V600E-driven papillary thyroid cancer that closely mimic the heterogeneous pathological progression of the disease seen in patients despite sharing a common driver mutation. We anticipate that these newly developed models will allow for the discovery of novel mechanisms that determine differences in disease progression among BRAFV600E-driven thyroid cancers and facilitate the testing of therapeutic interventions.Laboratory experimental models are essential for advancements in thyroid cancer translational research. In this study, we discuss the development and characterization of six cell-based models of BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a V600EBraf+/−/Pten+/−/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAF V600E is the most common driver mutation of PTC in adults and the second most common driver mutation in children [V600E mutation confers constitutive activation of the kinase, leading to sustained activation of the mitogen-activated protein kinase (MAPK) signaling pathway [V600E mutation with extrathyroidal extension, lymphatic and vascular invasion, local lymph node metastasis, higher clinical stage, persistent disease, reduced avidity for radioactive iodine, and lower survival rates in adults with PTC [Thyroid cancer is the most common endocrine malignancy and the most frequently diagnosed cancer among adolescents and young adults between ages 15–29 . Papillachildren ,9,10. Th pathway ,12. Clinwith PTC ,17,18. Iwith PTC ,20,21. Nwith PTC ,23,24,25Most PTCs are successfully managed with standard multimodal therapy. Patients undergo surgical tumor resection and lymph node dissection for localized disease, often followed by radioactive iodine (RAI) ablation, as adjuvant therapy in patients with high risk of recurrence or metastatic disease. Patients are then placed on thyroid hormone replacement therapy, with or without long-term thyroid hormone suppression, based on risk of recurrence. In the subset of patients with advanced and progressive RAI-refractory disease, oncogene-targeted therapies represent essential advancements in care and continue to be important areas of preclinical and clinical investigation in both adult and pediatric thyroid cancer.G12V-driven models of FTC [V600E-driven PTCs.It remains imperative to develop tumor-specific in vitro and in vivo preclinical models that recapitulate patient disease to prospectively evaluate treatments for efficacy and to decipher molecular mechanisms that confer therapeutic resistance. Although cell lines derived from human thyroid tumors provide a powerful tool to study novel therapies, research using human cells is limited to the tissue culture environment or xenograft models in immunocompromised hosts, which fails to recapitulate the interactions between tumor cells and the immune system in the host. Further, it was discovered that many previously reported thyroid tumor cell lines were either not unique or not actually of thyroid origin, further limiting the utility of some of these models . In conts of FTC  and sougV600EBraf+/−/Pten+/−/TPO-Cre mouse model of PTC. We have previously described a mouse model of PTC whereby thyroid-specific expression of V600EBraf induces development of thyroid tumors with 100% penetrance by five weeks of age [V600EBraf and homozygous Pten inactivation in mice to achieve concomitant MAPK and PI3K/Akt pathway activation. These mice develop PTCs that rapidly progress to PDTC with 100% penetrance and lethality by weaning [V600EBraf and heterozygous Pten. Three cell lines were isolated from well differentiated PTC tumors  and the other three cell lines were isolated from higher grade tumors with more invasive characteristics . This variability reproduces the clinical heterogeneity within the PTC patient population and, thus, allows for evaluation of characteristic features, in a subset of cell lines, that may be critical for disease progression. These cell lines represent novel and physiologically relevant research tools that can be used to evaluate therapeutic strategies and to illuminate factors that impact the progression of disease.In this study, we detail the establishment of six independent cell lines from the s of age . Additio weaning . These m weaning ,31. HereV600EBraf+/−/Pten+/−/TPO-Cre mice of a pure 129/SvImJ genetic background (Jackson Laboratory mouse strain #:002448 here on referred to as 129SvJ). B1865 and B1866 tumor cell lines were established from thyroid tumors of V600EBraf+/−/Pten+/−/TPO-Cre mice of a mixed genetic background. Thyroid tumors or WT thyroids were dissected and minced. The pieces were then resuspended in Ham’s F12 medium  supplemented with 10% fetal bovine serum , 2 mM L-Glutamine (Gibco), Penicillin/Streptomycin/Amphotericin B , 10 µg/mL Piperacillin sodium salt , and 10 µg/mL