Ratiometric delivery of combination chemotherapy can achieve therapeutic efficacy based on synergistic interactions between drugs. It is critical to design such combinations with drugs that complement each other and reduce cancer growth through multiple mechanisms. Using hyaluronic acid (HA) as a carrier, two chemotherapeutic agents—doxorubicin (DOX) and camptothecin (CPT)—were incorporated and tested for their synergistic potency against a broad panel of blood-cancer cell lines. The pair also demonstrated the ability to achieve immunogenic cell death by increasing the surface exposure levels of Calreticulin, thereby highlighting its ability to induce apoptosis via an alternate pathway. Global proteomic profiling of cancer cells treated with HA–DOX–CPT identified pathways that could potentially predict patient sensitivity to HA–DOX–CPT. This lays the foundation for further exploration of integrating drug delivery and proteomics in personalized immunogenic chemotherapy. Combination chemotherapy has played an active role in treating blood cancers such as acute myeloid leukemia (AML) and T-cell lymphoma (TCL). Over the past couple of decades, significant effort has gone into understanding the biological basis of blood cancers. This accelerated the development and use of drug cocktails in clinical oncology ,6,7,8,9.Although combination chemotherapy can reduce the primary tumor burden, presence of dormant or residual cancer cells can result in an aggressive disease relapse. This underscores the importance of achieving cancer-specific immunity in patients for minimizing chances of a relapse. However, the immunosuppressive nature of chemotherapy makes it a challenge to achieve antitumor immunity, especially when combined with immunotherapies. Conventional chemotherapy was always known to induce tumor cell death via non-immunogenic mechanisms such as apoptosis . HoweverWith the advent of personalized chemotherapy, global proteomic profiling is increasingly being carried out in patient-derived cell lines and tissues to provide insights into cancer classification, treatment regimens, and analyses of drug resistance ,39,40. HHere, we propose an effective and personalized immunogenic chemotherapy by conjugating doxorubicin (DOX) and camptothecin (CPT) at different ratios to the HA polymer backbone (HA–DOX–CPT). Previously, HA nanoparticles have demonstrated efficacy in treating solid tumors in vivo ,22,23. DWe first establish a broad spectrum of the biological response for multiple cell lines, namely leukemia and T-cell lymphoma, which are both drug-sensitive and resistant types. For this, we perform a systematic evaluation of HA–DOX–CPT at different ratios over a range of concentrations. The most synergistic ratios are then assessed for their immunogenic activity in different cell lines. We then identified the key signaling pathways, which, when activated, can overcome drug resistance and induce immunogenic cell death with enhanced therapeutic efficacy. For this, we executed a small-scale proteomic workflow to obtain the quantitative proteomics profile of a multidrug-resistant blood-cancer cell line: HL-60/MX2 treated with HA–DOX–CPT. We identified up/downregulated proteins externalized by HL-60/MX2 based on the combined exploitation of mass spectrometry and bioinformatics tools. Eventually, this enabled us to identify biological pathways that could potentially predict patient sensitivity to HA–DOX–CPT-based personalized immunogenic chemotherapy.2. Antibodies used for flow-cytometry analysis were obtained from Biolegend. Sephadex G-25 PD-10 columns were obtained from GE Healthcare Life Sciences  and dialysis cassettes with 3500 MWCO were obtained from Life Technologies, USA. All other chemicals used for this study were obtained from Fisher Scientific and were of the highest possible grade commercially available.Camptothecin (CPT) and 4-(dimethylamino)pyridine (DMAP) were purchased from Sigma-Aldrich . N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Life Technologies, Carlsbad, CA, USA. Doxorubicin hydrochloride (DOX–HCl) was obtained from LC Laboratories . Hyaluronic acid (HA) of 50 kDa MW was purchased from Creative PEGWorks . Cell Titer Blue was purchased from Life Technologies, USA. All cell lines—leukemia and T-cell lymphoma —were genSingle and dual-drug conjugates were synthesized via nucleophilic acyl substitution reactions . In totaThe NanoSight LM10 system  was used to assess the size of HA–DOX–CPT. For size analysis, the Nanosight system was supplemented with a fast video capture and Nanoparticle Tracking Analysis (NTA 2.3) software. The samples were measured by capturing videos set at a recording time of 30 s each with adjustments made to the manual shutter and gain at room temperature. Image processing was done using the NTA 2.3 software to record the size. For each sample, the instrument was recalibrated and the measurements were made in triplicates.Lyophilized HA–DOX–CPT was reconstituted in PBS buffer (pH 7.4) at 1 mg/mL and incubated in Slide-A-Lyzer MINI dialysis devices  at 37 °C for up to 5 days. The dialysis devices were inserted into microcentrifuge tubes with 1 mL PBS. In total, 100 µL of the release medium in microcentrifuge tubes was collected at the indicated time points and drug concentration was measured via fluorescence using the TECAN plate reader. Points for the cumulative release were obtained by dividing the amount of drug released each day with the mass for the initial input. All measurements were carried out in triplicates and the results are indicated as the mean ± SD.2 atmosphere. The Cell Titer-Blue® Viability Assay was used to measure the cell viability and was expressed as the percentage of viable cells relative to the survival of untreated cells (defined as the maximum cell viability). The combination index (CI) was then estimated from the dose–response curves plotted for the single and dual-drug conjugates. A value of CI less than 1 indicates synergism; CI = 1 indicates the additive effect; and CI > 1 indicates antagonism. The further a CI value is from 1, the more pronounced the drug interaction, i.e., synergism or antagonism.All 7 T-cell lymphoma and 7 leukemia cell lines were incubated with HA–DOX, HA–CPT, and HA–DOX–CPT  over a range of serially diluted concentrations for 48 h and 72 h, respectively. For the dual-drug conjugates, the treatments were set up with respect to DOX concentrations. This was followed throughout the study. The cell lines were seeded in a 96-well cell culture plate at 50,000 cells per well and maintained in RPMI media (20% FBS) at 37 °C in a 5% CO2 atmosphere. The cells were then incubated overnight with HA–DOX–CPT (5 µM DOX) and stained with anti-human Alexa Fluor® 647 Calreticulin, Alexa Fluor® 488 anti-Hsp70 or its isotype controls (Alexa Fluor® 647 Mouse IgG2a and Alexa Fluor® 488 Mouse IgG2a) prior to analysis via flow cytometry.Leukemia cell lines  and T-cell lymphoma cell lines  were seeded at a density of 50,000 cells/100 µL in RPMI media (20% FBS) and maintained at 37 °C in a 5% CODried samples were resolubilized in Covaris  DF buffer and break with focused ultrasound by the Covaris S220 instrument with a 180 s cycle and 10% peak-to-peak power. After the cell break down procedure, all proteins were precipitated from the extraction solution by the methanol/chloroform cold precipitation procedure and all buffers were removed. Protein pellets were resolubilized in TEAB (triethyl ammonia bicarbonate) buffer, pH 7.5, for a further FASP based on 10 kDa filters  with reduction and alkylation steps using a previously established filter-assisted digestion protocol . DigestePost digestion of TMT, the labelled set the samples were submitted for hiPH separation based on the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit . The peptides were divided into 40 fractions and dried in SpeedVac . The fractions were then resuspended in 0.1% formic acid solution before injection to the mass spectrometer. Tribrid Lumos Orbitrap  equipped with the UltiMate 3000 HPLC Nano tandem pump , followed by 50 cm of the PharmaFluidics analytical column . A gradient from 5 to 27% ACN in 0.1% formic acid over 90 min at 200 nl/min was applied to achieve separation. A home-made electrode junction at the end of a microcapillary column and sprayed from stainless steel 4 cm needles  was used to enable electrospray ionization by applying a voltage of 2 kV.The Lumos Orbitrap instrument was operated in data-dependent mode for the mass spectrometry methods. The mass spectrometry survey scan was performed in the Orbitrap in the range of 410–1800 m/z at a resolution of 6 × 104, followed by the selection of the twenty most intense ions (TOP20) for HCD-MS2 fragmentation in the Lumos Orbitrap using a precursor isolation width window of 0.8 Th, the AGC setting of 50,000, and a maximum ion accumulation of 200 ms. Singly charged ion species were not subjected to HCD fragmentation. Collision energy was normalized and set to 37 V, and an activation of the time of 1 ms was set. Ions in a 10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 60 s and subsequently each precursor ion was selected for the CID type of fragmentation in ion trap part of the instrument with both 25 V energy and a max of 50 ms ion accumulation time.Proteome Discoverer 2.4.  software was used to analyze raw data. The Sequest HT algorithm was used for assignment of MS/MS spectra by searching the data against a protein sequence database. The Bacillus Subtilis database and other known contaminants such as human keratins and common lab contaminants were also searched. Sequest HT searches were performed using a 10 ppm precursor ion tolerance and required each peptides’ N-/C termini to adhere with Trypsin protease specificity, while allowing for up to two missed cleavages. Furthermore, 6-plex TMT tags on peptide N termini and lysine residues (+229.162932 Da) were set as static modifications while methionine oxidation (+15.99492 Da) was set as a variable modification. A target-decoy database search was applied to MS2 spectra with a false discovery rate (FDR) of 1% on the protein level. Filtering was performed using a percolator (64-bit version) . For quap value of < 0.05 was considered significant.All experiments were carried out in triplicates and results are indicated as the mean ± SD unless otherwise indicated. All graphs have been generated and analyzed using Prism non-linear regression software . A Chemotherapeutic drugs, namely doxorubicin (DOX) and camptothecin (CPT), were incorporated onto the HA polymer backbone at various molar ratios . Nanoparticle Tracking Analysis (NTS) revealed the mean average size to be at 149 nm with a fairly polydisperse size distribution A. In vitHA–DOX–CPT was highly effective against both leukemia and T-cell lymphoma cell lines. For the leukemia cell lines, IC90 values for DOX in HA–DOX–CPT were at least 20-folds lower compared to HA–DOX alone and for CPT, the values were at least two-folds lower than HA–CPT alone . For theHA–DOX–CPT upregulated the surface exposure of Calreticulin (CRT), an inducer of immunogenic cell death in HL-60 and its multidrug-resistant variant HL-60/MX2. However, the levels remained at the baseline for MOLM-13 and were reduced for THP-1 . For theMS-based quantitative proteomic profiling was performed to compare the proteome expression levels of DOX-resistant HL-60/MX2 cells in four different conditions  vs. log2 (fold change of HA–DOX/HA–DOX–CPT R0.8) or log2 (fold change of HA–DOX–CPT R0.8/HA–DOX–CPT R15) was constructed to graphically display the quantitative data (p < 0.05). Points to the left of the left-most non-axial vertical line denote protein fold changes of HA–DOX/HA–DOX–CPT R0.8  in response to a drug is an important damage-associated molecular pattern (DAMP) for anticancer immunogenicity ,57. DOX Recent advances in proteomics provide a powerful approach to study the global cellular effects of a drug or drug cocktail. We quantified more than 4000 cellular proteins and identified a total of 48 proteins whose levels changed in response to HA–DOX and HA–DOX–CPT treatment with the multidrug resistant cell line HL-60/MX2. Principal component analysis revealed strong patterns within the dataset GO analysis pertaining to the molecular function revealed binding, catalytic activity, a transporter, and a transcription regulator activity as affected in response to treatment with HA–DOX–CPT . GO analOne of the hallmarks of cancer progression is chromosomal instability (CIN), a source of genetic variation in altered chromosomal structures or its number. Chromosomal reorganization was depicted as a key pathway during the GO analysis of treatment groups HA–DOX vs. HA–DOX–CPT. When compared with HA–DOX, chromatin assembly and disassembly was upregulated in the cells treated with HA–DOX–CPT. Many chemotherapeutic agents are known to upregulate CIN, which leads to the apoptosis of aberrant cells. However, contradictory findings while studying the effect of CIN on therapeutic response and drug resistance have also been reported . Some stDespite major advances in proteomics, the use of this technique in analyzing the efficacy of ratiometric drug delivery is still in its infancy. This is an excellent approach for the discovery of predictors not just for individual drug treatments but also for personalized combination therapies. Our proteomic studies in blood-cancer cells have revealed multiple candidates that might serve as biomarkers of prognosis and drug resistance. In the future, these findings can be utilized as a resource to guide the design of combination drug delivery while identifying mechanisms or pathways that promote multi-drug resistance and promote enhanced therapeutic efficacy. Future studies will be focused on validating the chemo-immunogenic effect of HA–DOX–CPT in drug-resistant tumor models in vivo. However, challenges exist in ensuring that the chemo-immunogenic effect of this combination at specific ratios is retained at the target site. Since DOX and CPT have relatively different pharmacokinetic properties, normalizing their circulation times to that of the nanoparticle carrier would be a first step. Incorporating the drug pair onto the carrier would involve a systematic approach to formulation design and optimization. In addition to ensuring the stability of these hyaluronic acid nanoparticles during circulation, achieving control over its drug release for the desired pharmacodynamic effects following its cell uptake would be vital. By integrating “proteomics” with “designer combinatorial NPs”, traditional cancer nanomedicine that focuses on targeting and disease management could undergo a paradigm shift for a whole-body level of effective immunoprotection against cancer relapse.


