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


Primary cell cultures are objects of intense research due to their high impact on molecular biology and oncology advancement. Initially, the patient-derived specimen must be subjected to dissociation and isolation. Techniques for tumour dissociation are usually reliant on the organisation of connecting tissue. The most common methods include enzymatic digestion , chemical treatment (with ethylene diamine tetraacetic acid and ethylene glycol tetraacetic acid), or mechanical disaggregation to obtain a uniform cell population. Cells isolated from the tissue specimen are cultured as a monolayer or three-dimensional culture, in the form of multicellular spheroids, scaffold-based cultures , or matrix-embedded cultures. Every primary cell line must be characterised to identify its origin, purity, and significant features. The process of characterisation should include different assays utilising specific (extra- and intracellular) markers. The most frequently used approaches comprise immunohistochemistry, immunocytochemistry, western blot, flow cytometry, real-time polymerase chain reaction, karyotyping, confocal microscopy, and next-generation sequencing. The growing body of evidence indicates the validity of the usage of primary cancer cell lines in the formulation of novel anti-cancer treatments and their contribution to drug development.Primary cancer cell lines are  Cell culture development significantly changed the area of life sciences and contributed to great advancements in medicine. Research with the use of cell lines is an essential procedure for modelling diseases, stem cell and cancer investigation, and the establishment of therapies . The firCell lines, as we know them today, play a remarkable role in cancer research. The application of cancer cell lines enabled a better understanding of tumour biology and improved drug development . The exaThe main aim of this review is to present the most recent data referring to primary cell lines isolation and characterisation processes. This article highlights the variety of isolation methods depending on the cells’ source and its next implementation. Moreover, we aimed to introduce different molecular methods of isolated cells’ characterisation. This review is a summary of the available knowledge in the field of primary cell lines culture.ex vivo cell populations deriving directly from resected tissue samples, most commonly from core biopsies, fine-needle aspirates, pleural effusions, resections, or autopsy specimens , whole organs, saliva, urine, faeces, and other body liquids . Those are gathered for different purposes: diagnosis, clinical treatment, or research . LaboratThe Clinical Trials Regulation EU No 536/2014 (CTR) modulates research on biological materials of human origin. According to those directions, obtaining biological samples must be held only with the “donors” written informed consent. Alternatively, an authorisation may be given on behalf of the donor by an eligible person or representative authority. Written informed consent should enable the right to withdraw the approval and alter the scope of the agreement. The person concerned can stipulate the use of his/her biological material and personal data at any time. However, when gathering “patients” information, it is hard to foresee the purpose and scope of personal data processing for future research. Thus, it should be allowed to consent to certain areas of study, depending on the “donors” wishes. Alternatively, it is advised to compose an additional article of the consent, stating that the donor permits to use his/her data outside of the clinical trial protocol.1.Research should commence only after an independent evaluation of its scientific value, including the significance of the aim and confirmation of ethical standards by the appropriate committee [Biospecimens are usually collected during surgery or examination . For somStaphylococcus aureus, bacteria, and fungi , methicillin-resistant nd fungi . For thend fungi . Authorsnd fungi . As far nd fungi . Also, and fungi .Techniques for tumour dissociation are usually reliant on the organisation of connecting tissue. To obtain a uniform cell population, the most common methods include enzymatic digestion, chemical treatment, or mechanical dissociation .2. The procedure of cell isolation with collagenase and hyaluronidase is also successful in breast cancer  is effective in breast and ovarian cancer . DNase gc cancer . Dispasec cancer . This en enzymes  may be u enzymes . Isolati enzymes . The use enzymes .2+ and Mg2+, that provide integrity for the cell surface and intracellular structural matrix. The use of chemical agents enables removing those ions, which causes the loosening of intercellular bonds in, i.e., epithelial cells. The best chemicals used for sequestration are ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and tetraphenyl boron complexes with potassium ions  and free flow of cytokines and growth factors throughout the tissue-like gel. Hydrogels can be natural and synthetic, and natural ones are made with polymers, such as collagen, fibrinogen, Matrigel, gelatin, or alginate. Artificial models usually use synthetic polymers made up of polyethene glycol, polylactic acid, and poly(vinyl acetate). Polymeric scaffolds are replicating the structure of the ECM very accurately, thus they are an essential tool to study cell-to-ECM