TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line . The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations , with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with  TP53 mutations, irrespective of histology [TP53 status of GCTs, combined with a pluripotent phenotype, high levels of PUMA and NOXA, and, often, low expression levels of CDKN1A (P21), result in a cellular disbalance and a favor towards apoptosis over DNA repair [TP53 mutations, or whether these mutations simply occur more in these tumors as these tumors harbor different intrinsic resistance mechanisms. To this end, we wanted to elucidate whether the removal of TP53 in a testicular GCT cell line can convey cisplatin resistance, and, thus, (partly) explain mediastinal GCT aggressiveness and treatment resistance. Firstly, we used the online cBioPortal tool to analyze an extensive GCT patient data set containing detailed clinical information, including treatment, tumor resistance, tumor stage, anatomical location, histology, and genetic mutations [TP53 background (TP53 mutant/loss in NCCIT from the mediastinum and TP53 wild-type in 2102Ep from the testis), were modified using a CRISPR/Cas9-mediated TP53 knock-out model system. We subsequently investigated the difference in cisplatin resistance in these testicular and mediastinal GCT cell lines. Using both GCT patient data characteristics and functional mechanistic cell line investigations, we show a role of P53 in GCT cisplatin resistance related to the anatomical location of the tumor.Germ cell tumors (GCTs) are the most common solid malignancies in young men ,2. Despiistology ,10. MoreA repair ,13,14,15A repair ,17,18,19A repair ,17. On tA repair ,17,20,21A repair ,7. WhileA repair . Apart fA repair ,17,19,23TP53 (and MDM2) alterations in cisplatin-sensitive and -resistant GCTs. Strikingly, while GCTs rarely harbor TP53 mutations, in line with their embryonic phenotype [TP53 are detected in cisplatin-resistant patients. Furthermore, we observe that MDM2 amplifications become increasingly abundant in patients with cisplatin-resistant GCTs. Note that, as expected, alterations regarding TP53 are often missense mutations or deep deletions. When comparing the disease-free survival of patients with alterations in the TP53 gene to patients with wild-type TP53 , we observed a highly significant  decrease in disease-free survival in patients harboring TP53 alterations  GCTs, we initially used the cBioPortal online tool. We investigated the MSKCC data set on refractory GCTs previously reported by Bagrodia and colleagues in 2016 . The rathenotype , alteraterations . As preve testis . Interest tumors A,B.TP53 status, whereas NCCIT carries a hemizygous one-base-pair deletion at nucleotide 949 (codon 272), resulting in a frameshift and a premature STOP codon at codon 347 .To study the difference between mediastinal and testicular GCTs, we used the well-established and -characterized NCCIT and 2102Ep GCT (EC) cell lines. While 2102Ep originates from the testis, NCCIT originates from the mediastinum, with a similar differentiation state . Furtherodon 347 A. This oMDM2 expression than both the 2102Ep cell lines, with 2102Ep resistance displaying the highest levels of MDM2 . RNA sequencing (RNA-seq) analysis showed that both the parental and resistant NCCIT cell lines had lower normalized  of MDM2 B, suppor of MDM2 C. In conll lines . Principll lines D,E. Thisterature ,16,23,27TP53 status [TP53 mutant status, the NCCIT cell line is considered to be inherently sensitive to cisplatin. Thus, we further compared the mutational status of the NCCIT cell line to the mutations found in refractory GCT patients , suggests that TP53 mutations could add additively to inherent cisplatin resistance mechanisms [TP53 in cisplatin resistance in the approach described. Therefore, we generated isogenic CRISPR/Cas9-mediated TP53 knock-out clones of both 2102Ep and NCCIT, as well as their resistant counterparts. Sanger sequencing, after mono-clonal picking and expansion, revealed mono-clonal sequence traces and a one-base-pair insertion at amino acid 48, resulting in a premature STOP codon at amino acid 51  profiling (TP53 and CDKN1A (P21) expression in both 2102Ep parental TP53 knock-out lines  and 2102Ep-resistant TP53 knock-out lines , which was also confirmed by Western blot (TP53 expression had no effect on the differentiation status. The miR371a-3p expression levels were checked because of its many implications in GCTs (mostly as a biomarker and marker of pluripotency in these tumors), together with the implications of P53 pathway regulation [TP53 knock-out clones, with cisplatin, we identified a clear significant  shift in cisplatin resistance when comparing the TP53 knock-out clone to its wild-type counterpart, with the 2102Ep-resistant TP53 knock-out clone demonstrating the highest cisplatin resistance  in the NCCIT-resistant TP53 knock-out clone compared to its NCCIT-resistant counterpart  shift in cisplatin resistance in the NCCIT-resistant TP53 knock-out clone compared to its NCCIT-resistant counterpart  [MDM4 alterations is not fully unexpected, as MDM4 is not able to directly ubiquitinate P53 and target it for proteasomal degradation; this is in stark contrast to MDM2, which is able to directly downregulate P53 through proteasomal degradation [MDM2 amplifications were mostly testicular in origin, whereas TP53-mutated tumors were primarily mediastinal [TP53 mutations in mediastinal GCTs could be due to a less favorable niche and more strict selection for these tumors [TP53 signaling pathway [TP53 mutations, and, thereby, a more treatment-resistant phenotype. In this study, we tried to elucidate if there was a causal link between the poor prognosis for mediastinal GCTs and their bias towards TP53 mutations. In line with cBioPortal analysis, both the Western blot and RNA-seq data indicated higher MDM2 protein levels and a higher MDM2 expression in the testicular 2102Ep GCT cell line compared to the mediastinal NCCIT GCT cell line, despite the lack of MDM2 amplifications in 2102Ep. This suggests a mechanism for treatment resistance within wild-type TP53 GCTs via MDM2 [MDM4 between the mediastinal and testicular cell lines. Finally, as P53 is known to regulate MDM2 and MDM4, and could, therefore, interfere with DNA-damage sensing, we studied the upstream activity of the DNA-damage signaling pathway in the TP53-mutated NCCIT cell line [TP53 mutation present in the NCCIT cell line to the mutational profile of TP53 mutations in refractory GCT patients, we noticed that the NCCIT mutant was located more distally within the protein and was unlikely to fully disrupt the function of the DNA-binding domain [TP53 in GCT cisplatin resistance and to interrogate the functionality of the mutant TP53 in the NCCIT cell line, we generated CRISPR/Cas9-mediated TP53 knock-out cell lines. As the loss of TP53 can contribute to chromosomal instability and, therefore, cisplatin resistance, we used GSA analysis to verify the genomic changes between the original and knock-out clones. No major copy number changes were identified related to the TP53 knock-out procedure and subsequent selection. However, small copy number alterations could be observed, possibly related to clonal selection and expansion. Interestingly, although rarely observed in testicular GCT patients, full knock-out of TP53 in the testicular EC cell line 2102Ep resulted in a significant increase in cisplatin resistance in both a parental and resistant background. This coincided with both the reduced expression levels of TP53 and CDKN1A (P21), as well as the reduced protein levels of P53 and P21, suggesting a strong dependency on intact TP53 for CDKN1A expression in this cell line. Strikingly, when we knocked out TP53 in the TP53 mutant mediastinal EC cell line NCCIT, we also observed an increase in cisplatin resistance in the resistant cell line only. The knock-out of TP53 resulted in a full loss of P53 at the protein level and a minor reduction at the mRNA level. Interestingly, the parental TP53 knock-out cell line showed reduced expression of microRNA371a-3p, while the resistant TP53 knock-out line showed an increase. It remains to be elucidated whether this NCCIT observation is a passenger effect or is possibly related to previous findings regarding the negative regulatory effect of this microRNA cluster on TP53 expression [TP53 knock-out is relevant in the context of the informativity of this molecular biomarker for GCTs, as recently reviewed by Leão and colleagues, as well as in the case of refractory disease [TP53 does not induce differentiation, excluding the possibility of increased resistance due to a differentiated phenotype [TP53 status of these cell lines, being wild-type, hemizygous mutant, or full knock-out, it did not interfere with the detection of the microRNA cluster 371a-3. In other words, the miR371a-3 cluster appears to retain its suitability as a GCT biomarker irrespective of the tumor’s TP53 status and, thus, also partially the level of cisplatin resistance [TP53 in the NCCIT-resistant (but not 2102Ep) cell line resulted in increased protein levels of P21, an effect not readily observed at the mRNA level, suggesting a negative effect on the P21 levels of the mutant TP53 present in the resistant NCCIT subclone. This is interesting in light of previous studies indicating increased tumor resistance, malignancy, and aggressiveness caused by the P53-independent upregulation of P21 in TP53 mutant tumors [Here, we present a study on the effect of the loss of t tumors . Of noteficance) . This loradation ,21,39. Siastinal . One coue tumors . As ment pathway . It coulvia MDM2 . This isvia MDM2 ,21,42. Well line ,27. Cellell line ,44,45. Oell line ,44,45. Wpression . The abs disease . In conthenotype . It is isistance ,47,48,49t tumors ,52,53,542. The