Several chemotherapy drugs are approved for ovarian cancer treatment in the neo-adjuvant/adjuvant setting as well as following relapse. These include carboplatin, paclitaxel, doxorubicin, topotecan, PARP inhibitors (PARPi), and gemcitabine. However, except for PAPRi, there are no predictive biomarkers to guide the choice of drug. The majority of chemotherapeutic drugs function by inducing DNA damage or inhibiting its repair. However, the association of DNA damage repair (DDR) pathway alterations with therapy response remain unclear. In this study, using a panel of 14 ovarian cancer cell lines, 10 patient ascites-derived primary cultures and bioinformatic analysis of The Cancer Genome Atlas (TCGA) ovarian cancer dataset, we identified the role of genomic/transcriptomic and/or functional alterations in DDR pathways as determinants of therapy response.Defective DNA damage response (DDR) pathways are enabling characteristics of cancers that not only can be exploited to specifically target cancer cells but also can predict chemotherapy response. Defective Homologous Recombination Repair (HRR) function, e.g., due to BRCA1/2 loss, is a determinant of response to platinum agents and PARP inhibitors in ovarian cancers. Most chemotherapies function by either inducing DNA damage or impacting on its repair but are generally used in the clinic unselectively. The significance of HRR and other DDR pathways in determining response to several other chemotherapy drugs is not well understood. In this study, the genomic, transcriptomic and functional analysis of DDR pathways in a panel of 14 ovarian cancer cell lines identified that defects in DDR pathways could determine response to several chemotherapy drugs. Carboplatin, rucaparib, and topotecan sensitivity were associated with functional loss of HRR  and mismatch repair. Two DDR gene expression clusters correlating with treatment response were identified, with PARP10 identified as a novel marker of platinum response, which was confirmed in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Reduced non-homologous end-joining function correlated with increased sensitivity to doxorubicin, while cells with high intrinsic oxidative stress showed sensitivity to gemcitabine. In this era of personalised medicine, molecular/functional characterisation of DDR pathways could guide chemotherapy choices in the clinic allowing specific targeting of ovarian cancers. Ovarian cancer accounts for ~300,000 new cancer cases globally each year and is one of the leading causes of death among gynaecological cancers, with a mortality rate of ~185,000 deaths per year . Whilst The disproportionate mortality rate in ovarian cancer continues to attract attention with the ambition to improve survival through the stratified provision of targeted therapies. The focus on exploiting defective DNA damage response (DDR) pathways has increased following the successful implementation of poly (ADP ribose) polymerase inhibitor (PARPi) therapy ,7. ThereMultiple studies have clearly demonstrated mutations in genes encoding proteins involved in the homologous recombination repair (HRR) pathway, e.g., BRCA, as determinants of response to both platinum agents and PARPis ,11. HoweThis study, therefore, aimed to explore the sensitivity of five chemotherapy drugs and one PARPi used to treat ovarian cancer: carboplatin, doxorubicin, gemcitabine, paclitaxel, topotecan, and rucaparib in a panel of 14 ovarian cancer cell lines and ascites-derived primary cultures from women with ovarian cancer and their association with the genomic, transcriptomic and functional alterations of DDR genes/pathways. We identified that HRR status was significantly associated with carboplatin and rucaparib response in agreement with previous studies ,13. NHEJA panel of 14 ovarian cancer cell lines was used to screen for sensitivities to chemotherapy and DDR pathway functional analysis. The clinical and morphological features of the cell lines are listed in v/v) with RPMI-1640 media supplemented with 20% fetal calf serum and 1% penicillin/streptomycin. Cultures were incubated at 37 °C, 5% CO2, 95% humidified air. The medium was replenished every 4–7 days until cultures reached 70–80% confluence. Cells were then passaged for continuous culture. All experiments were carried out at early passage (<4).Ethical approval for the collection of clinical material and data was granted (REC: 17/NE/0361), and written consent was taken. Malignant ascitic fluid was collected from patients undergoing cytoreductive surgery or symptomatic drainage of ascites, as previously described . AscitesCarboplatin, gemcitabine hydrochloride, doxorubicin hydrochloride, topotecan hydrochloride hydrate, and paclitaxel were supplied by Sigma Aldrich, UK. Rucaparib was a kind gift from Pfizer Global R&D . Carboplatin, gemcitabine hydrochloride and doxorubicin hydrochloride were dissolved in 0.9% saline and topotecan, paclitaxel, and rucaparib were dissolved in Dimethyl Sulfoxide (DMSO).v/v) and stained with 0.4% crystal violet. Colonies were counted, and the % survival for each treatment calculated from the relative plating efficiency of treated versus vehicle (0.5% DMSO/only media) controls. The LC50 values were calculated from individual experiments where the concentration causing a 50% decrease in cell survival was calculated by interpolation of the survival curves. The % Survival was also estimated from the individual survival curves at a selected concentration. The mean and standard deviation of the LC50 and % Survival values were calculated further from 3 independent experiments.Cells were plated at low density ranging from 50 to 4000 cells/well based on the plating efficiency (colonies formed/cells seeded × 100) of the cell lines and anticipated cytotoxicity of the drugs to get countable colonies (20–150) and allowed to adhere for 24 h. This was followed by treatment with different concentrations of drugs as a single agent for 24 h. The DMSO concentration was maintained at 0.5% for drugs dissolved in DMSO. Following 24 h of drug treatment, the medium was replaced with a drug-free medium and cells incubated until colonies >30 cells formed. Cells were fixed in methanol: acetic acid (3:1 w/v) trichloroacetic and stained with 0.4% SRB. Absorbance was measured at 570 nm using a BMG FLUOstar Omega plate reader, and the growth as a percentage of vehicle control was calculated  for each drug concentration.Primary cultures were seeded into 96-well plates (1000 cells/100 μL per well) and allowed to adhere overnight. Media was replaced with fresh medium containing increasing concentrations of each drug which was again replaced with fresh medium after 24 h and incubated for a further 5–6 days or until the control wells were subconfluent. Cells were fixed with 50%  (accessed on 12 January 2021).Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) data was accessed using cbioportal  (13) was used for validation of the correlation of gene expression with chemotherapy response.HRR was assessed by immunofluorescence microscopy, as previously described . Cells wNHEJ plasmid re-joining assay was performed as described previously by Bradbury et al. . Equimol® DNA Mini Kit  according to the manufacturer’s instructions. DNA was quantified using a NanoDrop™ ND-1000 Spectrophotometer . Using the HT 8-oxo-dG ELISA Kit II , samples were treated with DNase and alkaline phosphatase each for one hour at 37 °C as per manufacturer’s instructions. Equal quantities of DNA from each cell line were analysed for 8-OHdG levels. The absorbance (450 nm) measured following detection with horseradish peroxidase (HRP) and TACS-Sapphire™ colourimetric substrate corresponds to the antibody bound to the pre-coated 8-OHdG on the well. The absolute 8-OhdG levels were calculated using serial dilutions of the standard supplied with the kit.4.6.1 TP53 functional analysis. Pearson’s correlation coefficient analysis was performed to correlate 8-OhdG level/µg of DNA of each cell line with % survival at fixed concentrations of the drugs.Cells were seeded at 0.5 × 106 cell/mL in 100 mm dishes and cultured for two doubling times before harvesting, snap freezing and storing the cell pellet at −80 °C prior to DNA isolation. The snap-frozen cell pellets were rapidly thawed at 37 °C and DNA isolated using the QIAampt-test. Pearson’s correlation coefficients were calculated for all correlation analyses. Hierarchical clustering of Pearson’s correlation coefficients was performed to identify drug sensitivity clusters and the associated gene expression clusters. All statistical analysis was performed using Graphpad Prism 8 and statistical packages in R. The following p-value cut-offs were used: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.Statistical comparison of datasets between two defined groups was performed using a non-parametric 50 values ranging from 150-fold for rucaparib, 31-fold for carboplatin, 14-fold for gemcitabine, 11-fold for topotecan and paclitaxel, and 4-fold for doxorubicin  at a fixed drug concentration was used for further analysis (Last accessed on 12 January 2021). We observed a statistically significant positive correlation between the two parameters (p = 0.03), in comparison to the non-HGSOC subgroup. On the other hand, paclitaxel sensitivity was greater (3.5-fold) among cell lines grouped in the ‘non- HGS’ subgroup, although not statistically significant . UWB1.289, COV62, and NIH-OVCAR3 were the most sensitive to both drugs. NIH-OVCAR3 data was previously published by Bradbury et al. [p-value = 0.11). Conversely, a negative correlation was seen between doxorubicin and topotecan sensitivity   . No othe = 0.15) C.The wide spectrum of drug sensitivities across the cell line panel led us to investigate differences in the genomic landscape of DDR pathways as determinants of drug sensitivity. Firstly, the cell line profiles were confirmed by correlating the published CCLE RNA-Seq data with the targeted RNA-Seq data (in-house) for 2495 genes highlighting a positive correlation across most (91%) of the genes , confirmWhile CNA status did not show any strong association with chemotherapy response, the differences in mRNA levels were more likely to show variation across cell lines. Therefore, to understand the significance of differential mRNA expression of the 120 DDR genes with drug response, we correlated the log-transformed RSEM values as a measure of gene expression derived from the database with the % survival for each drug at a fixed concentration . Hierarcn = 105) and resistant (n = 43) groups of patients. While most genes followed a similar differential expression trend, as observed in the cell lines, the top five differentially expressed genes were XRCC4, RPA3, PARP11, RAD52, and PARP10  with platinum response status for 190 patients. We tested for any differential expression of 48 genes that formed the Clusters A and B B betweend PARP10 . PARP10 To assess the association between the functional activity of DDR pathways and response to chemotherapy, we first characterised the cells for their HRR ability using the RAD51 focus formation assay. Twelve of fourteen cell lines showed >2-fold increase