obtained for different doses in order to obtain dose-response curves.To use the GR metrics with our proliferation assay, TM Quantitative Peroxide Assay Kit  was used according to the manufacturer’s instructions. The optical density at 595 nm was measured with the Spark multimode microplate reader .For hydrogen peroxide detection, cell suspensions were centrifuged after plasma treatment, and the supernatant was collected and processed immediately. Pierce−1) just before imaging.Live cells were followed using IncuCyte S3 Live-cell Imaging System , using a 10× objective. During the incubation period, cells were maintained in 48-well plates with 250 μL of medium per well. Only 20% of this medium was carried from the treated microtube as described in For the combination with PARP-inhibitor olaparib , cells were trypsinized, diluted to the appropriate concentration and pretreated with 2 μM of olaparib for 2 h before plasma treatment (or radiation). Following plasma treatment, 50 μL of the treated cells were transferred to a 48-well plate containing 200 μL of fresh media. Cells pretreated with olaparib were also maintained in media containing 2 μM of olaparib during the six days of incubation. For the combination with radiation, the plasma treatment was performed first and then cells were exposed to 4 Gy of radiation, within 30 min. The Chou-Talalay method  for drugAfter treatment, cells were plated in 8-well chamber slides and fixed 24 h later with 10% formalin for 5 min. Cells were permeabilized with 0.25% Triton for 10 min, incubated in blocking solution  for 1 h and with the primary antibody overnight at 4 °C. Cells were washed and incubated with the secondary antibody for 1 h at room temperature and then washed again. Prolong containing DAPI was used for slide mounting and images were obtained using a Zeiss Observer Z1 microscope . Primary antibody used was phospho-H2AX (Ser139)  and secondary antibody was Alexa fluor-488 (1:750).t-test were used to compare different treatments with the control, to compare the effects of NTP between different breast cancer subtypes and to compare the influence of O2 on the antiproliferative effect of NTP in the γ mode. Pearson’s correlation test was performed to determine the correlation of the antiproliferative effect of NTP and RT. Data analysis, microscopy image analysis and statistical analysis were performed using homemade codes with Mathematica 10 .Student’s γ modes were compared when a small concentration of O2 is injected via the high-voltage electrode. Unlike previous works were O2 is injected within the plasma itself, our plasma device allows the injection of O2 downstream of the plasma. In the γ mode, O2 is in contact with the flowing afterglow and a significant increase of the antiproliferative capacity of the plasma occurs. This effect was observed on all cell lines, and highlights the potential benefit on cancer treatment of the precise control of gas composition via downstream injection.In this study, we were able to classify a large panel of breast cancer cell lines according to their sensitivity to NTP in three different discharge modes. Cell lines that are more sensitive to a discharge mode are also found to be more sensitive to other discharge modes. In addition, the Ω and 50 to compare cell lines classified in the aforementioned subgroups, no subgroup of cell lines could be identified as strongly sensitive or resistant to NTP. Hence this highlights the potential clinical benefit to a majority of patient, should NTP be used intraoperatively to treat the tumour bed after tumour resection.One of the aims of this work was to determine if a particular subtype of breast cancer could be particularly sensitive to NTP. Classification was selected with respect to their molecular subtype  and to their receptor status . Using the GRComparing the sensitivity of cancer cell lines to NTP with their sensitivity to RT, a good correlation was found: cell lines more sensitive to RT are also more sensitive to plasma. Combination of NTP and RT was also found synergistic on a subpopulation of cell lines. These results suggest that adding a relatively low dose of NTP to a patient’s therapeutic plan could allow to reduce the dose of RT required, therefore minimizing side effects without compromising efficacy. In addition, we demonstrated for the first time that NTP can synergistically be combined with olaparib, a PARP inhibitor. Olaparib being more and more used in the clinic, its role as a NTP sensitizer is a very important feature towards the eventual position NTP could hold in the arsenal of breast cancer treatment.


In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B  signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip Melanoma is a malignant tumour that arises from the malignant transformation of melanocytes. It particularly affects young adults as it is the third most frequent cancer in the 20–39 year old age range. It is the least common but the most aggressive skin cancer, accounting for the vast majority of skin cancer deaths . HistoriV600EBRAF is activated almost 500 fold [V600EBRAF also stimulates melanoma cell invasion in vitro and is important for tumour neo-angiogenesis in vivo. The identification of the signalling pathways that are central to melanoma initiation and progression inaugurated a new era of treatments of both metastatic and early stage melanoma, with novel agents targeting specific genomic alterations underlying melanoma initiation and progression . UltraVi500 fold , it tran500 fold , and it 500 fold . V600EBRV600BRAF mutations [In this context, BRAF inhibitors (BRAFi) have generated remarkable clinical responses in a high proportion of melanoma patients harbouring utations ,12,13. Iutations . These rutations  and withutations . Combinautations , vemurafutations , encorafutations . The comutations . Howeverutations ,21,22. Targeting specific oncoproteins crucial for tumour growth, such as BRAF, induces adaptive kinome responses that upregulate alternative kinase signalling to overcome the inhibitor treatment ,22,23. TThus, biomarkers of resistance mechanisms are searched to identify patients in need of novel drug combinations. In this study, we explored a top-down approach using an innovative multiplex kinase assay to identify kinome profiles of individuals not responding to therapy using baseline biopsy samples, and to further understand the mechanism of resistance using patient-derived cell lines characterised for sensitivity, intrinsic and acquired resistance to the MAPK inhibitors.−2 M, aliquoted and stored at −20 °C.Vemurafenib (PLX4032) was from Nuclilab , dabrafenib and trametinib were from Selleck Chemicals . All were dissolved according to the manufacturer’s recommendation in DMSO at 10V600EBRAF mutant human melanoma cell lines  (® HTB72™ melanoma cell line [d MM074)  used in ell line . The BRAell line ,26.2 atmosphere. For routine maintenance, cells were propagated in flasks, harvested by trypsinization (0.05% trypsin-EDTA)  and sub-cultured twice weekly. Cell count and volume were evaluated using a TC10™ Automated Cell Counter . All cell lines were regularly checked for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kit .Cells were grown in HAM-F10 medium supplemented with 5% heat-inactivated foetal calf serum, 5% heat-inactivated newborn calf serum and with L-glutamine, penicillin and streptomycin at standard concentrations  (culture medium) at 37 °C in a humidified 95% air and 5% COThis retrospective study was approved by the Central Ethics Committee of the Institut Jules Bordet (CE2023). All patients included in this study received information regarding the use of residual human corporal materials and clinical data for researches and signed an informed consent form prior to biopsy. Snap-frozen in liquid nitrogen biopsies  were collected from seven patients with metastatic melanoma harbouring the BRAF V600E mutation prior to the initiation of first-line therapy with vemurafenib. Biopsies were stored at −80 °C until use. Four patients were responders and three patients were non-responders to the therapy. Responders were patients who had achieved a partial or complete response as best response using Response Evaluation Criteria In Solid Tumors (RECIST) 1.1  or PET R® OCTTM embedded and cut in a microtome at −20 °C. Tissue sections of 5 µm were cut for histological evaluation (HE staining to assess the percentage of tumour) while 60 µm tissue sections  were cut for protein extraction. Resistant cell lines were cultured in the presence of 2 µM vemurafenib. Before lysis of cell lines, culture medium with or without 2 µM vemurafenib was removed from the cells and the cells were washed with ice-cold PBS prior to protein extraction.Frozen tissues were TekTissues and cells were lysed on ice with M-PER  lysis buffer containing 1/100 Protease Inhibitor Cocktail EDTA-free (Thermo Fischer Scientific) and 1/100 Phosphatase Inhibitor Cocktail (Thermo Fischer Scientific). After centrifugation , the supernatants were aliquoted, snap-frozen in liquid nitrogen and stored at −80 °C. The protein concentration was determined using the BCA Assay  with bovine serum albumin (BSA) as the standard. For each experiment, a never thawed aliquot was used.