resistant isogenic clones were generated by the group of Christoph Oing and Friedemann Honecker, University of Hamburg, Germany. These resistant cell lines were obtained by exposing the parental cell lines to cisplatin over a period of 6–9 months to increase the sublethal concentrations of these cells to cisplatin. Cisplatin dose was kept the same for two subsequent treatments and stepwise increased by 30–50%. Each cisplatin treatment was applied for 24 h in 80% confluent cells, followed by a medium change. Cells were allowed to rest with regular medium change over a period of 5–7 days until re-growth was detectable, and cells were re-plated afterwards. After one passage to regenerate, the next treatment was applied as mentioned above. Cells were passaged for a maximum of 7 cycles, followed by intermittent cryopreservation to prevent differentiation. For experimental use, obtained resistant subclones were cultured similarly to their sensitive parental lines without continuous cisplatin supplementation. The resistant subclones were maintained similarly to the parental cells .The parental (T)GCT cell lines used were previously reported and further characterized by us . NCCIT  and selecting the germ cell tumors  data set under testis tumors [TP53 and MDM2 in the gene query section. Subsequent analysis was performed using the tools provided by cBioPortal.Patient data were analyzed by using cBioPortal ,25 by vis tumors . We querSTAR (v2.5.3a) was used as aligner for RNA-seq data, using 2-pass mapping for each sample separately. Mapping quality plots were generated and checked based on sambamba flagstat (v0.6.7) statistics. Count files, with the number of RNA-seq reads for each gene were created with subread FeatureCounts (v1.5.2) and normalized for library size to counts per million (CPM).Cell lysates were made using RIPA buffer, followed by measuring protein concentrations according to Pierce BCA Protein Assay Kit . A total of 25 µg protein was loaded onto a 4–15% Mini-Protean TGX Stain-Free Protein Gel . After separation, the proteins were transferred to a 0.2 µm PVDF membrane with the Turbo Trans-Blot system . The following primary antibodies were added to the membranes: mouse anti-MDM2 (IF2) , mouse anti-MDMX , anti-β-actin antibody , mouse anti-human p53 , rabbit anti-human p21 Waf1/Cip1  and mouse anti-vinculin , as a loading control. After incubating the membranes overnight at 4 °C, either goat anti-mouse IgG(H+L) cross-absorbed, HRP  or goat anti-rabbit IgG (H+L) cross-absorbed, HRP  were added as secondary antibodies and the membranes were incubated for 2 h at RT. To detect the proteins, Clarity Western ECL substrate  was added to the membranes.TP53 , using the Neon Transfection System . To confirm a successful TP53 knock-out and to determine introduced mutations, of all the different cell lines, DNA was isolated using QuickExtract according to manufacturer’s protocol, followed by PCR with the following primers: forward: CAGTCAGATCCTAGCGTCGA and reverse: CACTGACAGGAAGCCAAAGG. Sequencing was performed by Macrogen Europe and knock-out efficiency was determined with the online ICE analysis tool by Synthego ICE analysis. Available online: https://ice.synthego.com/#/ (accessed on 15 September 2021).Both parental and resistant cells of NCCIT and 2102Ep were exposed to a ribonucleoprotein complex made of resuspension buffer R, 61 µM Cas9 protein and 100 µM gRNA duplex consisting of cRNA and tracrRNA, which was designed to target the gene of interest, High-quality total RNA was extracted from the above-mentioned cell lines using TRIzol reagent  according to the manufacturer’s instructions. Quantity and quality were assessed on Nanodrop One  and with Qubit 4 fluorometer .Targeted miRNA profiling was performed on diluted RNA (5 ng) using TaqMan MicroRNA Reverse Transcription Kit  and TaqMan Assays RNU48 (001006), hsa-miR371-3p (002124), and hsa-miR885-5p (002296) as described before . RNU48 wHPRT (hs02800695_m1), TP53 (hs01034249_m1), TP73 (hs01056231_m1), CDKN1A/P21 (hs99999142_m1), BBC3/PUMA (hs00248075_m1), PMAIP1/NOXA (hs00560402_m1), SOX2 (hs01053049_s1), POU5F1 (hs00999632_g1), POU5F1 (hs04195369_s1), POU5F1 (hs03005111_g1). The 2× TaqMan Advanced PCR Master Mix  was used and reactions were run in 96-well plates on QuantStudio 12K Flex System . HPRT was used as a housekeeping gene for normalization purposes and relative gene expression levels were computed as 2−ΔΔCt. Fold change between parental and knock-out clones was plotted.Diluted RNA (50 ng) was reverse transcribed using SuperScript IV reverse transcriptase . RT-QPCR was run using the following TaqMan gene expression assays: 2 overnight. Then, the following concentrations of cisplatin were made from 1 mg/mL cisplatin stock  diluted in fresh medium enriched with 0.9% NaCl: 32 µM, 16 µM, 10 µM, 8 µM, 6 µM, 4 µM, 2 µM, 1 µM and 0.33 µM, or, 32 µM, 25 µM, 20 µM, 17.5 µM, 15 µM, 12.5 µM, 10 µM, 5 µM and 2.5 µM, dependent on expected IC50. The medium in the wells was replaced with 100 µL of the corresponding cisplatin medium. The plate was incubated at 37 °C and 5% CO2 for 72 h.The sensitivity of the cells (both NCCIT and 2102Ep) to cisplatin was determined using a viability assay. To test the sensitivity of the cells to cisplatin, 10.000 NCCIT and 2102Ep parental cells and 4.000 2102Ep-resistant cells  were seeded in 100 µL RPMI or DMEM (respectively) medium per well in a 96-well plate. The plate was incubated at 37 °C and 5% CO® 2.0 Luminescent Cell Viability Assay kit  was used to lyse the cells to be able to measure the viability. Afterwards, 100 µL of the lysate was transferred to a Pierce white opaque 96-well plate (Thermo Fisher). The luminescence was measured on an ID3 Spectramax  or a FLUOstar Omega .After incubation of either 2102Ep cells or NCCIT cells with cisplatin concentrations, CellTiter-Glot-test or one-way ANOVA with a Tukey’s multiple comparisons post hoc test.The viability assays were performed with three technical replicates and at least on three separate occasions . Data were visualized and interpreted using GraphPad Prism 9. To extract IC50 values, concentrations were transformed to logarithms, non-linear S-curves were fit through the data set using a GraphPad algorithm to extract absolute IC50s and the S-curves were interpolated with y = 0.5 to derive the absolute IC50s. Statistic differences were calculated using either an unpaired Student’s Genomic changes between original and knock-out subclones were identified after mono-clonal expansion and a minimum of ~3 months of separate cell culture using Infinium Global Screening Array-24 v3.0 BeadChipGSA (GSA) profiling. Array data were obtained from the HUGE-F as a Genome Studio vs. 2.0.4  project using the hg38 reference genome.® 5.3.2 and GraphPad Prism 9.Data were visualized using cBioPortal (v3.7.12), Adobe Illustrator (2020), SnapGeneTP53 status in cisplatin resistance in GCTs, related to the anatomical location associated with molecular constitution. The results obtained show that the investigated cell lines, independent of intrinsic resistance, demonstrate a beneficial effect of the loss of TP53 regarding cisplatin resistance, as schematically represented in TP53, originally present in the commonly used NCCIT, is functional in the context of cisplatin sensitivity, as the knock-out of this mutant resulted in increased cisplatin resistance. The isogenic generated cell lines provide a novel informative model system to study the involvement of TP53 in the original cellular background (of NCCIT ad 2102Ep), and provide insight into the clinical behavior of GCTs. Moreover, we provide, to our knowledge, for the first time, insights into the functionality of the hemizygous TP53 mutant present in NCCIT, and, additionally, we developed a cell line harboring a bona fide TP53 null status. More GCT cell lines originating from both tumor sites , or even tumor-derived organoids from these sites, could provide more insights into the role of TP53 in the clinical behavior and chemotherapy response of these tumors. These data can aid in patient stratification for optimal clinical decision making, especially for mediastinal tumors in which TP53 mutations are more common. Patients could benefit from screening for intrinsic TP53 mutations in the primary tumor or acquired TP53 mutations in the refractory malignancies. This study illustrates the contribution of TP53 not only to known cisplatin sensitivity, but also as a potential target for acquired cisplatin resistance.In conclusion, this study, combining GCT patient data characteristics and functional mechanistic cell line investigations, illustrates the role of