in RAD51 foci formation following the induction of DNA damage and were classified as HRR competent (HRC) A. Howeven = 5) or unknown (n = 3). We identified a spectrum of sensitivities across the samples with the fold-difference between the highest and lowest GI50 value (dose of the drug causing 50% growth inhibition) ranging from 38-fold for topotecan, 26-fold for rucaparib, 21-fold for gemcitabine, 10-fold for doxorubicin, and 5-fold for carboplatin and paclitaxel  and rucaparib . In contrast, HRC cancers were more sensitive to doxorubicin   .A plasmid re-joining assay was used to estimate the NHEJ activity of the cell line panel. A wide spectrum of %NHEJ activity was observed across the cell line panel ranging from 0.2% (CAOV3) to 24.24% (NIH-OVCAR3) with a median of 4.3%. To identify any association with chemotherapy response, the NHEJ activity of each cell line was correlated with % survival at fixed drug concentration. The distribution of NHEJ activity highlighted two cell lines, OAW42 and ES2, as outliers in terms of correlation with chemotherapy response and hence were removed. The %NHEJ activity correlated positively with response to doxorubicin (as expected), gemcitabine, and paclitaxel and correlated negatively with carboplatin and rucaparib response. Survival following topotecan exposure did not correlate with NHEJ activity .Genomic analysis identified BER pathway alterations as the next most frequent alterations after HRR. The main endogenous lesion repaired by this pathway is oxidised guanine at the eighth-position resulting from oxidative stress. Therefore, we measured the baseline oxidative stress of each cell line by quantification of 8-OHdG levels/µg of DNA as an indirect measure of intrinsic BER pathway activation. The highest baseline 8OHdG levels were observed in the ES2 cell line (5.02 nM) and the lowest in the OAW42 cell line (1.43 nM) with a median concentration of 3.05 nM. Correlation with survival showed the strongest trend towards negative correlation with gemcitabine. Gemcitabine has been reported to enhance reactive oxygen species (ROS) production and consequently oxidative stress; therefore, this observation highlighted that it is likely that gemcitabine would be more cytotoxic in cell lines with poor BER ability resulting from oxidative DNA damage. There was a weaker correlation with cytotoxicity with other drugs .To understand the likely effect of MMR loss, we compared the sensitivities between MMR-competent (CP70-B1) and MMR-defective (CP70-A2) cell line pairs for their response to all six drugs. The CP70-A2 cells were significantly more sensitive to carboplatin (2.3-fold), topotecan (3.5-fold), and rucaparib (1.7-fold); the CP70-B1 cells showed greater sensitivity to doxorubicin (7.4-fold). There was no difference in sensitivity to gemcitabine and paclitaxel between the cell line pairs F.DDR pathways have been extensively studied across cancer types, and the success of PARPi in BRCA-associated cancers  has greaUtilising a panel of cell lines with different histologic subtypes, we identified that HGSOC cell lines  were more sensitive to carboplatin, gemcitabine, topotecan and rucaparib, while the non-HGSOC cell lines  were more sensitive to doxorubicin and paclitaxel. Several earlier studies have attempted to analyse the genomic/transcriptomic/histological sub-type characterisation of ovarian cancer cell lines or correlate with response to chemotherapy ,22,23. HGenomic profiling of 120 DDR/PARP family genes identified frequent CNAs in genes involved in HRR and BER function and several PARP family proteins, which were exclusive to HGSOCs. PARP10 amplification and/or increased expression correlated with sensitivity to carboplatin and rucaparib, which was confirmed in the TCGA ovarian cancer dataset where it associated with platinum response. While the role of PARP10 in alleviating replication stress  has beenA strong positive correlation was observed between carboplatin and rucaparib sensitivity, which could be explained by genomic loss of BRCA gene/HRR dysfunction in both the cell line panel and patient ascites-derived primary cultures. NIH-OVCAR3, as we have shown previously , was an Interestingly, intrinsic oxidative stress, which is an indirect indicator of baseline BER activity, measured in terms of 8-OHdG levels per µg of DNA, identified a negative correlation of survival with gemcitabine. Gemcitabine has a direct role in inducing oxidative stress by increasing ROS production . These oIn summary, our data highlights the importance of the genomic/transcriptomic as well as functional analysis of DDR pathways, which can guide the selection of chemotherapy choice for relapsed HGSOCs. As observed clinically, HRD cancer cell lines and primary cultures were more sensitive to carboplatin and rucaparib. Also, predictably cells with low NHEJ activity were more sensitive to doxorubicin, but interestingly, HRD cells were less sensitive to doxorubicin. Additionally, 8-OHdG levels, indicative of high intrinsic oxidative stress, was associated with increased sensitivity to gemcitabine. We also identified that common gene amplifications in ovarian cancers, like PARP10, associated with platinum response. This study, therefore, highlights the relevance of key DDR pathway alterations in determining chemotherapy response which demands further exploration in the clinic.Helleday T and Curtin NJ. Therapeutic Compounds (PARP inhibitors in homologous repair/BRCA defective cancer) WO 2005/012305 A2Boritzki TJ, Calvert AH, Curtin NJ, Dewji MR, Hostomsky Z, Jones C, Kaufman R, Klamerus KJ, Newell DR, Plummer ER, Reich SD, Steinfeldt HM, Stratford IJ, Thomas HR Williams KJ. Therapeutic Combinations Comprising PARP inhibitor WO/2006/033006Falcon S, Reaper P, Pollard J, Curtin NJ, Middleton FK and Chen T. Method for measuring ATR inhibition mediated increases in DNA damage. WO2014055756A1


Background and objectives: The chromosome locus 20q11.21 is a commonly amplified locus in colorectal cancer, with a prevalence of 8% to 9%. Several candidate cancer-associated genes are transcribed from the locus. The therapeutic implications of the amplification in colorectal cancer remain unclear. Materials and Methods: Preclinical cell line models of colorectal cancer included in the Cancer Cell Line Encyclopedia (CCLE) collection were examined for the presence of amplifications in 20q11.21 genes. Correlations of the presence of 20q11.21 amplifications with gene essentialities and drug sensitivities were surveyed on salient databases for determination of therapeutic leads. Results: A significant subset of colorectal cancer cell lines in the CCLE  bear amplifications of genes located at 20q11.21. Cancer-associated genes of the locus include ASXL1, DNMT3B, BCL2L1, TPX2, KIF3B and POFUT1. These genes are all amplified in the 12 cell lines, but they are variably over-expressed at the mRNA level, compared to non-amplified lines. 20q11.21 amplified cell lines are sensitive to various tyrosine kinase inhibitors and are resistant to chemotherapy drugs targeting the mitotic apparatus and microtubules. CRISPR and RNAi dependencies screening revealed, besides the β-catenin and KRAS genes, a few recurrent gene dependencies in more than one cell line, including YAP1 and JUP. Conclusions: Cell line models of colorectal cancer with 20q11.21 gene amplifications display dependencies on the presence of specific genes and resistance or sensitivity to specific drugs and drug categories. Observations from in vitro models may form the basis for clinical drug development in this subtype of colorectal cancer. Genetic lesions conferring synthetic lethality to certain drugs or categories of drugs could be discovered with this approach. Development of targeted therapies of cancer based on underlying molecular defects that drive carcinogenesis has improved cancer patient outcomes in recent years. New candidate drugs are often first tested in preclinical in vitro models consisting of cell lines cultured in highly artificial conditions . Thus, tERBB2, encoding for HER2 receptor protein and leading to sensitivity to monoclonal antibodies and small-molecule tyrosine kinase inhibitors blocking the receptor [ASXL1, DNMT3B, BCL2L1, TPX2, KIF3B and POFUT1. The expression of resulting mRNA in 20q11.21 amplified colorectal cancers is variable. Some of the amplified genes are rarely over-expressed at the mRNA level, while others, such as ASXL1, KIF3B and POFUT1, are over-expressed in most amplified cancers. ASXL1 is over-expressed in 88.9% of 20q11.21 amplified colorectal cancers in the TCGA cohort, while POFUT1 is over-expressed in 90.5% of 20q11.21 amplified cases in the same cohort [Amplification of specific loci is common in cancers and is often observed with tumor specificity, being more frequent in specific types of cancers. Perhaps one of the best-known amplifications with clinical therapeutic implications is observed in a subset of breast cancers at chromosome 17q. The locus includes oncogene receptor . In anotreceptor . In othehttp://www.cbioportal.org, accessed on 3 April 2021) is a user-friendly, open-access platform for genomic analysis of tumors and cancer cell lines [Cancer cell lines included in the current investigation constitute part of the Cancer Cell Line Encyclopedia (CCLE) collection . The cBill lines . Additioll lines  were anall lines . An Aneull lines . A chromll lines . The defll lines .The OncoKB knowledgebase is a database of cancer-related genes and classifies cancer-related genes as oncogenes or tumor suppressors . OncoKB www.cancerrxgene.org, accessed on 6 April 2021) was queried to obtain data on drug sensitivity of cell lines from colorectal cancer and other cancers with the 20q11.21 amplification [The Genomics of Drug Sensitivity in Cancer (GDSC) dataset . Cancer cell lines with this recurrent copy number alteration, independently of primary type, display resistance to several currently used chemotherapy drugs, including the microtubule poisons vincristine, vinblastine and docetaxel, the topoisomerase II inhibitors teniposide and epirubicin and the DNA poisons temozolamide and actinomycin D (50) than colorectal cancer cell lines without the amplification, although, due to the smaller size of the cohort, differences are not statistically significant except for Alisertib and EPZ004777  included in CCLE have amplifications of genes at the 20q11.21 locus, as assessed in cBioportal. All three genes from this locus that are listed as cancer-related at OncoKB knowledgebase  are amplified in the 12 cell lines . Other gomycin D . In addiPZ004777 .In contrast to promoting drug resistance, the cnaPANCAN363 copy number alteration (20q11.21 amplification) does not confer sensitivity to any of the 185 drugs tested in the Genomics of Drug Sensitivity in Cancer database (GDSC). However, individual colorectal cancer cell lines with the amplification display sensitivities to tested drugs . Drug camRNA expression of genes at 20q11.21 in 12 colorectal cancer cell lines with 20q11.21 amplification was compared with the corresponding expression in 12 randomly selected colorectal cancer cell lines from CCLE without amplification in the locus. Among