® 96 or PamChip® 12 serine/threonine (STK) and protein tyrosine (PTK) peptide microarray system from PamGene International B.V.  according to the instructions of the manufacturer, essentially as described in Rosenberger et al. [Kinase activity profiles were determined using the PamChipr et al. . Of noteData quantification of the array images at all exposure times and time points and visualization of the data were performed using BioNavigator software . BioNavigator automatically grids images taken by the PamStation and quantifies the signal and background of each peptide spot, providing a signal minus background value at multiple times throughout the incubation at multiple exposure times. These values were combined into one value for each time point. For the STK assay, peptides having a signal above the background in more than 80% of the arrays, i.e., 126/144 peptides were retained for the analysis. For the PTK assay, only peptides showing an increase of signal in time, essentially identifying phosphorylation kinetics in more than 80% of the arrays, i.e., 90/142 peptides, were retained for analysis.Values were 2log-transformed for visualization. To identify peptides that are significantly different between different conditions, statistical tests were performed, such as Student’s t-test, Paired t-test, one-way ANOVA or a mixed model analysis.https://string-db.org/) was used to create functional protein interaction networks. Kinome phylogenetic trees were made in Kinmap (http://www.kinhub.org/kinmap/).For upstream kinase analysis, kinases known to phosphorylate the phosphorylation sites in the peptides were identified in databases. Based on phosphorylation patterns and permutation tests, the kinases most likely to cause the effects were identified, using Bionavigator sofware . STRING 6 cells/dish) in culture medium. One day after plating, the culture medium was replaced by fresh medium and cells were grown for two more days. Then, cells were exposed to effectors or vehicle for 24 hours. Cells were lysed using a detergent cocktail and extracted proteins were analysed by Western blotting as previously described [® Dry Blotting System . Immunodetections were performed using antibodies raised against pAKT (Ser 473) , AKT , pSrc (Tyr416) , Src , Hepatocyte growth factor receptor MET , Epidermal Growth Factor Receptor (EGFR)  , pERK (Tyr 204) , ERK2  , and β-actin  .Cells were plated in Petri dishes  following the manufacturer’s indications. Immunostaining signals were digitalised with a PC-driven LAS-3000 CCD camera , using a software specifically designed for image acquisition . Immunoreactive band intensities were quantified using the software AIDA® Image Analyser 3.45 (Raytest®).Peroxidase-labelled anti-rabbit IgG antibody (1/5000) or peroxidase-labelled anti-mouse IgG antibody (1/5000)  were used as secondary reagents to detect corresponding primary antibodies. Bound peroxidase activity was revealed using the SuperSignal®) that assesses both serine/threonine kinase (STK) and protein tyrosine kinase (PTK) activities. The experimental workflow is represented in p < 0.05) between responders and non-responders. For PTK, a peptide derived from the Ephrin Receptor B4 (EphB4) had p < 0.05 between the two groups. For STK, one peptide , aka Mammalian target of rapamycin (mTOR)) contains the AKT phosphorylation site of mTOR, the other is a Protein Kinase C (PKC)-derived peptide and the third is derived from the Neurotrophic tyrosine kinase receptor type 3 (NTRK3)  [We performed first a kinome profiling of melanoma samples from patient tissues using a phosphorylation-based kinome array (PamChipelanoma) .The peptides present on the array are phosphorylated by kinases in the tissue lysates. To generate a hypothesis about kinases in the lysate responsible for this phosphorylation, without a priori assumptions, we performed an upstream kinase analysis. Based on the relatively small differences between the responders and non-responders, the upstream kinase analysis suggested an increased activity in responders of AKT, p70S6K , mTOR, PKA (Protein Kinase A) and/or PKC (Protein Kinase C) and members of the EGFR family B. Thus, V600EBRAF inhibitor (dabrafenib) with two purposes: (1) to gain a more comprehensive insight into the pathways that have an impact on the response to BRAF inhibitors, and (2) to distinguish responders from non-responders based on relative signal intensities (log fold change inhibited/non-inhibited) to overcome the differences in basal kinase activity between the patients.We evaluated the inhibition profiles obtained with ex vivo exposure of melanoma tumour lysates to a V600EBRAF, which is 7 and 9 fold less potent to BRAF WT and CRAF, respectively [Dabrafenib is an inhibitor of ectively . The oveectively .p < 0.05) (25 µM inhibitor was excluded to avoid off-target effects) were linked to the UniProt ID of their parental protein and subjected to a network analysis in STRING. Each protein was represented as a node with edged interactions. The analysis shows a strong connection between MEK (aka MP2K1) and AKT (PKB) and mTOR (Effect of dabrafenib addition to lysates on kinase activity was expressed as Log Fold Change (LFC) vs. the incubation without inhibitor. Statistical analysis of LFCs was used to identify a subset of peptides that showed significantly different inhibition between the responders and the non-responders at any of the inhibitor concentrations used . At 1 µMand mTOR D. Upstre50 in resistant cells as compared to parental cell lines , all with BRAF V600E mutations, were made resistant to the BRAF V600E inhibitor vemurafenib after chronic exposure for 12 weeks to gradually increasing concentrations of vemurafenib (0.1–2 µM) . Acquirell lines .p < 0.05) in the STK and PTK assays, respectively, suggesting that intrinsic resistance is not related to the activation of specific kinases.However, the cell lines with intrinsic resistance showed opposing effects. For example, the MM043 cells, an intestinal metastatic cell line with intrinsic resistance to vemurafenib, showed a very different STK activity profile as compared to the other cell lines A. StatisCells with acquired resistance to vemurafenib (except for MM164) show higher tyrosine kinase activity as evidenced by higher phosphorylation levels of peptide substrates, compared to the sensitive counterpart A. STK acNext, we analysed the protein expression of significantly regulated kinases to evaluate whether the comparative analyses of kinome profiles between the sensitive and the resistant cell lines are reflected in altered levels of phosphorylation of target proteins by Western blot C. An uprHence, these data further support that, first, altered expression of selected kinases is reflected in kinome profiling, second, acquired resistance occurs via a common mechanism of increased kinase activity.To identify common effects of acquired resistance on kinase activity, a linear mixed model  and random (cell line) factors) was used to identify the effects that are common to the four cell lines A. For vip < 0.01 were used for a STRING network analysis . The contribution of MEK, AKT and mTOR to acquired resistance is very cell line-dependent, although a strong link to STK activity via PI3K and PDK1 is suggested. Most of the kinases cited above with a significant change in activity between sensitive and resistant cells are also involved in the MAPK, PI3K and SRC networks and regulate cell survival and cell migration.Our next aim was to evaluate the effect of dabrafenib and trametinib, alone or in combination, on the kinase activity of cell lines with acquired or intrinsic resistance to vemurafenib in comparison with parental cells. Here too, the inhibitor(s) was added to the lysates just prior to the assay. We reported that dabrafenib is a more potent inhibitor of PTK activity than of STK activity, both in the cell line and in the melanoma tissue lysates. Here, we observed that 10 µM trametinib resulted in overall inhibition of STK activity, whereas a slight activation was observed on PTK A. Treatmp < 0.05) associated with the combined treatment for the acquired resistant and sensitive cell lines, respectively (eight peptides in common), and five for the intrinsic resistant cell lines. On STK, no interaction between dabrafenib and trametinib was found for the cell lines with acquired resistance, but there was interaction for the sensitive and intrinsic cell lines (11 peptides in common (p < 0.05) for intrinsic and 16 peptides for the sensitive cell lines). The network representations of these peptide sets show that many nodes are affected by the combination of dabrafenib and trametinib in the sensitive cell lines  is positively correlated with clinical response . PhosphoBiological and technological advances have been developed to identify new targets for drug development. RNA interference (RNAi) , genomicV600EBRAF inhibitor could identify robust common features that discriminate responders from non-responders. Furthermore, both in melanoma tissues and cell lines, the BRAF inhibitor was found to be more potent as a PTK inhibitor than as a STK inhibitor, in accordance with Tahiri et al, who found higher inhibition of PTK activity by vemurafenib in V600EBRAF melanoma than in BRAF WT [V600EBRAF mainly activates signalling pathways implicating tyrosine kinases.We evaluated the inhibition profiles obtained after ex vivo exposure of tumour tissue lysates to MAPK inhibitors and found that phosphorylation levels of several kinase substrates differed between patients who were responding and not responding to BRAF inhibition monotherapy. Inhibition of STK activity in sensitive samples by dabrafenib and trametinib in combination was stronger, indicating a high dependency on the BRAF pathway in these responder patients . However BRAF WT , indicatV600EBRAF inhibitor vemurafenib or with intrinsic or acquired resistance to the drug, and we showed significant differences in phosphorylation profiles between the two groups (sensitive vs. resistant). Interestingly, kinases within the MAPK, PI3K/AKT and SRC signalling networks α/β, insulin-like growth factor 1 receptor (IGFR1R) and RET), AKT and SRC.V600EBRAF inhibition therapy [Furthermore, PDPK1 and PIK3R1 (p85α regulatory subunit of PI3K), two main components of the PI3K/AKT pathway, were also present in the identified protein pattern. Several studies have reported a central role for the PI3K/AKT pathway in the resistance to  therapy ,43,44. JMoreover, EpoR (erythropoietin receptor), also displayed in the interaction network, can promote angiogenesis, melanoma cell survival and has been reported to counteract cisplatin-induced cell death ,46. InteV600E cells [In a next step, kinase profiling was validated by Western blot. The results revealed an upregulation of RTK (EGFR and c-MET) and an activation of MAPK, PI3K/AKT and SRC signalling networks, confirming our findings in patients as well as in cell lines C. Indeed0E cells .Thus our results clearly support the involvement of PI3K/AKT pathway activation in vitro and in vivo by various converging mechanisms.Furthermore, this technology offered us the opportunity to compare sensitive and resistant cell lines to BRAFi with the aim to dissect the global impact of adding MEKi on kinase activity. In sensitive cells, BRAFi induced a stronger kinase inhibition than the one obtained with MEKi alone. This could be in line with the finding that MEKi monotherapy is less effective than BRAFi monotherapy in terms of response rate . In resiLastly, cAMP/PKA are known to have a particular role in overcoming resistance to BRAF V600E inhibitors ,49,50, eIn conclusion and despite the limited number of patient biopsies, our study proposes a kinome profile using a reliable multiplex assay platform able to predict treatment outcome. Our findings will need a larger validation cohort in a prospective study to draw definitive conclusions. Nonetheless, one of the tumour tissue biopsies that we used in this study (518) originates from a patient under vemurafenib monotherapy without evidence of any recurrences for more than five years now, showing the long-term efficacy of such treatment in certain patients. Interestingly, our study showed that the following kinome profile, together with a relatively high cAMP/PKA pathway activity may explain this long-lasting tumour control.