interactions. Hydrophilic glass fibres are crucial models for designing 3D tumours and tracking cell migration, however, this model is still being explored. Organoids mimic the microenvironment of organs and are very useful for modelling human diseases by pluripotent stem cells derived from patients .The multicellular spheroids model includes techniques such as hanging drop microplates, magnetic levitation, and spheroid microplates. The last approach is especially unique due to its ability to grow freely without any scaffold. Spheroid microplates enable the growth of multicellular cultures because of the large volume. Moreover, cell growth has significant differences between cells grown as multicellular spheres and cultured in 2D culture. The cells from 3D culture showed multidrug resistance, displayed stem-like behaviours, and their motility was increased. The hanging drop method uses gravitation force to induce self-aggregation of cells that further make up spheroids. This approach offers high replicability of results. Magnetic levitation is performed by injecting magnetic nanoparticles into cells that induce cell aggregation to spheroids when exposed to magnetic force. This generates a concentrated environment where ECM can be synthesised, and further biochemical assays may be performed, i.e., western blotting .Cell lines’ characterisation is crucial to identifying cell line origin, purity, and features. The process of characterisation and authentication should include different assays related to specific (extra- and intracellular) markers. The researchers should confirm whether obtained cell lines meet their expectations because, during multiple passages, the cell line may lose its key characteristic features . Additioin vivo state as much as possible, thus fixation is the most significant step. Then the sample is processed in paraffin wax, which provides a sort of supporting media. After that, tissue fragments of 4 μm thickness are dissected and placed in a water bath at the temperature of 45°C. The specimen is laid on a microscope slide and dried at 37°C overnight for better adherence of tissue to the slide. The sample is rewaxed before immunohistochemical staining (Immunohistochemistry (IHC) is a leading method for cancer diagnosis in clinical pathology. IHC uses monoclonal and polyclonal antibodies to identify specific antigens in specimens . There astaining . IHC is staining . IHC maystaining .INSM-1 that regulates the expression of neuroendocrine markers (Western blot (WB) is a commonly used method to separate and identify proteins. Initially, a mixture of proteins is separated during electrophoresis. Then, the proteins are transferred to the membrane, which is incubated with specific antibodies. Incubation is followed by washing which causes a rinse of unbounded antibodies. Antibodies bounded to proteins are detected and visualised on the membrane. The thickness of each band depends on the amount of protein . WB is u markers .Flow cytometry (FC) is a prominent method used in immunology, molecular biology, and cancer research. This approach utilises single or multiple laser light to analyse cells and particles suspended in buffered solution. Each particle undergoes analysis under visible light scatter and fluorescent assessment. Visible light scatters evaluated particles in the forward direction, which displays the comparative cell size and at 90°, which shows the cell’s complexity and granularity. Fluorescent assessment includes cell transfection and fluorescent proteins’ expression, staining with fluorescent dyes, or fluorescently conjugated antibodies. Visible light scatters independent of fluorescence measurement. The most common purpose of FC is immunophenotyping. It enables analysis of cell culture for various parameters, such as T and B cell markers, monocyte markers (CD14 and CD11b), NK cell markers (CD56 and CD161), activation markers (CD69 and CD25), memory markers (CD45RO and CD27), cytokines presence , and antigen-specific markers (MHC). Cell line’s features can be analysed in terms of proliferation (markers Ki67 and CFSE) and apoptosis (the activity of caspases) . Like otImmunocytochemistry (ICC) utilises labelling cells with antibodies to determine population homogeneity and molecular profile. This method visualises individual cells and enables evaluating the distribution of molecular markers in the cell culture. Immunocytochemistry includes cell morphology and exposes subcellular localisation of a particular antigen. The experiment should consist of the negative control (without primary antibody) and positive control (with a cell known to express given antigen) to determine staining propriety. Immunocytochemistry often uses fluorescently tagged secondary antibodies to reveal the primary antibody bounded to an epitope on the investigating molecule. A secondary antibody can detect the heavy chain of the primary antibody. This procedure utilises prolonged incubation for the primary antibody, followed by a few washes to remove the remaining antibodies. Then shorter incubation is applied for a secondary antibody with subsequent washing. Afterwards, the sample is processed under microscopy . The immImmunofluorescence (IF) is a method of choice while working with adherent cell lines. It simplifies the isolation of the individual signal and cell lines usually are not vulnerable to damages, due to similar auto-fluorescence as they exhibit