the six genes located at 20q11.21 with potential cancer pathogenesis interest, BCL2L1, POFUT1 and KIF3B were over-expressed at the mRNA level in 20q11.21 amplified cell lines compared with non-amplified cell lines . Over-ex50 in amplified lines 11.6 µmol versus 42.5 µmol in non-amplified cell lines, p = 0.04). In contrast, other BCL2 family inhibitors examined such as venetoclax and navitoclax showed no sensitivity in amplified cell lines or even a trend for resistance compared to non-amplified lines. Colorectal cancer cell lines with the 20q11.21 amplification displayed resistance to the microtubule polymerization stabilizer epothilone B, compared with non-amplified cell lines . Z-LLNie-CHO, a γ secretase inhibitor of the Notch cascade, displayed a non-significant trend towards resistance in amplified cell lines .Based on the mRNA over-expressions of genes at 20q11.21, the sensitivity of cell lines to BCL-xL inhibitors, Notch inhibitors (Notch pathway is activated by POFUT1 enzyme) and mitotic spindle inhibitors was evaluated at the GDSC. A notable drug in these categories displaying sensitivity in amplified cell lines compared to non-amplified cell lines is the BCL2 family inhibitor WEHI-539  . CancersDependencies of 20q11.21 amplified cell lines on particular genes and their products as derived from CRISPR and RNAi arrays could inform development of therapies based on synthetic lethalities. Yes-associated protein 1 (YAP1), a transcription factor of the Hippo pathway, comes up in the dependency screening of 20q amplified colorectal cancer cell lines, confirming a key role of the pathway in colorectal cancer. YAP1 co-operates with transcription factors TAZ and TEAD in transcription of genes involved in proliferation following tissue damage and promoting regeneration in the gut . In coloDONSON, one of the discovered dependencies present in 2 of 7 tested amplified cell lines, is a gene of unknown function, playing a role in DNA replication and the stabilization and protection of stalled replication forks. Mutations of the gene are associated with the microcephaly-micromelia syndrome . In cancSNAP23, another dependency gene present in 2 of 7 tested amplified cell lines, encodes for one of the proteins of the cellular machinery for membrane fusion and exocytosis. It is also involved in cell signaling, promoting malignant cell motion, and through this mechanism, it may favor metastasis .HMGCS1 is a mevalonate precursor enzyme and catalyzes the conversion of two molecules of acetoacetyl-CoA to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), the precursor of cholesterol production . HMGCS1


Treatment with siHDAC6 diminished viability, clonogenic potential, and migration ability in GBM-derived cell lines. HDAC6 inhibition also reverted the mesenchymal phenotype, inhibited the Sonic Hedgehog pathway, restored primary cilium structure, and decreased autophagy. Thus, we confirm that HDAC6 is a good therapeutic target for GBM treatment.Glioblastoma multiforme (GBM) is the most common as well as the most aggressive malignant brain tumor, with an overall survival of almost 15 months. Histone deacetylase 6 (HDAC6), an enzyme related to the deacetylation of α-tubulin, is overexpressed in GBM. The aim of our research was to study the effects of HDAC6 silencing in GBM cells. We first confirmed the overexpression of HDAC6 in GBM tissue (Glioblastoma multiforme, the most common type of malignant brain tumor as well as the most aggressive one, lacks an effective therapy. Glioblastoma presents overexpression of mesenchymal markers Snail, Slug, and N-Cadherin and of the autophagic marker p62. Glioblastoma cell lines also present increased autophagy, overexpression of mesenchymal markers, Shh pathway activation, and lack of primary cilia. In this study, we aimed to evaluate the role of HDAC6 in the pathogenesis of glioblastoma, as HDAC6 is the most overexpressed of all HDACs isoforms in this tumor. We treated glioblastoma cell lines with siHDAC6. HDAC6 silencing inhibited proliferation, migration, and clonogenicity of glioblastoma cell lines. They also reversed the mesenchymal phenotype, decreased autophagy, inhibited Shh pathway, and recovered the expression of primary cilia in glioblastoma cell lines. These results demonstrate that HDAC6 might be a good target for glioblastoma treatment. Glioblastoma (GBM), or grade IV astrocytoma, is the most common form of malignant brain tumor as well as the most aggressive one. It corresponds to 14.6% of all brain tumors, 48.3% of malignant brain tumors, and 57.3% of gliomas, making GBM the third most common brain tumor . DespiteHistone deacetylase 6 (HDAC6) is a IIb class of HDACs and is the isoform that presents the highest expression in GBM . This prEpithelial-to-mesenchymal transition (EMT) is a cellular programme by which epithelial cells lose the expression of epithelial markers and acquire the expression of mesenchymal markers, increasing its migration capacity and its resistance to apoptosis. This mesenchymal state can be reverted in a process called mesenchymal-to-epithelial transition (MET). Epithelial-derived tumors have been seen to be the most lethal, such as breast, colon, pancreas, and kidney cancer . EMT canAutophagy is a process by which cellular components are degraded to maintain the cellular homeostasis and protect cells from metabolic stress. Autophagy provides amino acids and free fatty acids to the cells to obtain energy from tricarboxylic acid cycle (TCA) . The autIn this study, we aimed to know the effects of silencing HDAC6 in GBM cell lines. HDAC6 expression associated to clonogenicity, cell migration, and cell proliferation and also to EMT, Shh pathway activation, lack of a primary cilium, and to autophagy promotion. Our HDAC6 siRNA-silencing experiments demonstrate a reversal of all those cellular characteristics, making of HDAC6 a potential therapeutic target for GBM treatment.n = 7, parietal lobe: n = 7) and of GBM samples (n = 11) were a kind gift from Dr. Lanciego and Dr. Idoate, respectively, both coauthors of the study. Proteins were extracted with RIPA buffer, and RNA was extracted with TRIzol reagent with the help of an Ultra turrax . The present study was authorized by the Ethics Committee of the University of Navarra . Patients provided informed consent for the use of their samples for research. All samples were fully anonymized prior to accessing. Frozen sections of control brains  and GBM tissue (n = 40), we performed immunohistochemistry for these proteins at the Department of Pathology of the Hospital Complex of Navarra, Pamplona, Spain . Antibodies used in this experiment were: HDAC6 (1677558S) from Cell Signaling , and the p62 antibody was a kind gift from the Hospital Complex of Navarra. In order to compare the expression status of HDAC6 and the autophagic marker p62 between normal human brain tissue , T98G was obtained from the European Collection of Authenticated Cell Cultures , and U87MG was obtained from ATCC . Provenance of the cell lines is as follows: LN405 (DSMZ ACC 189)  are glio™ and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). U87MG was cultured in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX™, supplemented with 10% FBS, 1% P/S, and 4% non-essential amino acids (NEAA). NHA cell lines were cultured in AGM™ BulletKit™  and supplemented with AGM™ SingleQuots™ .LN405 and T98G cell lines present mutations in PTEN and TP53 genes, while U87MG cell line is mutated in PTEN but has a TP53 wild-type gene . The nor® siRNA Universal Negative Control #1  was transfected as a control group. At 72 h post-transfection, cells were harvested and seeded for other experiments, or proteins and RNA were extracted with RIPA buffer and TRIzol reagent, respectively. GBM cell lines were seeded in 6-well plates at a confluence of 150,000 cells/well. At 24 h post-seeding, cells were transfected with a mix of two different siHDAC6  following lipofectamine 2000 protocol. MISSIONNHA cell line was seeded in 6-well plates at a final confluence of 300,000 cells/well. The following day, cells were transfected following lipofectamine 2000 protocol. Plasmids transfected were: pcDNA3 LIC cloning vector 6A (#30124) and pcDNA-HDAC6-FLAG (#30482); both of them were acquired from AddGene .Transfected cells were plated in 96-well plates at a final confluence of 2500 cells/well. At 24 h post-plating, medium was changed with fresh medium. In order to know the starting viability, we added MTT -2,5-diphenyl-2H-tetrazolium bromide) reagent at a final concentration of 0.5 mg/mL and incubated for 90 min. After that, cells were washed with ice cold PBS, and 100 µL of DMSO were added to each well. Absorbance was measured at 550 nm in a Multiskan EX . The same procedure was done 72 h after medium change. 2. After that, cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 40 min. Fixed cells were then stained with crystal violet for 15 min. The following day, cells were counted using Colony Counter 560 .Transfected cells were plated in 6-well plates at a final confluence of 300 cells/well and were incubated for 10 days at 37 °C 5% COsiRNA-treated cells were seeded in 24-well plates at a final confluence of 250,000 cells/well for LN405 and T98G and 150,000 cells for U87MG. At 24 h post-seeding, a scratch was done with a 200 µL pipette tip, and cell culture medium was changed out for new medium with 2.5% FBS to halt the proliferation of the cells during the scratch wound healing assay. Photographs were taken at times 0, 24, and 48 h post-scratch. Closure of the scratch was then analyzed with ImageJ software. −ΔΔCt method.Total RNA was extracted from untreated cell lines, or siRNAs-treated GBM cell lines, following TRIzol reagent protocol. cDNA was obtained from 2 µg of RNA using SuperScript ™ II Reverse Transcriptase . RT-qPCR was performed in an iQ5 Multicolor real-time PCR detection system . The sequence and Tm of the different primers is described in Proteins were extracted from the untreated cell lines, from the siRNA-treated GBM cell lines, and from the plasmid-treated NHA cell line. Next, 20 µg of proteins were loaded in each well of an SDS-PAGE, immunoblotted, and incubated overnight with primary antibody: HDAC6 1:1000 , N-cadherin 1:1000 , Snail 1:1000 , Slug 1:1000 , LC3B 1:1000 , acetylated α-tubulin 1:10,000 , α-tubulin 1:10,000 , and β-actin 1:10,000 . Secondary antibodies were: α-mouse 1:20,000  and α-rabbit 1:2000 .Cells were seeded in 24-well plates at a final confluence of 50,000 cells/well. The following day, cells were transfected with p8Xgli-luciferase (wild type or mutated), pRLTK, and control plasmid or pGli1 at a ratio 4:1:5 in 0.5 µg following lipofectamine 2000 protocol. Two days after transfection, media were changed out for one with 0.5% FBS, and 24 h later, luminescence were measured using Dual Glo luciferase assay system  in a Glomax 20/20 luminometer . Gli-luciferase activity was measured in U87MG after siRNA treatment following the same protocol but changing the control or Gli plasmid by 25 pmol of siCTRL or siHDAC6.