Bladder cancer has a high incidence and mortality. Besides this, currently available therapies for this type of cancer have low efficacy and show considerable adverse effects, urging the need of new therapeutic approaches. Cold Atmospheric Plasma treatment presents itself as a promising alternative, having demonstrated antitumor effects against several types of cancer. The present work arises from a multidisciplinary team, namely, medical doctors and researchers, in an attempt to find new therapeutic strategies to fight bladder cancer. Therefore, our main objective is to evaluate Cold Atmospheric Plasma effects against bladder cancer, as well as the mechanisms by which it exerts its effects. The results obtained demonstrate that Cold Atmospheric Plasma treatment has a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line. This new approach using Cold Atmospheric Plasma for the treatment of bladder cancer presents enormous clinical benefits, since it is able to selectively treat the tumor tissue, sparing the normal urothelium, with an additional glaring positive economic impact, since it entails a decrease in the cost of therapy in comparison with conventional therapeutic options.m) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψ Bacillus Calmette-Guérin (BCG) [Bladder cancer is the 11th cancer with the highest incidence in the world . This thin (BCG) ,6. In thin (BCG) , like inin (BCG) ,8,9. FurPlasma is frequently referred to as the fourth state of matter, being a partially ionized gas composed of ions, electrons, and neutral particles ,11,12. QMore recently, Plasma Activated Medium (PAM) has been described, a water-based medium that is treated with CAP. PAM acquires characteristics similar to CAP, allowing the selective treatment of neoplasms , with twThere are only two studies that demonstrate the effect of CAP on bladder cancer, one in vitro  and the In this work, we propose to study the effects of CAP on bladder cancer, particularly in the high-risk/very-high-risk tumors because of high grade tumor (G3) or aggressive variants, for which BCG therapy or even cystectomy are the standard of care. For this, two bladder cancer cell lines were treated with CAP and its anti-tumor capacity were evaluated, as well as the mechanisms by which it exerts its effects, starting from the mechanisms proposed for CAP’s action.® CRL-1472™, Manassas, VA, USA) representative of grade III carcinoma and the TCCSUP cell line (ATCC® HTB-5™) representative of grade IV urothelial carcinoma. The cell lines used were obtained from the American Type Culture Collection (ATCC®) and maintained in a humidified atmosphere, 37 °C and 5% CO2. Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium, DMEM  supplemented with 1% antibiotic , 0.25 mM sodium pyruvate , and 10 or 5% FBS  for HT1376 or TCCSUP, respectively.Human bladder cancer cell lines with different molecular characteristics and belonging to different stages of the disease were used, the HT-1376 cell line  were studied using flow cytometry 24 h after the CAP treatment. The cells were plated under the same conditions described for the oxidative stress assays and the same CAP exposure times were also used. The viability and cell death profile, the cell cycle, and the mitochondrial membrane potential . Results are presented as the percentage of metabolic activity normalized to the control. The metabolic activity was evaluated by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay. MTT is a yellow tetrazolium reagent, that is reduced with the formation of purple formazan crystals in the presence of dehydrogenase enzymes located in viable mitochondria . Upon 24® Multimode Plate Reader, PerkinElmer®). Results are presented as the percentage of protein content normalized to the control.To evaluate the protein content, it was used the colorimetric sulphorhodamine B (SRB) assay, a negatively charged pink aminoxanthine dye that gives a measure of protein synthesis. The greater the number of cells, the greater amount of dye is taken up, and the greater the absorbance . Upon 240/G1 phase and cells in the G2/M phase have twice the DNA content than cells in the G0/G1 phase. Sometimes, it is still possible to identify a cell population in pre-G0/G1 that has less DNA, also called apoptotic peak. Upon 24 h after CAP treatment, cells were detached and 1 × 106 cells were centrifuged for 5 min at 1300× g. Cell cycle labeling was performed as previously described [0/G1, G0/G1, S, and G2/M.To evaluate the effect of CAP on the progression of the cell cycle in both bladder carcinoma cell lines, the labeling with propidium iodide (PI) was used. PI has the ability to interleave in DNA and allows different populations to be obtained in each of the phases of the cell cycle, but it also interleaves with RNA. Thus, in order to obtain a specific DNA labeling, it is necessary to remove the RNA enzymatically using RNase . Cells iescribed , using a−/PI−) and three different types of cell death: initial apoptosis (AV+/PI−), late apoptosis/necrosis (AV+/PI+), and necrosis (AV−/PI+). Upon 24 h after CAP treatment, cells were detached and 1 × 106 cells were centrifuged for 5 min at 1300× g. Then, cells were washed with PBS and incubated with 100 µL of binding buffer , 0.14 M NaCl and 0.25 mM CaCl2 ), 2.5 µL of AV-FITC , and 1 µL of PI  for 15 min at 37 °C, in the dark. After incubation, 400 µL of binding buffer was added and analysis was performed on a flow cytometer , using the excitation wavelength of 488 nm and emission wavelengths of 533 nm for the AV-FITC and 640 nm for the PI. Results are presented as the percentage of cells identified in each of the subpopulations: viable cells (V), cells in initial apoptosis (A), cells in late apoptosis/necrosis (A/N), and cells in necrosis (N).To determine the type of cell death induced by CAP in both bladder carcinoma cell lines, the double labeling with annexin V (AV) marked with FITC (fluorescein isothiocyanate) and PI was used. This assay is based on the affinity of AV for phosphatidylserine, which at the beginning of apoptosis is translocated from the inner layer to the outer layer of the plasma membrane, being exposed to the cell surface. PI is used in conjunction with AV since it is intercalated in DNA in cells that have impaired plasma membrane integrity . This dom), the fluorescent probe JC-1  was used. JC-1 is a lipophilic and cationic dye with the ability to form aggregates (A) or monomers (M), depending on the state of Δψm. The lower the aggregates/monomers (A/M) ratio, the lower the Δψm, which indicates mitochondrial dysfunction [6 cells were labeled with 5 µg/mL of the JC-1 probe  for 15 min at 37 °C, in the dark. Analysis was performed on a flow cytometer , using the excitation wavelength of 590 nm for the aggregates and 530 nm for the monomers. Results are presented as the variation in the ratio of the aggregates/monomers (A/M) fluorescence intensities, normalized to the control.To evaluate the effect of CAP on the potential of the mitochondrial membrane , which is internalized in the cells and accumulates preferentially in the cytosol. After deacetylation by intracellular esterases and oxidation in the presence of peroxide, a fluorescent product is formed 2′,7′-dichlorofluorescein (DCF) [The quantification of intracellular peroxides was carried out through the intracellular oxidation of the non-fluorescent probe DCFHin (DCF) ,41. To ein (DCF) . To dete5 cells were centrifuged for 5 min at 1300× g. Then the cells were washed with PBS, centrifuged again for 5 min at 1300× g and 1 mL of PBS was added. The cells were incubated with 5 µM of the probe DCFH2-DA  for 45 min or with 2 µM of the probe DHE  for 15 min or with 1 μM of the probe DAF-FM Diacetate  for 1 h at 37 °C, in the dark. Finally, the cells were washed with PBS, by centrifugation for 5 min at 1300× g. The reading was performed on a spectrophotometer  with excitation wavelengths of 485 nm and emission of 528 nm for DCFH2-DA, 530 nm and emission of 645 nm for DHE and 495 nm and emission of 515 nm for DAF-FM Diacetate. Results are presented as the mean of the fluorescence intensities (MFIs) normalized to the control.Upon 2, 6, and 24 h after CAP treatment, 5 × 10The hydroxyl radical was indirectly evaluated through the use of an inhibitor, D-mannitol, a scavenger for the hydroxyl radical ,44. The 5 cells were centrifuged for 5 min at 1300× g. Then cells were washed with PBS, centrifuged again for 5 min at 1300× g and 1 mL of PBS was added. The cells were incubated with 10 μM of orange mercury  for 15 min at 37 °C, in the dark. Finally, the cells were centrifuged for 5 min at 1300× g and the reading was performed on a spectrophotometer  with excitation wavelengths of 485 nm and emission of 590 nm. The results are presented as the MFIs normalized to the control.To determine the intracellular content of reduced glutathione (GSH), a non-enzymatic antioxidant defense, the fluorescent orange mercury compound was used. The probe reacts quickly with GSH, giving rise to a compound that emits fluorescence . Upon 2,Statistical analysis was performed using GraphPad Prism software version 7.00 for Windows . The evaluation of the normality of distribution of the quantitative variables was carried out according to the normality test of D’Agostino Pearson. Parametric tests were used in case of a normal distribution and non-parametric tests were used otherwise. Outliers were identified using the ROUT method. The comparison of the different groups of exposure times to CAP was made according to the one-factor Analysis of Variance (ANOVA) test  or according to the Kruskal Wallis test (otherwise). For multiple comparisons, in case of normal distribution, Dunnett test was used to compare each mean of the exposure time groups with the mean of the control group and Tukey test to compare all the means of all groups with each other, in case of non-normal distribution it was used Dunn’s test. To compare the different times after CAP treatment considering the different exposure times, the two-way ANOVA test was used, using the Tukey test for multiple comparisons. A significance of 0.05 was considered for all comparisons.In this study, we investigated the effects of CAP in two bladder cancer cell lines, the HT-1376 (grade III) and the TCCSUP (grade IV). Our focus was to assess cell proliferation, cell viability, and cell death profile, as well as the changes in the cell cycle, the mitochondrial membrane potential, and oxidative stress. In this context, we evaluated the reactive oxygen and nitrogen species involved in the CAP mechanism of action. To make it clearer, from now on, the period of time to which the cells are exposed to treatment with CAP will be called “exposure time,” and the period of time between the end of the exposure to CAP until the evaluation of any parameter at the cellular level will be called “time after CAP treatment.”In order to evaluate whether CAP inhibited cell proliferation, the two bladder cancer cell lines were exposed to CAP treatment for 15, 30, 60, 90, and 120 s. MTT and SRB assays were performed 24, 48, and 72 h after CAP treatment.In the MTT assay, the metabolic activity decreased in both cell lines depending on the exposure time and time