Ciprofloxacin Hydrochloride . Cells were then seeded into bind tissue culture flasks , and each flask was given an additional 500 μL of FBS. They were maintained at 37 °C in 5% CO2. After 24–48 h, the media were replaced, and the cells were maintained in 10% FBS F12 media supplemented with antibiotic for the next two weeks. WT thyrocytes were only cultured for at most one week before RNA was extracted to limit outgrowth of non-thyrocyte cells in culture. Then, antibiotic-free complete Ham’s 12 media was used. To rule out stromal cell contamination, all tumor cell lines were passaged at least 5 times after seeding and then genotyped using primers specific for BrafV600E to check for complete recombination , Pax8, Slc5a5 (Nis), and B2m were measured using pre-designed probes  and Taqman master mix . A total of four µL of cDNA from tumor samples and independent passages of each cell line and WT thyroids were run in triplicate on the QuantStudio 3 . B2m was used as an internal control to normalize the expression levels of thyroid specific genes. Data analysis was based on the delta-delta Ct method.Total RNA from all cell lines was extracted using the Direct-zol RNA Miniprep . Equal amounts of RNA template were reverse transcribed using the Verso cDNA synthesis kit . The differential mRNA expressions of Cells were plated, 300 per well, in quadruplicate into 96-well plates. At designated time points, cell growth was assessed using the Cell Titer-Glo luminescent cell viability assay . Luminescence was determined using the Varioskan Lux . Day 0 represents 18 h after plating. Inhibitor studies were conducted using Dabrafenib methanesulfonate salt  and Trametinib  dissolved in DMSO. To assess the relative viability of cells in the presence of single inhibitors, Dabrafenib, Trametinib, or vehicle (DMSO) were added 18h after plating. Relative viability was assessed using the Cell Titer-Glo luminescent cell viability assay, and concentrations of Dabrafenib 10 nM and Trametinib 1 nM were selected for viability assays of the complete cell line panel.To prepare total cellular protein lysates, 200,000 cells per well were seeded in six-well plates and allowed to attach for 24 h in complete medium. Cells were serum starved overnight before any specific treatment or stimulation. Then, cells were lysate in equal volumes of radioimmunoprecipitation (RIPA) buffer  supplemented with phosphatase and protease inhibitors  and 4× sample buffer  containing 200 mM DTT and boiled for 5 min. A small aliquot was taken after harvesting to measure protein concentration by BCA. To perform the SDS–polyacrylamide gel electrophoresis (PAGE), equal quantities of total cellular proteins from each sample were loaded into 10% acrylamide gels containing SDS-PAGE. Protein sizes were determined by comparison to a set of standards . To perform the immunoblot, proteins were transferred to a PVDF membrane pore size 0.45 μm  after methanol activation. Primary antibodies (1:1000) specific to pERK (9101S), ERK (9102S), pAKT (9271S), AKT (4685S), PTEN (9188T), E-cadherin (14472s), Vimentin (5741S), and β-actin (3700S)  were diluted in 5% bovine serum albumin (BSA) and 0.1% Tween-20 tris buffer (TBS-T) and incubated overnight at 4 °C. Specific binding of antibodies was detected with appropriate HRP-coupled secondary antibodies anti-rabbit (1:3000) or anti-mouse (1:5000) and visualized using enhanced chemiluminescence reagent . Protein expression levels were analyzed and compared by densitometry using iBright Analysis Software . Relative protein phosphorylation levels were obtained by normalizing the amount of phosphorylated protein to that of total protein. Original, uncropped Western blot membrane can be found in Organoids were grown using previously described methods . BrieflyMigration assays were performed in 24-well plates with cell culture inserts with 8.0 μm pores ; 75,000 tumor cells were seeded in the top of each insert in serum free Hams F-12 media. Complete 10% FBS Hams F12 media was added to the bottom chamber of the plate. Cells were then incubated at 37 °C for 18 h. The cells were then fixed using 4% Paraformaldehyde, and the cells were removed from the top of the chamber using cotton tipped applicators. The underside of the chambers was then stained with crystal violet. Images were taken on the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific).Phase contrast imaging was performed using the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific). H&E histology slides were imaged using the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific). Trichrome histology slides were scanned by the CHOP pathology core and images captured using Image Scope. For differential interference contrast (DIC) imaging live cells were imaged with a Zeiss Axiovert 200M