® 594 1:500  and α-rabbit AlexaFluor® 488 1:500 . After three washes of 5 min each, VectaShield  were added to coverslips. Finally, coverslips were mounted and visualized in a LSM 800 microscope . Cell lines were seeded in 24-well plates at a confluence of 50,000 cells/well. The well contained a sterilized coverslip. At 48 h post-seeding, medium was changed out for one without FBS, and, the following day, cells were fixed with 4% paraformaldehyde for 10 min. After that, cells were permeabilized with 0.025 Triton-X100 in PBS and blocked with blocking solution  for 1 h. Antibodies were incubated overnight: acetylated α-tubulin 1:500  and γ-tubulin 1:500 . Cells were washed three times with PBS, 5 min each wash, and then were incubated with secondary antibody: α-mouse AlexaFluorGroup I: negative control. It was transfected with a plasmid containing 8 times the mutated Gli1 promoter sequence (no Gli1 binding) followed by the firefly luciferase gene, the plasmid used as a loading control, renilla luciferase, and an empty control plasmid.Group II: experimental group. It was transfected with the plasmid containing 8 times the Gli1 wild-type promoter sequence followed by the firefly luciferase gene, the plasmid used as loading control, and an empty control plasmid.Group III: positive control. It was transfected with the plasmid that contained 8 times the sequence of the Gli1 wild-type promoter followed by the firefly luciferase gene, the plasmid used as loading control, and the Gli1-expression plasmid.The next three groups were created to define the basal state of Sonic Hedgehog activation:Cell lines were treated with 100 nM Bafilomycin A1 or DMSO as vehicle control for 6 h, and, after that time, proteins were extracted and run in a western blot. LC3B and β-actin expression was measured. The 72-h siRNA-treated GBM cell lines were then treated with 100 nM Bafilomycin A1, EBSS, EBSS + Bafilomycin A1, or DMSO as a vehicle control for 6 h. After that, proteins were extracted, and LC3B and β-actin was measured. GBM cell lines were seeded in 12-well plates and were transfected the next day with 50 pmol siCTRL or siHDAC6 and 1 µg of EGFP-LC3B plasmid following lipofectamine 2000 protocol. At 48 h after transfection, cells were treated with 100 nM Bafilomycin A1, EBSS, EBSS + Bafilomycin A1, or DMSO as a vehicle control for 6 h, and autophagosome formation was measured in a EVOS microscope. Autophagosomes were counted using ImageJ software.p < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 8.0 Software. Studies that contained more than two groups were analyzed with a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test. p = 0.0038) (p = 0.053). It should be noted that we were unable to detect this expression in three of seven samples from the temporal area and in four of seven samples from the parietal area, which could have increased the differences and the statistical power.RNA was extracted from frozen samples of healthy brains and glioblastomas, and a reverse transcription (RT) and a real-time PCR (qPCR) were performed to study the expression of HDAC6 in those samples. As a result, we obtained that HDAC6 was three times more expressed in glioblastoma tissue than in tissues of the temporal area of healthy brains ( 0.0038) . The difp = 0.0062), and the differences between the parietal zone of the brain and glioblastoma were almost significant (p = 0.062). Slug was also found four times more expressed in the tumor tissue than in tissues of the temporal zone (p = 0.0426) and did not give a significant difference between tissues of the parietal zone and glioblastoma (p = 0.31).Regarding the Snail and Slug genes, we obtained similar results as for the expression of HDAC6 . We obseAs explained, HDAC6 and the mesenchymal markers Snail and Slug were overexpressed in tumor tissue, as detected by RT-qPCR. To test the markers by western blot, proteins were extracted from healthy brain tissues and glioblastoma, and the expression of HDAC6 and N-Cadherin were quantified using β-actin as a loading control.The results obtained by the RT-qPCR were correlated with the data obtained by western blot. Tumor tissues showed overexpression of HDAC6 in comparison with the healthy tissues of the temporal and parietal zones . SimilarOnce we demonstrated that HDAC6 is overexpressed at the RNA and protein level in GBM, we performed immunohistochemistry against HDAC6 in healthy brain sections and in GBM sections from the Hospital Complex of Navarra. As we see in On the other hand, we also studied the autophagic marker p62  by immunIn order to compare the basal state of HDAC6 expression and some mesenchymal markers among the cell lines used in our study, two types of independent experiments were carried out. First, an extraction of mRNA from the four cell lines and reverse transcription of it was performed. Once the cDNA was obtained, an RT-qPCR was made to quantify and compare the expression levels of HDAC6, Snail, Slug, and N-Cadherin genes. . HDAC6 lFor detection of expression at the protein level, the protein extract was quantified and the same amount of protein from each cell line was loaded on an SDS-PAGE to make a western blot. As in the RT-qPCR, we observed that the levels of Snail and Slug in LN405 and T98G cell lines were increased . UnexpecRegarding the expression of HDAC6, at first glance, we witnessed a band in all cell lines and, surprisingly, this band was more intense in the non-tumor cells, which was not consistent with the data obtained both by RT-qPCR and western blot of the tissues. Interestingly, that same more intense band in the NHA line did not correspond to the height of HDAC6 but was slightly below the bands observed in the tumor cells . To checRT-qPCR showed Gli1 overexpression in glioblastoma cell lines . This leTo check the basal state of the activation of the Sonic Hedgehog pathway, cells were transfected with various plasmids, creating three different groups detailed in Materials and Methods .The day after transfection, the culture medium was changed to a medium that did not contain serum, to inhibit cell proliferation. The incubation time with serum-free medium was one day. After this time, the activities of the luciferases were measured with the Promega Dual-Glo luciferase kit . Of the four cell lines used in the experiment, only the U87MG cell line was shown to have the Sonic Hedgehog pathway activated, since it was the only cell line in which higher values of the firefly luciferase/renilla luciferase ratio were obtained in the group with the wild-type promoter versus the mutated one . In all Once we saw that the three tumor cell lines presented HDAC6 overexpression both at the mRNA and at the protein level, we also wanted to study if they had a well-formed primary cilium since HDAC6 has acetylated α-tubulin as its target, one of the structural proteins of the primary cilium. Therefore, we believe that the cilium is a target for HDAC6.To do this, we seeded the cells in 24-well plates in which a coverslip previously sterilized with ethanol had been deposited. The following day, the culture medium was changed to new medium lacking serum and, after another 24 h, the coverslips were fixed with 4% formaldehyde. Immunofluorescence was performed against acetylated α-tubulin and γ-tubulin to visualize the primary cilium, using the NHA cell line as a positive control.The primary cilium can be identified if the basal body is detected: positive for γ-tubulin, close to the axoneme, and positive for acetylated α-tubulin as well . Of the As we have previously seen in immunohistochemistry in healthy brain and glioblastoma sections, the autophagy marker p62 is overexpressed in tumor tissue . This leTo study this process, we created two experimental groups for each cell line. The first group was the control group without treatment. The second group was treated with 100 nM bafilomycin A1, a V-ATPase inhibitor, which prevents acidification of the autophagosome and its subsequent degradation, thus inhibiting the autophagic process. We used LC3B as autophagy marker in its lipidated variant, LC3BII, since it is the one found in the phagosome membrane.The reason why we decided to carry out these experimental groups was the dynamism of autophagy. Autophagosomes are in a constant process of creation and destruction. Therefore, if we block autophagy, we will see an accumulation of autophagosomes in the cell. This difference in autophagosomes between the control and the treated group would be equivalent to autophagic flux; that is, autophagosomes that should have been degraded in baseline conditions were not finally degraded due to the inhibitory action of the drug. Therefore, the greater the difference in the levels of LC3BII between the control group and the group treated with bafilomycin A1, the greater autophagic activity the cell line will present.Consistent with what was observed in the immunohistochemistry of autophagic markers in tissues, increased autophagy was observed in tumor lines LN405, T98G, and U87MG , a resulOnce we saw that the autophagic process was increased in tumor cell lines, we decided to study whether its blockade inhibits cell proliferation. To do this, we seeded cells from the three cell lines in 96-well plates, and the next day, they were treated with increasing concentrations of bafilomycin A1. Three days after treatment, an MTT was performed to visualize cell viability. We saw that all cell lines suffered a great decrease in cell viability in those conditions treated with bafilomycin A1 , which sAfter observing an increase in the expression of HDAC6 both at the mRNA level and at the protein level  in tumorFor this purpose, cells of the three tumor cell lines were seeded in 6-well plates. The next day, they were silenced with a mixture of two siRNAs that targeted HDAC6 mRNA (siHDAC6) or with a control siRNA (siCTRL) that has no affinity for any mRNA sequence. Silencing was done using the lipofectamine 2000 method. Four hours after incubation with the siRNAs, the medium was changed and allowed to incubate for 72 h. After this time, the cells were subjected to different experiments.RNA was extracted from the silenced cells with the control siRNA or with the mixture of HDAC6 siRNAs. This RNA was subjected to RT-qPCR to analyze the expression status of HDAC6 after silencing in the three cell lines. We observed that in the three cell lines there was a decrease in the expression of HDAC6 (between 50% and 80%) in the cells treated with siHDAC6 compared with those silenced with siCTRL .Once we saw that the silencing with siHDAC6 significantly decreased the expression of the HDAC6 mRNA, we wanted to see if this translated into a decrease in the protein level. For this, 72 h after treatment with siRNAs, protein extraction and a western blot were performed. This experiment confirmed the data obtained by means of RT-qPCR, since there was a decrease in the protein level of HDAC6 levels in the condition treated with siHDAC6 . FurtherAfter demonstrating that treatment with siHDAC6 decreases the expression of this protein, we studied the effect of silencing on proliferation and the ability to form colonies in tumor lines. To do this, we seeded the cells, and the next day, we performed the silencing with siRNAs of HDAC6 or the