Currently, no clinically useful biomarkers for radioresistance are available in head and neck squamous cell carcinoma (HNSCC). This study assesses the usefulness of Cell Line Microarray (CMA) method to enhance immunohistochemical screening of potential immunohistochemical biomarkers for radioresistance in HNSCC cell lines.Twenty-nine HNSCC cell lines were cultured, cell pellets formalin-fixed, paraffin-embedded, and arrayed. Radioresistance features of the cell lines were combined to immunohistochemical stains for p53, NDFIP1, EGFR, stem cell marker Oct4, and PP2A inhibitor CIP2A.Expression of p53, EGFR or CIP2A did not indicate intrinsic radioresistance in vitro. Stem cell marker Oct4 nuclear positivity and NDFIP1 nuclear positivity was correlated with increased intrinsic radioresistance.The usefulness of CMA in analysis of HNSCC cell lines and discovery of biomarkers is demonstrated. CMA is very well adapted to both testing of antibodies in a large panel of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC.The online version contains supplementary material available at 10.1186/s12885-021-08618-6. Radiotherapy has a major role in the treatment of head and neck squamous cell carcinoma (HNSCC). Albeit the general radiosensitivity of HNSCC, radioresistance of a subset of the tumors is a major clinical problem requiring further study both for an enhanced mechanistic understanding of radioresistance, and to overcome clinical radioresistance in patient therapy. Putative mechanisms responsible for clinical radioresistance include hypoxic environment, EGFR-pathway alterations, epithelial-mesenchymal transition, deregulation of p53, angiogenesis, and cancer stem cells . DespiteThe inability to correctly identify clinically significant molecular events in cell line studies remains a significant problem, since virtually all in vivo cancer studies are preceded by in vitro cell line investigation into cancer behavior and characteristics. The genomic and molecular diversity of different cell lines complicates the selection of cell lines for in vitro studies. Behavior of different cell lines may vary according to the planned experiment e.g. due to differences in protein expression or genomic features. Thus, selection from the multitude of available cell lines happens often by trial and error. Thus, there is a high demand for high-throughput screening method for cell line characteristics.Tissue microarray (TMA) methods have proven to be an excellent tool for profiling and screening tumor samples , 5. ReceIn this study, we aimed to assess a carefully selected panel of five immunohistochemical stains of putative radiotherapy biomarkers – p53, EGFR, Oct4, NDFIP1 and CIP2A – in head and neck cancer cell lines. Mutations of the tumor suppressor p53 occur frequently in HNSCC tumors and cell lines , 9. Whil2 in Dulbecco’s Modified Eagle’s Medium (DMEM), with 10% FCS, glutamine and antibiotics (penicillin and streptomycin). The intrinsic radioresistance of UT-SCC cell lines was previously determined using the 96-well clonogenic assay for radioresistance [The included 26 UT-SCC cell lines are summarized in Table sistance , 9, 22. Three previously established stable shRNA-transfected UT-SCC cell lines were included, two shCIP2A-silenced variants of UT-SCC-24A and one shCIP2A-silenced variant of UT-SCC-14A. shRNA cell lines were generated using pGIPZ lentiviral vectors consisting of a GFP tag and puromycin resistance (Open Biosystems). Cells transfected with lentiviral vector pGIPZ.NS shRNA containing non-silencing shRNA served as control cells expressing high CIP2A. Two stable cell lines for low CIP2A expression were generated using pGIPZ.shRNA (#556) and pGIPZ.shRNA (#557) containing targeting CIP2A antisense sequences TACATCAGCAGCAAGTTTG and TACTCAATGTCTTTATGTG, respectively. Lentivirus production followed the standard protocol. At the time of transduction, cell line confluency was approximately 40–50%. After infection, the cells were selected for puromycin resistance, and if the number of GFP-positive cells was low, the cells were sorted with FACS for further experiments. Finally, all cell lines were tested negative for replication competent viruses (RCV test) as well as for Mycoplasma, Acholeplasma, Entomoplasma and Spiroplasma . Western blot using anti-CIP2A antibody  was used to confirm the successful silencing of CIP2A expression  the supernatant was removed and 10 ml of buffered formalin was added for 48 h, after which the pellets were stored in PBS (+ 4 °C). Microfuge tubes were cut open, the pellet transferred into a tissue cassette, and submitted for paraffin embedding  with OptiView DAB kit and with 64-min CC1 preparation and 32-min antibody incubation. Mouse monoclonal anti-CIP2A antibody  was used. For PME-1, SET, LIMA1, NDFIP1, and Oct4 stainings, immunohistochemical stainings were done as previously described , 24. TheAll stainings were analyzed independently by at least two authors , and contradictory cases were discussed until consensus was reached. For microscopic photographs, slides were scanned using Pannoramic 250 Flash slide scanner. Using the CaseViewer software, photographs of either 10-fold or 20-fold magnification were exported in 300 dpi quality. Images were cropped in image editing software, while no color adjustments were performed.p53 staining was analyzed using a previously established 3-tier system consisting of deletion-like absence of p53 staining, wild-type pattern of staining, and mutation/amplification-associated aberrant-type overexpression. EGFR staining was scored in a 4-tier system  for cytoplasic/membraneous staining pattern. For CIP2A, the intensity of the cytoplasmic/membraneous staining was scored on a scale of 1 to 3 as weak/negative, intermediate or strong, taking into account the number of positive cells. In patient samples, CIP2A was uniformly present and was scored similarly based on cytoplasmic/membraneous staining intensity. Oct4 immunostaining was analyzed for the presence of positive nuclei. Oct4 positivity was present only in a subpopulation of cells, and thus the presence of strong nuclear immunoreactivity of individual cells was regarded positive. NDFIP1 staining was scored positive, when strong, uniform nuclear staining pattern was present, whereas cytoplasmic staining was not taken into account. A three-tier scoring system was used for uniform nuclear PME-1 and SET staining .p value < 0.05 was deemed significant.Cell line data, patient data, and results of immunohistochemistry were entered in SPSS 25 software. Dependence of cell line radioresistance to the staining results was analyzed using General Linear Model statistics. Both main effects and interactions were observed. Estimated marginal means were calculated and 95% confidence intervals determined using bootstrapping. Correlation was determined by Spearman’s method. Throughout, For the CMA construction, UT-SCC cell lines with previous data on radiosensitivity were preferentially selected. Altogether 26 UT-SCC cell lines were included, the majority of which were derived from male patients with oral cavity cancer Table . Six celp = 0.020), but no correlation between the expression of other biomarkers. Patient age and gender or tumor site, T class or nodal positivity were not directly linked with the expression of any investigated biomarkers.Immunohistochemical stains for p53, EGFR, Oct4, CIP2A, and NDFIP1 were analyzed (Fig. The associations between staining results and intrinsic radioresistance of the cell lines were analyzed. Expression of p53, EGFR, and CIP2A were not associated with radioresistance (Fig. To test the functionality of CMA in the validation of antibody specificity, and to study protein interactions in genetically modified cancer cell lines, three CIP2A shRNA-silenced cell lines were included in the CMA. CIP2A shRNA-silencing was confirmed by Western blot Fig. A and is Since CIP2A failed to predict radioresistance in the CMA and since CIP2A silencing did not affect other putative biomarkers expression, we were interested, whether CIP2A would have an association with other PP2A inhibitors and performed immunohistochemistry of two well-established endogenous PP2A inhibitors, PME-1 and SET, which are not pronouncedly identified as radiotherapy biomarkers (Supplemental Figure HNSCC consists of a genetically and behaviorally heterogeneous group of malignancies. The observed genomic instability is due to mutagenic insults on the mucosal lining of the upper aerodigestive tract such as tobacco and alcohol . AccordiPrior studies presenting similar methods of cell line array construction have mostly included relatively small amounts of cell lines, and have been constructed with other than head and neck cancers , 31, 32.In addition to efficiency, CMA provides other notable benefits as well. Use of exclusively cellular material allows strictly focusing the evaluation of IHC staining positivity on cancer cells and removing the majority of unspecific sources of unwanted IHC signaling positivity. This also provides a fixed, paraffin-embedded biobank of cell lines. It is possible to manipulate and modify the cell lines before constructing CMA, allowing e.g. molecular profiling of cell lines by inhibiting or enhancing the expression or effect of certain genes. This may also be applied to drug development and discovery studies, since cell lines may be exposed to drug of interest during culture. Moreover, in a CMA, all types of analysis available for FFPE patient samples can be used. As the CMA paraffin block is handled similarly to FFPE samples, cell line results can directly be compared with patient staining results. Our UT-SCC CMA included three CIP2A-shRNA-silenced cell lines, which demonstrate a highly successful silencing after lengthy cell line culture. CMA offers a powerful tool for the characterization of cellular effects of targeted silencing.A major weakness of cell line experiments in general and thus also of this study, is that cell line models are unable to take into account patient-related factors such as immunological responses, that may decisively affect the success of radiotherapy in HNSCC patients. However, the intrinsic radioresistance of the cancer cells is an important phenomenon in clinical radioresistance , 9, 22. Oct4 is a transcription factor essential for pluripotency in embryonic stem cells and testicular maturation. Oct4-related stemness has been found to have a role in radiotherapy resistance of various cancers, including HNSCC , 33–35. This study, in addition to confirming the feasibility of CMA methodology to screen for new predictive factors in head and neck cancer, revealed potential immunohistochemical biomarkers associated with radioresistance in head and neck cancer cell lines. Especially interesting for its potential clinical implications is the stem cell marker Oct4 which has been demonstrated to have a significant predictive role in previous studies , 14. In Additional file 1: Figure S1. Representative examples of low and moderate PME-1 and moderate and strong SET stainings.