platform using a 40× EC Plan Neo Fluar, 1.3 N.A., or a 63× Plan Apochromat, 1.4 N.A. objective  equipped with an Orca ER CCD camera . Images were captured using Slidebook 6 imaging software  using an Orca ER CCD camera (Hamamatsu).5 tumor cells  were pelleted by centrifugation, resuspended in 100 µL of a 1:1 mixture of Hams F-12 and Matrigel , and injected subcutaneously into the right hind flanks of 4–5-week-old male and female wild-type 129SvJ recipient mice. Tumor development was monitored weekly for 15 weeks. Mice were sacrificed due to tumor burden or loss of skin integrity as needed during that period or were collected finally at the end of the 15 weeks. Tumors were collected and fixed in 10% formalin for histological analysis.All animal experiments were performed at the Children’s Hospital of Philadelphia and approved by the IACUC. 5.0 × 10p-values of ≤0.05 were considered statistically significant. All data comparing more than two groups were analyzed via ANOVA analysis with post hoc analysis (indicated in figure legends).All data were analyzed using Prism 8 software . Differences with V600EBraf+/−/Pten+/−/TPO-Cre mice of a pure 129SvJ genetic background  or a mixed genetic background (cell lines B1865 and B1866). Tumors were collected from different sex animals and at a variety of time points from 3–24 weeks of age , Pax8, and Slc5a5 (Sodium Iodide symporter: Nis)  [Tg and Tshr expression levels were similar among the six cell lines but were very low compared to expression in WT thyrocytes. In contrast, Nis expression was variable between the six cell lines but was consistently much lower compared to WT thyrocytes. Like thyroid transcription factors, loss of Tg, Tshr, or Nis expression has been associated with PTC disease progression [Nis expression is particularly relevant clinically as it is associated with disease refractory to RAI ablation therapy [The thyroid epithelial lineage and differentiation status of the cell lines was determined by measuring the mRNA levels of thyroid-specific genes including er: Nis) A. Pax8 asgenesis ,34. The gression ,36,37,38 therapy ,40,41.We next sought to evaluate the proliferation rates of the cell lines in 2D culture using the Cell Titer Glo luminescence-based viability assay B. The CePten. To determine whether levels of PTEN protein expression correlated with basal pAKT levels, Western blot analysis was performed for PTEN. Consistent with their elevated basal pAKT expression, BB57, BB1845, B1865, and B1866 all showed reduced or no expression of PTEN protein  models of tumor growth may be more relevant as they better recapitulate the natural environment and structure of solid tumors. To characterize the growth of these novel cell lines in more physiologic conditions, we used an organoid forming assay D. BB342,Heterogeneity in cell morphology can be observed among the six cell lines A. DespitWe next evaluated the efficacy of MAPK specific inhibitors on the growth of these novel cell lines. Each cell line was treated with a variety of concentrations of either Dabrafenib (BRAF inhibitor) or Trametinib (MEK1/2 inhibitor) for 3 and 6 days . Both ofTo evaluate the growth-inhibiting effects of these drugs in a more physiologic environment, we established 3D organoid cultures of each cell line and assessed the size of the organoids following treatment with Dabrafenib, Trametinib, or DMSO control for 10 days C. AveragV600EBraf+/−/Pten+/−/TPO-Cre de novo tumors in the mouse thyroid gland precludes many in situ experiments. Therefore, the development of a subcutaneous model in immune competent animals would enable temporal models to study disease progression and to potentially serve as a future model for investigating targeted therapeutic interventions. The strengths of the subcutaneous tumor models coupled with the rapidly penetrant phenotype of our de novo V600EBraf+/−/Pten+/−/TPO-Cre murine model [The subcutaneous injection of tumor cells into murine hosts has been proven to be a valuable and convenient method for studying tumorigenesis. The very rapid growth and progression of ne model ,31 led uG12V-driven tumor cells, variable tumor penetrance was observed between each of the cell lines. BB19 and BB57 were the least penetrant, whereas BB342 had an intermediate penetrance and BB184-injected mice developed tumors with 100% penetrance  leads to the development of PTC in mice [Cancer cell lines harboring clinically relevant mutations derived from genetically engineered mouse models are invaluable tools for in vitro and in vivo preclinical research to drive advancements in our understanding of disease mechanisms and treatment approaches in thyroid cancer. Here, we sought to generate cell lines that would recapitulate the heterogeneity of PTC and that would allow us to examine factors that