from tissue sections . The immA confocal fluorescence microscope serves as a great tool for assessments in cells and tissues. Confocal microscopes generate high-contrast images through optical sectioning. It is achieved by a high-resolution objective lens which produces optical sections thinner than 1 μm without slicing the specimen. A single confocal image, also called a slice, is enough for quantification, although it is possible to use a series of confocal images to generate a 3D dataset. The 3D model enables the reconstruction and quantification of the whole sample volume. The use of fluorescent molecules is advantageous while assessing molecules’ localisation from various cellular compartments . In pracGene expression measurement in cultured cells by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is an essential technique used in molecular biology and medical research . This toKaryotype analysis includes pairing and ordering all organisms’ chromosomes, resulting in the genome-wide projection of the individual chromosomes. The preparation of karyotypes consists of staining, enabling exposure to chromosomes’ structural characteristics . KaryotyThere is an increasing demand for high throughput technologies for gene expression analysis of cell lines . In thisPrimary cell lines are pivotal for cancer research and have significantly contributed to understanding tumour biology, molecular processes, oncogene activation, and gene expression of individual patients. Primary cancer cultures are crucial for conceptualising therapeutic targets that may serve for future drug development or personalised cancer therapy. Patient-derived samples are currently extensively used for the improvement of existing treatments in personalised medicine. Moreover, the combination of primary cancer cell lines with advanced techniques (such as NGS) is a prospective cancer research tool that leaves the door open to identify novel biomarkers and oncogenes. Knowing the limitations and benefits of primary cell lines, scientists should pay more attention to the choice of model they will work on. It is important to also use established cell lines according to the purpose of the planned experiment. Nevertheless, the culture of primary cell lines is a key element in research towards personalised medicine, which is the future of cancer treatment.MR and OP wrote the manuscript with support from MM, IP, WMS, and TT. MR and OP declare an equal co-authorship. MM and IP participate in drafting the article and helped supervised the article. WMS and TT make substantial contributions to the conception and design of the article and give final approval of the version to be submitted and any revised version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRCA1 or BRCA2 that result in deficient homologous recombination repair (HRR). Use of olaparib, niraparib or rucaparib for the treatment of high-grade serous ovarian cancer, including in the maintenance setting, has extended both progression free and overall survival for women with this malignancy. While different PARP inhibitors (PARPis) are mechanistically similar, differences are apparent in their chemical structures, toxicity profiles, PARP trapping abilities and polypharmacological landscapes. We have treated ovarian cancer cell line models of known BRCA status, including the paired cell lines PEO1 and PEO4, and UWB1.289 and UWB1.289+BRCA1, with five PARPis  and observed differences between PARPis in both cell viability and cell survival. A cell line model of acquired resistance to veliparib showed increased resistance to the other four PARPis tested, suggesting that acquired resistance to one PARPi may not be able to be rescued by another. Lastly, as a proof of principle, HRR proficient ovarian cancer cells were sensitised to PARPis by depletion of BRCA1. In the future, guidelines will need to emerge to assist clinicians in matching specific PARPis to specific patients and tumours.Several poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors are now in clinical use for tumours with defects in BReast CAncer genes  BRCA1 and BRCA2 mutated tumours, with clinical benefits seen in patients with mutations in these DNA repair genes such as ovarian [BRCA1 methylated tumours are also sensitive to PARP inhibition [RAD51C, RAD51D, ATM and PALB2, where tumours are described as having a “BRCAness” phenotype [The advent of pharmacological inhibitors of the DNA repair enzyme poly (adenosine diphosphate-ribose) polymerase (PARP) has heralded major therapeutic advances for malignancies that have defects in components of homologous recombination repair (HRR) pathways ,2,3. The ovarian , breast  ovarian , prostat ovarian  and panc ovarian . BRCA1 mhibition , as are henotype ,9,10. Wihenotype ,12,13.BRCA mutations. By inhibiting PARP function in cells with deficient HRR, cells lose their ability to choose the default BER pathway to repair DNA damage, creating synthetic lethality that leads to cell death [PARP family members function in a number of cellular processes including the regulation of gene transcription, chromatin remodelling and DNA repair ,15. PARPll death ,19,20.+) in a process known as PARylation. While PARP1 is reported to conduct more than 90% of PARylation associated with DNA damage, PARPs 2, 3, 4, 5a and 5b also demonstrate PARylation activity [PARP1, PARP2, PARP3, PARP4  and tankyrases 1 and 2 (PARP5a and PARP5b) are amongst the most studied members of the PARP family . Catalytactivity ,22. Inhiactivity ,24.