control. At 72 h after the treatment, we trypsinized the cells, counted, and replated them both in 96-well plates to measure viability and in 6-well plates to perform a colony formation assay.For the proliferation assay, one day after plating cells, an MTT assay was performed to define the viability value at time zero and thus have a reference value. The MTT test was done on another plate 72 h after measuring the time zero plate. The value of time zero was subtracted from each value, and it was relativized to the results of the control group. As a result, we obtained that the silencing of HDAC6 inhibits cell growth , which mOn the other hand, seeing the oncogenic potential that HDAC6 has, we hypothesized whether, by silencing this protein, we could decrease the oncogenic activity of cell lines and decrease their clonogenic capacity. To do this, after seeding the silenced cells in the 6-well plates, they were incubated for 10 days so that they grew forming colonies. After this time, they were fixed with 4% formaldehyde and stained with crystal violet. The next day, the colonies formed during this time were counted. We saw that, as in the case of proliferation, the condition treated with siHDAC6 presented fewer colonies than the control condition . These rOnce we saw that the silencing of HDAC6 by interfering siRNAs decreases cell proliferation and the clonogenic capacity of tumor cell lines, we wondered if this silencing could also decrease cell migration. To study this effect, the same procedure was followed in the silencing as in the study of HDAC6 expression and of proliferation and clonogenic capacity after treatment with siCTRL or siHDAC6. In this case, the cells were trypsinized, counted, and seeded in 24-well plates until fully confluent. The next day, the monolayer was wounded and the culture medium was changed to another with 2.5% FBS to inhibit proliferation. When the wound was made, a photograph of the well was taken at time zero; at 24 and 48 h after the wound was made, photographs were taken again. HDAC6 silencing decreased the migratory capacity of the cells after wounding . This diWe have previously observed that the levels of some mesenchymal markers, such as N-cadherin, Snail, and Slug, are overexpressed in glioblastoma samples and tumor cell lines both at the mRNA level and at the protein level. We then wondered if we could inhibit its expression and thus revert the mesenchymal phenotype after HDAC6 silencing by siRNA.To do this, we made an RT-qPCR for Snail and Slug genes in the three cell lines, comparing their expression between the control group and the group in which we silenced HDAC6. We observed a decrease in Snail in all three tumor lines. In the case of Slug, the decrease was also observed but only in LN405 and T98G. The U87MG line did not show significant differences in the expression of this marker between the siCTRL and siHDAC6 groups .Once we saw the decrease in these markers at the mRNA level, we made the same comparison at the protein level to see if we also observed this decrease after HDAC6 silencing. We used the protein extracts used to study the protein expression of HDAC6 after treatment with the different siRNAs, and we performed a western blot against the same proteins as in the comparison by means of RT-qPCR. We observed that the Snail and Slug markers decreased in the LN405 and T98G cell lines, which are the ones with a more marked mesenchymal phenotype. In the case of U87MG, we did not detect a decrease in the Slug protein, while, as in the comparison between the cell lines in the basal state, we were not able to detect Snail E–G. ThesPrimary cilia are present in normal human astrocytes, but we have not been able to detect them in glioblastoma cells. Acetylated α-tubulin is one of the structural proteins of the primary cilium and is deacetylated by HDAC6. As we have seen in the comparison between cell lines and between healthy and tumor brain tissues, HDAC6 is overexpressed in glioblastoma, and this overexpression could lead to a decrease in acetylated α-tubulin levels and, consequently, to the absence of primary cilium in glioblastoma cell lines. Therefore, we believe that if we silence HDAC6, we could be able to restore the presence of the primary cilium in the glioblastoma cell lines. In order to test our hypothesis, we seeded cells in 24-well plates in which we had previously deposited a coverslip sterilized with ethanol. The next day, we did the silencing by dealing with siCTRL or the siHDAC6 mix. Two days after silencing, we changed the medium to serum-free medium to induce ciliogenesis and allowed to incubate for an additional day. After this time, we fixed the cells with 4% formaldehyde and performed immunohistochemistry against acetylated α-tubulin and γ-tubulin. We saw that none of the cell lines treated with siCTRL had cilium, corroborating what was seen in the comparison of the non-tumor cell line (NHA) with the glioblastoma cell lines. However, after HDAC6 silencing, a small subpopulation of cells presented a structure similar to the primary cilium, although we could not observe it in most of the cells . With thAs seen before, U87MG is the only one of the three cell lines in the study that exhibited activation of the Sonic Hedgehog pathway. To see if there was a relationship between the Sonic Hedgehog pathway and HDAC6 expression, we decided to study the expression of Gli1 by RT-qPCR in samples treated with siCTRL and with the mixture of siHDAC6 in U87MG cells.Gli1 expression decreased significantly when silencing HDAC6 compared to the control group , which sIn this case, we also saw a decrease in the quotient of firefly luciferase and renilla luciferase in the group in which we had silenced HDAC6 compared to the control group , which cImmunohistochemical analysis demonstrated that autophagic markers are overexpressed in glioblastoma samples compared to healthy brain tissue . FurtherIn order to answer our question, we reused the RNA samples extracted after silencing with siCTRL and siHDAC6 and performed an RT-qPCR to see if p62 expression had changed. In those samples in which we silenced HDAC6, p62 expression decreased. In conclusion, HDAC6 might be related to autophagy.Once we saw that HDAC6 had an effect on the expression of the marker p62 at the mRNA level, we tried to see if we really observed differences at the protein level after inhibition and stimulation of autophagy while silencing HDAC6. To do this, we seeded the cells and the next day, we performed the silencing with siCTRL or with the mixture of siHDAC6. Three days after silencing, different treatments were performed to observe autophagy in the different groups (siCTRL and siHDAC6). The first treatment was the control, which only carried DMSO; the second group was treated with bafilomycin A1 at a concentration of 100 nM; the third group was incubated with a nutrient-deficient medium, EBSS, to stimulate the autophagic process; and the last group was treated with 100 nM bafilomycin A1 in the EBSS nutrient-deficient medium. These treatments lasted 6 h. After this time, a protein extraction was carried out and was quantified the following day. With these protein extracts, a western blot was performed against the LC3B marker.In the LN405 cell line, a very low expression of the marker LC3BII was observed in basal conditions, which was increased when it was treated with the inhibitor bafilomycin A1 . This isIn the case of the T98G cell line, we obtained similar results to those obtained in LN405 cells. The expression of LC3BII increased in all the conditions in which we had carried out the treatment with bafilomycin A1, and the highest expression was found in the silenced condition with siCTRL and treated with EBSS + bafilomycin A1 . AlthougThe U87MG cell line also showed a decrease in autophagy when HDAC6 was silenced. Again, the difference in expression of LC3BII between the control and bafilomycin A1 groups was slightly lower in the conditions treated with the mixture of siHDAC6, while this difference was more noticeable between the EBSS and EBSS + bafilomycin A1 conditions . We can To give greater validity to the results, we decided to make an observation of the formation of autophagosomes by means of a fluorescence experiment. To do this, we seeded the cells, and the next day we co-transfected them with a plasmid that expressed the EGFP-LC3B fusion protein and with each of the siRNAs of the study, siCTRL, or siHDAC6. In this case, the period after the transfection in which we obtained the images was 48 h, since at 72 h, we observed great cell death. At 48 h after transfection, we performed the treatments with bafilomycin A1, EBSS, EBSS + bafilomycin A1, and DMSO as a control group. After 6 h incubation with the treatment, the autophagosomes were visualized in an EVOS microscope, and photographs of four random planes per well were taken, counting the amount of autophagosomes per cell in each of the conditions.The results we obtained corroborated the data obtained by western blot. In those conditions in which HDAC6 was silenced, the blockage of autophagy due to bafilomycin A1 treatment induced a lower number of autophagosomes per cell . This coWang et al.  observedAt the immunohistochemical level, we also studied the expression of different autophagic markers, such as p62 or LC3B. Autophagy is a regulatory mechanism usually increased in cancer cells to obtain the large amounts of energy needed for tumor development. An increase in autophagic markers in tumor tissue compared to healthy brain tissues is expected. This was the case with our immunohistochemical results of p62, a protein that showed overexpression in tumor tissue and is related to an advanced stage of the disease and to a worse prognosis in gliomas . HoweverThirdly, we also carried out a study of mesenchymal markers at the mRNA and protein levels. EMT is a reversible process that changes the cellular phenotype to a more undifferentiated phenotype and is usually accompanied by increased migration. In this case, using RT-qPCR, we studied the expression of the EMT-TFs Snail and Slug, seeing they were increased in glioblastoma. This overexpression has also been detected in other tumor types, such as breast , thyroidAfter having detected the overexpression of HDAC6 and mesenchymal and autophagic markers in tumor samples, we studied the expression levels of HDAC6 and of the mesenchymal markers Snail and Slug in our glioblastoma cell lines by RT-qPCR and western blot. Confirming the data obtained in tissue samples, we observed that both HDAC6 and mesenchymal markers were overexpressed in cell lines derived from glioblastoma. This result is consistent with previous studies conducted on other glioblastoma cell lines ,36. InteThe primary cilium, whose loss is associated with the dysregulation of several signaling pathways, such as the Shh pathway, is a structure responsible for capturing signals from the outside of the cell and for regulating the cell cycle. To verify the presence of the primary cilium in our four cell lines, we performed immunofluorescence against acetylated α-tubulin, a structural protein of the cilium. Only NHA cells presented a