In the absence of new therapeutic strategies, chemotherapeutic drugs are the most widely used strategy against metastatic breast cancer, in spite of eliciting multiple adverse effects and having low responses with an average 5-year patient survival rate. Among the new therapeutic targets that are currently in clinical trials, here, we addressed the association between the regulation of the metabolic process of autophagy and the exposure of damage-associated molecular patterns associated (DAMPs) to immunogenic cell death (ICD), which has not been previously studied. After validating an mCHR-GFP tandem LC3 sensor capacity to report dynamic changes of the autophagic metabolic flux in response to external stimuli and demonstrating that both basal autophagy levels and response to diverse autophagy regulators fluctuate among different cell lines, we explored the interaction between autophagy modulators and chemotherapeutic agents in regards of cytotoxicity and ICD using three different breast cancer cell lines. Since these interactions are very complex and variable throughout different cell lines, we designed a perturbation-based model in which we propose specific modes of action of chemotherapeutic agents on the autophagic flux and the corresponding strategies of modulation to enhance the response to chemotherapy. Our results point towards a promising therapeutic potential of the metabolic regulation of autophagy to overcome chemotherapy resistance by eliciting ICD. Breast tumors are the leading cause of cancer-related death per year in the female population ,2. AccorIn the past decade, the dichotomy of apoptosis (tolerogenic)—necrosis (immunogenic) cell death has been left behind and, instead, cell death is classified in a broad spectrum of possible subroutines, implying that each has an associated degree of immunogenicity . ImmunogAutophagy is a highly regulated catabolic process that encompasses the lysosomal degradation of cytoplasmic components. There are three different types: (1) Microautophagy, in which lysosomes derive membrane invaginations to capture cytoplasmic material; (2) Chaperone-mediated autophagy, in which proteins with a specific pentapeptide signal are recognized by the heat shock protein 70 (HSP70) and subsequently translocated into the lysosome; and (3) Macroautophagy , where cytoplasmic content, including organelles, is engulfed in double-membrane vesicles denominated autophagosomes (AP) that are fused with lysosomes in order to become autophagolysosomes (AL), the cellular microcompartment where degradation occurs . AutophaIn cancer, autophagy has a complex context-dependent role since it is associated both with tumor promotion and tumor suppression, varying among different models, types of cancer and its associated tumor microenvironment . On one Moreover, there is evidence of the importance of autophagy in the exposure/secretion of ICD associated DAMPs. For instance, ATP secretion prior to cell death was associated with the autophagic flux ,22,23, ePrecisely, we wondered if there is a specific autophagic phenotype as a determinant of the immunogenicity of cell death subroutines. The identification of such phenotype could orientate the design of combined therapeutic strategies of autophagic flux modulation and chemotherapeutic agents. Furthermore, it is important to ascertain if those modes of action hold true for different types of tumor cells with different basal autophagy levels and different response profiles to autophagic flux modulation agents. Therefore, we propose here a multiparametric study to describe the effect on cytotoxicity and immunogenicity through the modulation of the autophagic flux in cells treated with chemotherapy.In the study of autophagic flux, in vitro approaches have focused on LC3 protein fluorescent sensors, given that LC3 is specifically incorporated into the autophagosomal membranes. Upon pro-autophagic stimuli, such as nutrient deprivation, the LC3 sensor redistributes from a diffuse (LC3-I) to a clustered pattern (LC3-II), due to the formation and agglomeration of AP. In this sense, it is important that not all LC3-II is present on autophagic membranes and some population of LC3-II seems to be ectopically generated in an autophagy-independent manner, therefore measuring the autophagic flux is a better approach to precisely evaluate the autophagic activity ,27.Autophagic flux can be monitored by the quantification of fluorescent puncta on a single cell basis as we previously did . HoweverIn addition, the single LC3-GFP approach does not enable the differentiation between AP and AL, therefore, novel sensors with two fluorescent sensors associated with LC3 in tandem were devised. As such, one sensor fluorescence is quenched at low pH  while the other is stable (mCherry) at this condition. Using these validated sensors of autophagic flux, it is possible to identify whether a phagosome has already been fused or not with a lysosome . Still, Systems biology acts as a hatchet to unveil the underlying principles governing biological processes through the definition of how its components are organized and interlinked. By implementing various algorithms to analyze a network, the core modulators can be detected, and the dynamics of these core sets of modulators can be studied through mathematical modeling . These sIn the present study, we report the implementation and validation of an image analysis platform to describe the autophagic flux in breast cancer cells using the mCHR-GFP tandem LC3 sensor. The ratio of AL/AP reports dynamic changes of autophagic flux in response to starvation-induction and bafilomycin-mediated degradation blockade. These breast cancer cell lines display heterogeneous basal levels of autophagy and a differential response to drugs inducing known perturbations of autophagic flux. The perturbation-based dynamic modeling of the autophagic sensor indicates cell-line specific modes of action of chemotherapeutic agents on the autophagic flux. This study represents a first approach to explore the influence of autophagy in tumor chemotherapy-induced cytotoxicity and immunogenicity through the application of systems biology and perturbation-based models. Our results point out a promising therapeutic potential of the regulation of autophagy to overcome chemotherapy resistance by eliciting ICD.The use of tandem fluorescent sensors is the most widely used technique for monitoring autophagy since it allows for the quantification of two different cellular compartments simultaneously. This approach takes advantage of the low resistance of GFP fluorescence to acidic pH environments in contrast to the stable fluorescence of mCherry to those environments . To simuIn order to monitor the autophagic flux in living breast cancer cells, we aimed to produce cell lines with stable expression of the mCHR-GFP-LC3 tandem sensor. To achieve this, the gene construct was introduced into three different breast cancer lines using the triple transduction procedure. Briefly, the vector was assembled into HEK293T cells by the transduction of the pBABE-pure plasmids mCherry-EGFP-LC3B, pMD2.G and pCL-Eco with polyethyleneimine . The vesCells in the supplemented culture medium did not show any evident changes in the distribution and intensity of the sensor. However, when treated with bafilomycin, an accumulation of white fluorescent puncta was observed over time. This accumulation represents the basal autophagic flux of each cell line upon degradation blockade of AP A. On theIn order to confirm that the mCHR-GFP-LC3 sensor expressed in our breast cancer cell lines is able to report autophagic flux perturbations, we assessed the effect of several compounds which were extensively described as autophagy modulators. This includes the mTORC 1 inhibitor rapamycin, the mTORC1/2 inhibitor Torin 2, the USP10/13 inhibitor Spautin, the PI3K inhibitor Wortmannin and the lysosomotropic compound hydroxychloroquine (HCQ). All of these compounds are widely used as autophagic flux regulators in vitro and in vivo and were studied as stand-alone or combined therapies against different types of tumors. For this reason, we evaluated the changes over time reported by the autophagy sensor upon treatment of breast cancer cell lines with different concentrations of these drugs through a kinetic fluorescence microscopy assay. Autophagy inhibitors were evaluated in complete and starvation medium to assess the effect of these substances over basal and induced autophagy respectively, whereas the autophagic inducers were evaluated in complete medium only. The concentration of each autophagic modulator was selected taking into account the toxicity 72 h after treatment (data not shown).Rapamycin treatment caused a clear change in the cellular distribution of the sensor fluorescence for all three cell lines A, RAP. WSpautin-1 produced changes in sensor intensity in all three cell lines. There was a decrease in both APs and ALs, which was appreciable in complete medium A, SPA anThe results obtained with all these autophagy modulators are consistent with the expected regulator activity previously reported for each of them. This further demonstrates the ability of our sensor to differentially report a wide variety of known perturbations to autophagy, which proves to be heterogeneous among different breast cancer cell lines.The gold standard strategy to categorize cellular death as immunogenic relies on in vivo tests involving the vaccination of immune-competent mice with syngeneic cancer cells after they were treated in vitro with the drugs to be evaluated. Nonetheless, this approach cannot be used to test human cancer cell lines and is expensive and difficult to implement as a screening test. An alternative method to assess immunogenic cell death consists of the determination of DAMPs secreted or exposed by cells prior and during cell death. Traditionally, calreticulin exposure to the plasma membrane, extracellular ATP secretion and extracellular HMGB1 release were determined for this purpose .In order to evaluate the secretion/exposure of immunogenic cell death associated DAMPs exposed or released by the interaction of autophagy regulators and chemotherapeutic drugs, we performed a cytotoxicity assay in which breast cancer cells were treated with chemotherapeutics drugs alone, autophagy regulators alone and all the possible combinations of those groups of drugs. After 24 h of treatment, calreticulin exposure was determined on live cells, and after 72 h of treatment, the live-cell percentage was measured along with the ATP concentration in the culture supernatant. We compared these immunogenicity profiles with the changes observed in AL, AP and the AL/AP ratio at 48 h post-treatment. In general, the autophagy regulators had minimal effects on cytotoxicity , calretiRegarding paclitaxel, in the MCF-7 cell line, this drug displayed a strong synergism with rapamycin, and a slighter synergism with Torin-2, HCQ and wortmannin. From those interactions, rapamycin/HCQ also caused an increment in calreticulin exposure. In the T-47D cell line, there were no evident synergistic interactions, and HCQ and Spautin-1 practically nullified the cytotoxic activity of this drug. Nonetheless, Torin-2 and wortmannin did potentiate calreticulin exposure while HCQ caused an increase in ATP secretion despite antagonizing cell death and calreticulin exposure. In the MDA-MB-468 cell line, paclitaxel did not show any strong interactions for cytotoxicity