after CAP treatment, as shown in p = 0.0002) decrease in metabolic activity with a 120 s of exposure time in the HT-1376 cell line (p < 0.0001) decrease in metabolic activity with an exposure equal to or higher than 60 s  and 120 (p < 0.0001) seconds in the case of the HT-1376 cell line (p = 0.0096) or higher than (p < 0.0001) 15 s for TCCSUP cell line (p < 0.0001) and 0.91 ± 0.15% (p < 0.0001), respectively. For 48 h after CAP treatment, the longer the exposure time, the lower the metabolic activity with differences statistically significant for exposure times of 90 (ell line a and expell line b. The 90p = 0.0040) or higher than (p < 0.0001) 15 s of exposure (p < 0.0001). In the TCCSUP cell line, 15 s of exposure was enough to significantly (p < 0.0001) decrease the cells metabolic activity (p < 0.0001).Likewise, for 72 h after CAP treatment, in the HT-1376 cell line, an exposure time-dependent response was observed, with differences statistically significant for exposure times equal to (exposure a. With 1activity b. For exp < 0.0001) decrease in cell metabolic activity compared with the 24 and 48 h after CAP treatment. The TCCSUP cell line shows to be more sensitive to CAP treatment since, only 24 h after CAP treatment and with an exposure time of 90 s, its metabolic activity is reduced by more than 50%. Upon 48 and 72 h after CAP treatment and with exposure times of 60 (for 72 h after CAP treatment), 90 and 120 s (for 48 and 72 h after CAP treatment), the metabolic activity decreases to values close to 0%  or higher than (p < 0.0001) 30 s of exposure  decreased only with an exposure time of 120 s (p < 0.0001) for all exposure times (p < 0.0001) and 4.81 ± 0.26% (p < 0.0001) respectively.For 48 h after CAP treatment, the protein content of the HT-1376 cell line significantly (of 120 s c. Meanwhre times d. The 90p = 0.0077) or higher than (p < 0.0001) 30 s of exposure in the HT-1376 cell line (p < 0.0001). As for the TCCSUP cell line, all exposure times significantly (p < 0.0001) decreased the protein content to less than 50%  or higher than (p < 0.0001) 30 s for HT-1376 cell line (p < 0.0001) for TCCSUP cell line (p < 0.0001), as well as the necrotic cells (p = 0.0040) significantly increased with 120 s of exposure time (p = 0.0495) or higher than (p = 0.0003) 60 s, the percentage of cells in late apoptosis significantly increased, as well as the percentage of cells in necrosis (p = 0.0027) for 120 s of exposure time  increase of cells in the S phase for all exposure times (p < 0.0001) increase of cells in the S phase for exposure times of 30 and 60 s. For 120 s the cells in S phase slightly increase (p = 0.0246) compared to control. In addition, in the TCCSUP cell line, after 120 s of CAP treatment, there is a tendency for an increase in the pre-G0/G1 apoptotic peak (As shown in re times c. Regardtic peak d.m) in the two cell lines were determined by flow cytometry, 24 h after CAP treatment, using the probe JC-1. The cells were exposed to CAP treatment for 30, 60, and 120 s. It should be noted that the lower the aggregates/monomers (A/M) ratio, the lower the Δψm, which indicates mitochondrial dysfunction. In order to understand whether CAP induces mitochondrial dysfunction, the effects of CAP in the mitochondrial membrane potential  seconds in the case of the HT-1376 cell line and for all exposure times (p < 0.0001) in the TCCSUP cell line  also decreased the Δψm 2 times. Furthermore, 120 s of exposure drastically decreased the Δψm 5.9 times in the TCCSUP cell line. In both cell lines, the longer the exposure time, the lower the Δψell line e. The TCIn order to evaluate the alterations induced by CAP treatment in the oxidative stress by reactive oxygen and nitrogen species involved in the CAP mechanism of action, the two bladder cancer cell lines were exposed to CAP treatment for 30, 60, and 120 s. The intracellular content of peroxides, superoxide radical, as well as GSH, and nitric oxide were evaluated 2, 6, and 24 h after CAP treatment. In addition, the influence of hydroxyl radical was evaluated 24 h after CAP treatment through the presence of an inhibitor of this reactive specie, D-mannitol.p = 0.0320) increased compared to control with 30 s of exposure and 2 h after CAP treatment. However, for longer exposure times and longer times after CAP treatment, intracellular content of peroxides significantly (p < 0.05) decreased  decreased compared to control with 60 and 120 s of exposure. For 24 h after CAP treatment and with all exposure times, there was a significant (p < 0.0001) decrease to approximately half of the intracellular content of peroxides, comparing with control  increased compared to control with 60 s of exposure and 2 h after CAP treatment, while no statistical differences were observed in the remaining exposure times and times after CAP treatment  and 120 (p < 0.01) seconds relative to control. However, for the longest time after CAP treatment (24 h), superoxide levels significantly decreased (p = 0.0480) compared to control with 120 s of exposure. , the longer the exposure time, the higher the intracellular content of superoxide radical with differences statistically significant for exposure times of 60  increased compared to control with 60 and 120 s of exposure and with both 2 and 6 h after CAP treatment, while for 24 h after CAP treatment no differences were observed  decreased compared to control with 60 and 120 s of exposure and with both 6 and 24 h after CAP treatment, while for 2 h after CAP treatment no differences were observed  decreased the metabolic activity with 60 and 120 s of exposure time compared to CAP treatment alone (p < 0.05), but with 30, 60, and 120 s of exposure time  (https://cancer.sanger.ac.uk/cell_lines) both cell lines used express RB1 and TP53 mutations. RB1 and P53 proteins are involved in major signaling pathways that regulate G1 phase, influencing the decision on whether or not a cell enters in S phase [After evaluating the alterations on the cell cycle, CAP treatment induced cell cycle arrest in the S phase in both cell lines. Vandamme et al. as already demonstrated cell cycle arrest in S and GM phases . Volotskreatment ,68. Inte S phase . However S phase . The cel S phase . TCCSUP As previously reported, CAP treatment induces depolarization of the mitochondrial membrane and thus mitochondria-mediated cell death ,71. In oCAP has the ability to selectively induce cell death in cancer cells, in detriment of the normal ones ,17,72,73In the present study, this imbalance in the cellular stress caused by CAP was studied in order to understand which RONS plays a key role in the CAP mechanism of action. We observed that the intracellular content of peroxides decreased in both cell lines depending on the exposure time and time after CAP treatment, contrary to what has been reported by other authors, that have seen an increase in the intracellular content of peroxides after CAP treatment ,75. ThisOn the other hand, the intracellular content of superoxide radical increased for shorter times after CAP treatment in both cell lines, being more evident in the TCCSUP cells. These results demonstrate that the superoxide radical may be an important player in the CAP mechanism of action. Superoxide radical can be converted into hydrogen peroxide or can react with nitric oxide and form peroxynitrite radical that is a powerful oxidant capable of inducing membrane damage, DNA fragmentation, lipid peroxidation, cytochrome C release, and, as a consequence, apoptosis and necrosis ,76,77.Regarding nitric oxide, our results demonstrate an increase in the intracellular content of nitric oxide in the HT-1376 cells for shorter times after CAP treatment, while in the TCCSUP cells there was a decrease for longer times after CAP treatment. On one hand, as mentioned above, nitric oxide can be involved in the formation of peroxynitrite radical, and, on the other hand, it can have antitumor effects itself, but the mechanisms by which nitric oxide exerts its cytotoxic effects are not entirely clear . Xia et After evaluating the intracellular content of GSH, an antioxidant defense, the results demonstrated no differences between control and CAP exposure times, for any time after CAP treatment. In contrast, Ishaq et al. demonstrated, in melanoma cells, a decrease in the intracellular content of GSH depending on the CAP exposure time . These rTaken into account the complexity involved in the cell oxidative stress, it is important and mandatory to evaluate other reactive species like the peroxynitrite radical mentioned before, and the other antioxidant defenses, in order to understand how the imbalance in the oxidative stress caused by CAP treatment mediates cell death response. In addition to assessing intracellular content, it is of utmost importance to evaluate the presence of RONS in the extracellular environment in order to comprehend which RONS are delivered by CAP to the cells and which ones play a key role in the CAP mechanism of action.So far, the present study is the first one to study the effects of CAP treatment in two different bladder cancer cell lines and to demonstrate the greater sensitivity of the higher-grade cell line to CAP treatment. However, more detailed studies highlighting the effects of CAP in bladder cancer and normal cells are needed, in order to completely understand the whole picture of CAP treatment against bladder cancer. With good results, in the future, CAP-based therapies might be an alternative or adjunctive to the actual standard of care. Their applications may be held by different ways. For example, PAM could have a place as sole adjuvant treatment, or as a replacement to saline in the surgical irrigation, or even in the post-operative irrigation. On the other hand, CAP could be applied after a cystectomy or a nephroureterectomy, directly on the surgical bed, to treat microscopic positive margins.In our work, we demonstrated the effects of CAP on two bladder cancer cell lines, corresponding to two different stages of the disease. We found a decrease in metabolic activity and protein content for both cell lines. Additionally, there was a significant decrease in cell viability and a cell cycle arrest in both cell lines, mainly in the S phase. One of the main mechanisms of action described for CAP is RNOS production. In the present work, we observed an increase in superoxide and nitric oxide, without changes in GSH levels and with a decrease in peroxide values. The conversion of these reactive species to others such as peroxynitrite is very likely. Another CAP known mechanism of action is the decrease in the mitochondrial membrane potential, which occurred significantly in both cell lines. Some of the effects described are time- (both exposure time and time after CAP treatment) dependent.There is a need to develop new treatments for bladder tumors. CAP-based therapies may be an alternative in the future. This work is one of the firsts to demonstrate possible antitumoral effects of CAP in urothelial neoplasms, being more pronounced in the more advanced disease cell line (TCCSUP). More works are needed to know better the antitumoral mechanisms and to establish the optimal exposure time to each stage of the disease.