well-formed primary cilium. The three glioblastoma cell lines lacked a primary cilium. These data agree with previous studies carried out in patient tissues and in glioblastoma-derived cell lines ,7,37: deThe Shh pathway is involved in several cellular processes, such as embryonic development, cell division, and cancer ,46. To cThe baseline state of autophagy was the last comparison we made between the NHA and the glioblastoma cell lines. Autophagy is generally used by tumor cells to obtain the energy necessary to maintain the high rate of proliferation. We observed that in the three cell lines derived from glioblastoma, this process was more active than in the normal cell line. This corroborates our previous results obtained by immunohistochemistry, in which we saw that an autophagic marker, p62, presented a greater staining in tumor tissue than in healthy tissue. However, these results contrast with the data obtained by Huang et al., in which they show that as the grade of the astrocytic tumor increases, autophagy decreases ; but theWith these data, we have been able to determine that the glioblastoma cell lines used in our study exhibit HDAC6 overexpression, which we believe leads to defective ciliogenesis. Furthermore, although the three tumor cell lines express the mesenchymal markers Snail and Slug at the mRNA level, only U87MG cells lack an increase in their expression at the protein level, presenting a more epithelial phenotype than LN405 and T98G cells do. Of these three cell lines, only U87MG shows activation of the Shh pathway in the basal state despite being the most epithelial one. Furthermore, the glioblastoma-derived cell lines show overexpression of the autophagic process compared to the NHA cells. After having verified that HDAC6 is overexpressed in glioblastoma, we wanted to silence its expression using a siRNA to infer the effects this protein might contribute to the development of this tumor. After using the control siRNA or the mixture of HDAC6 siRNAs, we measured the expression of HDAC6 both at the mRNA and protein levels, and we observed that the HDAC6 siRNA mixture was effective in reducing the expression of this protein. Decreased HDAC6 expression was associated with increased expression of acetylated α-tubulin, since this is a target of HDAC6. With this experiment, we observed that our silencing system not only decreased the expression of HDAC6 but could also decrease its activity. Soon after, we carried out several experiments to study the effect of HDAC silencing on different processes that promote the development of glioblastoma.One of the effects of HDAC6 silencing in glioblastoma is the inhibition of cell proliferation and, therefore, the inhibition of tumor growth. These results have been observed in different tumor types, such as cholangiocarcinoma , lung caAlso, silencing of HDAC6 decreased cell migration, as has already been demonstrated in colon cancer ,55 afterHDAC6 is necessary for the induction of EMT after treatment with TGFβ in lung cancer  and in aThe primary cilium appears to be a tumor-suppressor structure in glioblastoma, since normal astrocytes have a perfectly formed primary cilium; but it is absent or aberrantly formed in glioblastoma cells ,37. HDACDespite having observed Gli1 overexpression in our three glioblastoma cell lines, the Shh pathway was active only in U87MG cells. Therefore, we decided to study the effect of HDAC6 silencing on the Shh pathway in this cell line. HDAC6 silencing decreased the level of Gli1 mRNA expression, which resulted in a reduction in the Shh pathway. Our results corroborate those of Yang et al.  in the sLastly, we observed that after the silencing of HDAC6, the expression of the autophagy marker p62 and the accumulation of LC3BII also decreased after bafilomycin A1 treatment. These data indicate less autophagy when HDAC6 activity was decreased. In addition, the number of autophagosomes formed in cells after HDAC6 silencing was lower than that of cells treated with siCTRL when autophagy inhibition had been performed by treatment with bafilomycin A1 both under normal conditions and in the absence of nutrients. These data are partially corroborated by Lee J.Y. et al. , who conFrom these results, we can assume that HDAC6 fulfills several functions in the development and growth of glioblastoma. This protein is involved in cell proliferation and clonogenicity as well as in the migratory capacity of tumor cells, increased after deacetylation of α-tubulin. We have also observed that HDAC6 inhibition decreases the levels of EMT-TFs Snail and Slug in mesenchymal glioblastoma cell lines and increases the expression of acetylated α-tubulin, the target of HDAC6 and possible epithelial marker, in the three cell lines used in the study. The increased expression of acetylated α-tubulin after HDAC6 inhibition resulted in the appearance of structures similar to the primary cilium in glioblastoma cells, structures that are not present at baseline. Furthermore, the primary cilium is related to a more epithelial-cell phenotype in various tumors. HDAC6 silencing also decreased the activation of the Shh pathway in U87MG cells, the only cell line in which the Shh was active under baseline conditions. Finally, we observed that HDAC6 promotes the autophagic process and decreases it after its inhibition with the mixture of HDAC6 siRNAs. We thus believe that, after the decrease in autophagy, cells cannot obtain all the energy they need for cell growth and proliferation, and, therefore, HDAC6 inhibition has a tumor-suppressing function.We have demonstrated HDAC6 is involved for the proliferation and migration of GBM cells by the negative regulation of primary cilium formation. This could also be considered as if HDAC6 might regulate differentiation of GBM cells. But our experiments cannot answer that question so far. They represent only an initial point from which to advance further to solve the question. More research is needed to finally figure out a molecular mechanism that regulates the primary cilium formation/differentiation by HDAC6 in glioblastoma cells.Noorani et al.  demonstrAbout the concept of essentiality of genes, we have reports that compare different knock-down expression techniques. Dempster et al.  analyzedSaccharomyces cerevisiae genes and grouped them into four categories [Essential genes have been defined as those required for the survival of an organism or a cell . Some oftegories : from coHDAC6 might be, having taken all these notes into account, an example of a non-essential, or at best, of a conditional essential, gene that plays a role in cell survival and proliferation. It is also important to show that HDAC6, far from being an essential gene in its entirety, could be an essential gene for some specific functions, as has been shown in inflammatory breast cancer . The impHDAC6, as well as mesenchymal and autophagic markers, are overexpressed in glioblastoma samples and cell lines. Glioblastoma cell lines lack a primary cilium and present increased autophagy compared with normal human astrocytes. HDAC6 silencing decreases cell proliferation, clonogenicity, and migration in glioblastoma cell lines; restores the primary cilium, reverts the EMT phenotype, and inhibits autophagy; and also inhibits the Shh pathway in U87MG cells. As a whole, we may conclude that HDAC6 seems to be a good target for glioblastoma treatment.


Radio-chemotherapy with 5-flu orouracil (5-FU) is the standard of care treatment for patients with colorectal cancer, but it is only effective for a third of them. Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy. In order to identify the mechanism of acquired resistance, 5-FU chemoresistance was induced in CRC cell lines by passaging cells with increasing concentrations of 5-FU. To study global molecular changes, quantitative proteomics and transcriptomics analyses were performed on these cell lines, comparing the resistant cells as well as the effect of chemo and radiotherapy. Interestingly, a very high proportion of downregulated genes were annotated as transcription factors coding for Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (KZFPs), the largest family of transcriptional repressors. Among nearly 350 KRAB-ZFPs, almost a quarter were downregulated after the induction of a 5-FU-resistance including a common one between the three CRC cell lines, ZNF649, whose role is still unknown. To confirm the observations of the proteomic and transcriptomic approaches, the abundance of 20 different KZFPs and control mRNAs was validated by RT-qPCR. In fact, several KZFPs were no longer detectable using qPCR in cell lines resistant to 5-FU, and the KZFPs that were downregulated only in one or two cell lines showed similar pattern of expression as measured by the omics approaches. This proteomic, transcriptomic and genomic analysis of intrinsic and acquired resistance highlights a possible new mechanism involved in the cellular adaptation to 5-FU and therefore identifies potential new therapeutic targets to overcome this resistance.The online version contains supplementary material available at 10.1186/s12885-022-09417-3. Colorectal cancer (CRC) is the second deadliest cancer worldwide after lung cancer , and is de novo thymidyl which is necessary for DNA replication and repair [5-FU exerts its effect by inducing DNA damage then cell death by three different mechanisms . These cd repair –22. Leucd repair . 5-FU isd repair –28. It nd repair , but alsd repair , 35 as wd repair . Moreoved repair , 33. dUTd repair . Repair d repair , 38. Thed repair .Despite our understanding of the mechanism of action of 5-FU, drug resistance remains a significant limitation to the clinical use of 5-FU, as both intrinsic and acquired chemoresistance represents the major obstacles for the success of 5-FU-based chemotherapy , 40. Intet al. divide CRC into three groups according to their phenotype: A, proliferative, B, epithelial and C, mesenchymal [Several comparisons exist between genomic characteristics of CRC and the response to 5-FU. For example, CRC with a defect in mismatch repair (MMR) genes due to microsatellite instabilities (MSI+) would be more resistant to 5-FU because of their inability to recognize and/or react to the incorporation of 5-FU in DNA, but it cannot alone explain the resistance to 5-FU , 54. In enchymal . Later, enchymal . Neverthenchymal , 56.In this study, we investigated intrinsic and acquired resistance to chemotherapy and radiation therapy in CRC cell lines to highlight molecular mechanisms potentially involved in this resistance, and identified proteins from the Krüppel-associated box (KRAB) domain containing zinc-finger proteins (KZFPs) as downregulated in colorectal cancer cell lines that are resistant to 5-FU.2. The RRIDs of each cell line are listed in the Key Resources Table. The genomic identity of each cell line is described in Table CRC cell lines were purchased from the American Type Culture Collection (ATCC). Caco-2/15 (ATCC® HTB-37), DLD-1 (ATCC® CCL-221), HCT-116 (ATCC® CCL-247), HT-29 (ATCC® HTB-38), SW480 (ATCC® CCL-228) and SW620 (ATCC® CCL-227) cell lines were grown as adherent cells in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% amphotericin B, at 37 °C with 5% COwww.graphpad.com).To establish the acquired resistant cell lines, DLD-1, HCT-116 and HT-29 were grown in medium supplemented with increasing concentrations of 5-FU for 8 months. The cells underwent three passages in increasing concentrations of 5-FU: 2 μM then 5 μM then increasing by 5 μM at each step, up to 50 μM. The resistant cells are maintained in a medium supplemented with 50 μM 5-FU. For the cell viability assays, 20,000 sensitive and resistant cells were grown in 6-well plate (D0) for two days. They were treated with either 10 μM uracil , or 10 μM 5-FU or 50 μM 5-FU at D + 2. There were counted using a hemocytometer at D + 2, D + 4 and D + 7 . Dose-response curves were obtained with GraphPad Prism 8.1.2 for Windows  control, (2) chemotherapy alone, (3) radiation therapy alone and (4) chemotherapy combined with radiation therapy. 