with autophagy modulators. However, rapamycin and HCQ led to an increase in calreticulin exposure although no combination elicited a greater ATP secretion than paclitaxel alone. It is of note that paclitaxel-induced ATP secretion in all three cell lines occurred, with strong increments when used in combination with HCQ in MCF-7 and T-47D cell lines. On the other hand, Spautin-1 antagonized the cytotoxic effect of paclitaxel, which was also reflected by a decrease in calreticulin and ATP levels for that condition in all three cell lines.In respect of cisplatin, in the MCF-7 and T-47D cell lines, the impact of autophagy modulators on cisplatin cytotoxicity was similar, with a less pronounced antagonism when treated together with rapamycin and Spautin-1 in the T-47D cell line. Furthermore, in both these cell lines, no ATP secretion was detected in any cisplatin combination and there were no concomitant appreciable interactions regarding calreticulin exposure, except for HCQ which potentiated calreticulin exposure in the MCF-7 cell line, in spite of having no impact on cisplatin cytotoxicity. Conversely, in the MDA-MB-468 cell line, cisplatin displayed remarkable synergisms with rapamycin, Torin-2 and HCQ, although, Spautin-1 antagonized cisplatin-induced cell death, as observed with the other two cell lines. Additionally, HCQ potentiated cisplatin-induced calreticulin exposure and was the only autophagy modulator that enabled ATP secretion when combined with cisplatin.With respect to epirubicin, in the MCF-7 cell line, rapamycin showed a slight synergism and Spautin-1 displayed a strong antagonism. As for calreticulin determination, no combination elicited a higher exposure than epirubicin alone and Spautin-1 actually completely blocked calreticulin translocation, an expectable outcome due to the strong antagonism in cytotoxicity. In the T-47D cells, results were different as shown by an antagonism in cytotoxicity with rapamycin but an evident synergism with HCQ and, once again, a strong antagonism with Spautin-1. Despite cytotoxicity antagonism, rapamycin and Spautin-1 caused an increase in calreticulin exposure compared to the epirubicin treatment alone. Cytotoxicity interactions with the MDA-MB-468 cell line were similar to those found in the MCF-7 cell line, however, rapamycin and wortmannin induced a higher exposure of calreticulin, compared to epirubicin alone. Lastly, ATP secretion was increased in epirubicin and HCQ combined treatment for all three cell lines, highlighting the importance of this combined treatment, despite cytotoxicity interactions and calreticulin translocation.These results confirm that cytotoxicity and immunogenic cell death associated with DAMP exposure/secretion interaction profiles with autophagy regulators are cell line-specific and that the same combination of drugs might be synergistic in one cell line and antagonistic in another cell line.In addition to being a physiological metabolic process, autophagy serves as a response to different types of cellular stress such as hypoxia, nutrient deprivation, or stress-induced by chemotherapeutic compounds. Moreover, autophagy plays an active role in the determination of multiple cell death subroutines . Since cThe results showed evident changes in the sensor fluorescence distribution and intensity when compared to the untreated control A. EpirubThese results suggest that the sensor is able to report changes in autophagic flux induced by chemotherapy. Therefore, to infer the specific mode of action of each chemotherapeutic perturbation on the autophagic flux, we performed double treatments of chemotherapy and drugs with known mechanisms of action on the autophagic machinery.Quantitative measurement of APs and ALs for all the double perturbations mentioned above confirmed that the variations in autophagy induced by these chemotherapeutic compounds are cell line-specific. The perturbation-response profiles for a specific treatment condition differed among the three cell lines , hinderiGiven the large amount of data obtained and its high complexity, a systems biology approach was required. Hence, we developed a family of mathematical models representing alternative hypotheses of the mode of action of each chemotherapeutic drug over the different steps of the autophagic flux. This data-driven approach has the potential to help us identify the model with the modes of action that better fit the experimental data for each breast cancer cell line. We started by proposing a network topology of the mCHR-GFP-LC3 sensor metabolic pathway. Using this basal topology, we introduced variations considering different single modes of action for each chemotherapeutic drug and all combinations thereof. Afterward, the family of network topologies was converted into a family of SBML mathematical models using CellDesigner. The SBML models were imported in COPASI to perform the parameter estimation task for each of them. The lower objective function value of the parameter estimation was the metric to indicate the fitness of each model to the experimental data.The best-fitted model for each cell line provides insight regarding which combination of modes of action is the most congruent with the experimental perturbation data B. Our moThis data-driven approach indicates that the mCHR-GFP-LC3 sensor reports the modes of action of chemotherapeutic perturbations on the autophagic flux and suggests specific modes of action for epirubicin, cisplatin and paclitaxel across three different breast cancer cell lines.The formation of the AP comprises three basic steps: initiation, nucleation and elongation. The central metabolic regulator of autophagy is mTOR, which acts through two different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes require the presence and interaction of the proteins Deptor, Tel2, Tti1 and mLST8. Moreover, mTORC1 additionally requires the RAPTOR and PRAS40 proteins, while mTORC2 requires the Rictor and mSin1 proteins . In the After the initiation of autophagy, the next step is nucleation. In the endoplasmic reticulum membrane, the ULK complex activates another important complex, the phosphatidylinositol kinase type 3 complex (PI3K class 3), which is composed first of all by the vesicular protein sorting 34 kinase (Vps34), Vps15, the beclin-1 protein and finally the ATG14 protein. Once activated, this complex catalyzes the conversion of phosphatidylinositol to phosphatidylinositol triphosphate which is essential for the next step and to recruit the protein machinery necessary to continue the pathway . Two widIn the third phase of the autophagic flux, two ubiquitin-like conjugation systems function at a late step of AP function: ATG12-ATG5 and ATG8-phosphatidylethanolamine (PE). ATG12 is activated by ATG7 and then conjugated to ATG5. The enzyme ATG10 catalyzes this conjugation. The new protein ATG12-ATG5 is non-covalently associated with ATG16. On the other hand, ATG8 and its mammalian homologs the microtubule-associated protein 1 light chain 3 (MAP1LC3), also known as LC3, and the γ-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) are synthesized as precursors with an additional sequence at C terminal end that is eliminated by proteases ATG4A-D, then LC3/GABARAP is conjugated to the amino group of the phosphatidylethanolamines present in the membrane of the phagophore that is expanding, and this conjugation is in charge of ATG7 and ATG3 activation, which leads to LC3/GABARAP up to the phospholipid. The aforementioned ATG12-ATG5-ATG16 complex participates actively in the conjugation of LC3/GABARAP to the phosphatidylethanolamine ,46,47.Once the AP is formed, it migrates through the cytoskeleton to the lysosome for the fusion to take place. It was speculated that the vacuolar ATPase that keeps the pH low within the lysosome served additionally as a receptor for the AP. This is because the use of an inhibitor of this ATPase such as bafilomycin-A2 causes AP-lysosome fusion blockade. However, the selective fusion mechanism of these two organelles is not known with certainty . In thisDespite these advances in the modulation of autophagy, there is still a gap in the understanding of the interactions between chemotherapy and modulators of autophagy. Indeed, autophagy perturbing agents can inhibit or activate different steps of the pathway but their potential side effects must be considered in the interpretation of the secondary consequences of autophagy perturbation, especially in long-term studies as ours. For example, lysosomotropic compounds can increase the rate of AP formation by inhibiting MTORC1, as activation of lysosomally localized MTORC1 depends on an active V-ATPase. HCQ treatment may cause an apparent increase in the formation of APs possibly by blocking fusion with the lysosome as observed by the reduction of colocalization of LC3 and LysoTracker™ despite the presence of APs and lysosomes. However, it can also block AP-lysosome fusion in HeLa and MEFs but this mechanism could be cell-type specific, as other studies report that CQ prevents AL clearance and degradation of cargo content, but not AP-lysosome fusion .Cellular metabolism and other physiological processes such as autophagy are highly complex, and classic mechanistic strategies to study biological phenomena are becoming less effective with the advent of technologies that engender a large amount of information. One way to infer the mechanism of action of an unknown perturbation on a metabolic pathway is to use perturbation-based modeling, a strategy in which simple modifications are introduced to a highly complex model followed by time-course monitoring of the effect upon the system. After accumulating data about the impact of many simple perturbations, as well as the combination of some of these, it is possible to mathematically model the effect of known perturbations, with the advantage that the model is robust enough to infer the effect of unknown perturbations .After validating the mCHR-GFP tandem LC3 sensor capacity to report dynamic changes of the autophagic flux in response to external stimuli  and demoPaclitaxel was able to induce ICD-associated DAMPs as a solo treatment in all three breast cancer cell lines, although this was more evident in the T-47D cell line. This induction was also reported through in vitro and in vivo models in ovarian cancer . InteresCisplatin was not able to release/expose ICD-associated DAMPs in two of three breast cancer cell lines when exposed on its own, which was expected since this drug was previously categorized as a non-ICD inducer . SurprisAs for epirubicin, it is recognized as a bona fide ICD inducer  and, remOur perturbation-based approach indeed enabled us to propose alternative modes of action of the chemotherapeutic drugs on the autophagic flux. As discussed above, chemotherapy was reported to be pleiotropic and have a context-dependent effect on autophagy. Upon this conundrum of different activities for an individual chemotherapeutic drug, it becomes critical how the cell senses the stress signals elicited by chemotherapy. Therefore, the specific configurations of critical pathways such as the autophagy pathway are paramount in cell fate decisions. In order to validate these model-driven hypotheses for those specific configurations of autophagy and their association to chemosensitivity a larger functional genomics study could be conducted with cell lines to identify the critical proteins involved in each of those configurations