®) was the first PARPi endorsed by the Food and Drug Administration (FDA) for the treatment of advanced germline BRCA-mutated ovarian cancer in 2014, followed by rucaparib (Rubraca®) for use to treat the same indication in 2016 [®) was endorsed in 2020 by the FDA as maintenance for advanced epithelial ovarian, fallopian tube or primary peritoneal cancer where patients have had complete or partial response to first-line platinum-based chemotherapy [®) became licensed by the FDA in 2018 for the treatment of BRCA-mutated HER2-negative breast cancers [BRCA mutations or mutations in other HRR-related genes such as PALB2 or RAD51C, loss of BRCA1 methylation that re-establishes HRR proficiency, the increase in expression of drug efflux pumps such as the MDR1 (p-glycoprotein) gene, aberrant replication fork protection and down-regulation of PARP proteins themselves, possibly as a result of PARP trapping [Olaparib  have defects in genes that function in HRR . We use BRCA2 mutant PEO1 cells were responsive to olaparib, niraparib and talazoparib over the dose curve compared with wild-type (WT) PEO4 cells, but not to rucaparib and veliparib in the context of cell viability  in PEO1 and PEO4 A, as weliability . Velipar2 status .BRCA1 mutant UWB1.289 cells were more responsive to all five PARPis tested based on LC50 data, compared to the paired cell line UWB1.289+BRCA1  [The human HGSOC cell lines UWB1.289, UWB1.289+BRCA1  and OVCAWT BRCA2 ,55. Thisstralia) ,52. Cleastralia) . Furtherstralia) . The A272 atmosphere.All cell lines were grown in RPMI 1640  supplemented with 10% FBS , with the exception of the UWB1.289 and UWB1.289 + BRCA1 cell lines which were maintained in 50% RPMI 1640 , 50% MEGM , supplemented with 3% FBS at 37 °C in a humidified 5% COTM Mycoplasma Detection Kit .Cell line authentication was performed by the Australian Genome Research Facility (AGRF) Melbourne, Australia, by short tandem repeat (STR) profiling using the GenePrint-10 System which co-amplifies ten loci, including the ASN-0002 loci  as well as D21S11. All cell lines tested negative for mycoplasma with the MycoAlertBRCA1 siRNA #13 , Chadstone, VIC, Australia), BRCA1 siRNA #14  or a non-silencing negative control  using HiPerfect transfection reagent (Qiagen). After 18 h, siRNA transfected cells were re-seeded for MTS or clonogenic assays. RNA was extracted 48 h post transfection using the RNeasy Mini kit  and 500 ng converted to cDNA using the SuperScript™ IV First-Strand Synthesis System . Quantitative real-time PCR (qRT-PCR) was performed using the TaqMan Fast Advanced Master Mix Kit  with Taqman assays BRCA1  and hydroxymethylbilane synthase (HMBS) endogenous control  on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). Each experiment was performed in triplicate and repeated at least three times, with data reported as the mean ± S.E.M.Cells were seeded into 6-well plates  for 24 h followed by transfection with 20 nM Cells were seeded into 96-well plates  and treated with niraparib, olaparib, rucaparib, talazoparib  or veliparib  for 5 days before being assessed for cell viability using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay . This assay measures cellular metabolic activity and is a surrogate for cell viability. All results using this assay are described in the context of cell viability. Each experiment was performed in triplicate and repeated four times, with data reported as the mean ± SEM.Relative lethal concentration 50 (LC50), the concentration required to bring the dose curve halfway between the top and bottom plateau of the curve, was calculated using GraphPad Prism 9. To address the issue that some of the drugs tested did not achieve total loss of cell viability at high levels, data were normalised to vehicle alone (100%) and the highest drug concentration where a plateau was observed (0%). A non-linear regression curve was fitted to the data and LC50 concentrations extrapolated .w/v crystal violet in 25% v/v methanol for 5 min [Clonogenic cell survival assays measure the ability of a single cell to grow into a colony post an intervention, in this case treatment with a PARP inhibitor or down-regulation of a gene. All results using this assay are described in the context of cell survival. Cells were seeded into 6-well plates at a density of 1000 cells/well for UWB1.289+BRCA1 and OVCAR-3, 2000 cells/well for UWB1.289, 750 cells/well for PEO1, 1500 cells/well for PEO4, 200 cells/well for A2780 and 180 cells/well for A2780veliR. Cells were then treated with niraparib, olaparib, rucaparib, talazoparib or veliparib for 8–21 days. Cells were fixed with 100% methanol for 20 min, rinsed briefly with water and stained with 0.5% or 5 min . Colonieor 5 min . A Jupytor 5 min .post hoc test was used to test for multiple comparisons between cell lines at discrete drug dosages. For all analyses, p < 0.05 was considered statistically significant.IBM SPSS software version 27.0  was used for statistical analyses. All results are expressed as the mean ± SEM from at least three independent experiments unless otherwise stated. One-sample t-tests were used to assess the efficacy of gene down-regulation. Independent samples T tests were used to compare plating efficiency in cell survival assays. Two-way ANOVA were used to compare paired cell lines over dose courses for different PARPis. One-way ANOVA with Tukey’s