R ranging from 0.35 to 0.54. ANBL-6 was the “best” line, markedly exceeding all others (p < 2.2e−16). Notably, some widely used cell lines  scored poorly in our patient similarity ranking . Lines cultured with interleukin-6 showed significantly improved correlations with patient tumor (p = 9.5e−4). When common MM genomic features were matched between cell lines and patients, only t and t led to increased transcriptional correlation. To demonstrate the utility of our top-ranked line for preclinical studies, we showed that intravenously implanted ANBL-6 proliferates in hematopoietic organs in immunocompromised mice. Overall, our large-scale quantitative correlation analysis, utilizing emerging datasets, provides a resource informing the MM community of cell lines that may be most reliable for modeling patient disease while also elucidating biological differences between cell lines and tumors.Multiple myeloma (MM) cell lines are routinely used to model the disease. However, a long-standing question is how well these cell lines truly represent tumor cells in patients. Here, we employ a recently described method of transcriptional correlation profiling to compare similarity of 66 MM cell lines to 779 newly diagnosed MM patient tumors. We found that individual MM lines differ significantly with respect to patient tumor representation, with median  The past 20 years have seen remarkable advances in multiple myeloma (MM) biology and therapy. Many of these discoveries originated with studies performed in MM cell lines. However, there are long-standing questions about the reliability of MM cell lines as models for true disease in patients. One major issue is that while the large majority of MM tumor cells reside within the bone marrow niche, essentially all MM cell lines have been derived from disease growing outside the bone marrow . These pDespite these caveats, cell lines remain the workhorse of MM research. To mitigate these limitations, several groups have developed cell lines that remain dependent on interleukin-6 (IL-6) in culture media , 2, 6. ITaken together, significant questions remain about both the qualitative and quantitative differences between MM cell lines and patient tumors. Furthermore, it remains unclear whether specific cell lines are more representative of patient tumors than others. Here, we aim to address these questions. Our work extends from our recent study , where wwww.keatslab.org) and MM patient tumors within the MMRF CoMMpass study (research.themmrf.org). Here, we employed our transcriptional correlation profiling approach to perform 51,414 individual correlations of cell lines versus patient tumor. We confirmed that MM cell lines and patient tumors display broad transcriptomic differences. However, we did identify cell lines, in particular ANBL-6, that appear to be more representative of patient disease than others. In contrast, some widely used lines scored relatively poorly in our ranking of similarity to patients. We further characterized additional features to aid in increasing similarity of cell lines to patient tumor. Here, we provide a resource for cell line selection in MM research while also elucidating underlying biological signatures distinguishing cell lines and patient tumors.As the TCGA primarily includes data on solid tumors, our prior publication did not include MM. Fortunately, the Multiple Myeloma Research Foundation (MMRF) has addressed this gap in knowledge. The MMRF has sponsored a comprehensive transcriptomic resource of MM cell lines (www.keatslab.org) and CoMMpass IA13 patient datasets (research.themmrf.org) and normalized via variance stabilizing transformation [See details of analysis in Supplementary Methods. Briefly, annotated read count data were obtained from Keats lab cell line (ormation . The topormation .scid gamma (NSG) mice. Tumor burden was monitored by bioluminescent imaging.See details of analysis in Supplementary Methods. Briefly, ANBL-6 cell lines were stably transduced with a lentiviral construct that stably expressed enhanced firefly luciferase and was implanted into female 6–8 week old NOD We began by obtaining RNA-seq read count data for 66 MM cell lines (Keats lab resource) and CD138+ enriched tumor cells from 779 newly diagnosed MM patients (CoMMpass release IA13). As in our prior study  (see “MaR = 1) means that a cell line is an exact representation of the patient tumor. We show individual correlation plots in Fig. As in our prior study, we focused our analysis on the top 5000 most variable genes across samples expressed consistently at >1 counts per million, with the reasoning that these genes are most likely to be biologically informative for similarity assessment (see Supplementary Methods). A workflow for our analysis is shown in Fig. R when correlated versus each patient in the CoMMpass database. We can draw some initial conclusions from this dataset. First, it is clear that none of the MM cell lines approached a perfect representation of patient tumor, as the median R values range from 0.35 to 0.54 . Consistent with this conclusion, principal component analysis of overall transcript expression demonstrated that MM cell lines form a distinct cluster from patient tumors . In parallel, the cell lines MMM.1 and FR4 appeared markedly below other lines .The violin plots in Fig. ors Fig. . Second,p < 2.2e−16). Similarly, MMM.1 and FR4 led to significantly worse correlations versus all other lines  than others. Notably, we compared our cell line rankings to the frequency of use of MM cell lines in the literature  Fig. . As expeDecades of anecdotal experience have suggested that cell lines may demonstrate phenotypic “drift” when cultured in different laboratories. Recent large-scale, multiomic studies have systematically confirmed and quantified these effects , 19. TheWe were encouraged to find strong consistency between the rankings generated from both cell line datasets Fig. . Upon vip = 9.5e−4) , we do note that lines cultured in IL-6 show a clear enrichment in the top half of the rankings . This finding suggests that coculture with critical microenvironment factors can at least partially drive cell lines to a more patient-like phenotype, even if not strictly required for cell growth.We noted that ANBL-6, our top-ranked line, was initially characterized as being dependent on IL-6 . We ther−4) Fig. . While tngs Fig. . Furtherp = 0.0014 and 1.7e−5 versus stage 2 or stage 1, respectively). Similarly, patients with higher M spike at baseline also showed greater tumor similarity to cell lines  capacity. As a corollary, patients with more aggressive disease may therefore have tumors with more similarity to cell lines. To test this, we first analyzed newly diagnosed patients in CoMMpass based on international staging system (ISS) stage at diagnosis Fig. . We indenes Fig. .n = 81) and obtained their overall transcriptional correlations to all cell lines, in comparison with our prior newly diagnosed analysis (n = 779). Here we also found significantly increased correlation with the relapsed versus newly diagnosed patients (p = 0.0015)  Fig. . Taken tWhile these results help clarify the basis of MM cell line phenotypes, we next used Gene Set Enrichment Analysis to further delineate biological signatures that mostly distinguish cell lines and patient tumors Fig. . We founFurthermore, it has been reported that MM cell lines frequently downregulate immunoglobulin synthesis compared with patient tumors , 23. We Over the past 10 years, large-scale whole genome and whole exome sequencing studies have revealed numerous mutations found recurrently in MM –30. ThesKRAS and NRAS, are mutated at similar frequency in both cell lines and patient samples  demonstrating consistency between these key sequence variants. TP53 mutations were markedly more common in cell lines (55.9% versus 4.1% in patients), potentially consistent with the more aggressive growth phenotype of cells in vitro. Other commonly mutated genes in patient tumors, as characterized by Walker et al. [Here we took advantage of whole exome sequencing data in CoMMpass and the Keats lab cell line database to investigate the relative frequency of mutations in both sample sets Fig. . We firsNRAS and KRAS (codons 12/13/61)—improved global transcriptomic correlations when matched between cell lines with patients carrying the same lesion. Our analysis confirmed that matching of t and t cell lines indeed improved correlations to patients with the same alteration as compared with those without   or activating RAS mutations , , and , as well activating mutations of ly) Fig. , left. Hons Fig. , right. t(14:16)/t and t, respectively. As indicated by several matches with a false discovery rate >0.05, we found a weaker match of any cell lines to the CD-1 subtype, characterized by t disease. We also noted several cell lines best matching to the “proliferative” subtype. However, in contrast to patient tumors, almost all cell lines showed at least moderate expression of the PR signature, potentially consistent with the ability to propagate rapidly in vitro. Finally, we noted that none of the cell lines showed strong matches to the “low bone” (LB), CD-2, and hyperdiploid (HP) gene expression signatures. The latter finding particularly emphasizes