and to identify the gene expression signatures that could be used to correlate these findings with the chemosensitivity of human tumors.This approach also enabled us to look for a common autophagic phenotype related to the exposure or release of ICD-associated DAMPs. Some of the most potentially immunogenic combinations such as PAC+HCQ, PAC+RAP in the MCF-7 cells and PAC+TOR, EPI+RAP or EPI+SPA in T-47D cells have in common the accumulation of APs. This suggests that an active AP formation with an impaired fusion to the lysosome represents a potential autophagic phenotype related to increased cytotoxicity and immunogenicity. Future experiments using single-cell technologies should be designed to correlate DAMP exposure and sensors of autophagic flux to validate this hypothesis. In vivo experiments monitoring endogenous LC3 expression and clustering, and the characterization of the immune infiltrate by immunohistochemistry and flow cytometry may contribute to assess whether the accumulation of APs upon treatment correlates with ICD in order to postulate combinatorial strategies designed to favor this autophagic phenotype.This study is the first to exploit systems biology and perturbation-based models in order to scan the association between autophagy and chemotherapy-induced cytotoxicity and immunogenicity. Our results point towards a promising therapeutic potential of the metabolic regulation of autophagy to overcome chemotherapy resistance by eliciting ICD. Furthermore, treatment combinations elicited diverse ICD markers induction and autophagic flux perturbations among the different cell lines, highlighting the importance of evaluating these therapeutic strategies according to the type of cancer.HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% heat-inactivated fetal bovine serum  and 1X antibiotic-antimycotic solution (Gibco 15240062).2.MCF-7, T-47D, and MDA-MB-468 breast cancer cell lines were obtained from the National Cancer Institute (NCI) collection and cultured in Roswell Park Memorial Institute medium  supplemented with 10% FBS (Gibco), 1X GlutaMAX (Gibco 35050061), and 1X antibiotic-antimycotic solution (Gibco). All cell lines were cultured at 37 °C in 5% CORetroviral particles were generated in 60% confluent HEK293T cell monolayers by triple transfection with polyetherimide (Polysciences 23966) of the transfer plasmid pBABE-puro mCherry-EGFP-LC3B   in RPMI 1640 10% FBS for 2 days. Cell sub-populations with homogeneous levels of expression of the mCherry-EGFP-LC3B construct were isolated for each cell line by fluorescence-activated cell sorting in a BD FACSJazz™ cell sorter . Sorted cells were cultured in RPMI 1640 10% FBS with 2 µg/mL puromycin.Autophagy activators rapamycin  and Torin-2 (Sigma-Aldrich SML1224) were dissolved in dimethyl sulfoxide . Autophagy inhibitors wortmannin (Sigma-Aldrich W1628), bafilomycin A1 (Sigma-Aldrich B1793) and Spautin-1 (Sigma-Aldrich SML0440) were dissolved in DMSO , while the hydroxychloroquine sulfate salt (Sigma Aldrich H0915) was dissolved in deionized water.Epirubicin and cisplatin were a gift from the Oncologic Pharmacy of the Calderon Guardia Hospital from the Social Security System of Costa Rica, as aqueous and DMSO solutions, respectively. Paclitaxel was dissolved in DMSO (Sigma-Aldrich T7402).MCF-7 cell line was treated using the following chemotherapeutic drugs concentrations: epirubicin 1.70 µM, cisplatin 12.5 µM and paclitaxel 7.70 µM. The T-47D cell line was treated using the following drug concentrations: epirubicin 0.85 µM, cisplatin 25.0 µM and paclitaxel 7.7 µM. MDA-MB-468 cell line was treated using the following drug concentrations: epirubicin 0.85 µM, cisplatin 1.60 µM and paclitaxel 7.7 µM. These concentrations are similar to those reached by these drugs in the intratumoral microenvironment and used by cytotoxicity assays . Drug coBreast cancer cell lines with stable expression of the mCherry-EGFP-LC3B sensor were seeded in 96-well plates , after 24 h incubation the medium was replaced with one containing the respective treatments. Images were taken in the green (GFP) and red (mCherry) channels every 3 h for 12 h in the Cytation 3™  automated fluorescence microscope. In order to induce autophagy, the Krebs-Henseleit (Sigma Aldrich) starvation medium was used, and bafilomycin A1 (Sigma Aldrich) was used at 50 nM to cause a blockade in the autophagic flux.In order to quantify AP and AL in a time-resolved manner, an image analysis protocol was made in Cell Profiler software (ver 3.0.0). It was adjusted for the automatic segmentation of cells and the quantification of mCherry positive GFP positive AP and mCherry positive GFP negative AL.Cell death was determined using a Hoechst 33,342  and propidium iodide  stain assay. Cells were seeded in 96 well plates (Greiner Bio-One µclear), incubated for 24 h and then treated for 72 h with a pre-established concentration of each chemotherapeutic treatment alone, autophagy modulator alone, or combinations of those. Once the incubation time had elapsed, Hoechst  and propidium iodide  were added to the medium of each well. Images in the red  and blue  channels were taken. With this information, the live-cell percentage was determined using the Cell Profiler image analysis software, through an image analysis pipeline designed to count total and dead cells. Live cells were calculated by dividing PI negative cells by the total amount of cells in each well. Cell death interactions (synergism vs. antagonism) were estimated by comparing the multiplied effect of each treatment alone with the real effect elicited by the combined treatment.Plasma membrane Calreticulin exposure was determined using a monoclonal anti-human calreticulin allophycocyanin (APC)-conjugated antibody . Cells were seeded in 24 well plates and incubated for 24 h, afterwards the medium was replaced with a new medium containing the different treatments. After 24 h of incubation, the medium was removed and each well was washed using phosphate-buffered solution pH 7.4  to eliminate floating dead cells. Then, cells were trypsinized and fixated using 2% paraformaldehyde (Sigma Aldrich P6148) for 15 min at room temperature. Once fixed, cells were treated with the anti-human calreticulin antibody and PI (1 µg/mL) for 1 h at 37 °C in the dark. Later, the APC fluorescence was quantified in PI negative cells using a BD Accuri™ C6 flow cytometer (BD Biosciences), which was proportional to the amount of calreticulin exposure in the plasma membrane in live cells. Results were expressed as the fold increase in the signal compared to the untreated control.At the end of the cytotoxicity assay, centrifuged supernatant was collected to measure ATP secretion. For this purpose, we used an ATP determination kit (Invitrogen™ A22066). Samples were analyzed in parallel to an ATP calibration curve . The tests were performed as recommended by the manufacturer. Results were expressed as the fold increase in the signal compared to the untreated control.−6. Finally, we proceeded to adjust all the generated mathematical models using that programmed Parameter Estimation task to obtain all the objective values for each proposed model. The hypothesis was ranked according to their degree of fitting to the experimental data to select the model with the highest likelihood to explain the specific mode of action of chemotherapy over the autophagic flux in each cell line”.“A basic topology of the autophagic flux was designed in Cell Designer software (ver 4.4), which we later exported in SBML format to be used in Copasi (ver 4.24). The model consisted of a single compartment and 2 core species, including the phagophore at the cytoplasm (CYT), the autophagosomes (AP), the autophagolysosomes (AL) and the degraded ALs (sink). We imported the model in COPASI  and inclp < 0.05 were considered statistically significant. Three independent replicates were conducted for each experiment.The data represented in heatmaps corresponds to the median value of the performed replicates. For the cytotoxicity quantifications, results were expressed as the mean value ± standard deviation (SD). The differences between groups were analyzed by a one-way variance (ANOVA). Differences at


Nitrogen-containing heterocyclic rings are common structural components of marketed drugs. Among these heterocycles, imidazole/fused imidazole rings are present in a wide range of bioactive compounds. The unique properties of such structures, including high polarity and the ability to participate in hydrogen bonding and coordination chemistry, allow them to interact with a wide range of biomolecules, and imidazole-/fused imidazole-containing compounds are reported to have a broad spectrum of biological activities. This review summarizes recent reports of imidazole/fused imidazole derivatives as anticancer agents appearing in the peer-reviewed literature from 2018 through 2020. Such molecules have been shown to modulate various targets, including microtubules, tyrosine and serine-threonine kinases, histone deacetylases, p53-Murine Double Minute 2 (MDM2) protein, poly (ADP-ribose) polymerase (PARP), G-quadraplexes, and other targets. Imidazole-containing compounds that display anticancer activity by unknown/undefined mechanisms are also described, as well as key features of structure-activity relationships. This review is intended to provide an overview of recent advances in imidazole-based anticancer drug discovery and development, as well as inspire the design and synthesis of new anticancer molecules. According to the 2020 World Cancer Report, slightly over 18 million new cases of cancer and nearly 10 million cancer-related deaths occurred across the globe in 2018 . While n1), bendamustine hydrochloride (2), fludarabine phosphate (3), nilotinib (4), and ponatinib (5), contain imidazole and fused imidazole as structural components   due to tldehydes , while tldehydes  or its mldehydes . Purine ldehydes ,28,29. Ildehydes . Since tMicrotubules are the cylindrical components of the cytoskeleton (composed of α- and β-tubulin heterodimers) which are critical players in various cellular functions including the maintenance of cell shape, cell signaling, vesicular transport, and cell division. Because they are the primary component of the mitotic spindle, microtubules play an important role in the proliferation of cancer cells, thus making microtubules one of the most attractive targets for anticancer drugs (reviewed in reference ). Microt50 values in the 80–1000 nM range. Among these, compound 6 (see 6–19) showed the highest potency, with IC50 values from 80–200 nM against HCT-15, HT29, HeLa, and MDA-MB-468 cells, while compound 7 exhibited good potency against HeLa and HCT-15 cells . In terms of the SAR, the placement of an aliphatic group on the imidazole nitrogen and the replacement of imidazole with an amide or ester group led to a loss of activity. Compound 6 was a better inhibitor of porcine brain tubulin polymerization (IC50 = 0.4 µM) than either colchicine (IC50 = 7.5 µM) or combretastatin A-4 (IC50 = 1.1 µM). At its IC50 concentration, compound 6 caused arrest of MDA-MB-468 cells in the G2/M phase of the cell cycle. In an MDA-MB-468 breast cancer xenograft model performed in nude mice, i.p. administration of compound 6 (60 mg/kg every other day for 21 days) suppressed tumor growth by 77% compared to control, without causing obvious weight loss in the animals.1-Substituted-2-aryl imidazoles were synthesized by Li et al. as