500,000 DLD-1, HCT-116 and HT-29 cells were grown in 100 mm Petri dish for 48 h. Cells were either (1) treated with uracil 10 μM for 1 and 24 h, or (2) treated with 5-FU 10 μM for 1, 4 and 24 h, or (3) irradiated with the corresponding LD50 using the irradiator X-RAD 225 XL (Precision X-ray) and lysed after 1, 4 and 24 h post-irradiation, or (4) treated with 5-FU 10 μM for 24 h, irradiated with the corresponding LD50 and lysed after 1, 4 and 24 h post-irradiation.Three experimental conditions were tested: 1) control, 2) chemotherapy alone and 3) chemotherapy combined with radiotherapy. 250,000 DLD-1, HCT-116 and HT-29 cells were grown in 100 mm Petri dish for 72 h. They were treated with either 10 μM uracil (condition 1) or 10 μM 5-FU (condition 2) for 24 h. Medium was changed and cells were irradiated with corresponding LD50 using an X-ray irradiator  (condition 3). The device was operated as per manufacturer’s settings for cultured cells . The same conditions with the corresponding resistant cell lines were conditions 4 to 6. All the experimental conditions were described in Fig. Sx g at 4 °C for 5 min. The pellets were lysed in 8 M urea 10 mM HEPES pH 8.0 and centrifuged at 16000 x g at 4 °C for 10 min. The amounts of protein in the supernatants were measured with Thermo Scientific™ Bicinchoninic acid (BCA) protein assay kit (#23225) and an amount of 50 μg of proteins was kept for each sample. The volumes were adjusted to 100 μL with 100 mM TEAB dissolution buffer (triethyl ammonium bicarbonate #90114).The cells were washed three times in cold PBS 1X, harvested in cold PBS 1X and centrifuged at 500 x g at 4 °C for 10 min, and then the pellets were dried.Five μL of 200 mM TCEP reducing agent (Bond-Breaker™ Tris-(2-carboxyethyl)-phosphine solution #77720) were added to each sample, which was then incubated at 55 °C for 1 h. Five μL of 375 μM CAA alkylated agent (chloroacetamide) were added to each sample, which was then incubated at room temperature in the dark for 30 min. Six volumes of chilled acetone was added, and proteins were precipitated at −20 °C for at least 4 h. The samples were centrifuged at 8000 The pellets were resuspended in 100 μL of 50 mM TEAB. 1.25 μg of 12.5 ng/ml Trypsin Gold (Promega #V5280) modified in 50 mM acetic acid was added then the samples were digested at 37 °C under agitation overnight.Each TMTsixplex label reagent (#90066) was equilibrated at room temperature, dissolved in 41 μl anhydrous acetonitrile and added in a 50 μg-peptide sample  and loaded onto a trap column with a constant flow of 4 μL/min . Peptides were then eluted onto an analytical C18 Column  over a 2-h gradient of acetonitrile (5–37%) in 0.1% formic acid (FA) at 500 nL/min while being injected into a TimsTOF Pro Mass Spectrometer coupled with a Captive Spray nano-electrospray source . Data were acquired using data-dependent auto-MS/MS with a 100–1700 m/z mass range, with PASEF enabled with a number of PASEF scans set at 10 (1.27 s duty cycle) and a dynamic exclusion of 0.4 min, m/z dependent isolation window and collision energy of 42.0 eV. The target intensity was set to 20,000, with an intensity threshold of 2500.Feature extraction, database searching, and quantitation were performed with the MaxQuant version 1.5.2.8 software  and the The same experimental conditions as “Proteomics – Experimental conditions” section were used for transcriptomics experiments.Total RNA from the six conditions for the three CRC cell lines in triplicate was extracted using RNeasy RNA isolation kit (#74104). The RNA concentrations were measured by NanoDrop and RNA qualities were evaluated on 1.2% agarose gel. The 54 samples were sent to McGill University and Genome Quebec Innovation Center.Libraries were generated from 250 ng of total RNA. mRNA enrichment was performed using the NEB Next® Poly(A) mRNA Magnetic Isolation Module . The cDNA synthesis was performed using NEBNext RNA® First Strand Synthesis and NEBNext® Ultra™ II Directional RNA Second Strand Synthesis modules . The final steps were performed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® . PCR adapters and primers were obtained from New England Biolabs. The libraries were quantified by Kapa Illumina GA with Revised Primers-SYBR Universal Fast Kit . The average size of the RNA fragments was determined using the LabChip GX instrument . Sequencing of the libraries was performed using NovaSeq 6000  using an S4 PE100 protocol.https://broadinstitute.github.io/picard/). RNA read quality was assessed using FastQC 0.11.8 (30254741) and the Trimmomatic 0.36 tool (24695404) was used to preprocess the data [RNA-seq analysis was carried out as described previously . Brieflythe data . The reathe data . The hum6 tool 245404 was www.graphpad.com). Venn diagrams were generated using Venny 2.1.0 [All experiments were performed on biological replicates. Sample sizes for each experiment are reported in the corresponding figure legends and methods. Statistically significant differences between control and experimental groups were determined using a test which is reported in the corresponding figure legends and methods, and were calculated using GraphPad Prism 8.1.2 for Windows  and half maximal inhibitory concentration (IC50), respectively. These cell lines had different genetic profiles and the mutations in key genes involved in CRC development have been characterized as well as the proteomic subtypes and the consensus molecular subtypes (CMS) (Table Radiation therapy combined with 5-FU-chemotherapy is the current standard of care treatment in the neoadjuvant setting for patients (TNM stages II and III) . To asseS) Table .50 (irradiation treatment) and IC50 (5-FU treatment) values for all six cell lines were different and did not correlate  were counted after an 8-day or 12-day incubation , HCT-116 (IC50: 4.5 μM) and then HT-29 (IC50: 2.7 μM)  administered for 24 h was measured for seven days to ensure that resistance was acquired in these cell lines  were treated under three different conditions  then were extracted in triplicate. All those different conditions were then mixed differentially in order to allow comparison between the different cell lines and the different treatments, resulting in different 6-plex TMT mixes Table S. HierarcChanges in gene expression at the transcriptional level in the different cell lines under different conditions was analyzed by RNAseq. To achieve this, total RNA from the three conditions  for the three CRC cell lines either sensitive or following acquired resistance to 5-FU were extracted in biological triplicates. Sequencing of the 54 libraries was performed using NovaSeq 6000 (Illumina) using an S2 PE100 protocol. The number of reads varied between 56,377,534 and 240,114,519 for each sample. The readings were aligned to the human transcriptome constructed from the annotations from ENSEMBL (Homo sapiens version 98) (Table SRegulation of DNA recombination (GO:0000018) represented by H1FX, KPNA2, RIF1, HIST1H1C and RPA2  included RPS29, MRPS28, RPLP1 and RPL37A  [Mitotic cell cycle process (GO:1903047) (Table SSecretory granule membrane (GO:0030667) can be observed   with SFNFinally, we were interested in proteins and transcripts modulated after the induction of 5-FU-chemoresistance in the three cell lines Fig. J, K.At the proteome level, down-regulated proteins were found in the three cell lines such as PDCD10, OCIAD2, THOC2, STX5, TRMT61A, DIDO1, FLOT1, LEMD3 or even the pro-apoptotic protein BAX  [50 = 14.05 μM in their study, against 2.7 μM in ours). The HT-29 cell line has an MSS phenotype, which does not exhibit microsatellite instability. This phenotype has repeatedly been associated with normal expression of genes involved in mismatch repair (MMR) and with a good response to 5-FU treatment [al. had defined an LD50 of 2.3 ± 0.98 Gy for HCT-116 cell line [K-RAS gene, a mutation carried by almost 50% of patients with CRC [K-RAS G13D) , SW480 (1.4 μM), HT-29 (2.7 μM), HCT-116 (4.5 μM), SW620 (5.2 μM) and DLD-1 (9.3 μM)  . The onlreatment , 80. Thiell line  which isell line , 83 and ell line  and HT-2ell line  cell linell line . This paell line  and can with CRC  and by H pathway , 90 and et al.’ study, morphological changes were observed between the sensitive lines and the corresponding resistant CRC cell lines  on the proteome and transcriptome were evaluated. 5-FU, by its mechanism of action, interferes with replication but also with DNA repair. It is also incorporated into RNA and DNA, causing damages. The result of radiotherapy is to induce cell death by causing DNA damages, and the cellular responses are staggered over time: early recognition of damage (a few seconds), signaling and repair of lesions (a few tens of minutes), induction or repression of genes (a few hours) then cell death (a few hours to a few days). It has been estimated that an irradiation dose of 1 Gy causes on average 1000 single-strand breaks (SSB) per cell and 40 double strand breaks (DSB) [Secondly, we induced a 5-FU-chemoresistance in the DLD-1, HCT-116 and HT-29 lines in order to study the mechanisms involved in acquired chemoresistance, a phenomenon which is observed in many patients during their treatment. To achieve resistance in cell lines, we followed the protocol described in Fig. ks (DSB) . The synet al. study [versus TMT; RKO cell line much more 5-FU sensitive than HCT-116, DLD-1 and HT-29 cell lines [We performed TMT-6plex and RNA sequencing experiments to assess proteomic and transcriptomic changes, respectively, in sensitive and resistant CRC cell lines in response to chemotherapy and radiation treatments. As expected, the first observation was that the number of proteins and transcripts modulated in response to 5-FU treatment was proportional to the level of intrinsic sensitivity to 5-FU of the three CRC cell lines, which was also more obvious at the transcriptional level) Fig. . At the l. study  but the ll lines ). On theWe then looked in more detail at the effect of 5-FU on the proteo-transcriptome of CRC cell lines Fig. . The DLDFinally, we were interested in proteins and transcripts modulated after the induction of 5-FU-chemoresistance in the three cell lines (Fig. Additional file 1.Additional file 2.Additional file 3.Additional file 4.