that no MM lines well recapitulate the common HP genotype. This result underscores a major difference between available in vitro MM models and in vivo disease, while also suggesting that HP disease may be highly dependent on the tumor microenvironment.Microarray-based transcriptome profiling by Zhan et al.  previoust and t/t signatures, similar to that noted above, but did not identify other prominent drivers of similarity across the landscape. We did note, however, that ANBL-6 clustered closely with other highly ranking cell lines MM.1S, MM.1R, ALMC-1, and ALMC-2, suggesting that these lines may carry a common transcriptional signature driving similarity to patient tumor.We further assessed whether unbiased hierarchical clustering of MM cell line transcriptomes across the 5000 genes used in Fig. Our overall rankings Fig.  suggest scid gamma (NSG) mice, may carry significant advantages for preclinical modeling if tumor cells home to hematopoietic tissues including bone marrow [Furthermore, while ANBL-6 has been used in many prior studies Fig. , the vase marrow . In thisTo our knowledge, it has not been tested whether ANBL-6 homes to hematopoietic tissues in a disseminated mouse model after IV implant. We therefore used lentiviral transduction to stably express luciferase in ANBL-6 cells and injected 1e6 cells into a pilot cohort of four NSG mice. In parallel, we injected 1e6 luciferase-labeled MM.1S cells into a separate cohort as a control, as these cells are well known to home to bone marrow in NSG mice . EncouraHere we present the first large-scale quantitative comparison of MM cell lines and primary patient tumors. Our results, using global transcriptome profiles as a proxy for overall biological state, quantitatively confirm long-standing suspicions that MM cell lines are indeed very different than patient tumors. Through these analyses, we describe biological factors that drive increased or decreased similarity between cell lines and patients, as well as outline strategies to potentially improve the quality of in vitro studies toward representing in vivo patient disease.One potential explanation for the overall low correlations between cell lines and patient transcriptomes is that cell lines are typically derived from multiply relapsed, extra-medullary samples that have lost dependency on the bone marrow microenvironment. This biological setting is a significant contrast to the newly diagnosed, marrow-based disease included in CoMMpass. Against this background, though, we argue that it is still certainly possible to improve the quality and relevance of in vitro studies by incorporating our rankings and other findings here. Even though many of our comparative analyses  show relatively modest absolute increases in global transcriptional correlation, these increases remain highly statistically significant and involve biologically relevant changes over hundreds of genes.We note that our analysis here is only enabled by recent, large-scale RNA sequencing-based studies in MM. Prior microarray-based expression profiling analyses of patient samples , for exaSimilar to our prior pan-cancer analysis of the TCGA and CCLE , here weNRAS and KRAS sequences [Our results specifically indicate that the cell line ANBL-6 sits significantly above all other cell lines in terms of patient similarity. ANBL-6 was isolated from peripheral blood of a relapsed MM patient and initially characterized as having typical malignant plasma cell immunophenotype, a  translocation, and lambda light chain secretion , and latequences , 33. We equences , therebyThe overall conclusions here will not necessarily be surprising to MM researchers given the years of anecdotal experience and knowledge of MM biology. However, like recent studies systematically investigating differences in cell line phenotype across different laboratories , 19, theSupplementary MaterialsDataset S2Dataset S1


MDM2/MDMX proteins are frequently elevated in hormone receptor-positive (ER+) breast cancer. We sought to determine the antitumor efficacy of the combination of ALRN-6924, a dual inhibitor of MDM2/MDMX, with chemotherapy in ER+ breast cancer models.TP53 status in ALRN-6924 efficacy. ER+ breast cancer cell lines (MCF-7 and ZR-75-1) were used to investigate the antitumor efficacy of ALRN-6924 combination. In vitro cell proliferation, cell cycle, and apoptosis assays were performed. Xenograft tumor volumes were measured, and reverse-phase protein array (RPPA), immunohistochemistry (IHC), and TUNEL assay of tumor tissues were performed to evaluate the in vivo pharmacodynamic effects of ALRN-6924 with paclitaxel.Three hundred two cell lines representing multiple tumor types were screened to confirm the role of TP53 (WT-TP53) cancer cell lines, but not mutant TP53. On ER+ breast cancer cell lines, it was synergistic in vitro and had enhanced in vivo antitumor activity with both paclitaxel and eribulin. Flow cytometry revealed signs of mitotic crisis in all treatment groups; however, S phase was only decreased in MCF-7 single agent and combinatorial ALRN-6924 arms. RPPA and IHC demonstrated an increase in p21 expression in both combinatorial and single agent ALRN-6924 in vivo treatment groups. Apoptotic assays revealed a significantly enhanced in vivo apoptotic rate in ALRN-6924 combined with paclitaxel treatment arm compared to either single agent.ALRN-6924 was active in wild-type The significant synergy observed with ALRN-6924 in combination with chemotherapeutic agents supports further evaluation in patients with hormone receptor-positive breast cancer.The online version contains supplementary material available at 10.1186/s13058-021-01406-x. TP53 remains the most frequently mutated gene observed in human malignancy [TP53 gene or through oncogenic activity of the E3 ubiquitin-protein ligase MDM2 [Key functions of the cellular tumor suppressor p53 are to prevent the development of malignancy and stop the growth and expansion of abnormal cells through repair of DNA damage, arresting cell cycle, induction of apoptosis, and controlling angiogenesis . During lignancy . p53 is ase MDM2 . Two difase MDM2 –7.MDM2 gene and/or overexpression of its protein have been found to be a main driver of malignancy in many tumor types, especially in tumors with retained wild-type TP53 status (WT TP53) [TP53 status [TP53 status but this differs across tumor histologies [TP53 tumors [Amplification of the WT TP53) . A previWT TP53)  and majo3 status . Additiotologies . MDM2 intologies  and weretologies . They hatologies . Interestologies . ALRN-693 tumors –18. ALRN3 tumors .Paclitaxel is one of the most commonly used standard of care chemotherapeutic agents in breast cancer patients . It induTP53 status by testing cell lines in vitro and in vivo. Our findings indicate that ALRN-6924 acts synergistically with both paclitaxel and eribulin in preclinical breast cancer models.Resistance to chemotherapeutic agents is a major concern when treating breast cancer patients, especially with taxanes . MDM2 isALRN-6924 was kindly provided by Aileron Therapeutics Inc. . RG7112 and RG7388 (idasanutlin) and paclitaxel were purchased for in vitro studies from MedChemExpress . For in vivo paclitaxel efficacy studies, the agent was obtained from MD Anderson Cancer Center pharmacy. This source of paclitaxel was later changed to the National Cancer Institute for pharmacodynamic experiments. Paclitaxel was prepared in 15% ethanol, 15% kolliphor , and water for in vivo studies. Eribulin was obtained from the MD Anderson Cancer Center pharmacy. Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich . All drugs used for in vitro studies were initially prepared in DMSO prior to dilution in a tissue culture medium.n = 233 cell lines) and a focused Horizons Discovery OncoSignature panel , were employed. Total 302 tested cell lines representing multiple solid and hematologic tumor types were composed of 100 cell lines with WT TP53 and 202 cell lines with mutant or null TP53  or ATP levels were measured by adding Perkin Elmer ATPLite (Horizons). Relative cell proliferation was assessed by changes in nuclear dye uptake or ATP levels compared to the untreated cell plate and presented as the effective concentration to inhibit 50% of cell proliferation (IC50).Two commercial cancer cell line panels, the Eurofins OncoPanel . These ER+ breast cancer cell lines were obtained from American Tissue Culture Collection . Except for ZR-75-1 cells that were cultured in RPMI-1640 medium, other cell lines were cultured in Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% fetal bovine serum (FBS) at 37 °C and humidified in 5% CO2. Cells were seeded in 96-well plates overnight at a density of 5000–10,000 cells per well based on cell line growth characteristics. Cells were treated with ALRN-6924 single agent in a dose range of 0.64–50,000 nM for 3 days. Sulforhodamine B staining (SRB) assay was performed to determine cell survival rate [50 was determined based on the sigmoid drug-inhibition curve using GraphPad Prism v7.03 software.In an independent screen of ER+ breast cancer cell lines, 6 cell lines were used, including MCF-7, ZR-75-1 (WT TP53), and HCC-1954, SK-BR-3, BT-474, CAMA1 . Then we had 5 serial dilutions at 5-fold from the highest combo doses. In