potential tubulin-targeted anticancer agents . The tar8) where structural modification of the 3,4,5-trimethoxyphenyl group in this compound led to improved anticancer activity dioxole, 5-methoxy-2,3-dihydrobenzo[b]dioxene, or 6-methoxy-3,4-dihydro-2H-benzo[b]dioxepine, the activity was greatest with the dioxole ring-containing compound and decreased with increasing ring size. Incubation of compound 9 with bovine brain tubulin at a concentration of 10 μM resulted in nearly complete inhibition of tubulin assembly. The X-ray crystal structure of compound 9 bound to the tubulin/stathmin-like domain of RB-3/tubulin tyrosine ligase complex confirmed that 9 binds to the colchicine site on β-tubulin. When given at a dose of 30 mg/kg every other day for 15 days by i.p. injection, compound 9 inhibited tumor growth by 73.9% in an A375 murine melanoma xenograft model without significant weight loss.Wang et al. designed novel anticancer compounds based on the clinical drug candidate VERU-111  conducted by Wang et al. reported structure-activity relationship (SAR) studies around the 3-indole moiety of this molecule colchicine (5 μM) to tubulin by 91%. Incubation with this compound at a concentration of 0.5 μM resulted in an increase in the percentage of A549 cells in the G2/M phase.Wang et al. synthesized a series of benzimidazole sulfonamides containing a substituted pyrazole ring at the C2 position . These d50 values of <10 μM against these cell lines. Among these compounds, SKLB0533 (23) was the most potent, displaying IC50 values of 0.08, 0.06, and 0.10 μM against HepG2, HCT116, and A549 cells compared to the standard compound myoseverin . In general, substitution with 2-propyl, 3-pentyl, or cyclopentyl groups at N9 resulted in increased antiproliferative activity compared to N9-tetrahydropyranyl derivatives. Compound 23 inhibited the assembly of porcine tubulin in vitro in a dose-dependent manner and caused the dose-dependent accumulation of HCT116 and SW620 cells in the G2/M cell cycle phase . Moreover, compound 23 displayed no activity against a panel of 420 kinases when tested at a concentration of 1 µM, suggesting its specific tubulin inhibitory activity. This compound displayed high activity against a panel of other colorectal cancer cell lines . In a murine xenograft model employing HCT116 cells, oral administration of compound 23 at 40 or 80 mg/kg once daily for 28 days resulted in tumor growth inhibition (TGI) of 39.9% and 71.6% compared to the control group, respectively, while the TGI for the group receiving oral capecitabine at 540 mg/kg once daily for 28 days was 61.6%.Zhang et al. synthesized aminomethyl dimethylpyridinones as potential anticancer agents against HepG2, HCT116, and A549 cancer cell lines and also evaluated their tubulin polymerization inhibitory activity . Most of23 anthracene-induced rat mammary carcinoma model caused a reduction of cancer markers (carcinoembryonic antigen and cancer antigen 15–3) and resulted in lower levels of the liver enzymes aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase compared to rats in the mammary carcinoma control group. In terms of the SAR for these compounds, imidazopyridine dimers were more active compared to imidazole dimers.A series of imidazole and imidazopyridine dimers synthesized by Meenakshisundaram et al. was tested for cytotoxicity against HeLa, MDA-MB-231, and ACHN cancer cell lines . CompounN-propargyl biaryl ketones (scaffold 223) with 4-methylpiperidine under microwave irradiation [224 was the most potent analog against the LN-405 cell line, displaying an IC50 value of 10 µM, while compound 225 exhibited an IC50 value of 75 µM against this cell line. Compound 224 was selectively cytotoxic for the LN-405 cell line compared to the fibroblast cells (IC50 = 480 µM). Results from a comet assay in LN-405 cells treated with a 10 µM compound 224 showed that this derivative caused DNA damage (affecting 14% of the cells) compared to the control (where DNA damage was observed in 0.7% of the cells). In silico studies using Schrödinger suggested that the target compounds are likely to be orally available and should penetrate the blood-brain barrier.Güçlü et al. developed a novel method for the synthesis of imidazopyridine derivatives by reacting 4-substituted-adiation . The der226 displayed IC50 values of 9.59, 12.12, and 10.10 μM against MDA-MB-231, MCF-7, and T47D cell lines, respectively, while compound 227 displayed IC50 values of 8.13 and 9.61 μM against MCF-7 and T47D cell lines. In general, compounds synthesized from cyclohexyl isocyanides had higher potencies than derivatives synthesized from tert-butyl isocyanides. The placement of a methyl group at positions 5, 6, or 7 of the imidazopyridine core increased the anticancer activity of compounds obtained from cyclohexyl isocyanides, while insertion of chlorine at C6 caused the loss of activity against all three cell lines.Khalili et al. synthesized a library of styrylimidazopyridines and evaluated them for activity against three breast cancer cell lines . Compoun228–230 exhibiting IC50 values in the range between 2.55–8.11 μM against these cancer cell lines. Among these, 229 and 230 were the most potent against the A549 cell line, displaying IC50 values of 2.55 and 3.69 μM against this cell line, respectively. In terms of the SAR, imidazopyridines possessing amide functionalities were more potent compared to derivatives containing a sulfonamide moiety.Chitti et al. reported the synthesis of imidazopyridine analogs as potential antiproliferative agents against A549, HeLa, and B16F10 cancer cell lines and also performed counter-screening using the HEK-293 cell line . A subse231 displayed the greatest potency against the HCT116 cell line (IC50 = 1.21 μM). While the structure-activity relationship is complex, the inclusion of an electron withdrawing group on the C3 phenyl group and on the terminal aniline generally resulted in increased potency. Compound 231 caused a dose-dependent increase in the percentage of HCT116 cells in the G0/G1 phase of the cell cycle at concentrations of 0.5–2.5 µM and increased the formation of reactive oxygen species in these cells at 1.0 and 2.5 µM concentrations.A series of imidazopyridine-arylaminopropenone conjugates synthesized by Mani et al. were tested for activity against A549, HCT116, B16F10, BT474, and MDA-MB-231 cancer cell lines . Among t232 displayed an IC50 value of 5.98 µM against A549 cells, while compound 233 exhibited IC50 values of 6.24 µM, 6.54 µM, and 4.14 µM on DU-145, HeLa, and A549 cell lines, respectively. Compounds bearing an amide group on the diazepane nitrogen were generally more active when compared to analogs bearing a sulfonamide moiety at this position.Mantipally et al. synthesized a series of novel homopiperazine linked imidazopyrimidine derivatives as candidate cytotoxic and antimicrobial agents . Evaluat235) from 1-(4-methoxyphenyl)-3-(1-methyl-1H-imidazol-4-yl)-1H-pyrazol-5-amine (234) and “active methylene” compounds, such as ethyl benzoylacetate and pentane-2,4-dione, using microwave irradiation [50 values in a narrow range (16.6–19.3 μg/mL). Compounds 236 and 237 displayed IC50 values of 16.6 and 18.1 μg/mL, respectively.El-Borai et al. synthesized imidazolyl pyrazolopyridine derivatives (scaffold adiation . Compari238 (see 238–241) displayed IC50 values of 4.3, 10.6, and 8.3 μM against A549, MCF-7, and HeLa cells, respectively, while exhibiting an IC50 value of 67.3 μM against the L929 cell line. In terms of the SAR, 2-alkyl-substituted compounds were less potent compared to 2-aryl-substituted compounds. In addition, compounds lacking a quinone moiety were inactive on the cancer cell lines tested.Liu et al. synthesized morpholino ethyl-substituted naphthoquinone imidazoles that were tested for in vitro activity against MCF-7, HeLa, and A549 cell lines, as well as for their effects on the L929 normal mouse fibroblast cell line . Compoun239 and 240 displayed IC50 values of 0.022, 1.12, 1.07 µM and 0.022, 1.13, and 0.536 µM against PC-3, HeLa, and A375 cells, respectively, while indole-substituted compound 241 displayed IC50 values of 0.128, 0.059, and 0.212 µM against these cell lines, respectively. In general, phenyl vinyl substitutions at the C2 position of imidazole resulted in compounds that were more active against PC-3 cells; however, compounds bearing indole vinyl substituents were more active towards A375 and HeLa cell lines. In A375 cells, both compounds 239 and 241 decreased the expression of survivin and Bcl-2 and increased the percentage of annexin V positive cells at 1 µM concentrations. In a subcutaneous solid tumor model using Ehrlich’s ascites carcinoma cells, intraperitoneal administration of compound 241 at a dose of 0.6 mg/kg/day for 10 consecutive days resulted in 53.12% reduction in tumor progression compared to the vehicle control group.Wei et al. synthesized a novel series of arylvinyl-naphthoimidazolium iodide and bromide derivatives and screened these compounds for antiproliferative activity against PC-3, HeLa, and A375 cell lines . Compoun242, 243, and 244 (see 242–253) exhibited IC50 values against HepG2 cells of 8.14, 9.58, and 9.77 µM, respectively, while all other compounds displayed IC50 values greater than 10 µM. Compounds 242 and 244 displayed selectivity indexes of 27.47 and 13.62 against HepG2 cells compared to Vero cells.Hebishy et al. reported the preparation of bis- and poly(imidazoles) via a three-component reaction among 1,2-diketones, aldehydes, and ammonium acetate using 10 mol% of a ZnO nanocatalyst under conventional heating, as well as microwave irradiation . The tar245 and 246. Compound 245 displayed 83.3% inhibition against MCF-7 cells and compound 246 exhibited 77.3% growth inhibition against HCT116 cells when tested at a concentration of 50 μg/mL. The standard drug doxorubicin exhibited 93.3% and 97.2% inhibition against these cell lines, respectively, when tested at the same concentration. Unsubstituted quinoxaline compound 245 displayed the best activity; substitution of the phenyl group linked to the imidazole ring in compound 245 with electron-withdrawing or electron-releasing groups led to a decrease in potency. Among the 6-Cl and 7-Cl-quinoxaline series, the 4-methoxyphenyl substitution in the former series showed the highest potency against both cancer cell lines tested.Ghanbarimasir et al. synthesized and evaluated the activity of 2-aminoimidazole-quinoxaline hybrids against MCF-7 and HCT116 cancer cell lines, along with their effects on a normal HFF cell line . The mos247 and 248) exhibited IC50 values against MCF-7 cells of 10.22 and 14.80 µM, respectively. Compounds substituted with electron-withdrawing groups as in 247 and 248 were more potent than compounds bearing electron-releasing groups.The anticancer activity of imidazole derivatives prepared from guanylhydrazone and phenylglyoxal was evaluated in vitro against MCF-7 and HepG2 cancer cell lines by Yavuz et al. . The syn249 and 250 displayed the greatest potency against the HL-60 cancer cell line, with IC50 values of 0.183 and 0.297 μM, respectively. Regarding substitution on the imidazopyrazole system, compounds having alkyl substitutions at C2 and alkylamines at C3 displayed the highest potency.Demjén et al. synthesized a library of imidazopyrazole-7-carboxamides and measured their activity against 4T1, MCF-7, and HL-60 cancer cell lines . Compoun251 and 252 displayed IC50 values ranging from 1.6–8.4 μM against the four cancer cell lines tested. Compound 251 showed the greatest potency against the HeLa cell line with an IC50 value of 1.6 μM. In terms of the SAR for these molecules, compounds possessing heteroaryl groups at R1 in scaffold 253 displayed superior potency compared to compounds possessing aryl rings at this position, while compounds displaying a chlorine at R2 generally exhibited inferior potency compared to compounds with a hydrogen atom at this position. Treatment of HeLa cells with compound 251 at a concentration of 5 μM caused a 40% decrease in the mitochondrial membrane potential and a 4-fold increase in the level of reactive oxygen species.Shaik et al. reported the synthesis of benzoimidazothiazole-propenone conjugates as potential antiproliferative agents against HeLa, HepG2, A549, and MCF-7 cancer cell lines . Compoun50 values below 20 μM) on MCF-7 and A549 cell lines when compared to artemisinin and dihydroartimisinin . Artemisinin-imidazole hybrid compound 254 (see 254–265) exhibited an IC50 value of 9.78 μM against MCF-7 cells, while artemisinin-indole hybrid 255 displayed IC50 values of 5.25 and 6.17 μM on MCF-7 and A549 cell lines, respectively. In general, derivatives bearing an indole substitution instead of imidazole displayed slightly greater potency. Compound 255 showed outstanding reversal activity in MCF-7/ADR cells resistant to adriamycin, displaying a reversal fold of 117.Hu et al. synthesized a series of artemisinin-imidazole and artemisinin-indole hybrids which were tested in vitro against A549, MCF-7, HepG2, and MDA-MB-231 cancer cell lines and were counter-screened against a normal LO2 cell line . Most of256) and the related cyclopentenyl nucleoside 258, a series of fluorocyclopentenyl pyrimidines and purines were synthesized and tested against six cancer cell lines [258 was the most active against these cell lines , with IC50 values ranging from 0.18–0.76 µM, while the addition of a fluorine atom at the 5-position of the pyrimidine ring resulted in a significant loss of potency. For the purines, fluorocyclopentenyl analog 257  and its N6-methyl derivative  displayed potency against the six cancer cell lines similar to or slightly lower than neoplanocin A , but the corresponding derivative bearing an oxygen atom bound at C6 lacked activity. Addition of the bulkier cyclopropylmethyl group to the N6 amino group resulted in a loss of activity, as did addition of an amino group to C2 of the purine ring. The phosphoamidate prodrug of compound 257 was 1.6- to 17.7-fold less active than 257 itself against the cancer cell lines, leading the authors to speculate that anticancer action is through inhibition of S-adenosylhomocysteine hydrolase by 257 rather than interference with DNA or RNA polymerase by the triphosphorylated metabolite of 257.Based on the strong antitumor activity of the natural product neoplanocin A (ll lines . Among t259) was synthesized by Pan et al. [261) via an intramolecular Heck reaction starting from 260. The target compounds were tested for activity against the BGC-823, A549, HCT116, HepG2, and A2780 cancer cell lines. Derivatives 262, 263, and 264 displayed IC50 values of 20.0, 6.23, and 3.80 µM against the HCT116 cancer cell line, whereas the IC50 value of ceratamine A was 12.4 µM. Interestingly, compounds with substitutions possessing bulky groups, electron-withdrawing groups, or electron-donating groups on the benzyl substituent attached to the nitrogen atom of the azepine ring generally displayed decreased potency against the other four cell lines tested. Compound 265, which contains a 3,4,5-trimethoxyphenyl substitution corresponding to the 3,5-dibromo-4-methoxyphenyl group of 259, was active against all five cancer cell lines tested, with IC50 values ranging from 8.56–12.9 µM. The removal of the benzyloxymethyl group from the imidazole ring resulted in a dramatic loss of potency against the cell lines.A novel series of imidazoazepine derivatives inspired by the natural product ceratamine A  displayed the greatest activity, with IC50 values of 2, 1, and 4 μM against the three cell lines, respectively. By comparison, the standard drug 5-FU exhibited IC50 values of 30, 4, and 3 μM against the Huh7, HCT116, and MCF-7 cell lines. Compound 266 also displayed activity against HepG2, MAHLAVU, and FOCUS hepatocellular carcinoma cells , while 5-FU exhibited IC50 values of 5, 10, and 4 μM against these three hepatocellular carcinoma cell lines, respectively.Tuncbilek et al. synthesized β-ll lines . Of thes267) and tested them for activity against CCRF-CEM and MCF-7 cancer cell lines [268 and 269 displayed the greatest potency against CCRF-CEM cells, with EC50 values of 6.3 and 6.5 µM, respectively. Against MCF-7 cells, the greatest potency was observed for compounds 270 and 269, which both exhibited EC50 values of 80 µM against this cancer cell line. By comparison, doxorubicin displayed an EC50 value of 1.4 µM against both CCRF-CEM and MCF-7 cells. These proxyphylline analogs were also tested for antifungal activity against Candida albicans, with compounds 270 and 269 exhibiting the best antifungal activity.Borowiecki et al. synthesized analogs of proxyphylline  and down-regulation of N-cadherin and vimentin  was observed in A549 cells incubated with compound 273 at concentrations ranging from 0.5–2.0 µM. Dose-dependent inhibition of the migration of A549 cells was observed in a wound healing assay when compound 273 was incubated with these cells at concentrations ranging from 0.25–1.0 µM.Zhao et al. synthesized 6-chloro-2-propylthio-8,9-dihydro-7H-purine derivatives as potential antiproliferative agents and evaluated their activity against A549, MGC-803, PC-3 and TE-1 cell lines . Of the 275 and 276 displayed the lowest IC50 values  against the A549 cell line (the IC50 of 5-FU was 83.03 μM); compound 275 exhibited a selectivity index of 32 and compound 276 displayed a selectivity index of 5.44 compared to HPBMC cells. For all other target compounds against the other cell lines, IC50 values were > 100 µM. In general, IC50 concentrations of compounds 275 and 276 increased the percentage of caspase 3/7 activation in the cancer cell lines to a greater degree than 5-FU.Shaaban et al. synthesized purines containing a pyrazole group as potential anticancer agents against A549, MCF-7, HepG2, Caco-2, and PC-3 cancer cells . Counter277 and 278 displayed IC50 values ≤ 2.21 and ≤ 1.55 μM, respectively, against the NCI-H460, HL-60, K562, and MCF-7 cell lines, with 278 displaying a selectivity index of > 250 for HL-60 cells compared to MRC-5 cells. Compound 279 displayed the greatest potency (IC50 = 0.39 μM) against the K562 cell line. Compound 278 also induced apoptosis in HL-60 cells at concentrations of 5 µM and 50 µM.Salas et al. synthesized a series of 31 purine derivatives as potential cytotoxic agents against CFPAC-1, NCI-H460, HL-60, Caco-2, HCT116, K562, MCF-7, MRC-5 cancer cell lines and normal MRC-5 cells . The com280 displayed IC50 values of 2.0 and 3.8 μM, respectively, against these cancer cell lines. Compounds 281 and 282 exhibited IC50 values of 0.5 and 0.4 μM, respectively, against HepG2 cells. In terms of the general SAR trends, compounds possessing a purinone core were typically more active against the HepG2 cells compared to target compounds containing an imidazotriazinone core, and introduction of fluorine atoms on the benzyl substituent at N9 and the presence of a longer alkyl group at the C2 position of the purinone core conveyed increased anticancer activity.Liu et al. synthesized purinone and imidazotriazinone derivatives as potential antitumor agents and tested these compounds against HepG2 and U-118 MG cancer cell lines . CompounH-purin-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (compound 283) displayed the greatest potency. Compound 283 displayed EC50 values of 4.26, 5.54, and 2.80 μM against these cell lines, respectively, while the standard drug 5-FU exhibited EC50 values of 1.50 and 2.40 μM against MCF-7 and HCT116 cells. Compound 283 also displayed low cytotoxicity against RFP TERT normal fibroblasts (EC50 = 54.0 μM) and induced apoptosis in 58% of MCF-7 cancer cells when incubated with these cells at a concentration of 30 μM for 24 h, while apoptosis was observed in less than 10% of the vehicle control cells.Fernández-Sáez et al. synthesized purine derivatives substituted with various heterocyclic rings and evaluated their activity against MCF-7, A375, and HCT116 cancer cell lines . Among t8 show outstanding efficacy in murine cancer models [8 for treating metastatic, castration-resistant prostate cancer . Kinases are popular drug targets, but there have been selectivity issues due to their structural and sequence similarity. Considering the covalent binding of Nek2 inhibitors 85 and 87 to their putative target [125 on MCM2, a promising new target, 127 is considered an exciting, potent anticancer candidate considering its efficacy in vivo at doses of just 1 mg/kg [141 displays in vivo anticancer efficacy [144 displays potent p53-MDM2/HDAC inhibitory activity and good activity in a murine xenograft model [150 shows exceptionally tight binding to WDR5, strong inhibition of MLL1 histone methyltransferase activity, and excellent activity against MYC-driven cancer cell lines [150 and its derivatives is, therefore, needed. Cutting edge drug discovery approaches, such as NMR-based fragment screening, also represent attractive strategies for the exploitation of WDR5, and, by extension, MYC, for cancer therapy [191 is undergoing phase II clinical trials for the treatment of gastrointestinal cancer refractory to current treatments [Cancer is one of the leading causes of mortality worldwide, placing a huge burden on the healthcare system and exacting a staggering human toll. Despite the many drugs available for treatment of various types of cancer, the side effects, resistance profiles, and variable efficacy of these drugs provides the motivation to discover new anticancer compounds. As illustrated in this review article, considerable attention has been devoted to the synthesis and anticancer evaluation of imidazole and fused imidazole derivatives in recent years. This review summarizes the in vitro and in vivo efficacy studies performed with these derivatives, mechanistic studies conducted on these candidates, and the SAR of the series presented. The work recapped here demonstrates that these imidazole-containing derivatives display anticancer activity through a wide range of mechanisms. While it is impossible to predict with certainty the candidates and approaches described in this review that will have the greatest impact on the treatment of cancer, the following future outlook identifies molecules and strategies that appear to be particularly promising to the authors. Although extensive work on antimicrotubule agents as anticancer compounds has been performed in the past, antitubulin candidates related to compound r models ,36, sugge target , such coe target . Given t 1 mg/kg  if off-t 1 mg/kg . The duaefficacy , while tft model , making ll lines . Further therapy . While neatments , indicatOur understanding of cancer biology continues to increase, but the development of effective, less toxic therapeutics remains a significant challenge, with drug resistance continuing to represent a major obstacle. Selective attack of key processes/enzymes of vital importance to specific cancers (targeted therapy) has already improved clinical outcomes in cancer ; the wor