the combination of ALRN-6924 with eribulin, 5000 nM and 80 nM were chosen as the highest doses respectively . To evaluate combination efficacy, a combination index (CI) was determined using the Chou-Talalay drug combination model (CI < 1.0 (curve left-shift): synergistic; CI = 1.0, additive; CI > 1.0 (curve right-shift): antagonistic). Experiments were performed in triplicate and repeated three times.Two ER+ breast cancer cell lines with WT 3 for MCF-7 and 5 × 103 for ZR-75-1 per well. Drug treatment started following 24 h of incubation, with changing of culture media and drugs at least twice a week. Each treatment group was tested in duplicate. Culturing and treatment of cells continued for 2–3 weeks. At the end of the experiment, the colonies were fixed in 10% buffered formalin and stained with 0.05% crystal violet in 25% methanol. Total colony area was measured using NIH ImageJ v1.52a software .Cells were seeded in 60-mm plates at a density of 1 × 10Pierce BCA protein assay kit  was utilized to obtain protein concentration of cell lysates. Membranes were blocked with 0.1% casein blocking buffer following separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer of proteins onto a 0.2-μm nitrocellulose membrane . Next, immunoblotting was performed by probing membranes with antibodies (anti-Mdm2 (#86934), anti-p53 (#9282), anti-p21 (#2947), anti-p-Histone-H3 (Ser10) (#29237), anti-c-Myc (#9402), anti-LC3A/B (#12741), anti-p-Rb (Ser807/811) (#8516), anti-Rb (#9309), anti-p-S6 (Ser235/236) (#4858), and anti-S6 (#2217)). All described antibodies were purchased from Cell Signaling Technology . Anti-β-actin antibody (#A5441) was purchased from Sigma-Aldrich . Immunoblotting signals were visualized by Odyssey IR imaging system . Image Studio software v4.0  was used for the analysis of the bands.To evaluate for apoptosis, we utilized the Annexin V Apoptosis kit . Cells were treated for 72 h with either vehicle, ALRN-6924 10 μM, paclitaxel 1 μM, or combination of both followed by collection of both floating and attached cells. These concentrations were chosen because of findings of p53 reactivation and apoptosis on immunoblotting, respectively. Cells were then probed with both Annexin V and propidium iodide (PI). Labeled cells were analyzed by flow cytometry in the MDACC Flow Cytometry and Cellular Imaging Core. Annexin V-positive cells were counted.To determine the effects of the combination of ALRN-6924 and paclitaxel on the cell cycle, we used MCF-7 and ZR-75-1 cell lines. Cells were plated and treated the following day in duplicates for 24 h with either vehicle, ALRN-6924 10 μM, paclitaxel 1 μM, or combination of both. Samples were collected, fixed, stained with PI  according to manufacturer protocol, and analyzed by flow cytometry in the MDACC Flow Cytometry and Cellular Imaging Core Facility. We evaluated the percentages of cells in each cell cycle phase: subG1, G0-G1, S, and G2-M.nu/nu mice  at a density of 5 × 106 in both MCF-7 and ZR-75-1 xenograft models. All mice were implanted with 60-day 17β-estradiol pellets  subcutaneously prior to inoculation of the tumor. Treatments began when tumor diameter reached between 150 and 200 mm3. The mice were euthanized at the end point of the study or when tumor diameter reached 1500 mm3.All animal experiments were approved by the Animal Care and Use Committee of MD Anderson. Cells were inoculated into the 4th mammary fat pad of 6- to 8-week-old female athymic BALB/c n = 6–8 in MCF-7, and n = 5 in ZR-75-1 xenograft combination therapy experiment). ALRN-6924  was dosed twice weekly and paclitaxel (10 mg/kg) was dosed weekly, with paclitaxel administered 6 h prior to ALRN-6924. All drugs were administered intravenously via the tail vein over a 26–28-day treatment window.In order to determine in vivo antitumor efficacy of ALRN-6924 in combination with paclitaxel, mice were randomized into 6 groups  was conducted by the MD Anderson Functional Proteomics core facility as previously described . DiffereXenograft tumor samples were formalin-fixed and paraffin-embedded (FFPE). Tumor tissue on hematoxylin and eosin (H&E)-stained slides was confirmed by a board-certified pathologist. Additionally, the presence of tumor necrosis was estimated and given as percentage of the entire tumor.2) of positive tumor cells were evaluated for TUNEL staining. For digital image analysis, IHC slides were scanned at 20× using Aperio AT2 scanner (Leica Biosystems), and the images were analyzed in HALO software (Indica Labs), using a cytonuclear algorithm. Distinguished necrotic areas and artifacts were excluded. All specimens but one (p21: insufficient staining) could be evaluated for IHC. The IHC evaluation was performed by a board-certified pathologist.Immunohistochemistry (IHC) was performed on 4-μm sections using the following antibodies: anti-Ki67 , anti-cleaved PARP (Asp214) , anti-p21 , anti-p53 , and anti-TUNEL . For Ki67, p-Histone-H3, and cleaved PARP, the percentage of all positive tumor cells were estimated regardless of staining intensity. The percentage of positive tumor cells and their staining intensity were evaluated for p21 and p53 resulting in an H-score (range 0–300) . TUNEL sUnsupervised hierarchical cluster analysis was performed in order to generate MCF-7 heat maps of differentially expressed proteins (DEPs) with false discovery rate (FDR) 0.05 (ALRN-6924 vs vehicle and ALRN-6924 plus paclitaxel vs paclitaxel).t-test was carried out for comparison between two groups.For in vitro studies, Student’s Treatment/control ratio (T/C %) was performed day 27 or day 29, depending on the study by using the formula: (median tumor volume of treatment group/median tumor volume of control) × 100. Antitumor activity was defined as ≤ 40% [3) = ((width)2 × length)/2. Change in tumor volume was obtained by using the formula: (TVDayX − TVDay0)/TVDay0. One-way ANOVA was carried out for in vivo studies in order to determine statistical significance.Tumor volume (TV) was obtained by using the formula: TV  compared to single agent treatment alone in both cell lines , but this was not enhanced compared to the effect of ALRN-6924 monotherapy. Apoptosis was also enhanced with the combination compared to vehicle in MCF-7 cells, but this was not greater than observed in paclitaxel alone  compared to the vehicle control  at Ser10 residue is a cell cycle marker of the G2-M phase and is associated with chromosome condensation during mitosis . Interesp = 0.003, p = 0.005, and p = 0.03 respectively)  with paclitaxel (10 mg/kg) and combination of ALRN-6924 (10 mg/kg) with eribulin (0.3 mg/kg) effectively inhibited tumor growth and had superior antitumor efficacy compared to single agents alone (p = 0.01) , and demonstrated greater accuracy . p-HH3 lIn a separate study, we examined the target specificity of MDM2/X inhibitors. We created MDMX knockout cell lines in OCI-AML3 cells Fig. SA, B. TheAnother question we have not specifically addressed is the effect of ALRN-6924 on the tumor microenvironment. Cell line-derived xenografts have mouse stroma. They are not the optimal model to assess the effect of ALRN-6924 on the tumor microenvironment. Further, our RPPA assay has been optimized to detect human markers and is focused on tumor markers, and thus, our current studies are not conducive to address this question. Therefore, we plan to test the effect of ALRN-6924 on the tumor microenvironment in ongoing clinical trials.mTOR pathway is one of the classical canonical pathways involved in cancer cell proliferation and metastasis . EmerginAdditional mechanistic studies are required in order to explore and determine the interplay between ALRN-6924 and paclitaxel. The question also remains on how the combination of ALRN-6924 and paclitaxel or eribulin affect other additional p53-dependent and independent pathways, potentially revealing additional affected pathways.TP53 WT and mutant p53 (TP53 MT) can affect each other, one being “dominant-positive” effect, where TP53 WT activity suppresses TP53 MT activity, or “dominant-negative effect” where TP53 MT allele inactivates TP53 WT allele [TP53 MT and TP53 WT; further studies are needed in order to determine if this combination can rescue TP53 WT and suppress mutant p53 activity in order to enhance paclitaxel or eribulin sensitivity.Changes in a single residue by hot-spot mutations can impair p53 activity which transforms p53 into a gain-of-function oncoprotein . Two sceT allele . We did Mdm2 amplification [TP53 WT but also in TP53 MT tumors [Notably, our studies have been conducted in immunocompromised mice, not allowing us to assess the immune effects of Mdm4/Mdm2 inhibition. Recently, there have been anectodal reports of hyperprogression on immunotherapy in patients with fication . The rolfication , 53. In T tumors . Thus, fTP53 WT status. Reactivating p53 in order to enhance antitumor efficacy of chemotherapy is an attractive option. Mdm4 and Mdm2 proteins known to regulate TP53 levels are frequently elevated in hormone receptor-positive breast cancer. In this study, we demonstrated the antitumor efficacy of ALRN-6924, a dual inhibitor of Mdm4 and Mdm2, in ER+ breast cancer cell line models and identified significant synergy with combination of ALRN-6924 with chemotherapeutic agents. These results support the clinical trial of ALRN-6924 in combination with paclitaxel  in patients with advanced, metastatic, or unresectable solid tumors.A large portion of hormone receptor-positive breast cancer have Additional file 1: Supplementary Figure 1. ALRN-6924 combined with eribulin synergistically inhibit clonogenic capacity of MCF-7 breast cancer cell line. MCF-7 colony formation assay treated with either vehicle, ALRN-6924, paclitaxel, or combination of both. Total colony area was measured by using NIH ImageJ software. Mean +/- SD is demonstrated. Supplementary Figure 2. In vitro validation of in vivo functional proteomic changes after treatment with ALRN-6924 and paclitaxel. A. MCF-7 cells were treated with vehicle, ALRN-6924 or paclitaxel in a dose-dependent fashion for 24 hours followed by immunoblotting with indicated antibodies. B. Quantification of relative p53 levels by standardizing to β-actin. C. Quantification of relative p21 levels by standardizing to β-actin. D. Quantification of relative c-Myc levels by standardizing to β-actin. E. Quantification of relative p-Rb (Ser807/811) levels by standardizing to β-actin. F. Quantification of relative LC3-I/LC3-II ratio levels by standardizing to β-actin. G. Quantification of relative LC3 levels by standardizing to β-actin. H. Quantification of relative p-S6 (Ser235/236) levels by standardizing to β-actin. Supplementary Figure 3. Immunoblotting of signaling pathways affected by ALRN-6924 combined with paclitaxel. A. MCF-7 cells were treated with vehicle, ALRN-6924, paclitaxel or combination of both in a dose-dependent fashion for 24 hours followed by immunoblotting with indicated antibodies. B. Quantification of relative p53 levels by standardizing to β-actin. C. Quantification of relative p21 levels by standardizing to β-actin. D. Quantification of relative p-Rb (Ser807/811) levels by standardizing to β-actin. E. Quantification of relative LC3-I/LC3-II ratio levels by standardizing to β-actin. Supplementary Figure 4. MDMX knockout cell line models. A. Immunoblotting of MDMX. Two MDMX knockout clones  were established in Myeloid leukemia cell line OCI-AML3 cells. MDMX was blotted with anti-MDMX antibody. B. Cell proliferation assay. Cell proliferation of two MDMX knockout cell line clones and control cell line was examined using the same methods as described in Fig. Supplementary Table 1. Cell line panels for drug screening. Supplementary Table 2. IPA analysis of MCF-7 ALRN-6924 + Paclitaxel vs Control.


High RAC1B and/or low RAC1 abundance also correlated closely with a poor invasion potential, and vice versa, as revealed by measuring random cell migration (chemokinesis), the preferred mode of cellular movement in cells that have undergone mesenchymal transdifferentiation. We propose that a high RAC1B:RAC1 ratio in BC cells is predictive of an epithelial phenotype, while low RAC1B along with high RAC1 is a distinguishing feature of the mesenchymal state. The combined quantitative assessment of RAC1B and RAC1 in tumor biopsies of BC patients may represent a novel diagnostic tool for probing molecular subtype and eventually predict malignant potential of breast tumors.Breast cancer (BC) is a heterogenous disease encompassing tumors with different histomorphological phenotypes and transcriptionally defined subtypes. However, the non-mutational/epigenetic alterations that are associated with or causally involved in phenotype diversity or conversion remain to be elucidated. Data from the pancreatic cancer model have shown that the small GTPase RAC1 and its alternatively spliced isoform, RAC1B, antagonistically control epithelial–mesenchymal transition and cell motility induced by transforming growth factor β. Using a battery of established BC cell lines with either a well-differentiated epithelial or poorly differentiated mesenchymal phenotype, we observed subtype-specific protein expression of RAC1B and RAC1. While epithelial BC lines were RAC1B Breast cancer (BC) is a heterogeneous disease with at least twenty histological subtypes of invasive breast cancer, based on growth patterns and morphology, and three biological subtypes, characterized by the expression of estrogen receptor (ER) and progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), or the lack of all three receptors (triple-negative BC (TNBC)). The “omics“ technologies have confirmed and further stratified molecular heterogeneity of this disease into biologically and clinically meaningful subtypes, including at least six intrinsic subtypes  and four TNBC molecular subtypes  , and theCentral to the development of malignant breast cancer subtypes is the process of epithelial–mesenchymal transition (EMT) that endows cancer cells with the ability for invasion and metastasis, cancer stem cell generation, and drug resistance ,5. RecenCDH1) and inhibited that of mesenchymal and invasion-associated genes  [BC shares in common with pancreatic ductal adenocarcinoma (PDAC) a couple of similarities, i.e., the desmoplastic stroma and the marked subtype diversification into epithelial/classical/luminal, quasi-mesenchymal/squamous, and basal-like subtypes . Howeverd SNAI2) , enhanced SNAI2) ,10, and d SNAI2) ,10 and Td SNAI2)  cells. Od SNAI2) . Given tA total of eight breast cancer cell lines with different tumor origin and transcriptional subtype were employed: five invasive ductal carcinomas (IDCs)—BT-20 , MCF-7 , MDA-MB-435s , T-47D , and ZR-75-1 —and three adenocarcinomas—MDA-MB-231 , MDA-MB-468 , and SKBR-3 . MCF-7, T-47D, and ZR-75-1 cells are luminal A-type and ER and PR-positive, whereas SKBR-3 cells are HER2-positive . Among tThe procedure of quantitative real-time RT-PCR (qPCR) and the primer sequences used for amplification of RAC1B, RAC1, and GAPDH as an internal control, were described in detail elsewhere ,9,10. Thp < 0.05)-calculated with the unpaired two-tailed Student’s t test–denoted by bars above the respective cell lines. The antibodies used were Rac1b , E-cadherin (#610181) and Rac1 (#610650) , GAPDH , Vimentin , and HA .Cell lysis and immunoblotting were essentially performed as described previously ,9,10 wit® DP system  to measure random/spontaneous cell migration  according to previous descriptions [We employed the xCELLigenceriptions ,10,11. Eriptions ,9,10. MDriptions . BT-20 cBC cell lines with two different phenotypes, i.e., epithelial  and mesenchymal  as well as a cell line with an as yet poorly characterized phenotype BT-20) were employed for this study. Initially, the phenotypic classification was confirmed by immunoblotting of the epithelial marker E-cadherin and the mesenchymal marker vimentin, as it is well documented that loss of E-cadherin expression and concomitant increases in vimentin are signs of an EMT and hallmarks of BC progression. We observed that MCF-7, T-47D, and MDA-MB-468 cells were positive for E-cadherin and negative for vimentin, while the reverse was true for MDA-MB-231 and -435s cells . Our res were empThe six cell lines were then analyzed for expression of RAC1B and RAC1 by immunoblotting A. MCF-7,Given the finding of high RAC1B and low RAC1 expression in well-differentiated BC cell lines, and vice versa, we addressed the question of whether the ratio of both isoforms is predictive of their invasive potential. Using impedance-based xCELLigence technology, we monitored in real-time the rate of random migratory activity (chemokinesis) as this is associated with—and therefore a good readout for—the mesenchymal/c-EMT phenotype. As strong constitutive chemokinetic activity of both MDA-MB-231 and -435s cells has already been demonstrated in a previous study , we usedhigh) by RNA interference (low) (low) by RNA interference. Knocking down RAC1 strongly decreased their chemokinetic activity compared to control cells  or RAC1Q61L  in MDA-MB-435s or MDA-MB-468 cells, respectively. Rendering RAC1 inactive clearly repressed random cell migration in MDA-MB-435s cells in a dominant-negative fashion  but no by RAC1B . HoweverThese data provide functional proof of the roles of RAC1B and RAC1 as an invasion inhibitor and promoter, respectively, and are in support of a tumor suppressor function of RAC1B in both PDAC and BC. In these tumor entities, RAC1B is also a prominent example for a splice product that is tumor-protective as opposed to the parental isoform, RAC1 . Our resWe have determined here the relative expression levels of the RAC1B and RAC1 proteins among different BC cell lines; however, the ultimate biological activity of both isoforms is also determined by their GTPase activity. The constitutive activity of RAC1B resulting from the inclusion of exon 3b ,25 mightOf particular interest to clinicians are basal-like carcinomas, due to their poor prognosis, higher incidence in younger women, and resistance to endocrine therapy for hormone receptor-positive or trastuzumab for HER2-positive forms of the disease. As histology is not consistently distinctive, the identification of novel clinically practical biomarkers that act as surrogate measures of subtype identity or disease states is urgently needed to improve the accuracy of diagnosis or risk assessment (via prognostic biomarkers) and to inform the clinician on the optimal therapeutic strategy (via predictive biomarkers) . Based o