contribute to disease progression. PTC is the most prevalent form of thyroid cancer, and incidence is steadily increasing . BRAFV60tric PTC ,11,12,45tric PTC ,23,24,25 in mice ,31. In aV600EBraf+/−/Pten+/−/TPO-Cre mouse thyroid tumors. These cell lines share the same initiating driver mutations but represent a heterogenous population regarding sex, age, and histologic differentiation status. Additionally, we shown that these cell lines can be used for a variety of in vitro applications including mechanistic studies of targeted inhibitors and formation of 3D organoid cultures, while also demonstrating successful transplantation into syngeneic hosts for in vivo studies. Altogether, our results demonstrate heterogeneity within these cell lines that recapitulate differences observed clinically in the patient population. This novel cell line panel provides an innovative research tool to identify differences among tumor cells with heterogenous responses to treatment and/or invasive behavior despite sharing the same initiating driver mutations. Previous studies have indicated that progression from PTC to PDTC or ATC may be facilitated by mutations leading to the activation of the PI3K/Akt pathway co-occurring with BRAF mutations [Pten (In this study, we described six novel independent murine PTC cell lines that were derived from utations ,23,24,25ns [Pten . These cEpithelial-to-mesenchymal transition is associated with loss of E-cadherin expression resulting in loss of cellular adhesion and polarity that leads to an invasive cell phenotype with increased metastatic potential ,46,47.PrThe current understanding of EMT in thyroid carcinomas suggests that EMT is implicated in the progression from well-differentiated thyroid cancers to PDTC and ATC ,50,51,52In this study, we sought to validate a syngeneic subcutaneous tumor model. This provides a readily accessible method of exploring tumorigenesis that recapitulates the complex interactions between tumor cells, the immune system, and physiologic components of the tumor microenvironment. Previous studies have shown that immune cell recruitment, particularly macrophages, plays a role in thyroid cancer pathogenesis ,54,55. FV600EBRAF mutation is very common in adult PTC and moderately common in pediatric PTC, the mechanisms behind the disparity in clinical outcomes between these two populations is not well-understood. We believe that these cell lines model the heterogeneity seen in the population of patients with BRAF-driven PTC and provide a novel platform to investigate features critical to PTC disease progression and response to therapies. These tools provide a valuable platform for more reductionist and mechanistic approaches to investigate Braf-driven disease progression.Additionally, this model could be used to evaluate the role of age at onset of disease in PTC development and progression by injecting hosts of different age equivalents to compare penetrance, disease progression, and metastasis ,59,60,61V600E-driven papillary thyroid cancer represents an advancement in the development of much-needed validated preclinical models for thyroid cancer research. We anticipate that these cell lines will provide a powerful platform for in vitro and in vivo studies that will lead to the development of precision diagnostic and treatment strategies for patients with newly diagnosed, advanced, or treatment-refractory thyroid cancer. Ongoing work in the laboratory is seeking to identify molecular mechanisms that drive heterogeneity observed in morphology and responses to therapy of these cell lines, despite sharing identical driver mutations.The establishment of six independent cell lines modeling BRAF


MYC is one of the most frequently altered driver genes in triple-negative breast cancer (TNBC). The aim of this study was to evaluate targeting MYC for the treatment of TNBC.The anti-proliferative and apoptosis-inducing effects of the recently discovered MYC inhibitor, MYCi975 were investigated in a panel of 14 breast cancer cell lines representing the main molecular forms of breast cancer.p = 0.047, r = − 0.5385) or ELISA , i.e., response to MYCi975 decreased as endogenous MYC levels increased. MYCi975 also induced variable levels of apoptosis across the panel of cell lines, ranging from no detectable induction to 80% induction. Inhibition of proliferation and induction of apoptosis were greater in TNBC than in non-TNBC cell lines . Finally, combined treatment with MYCi975 and either paclitaxel or doxorubicin resulted in enhanced cell growth inhibition.IC50 values for growth inhibition by MYCi975 varied from 2.49 to 7.73 µM. Response was inversely related to endogenous MYC levels as measured by western blotting (Our findings open the possibility of targeting MYC for the treatment of TNBC. Based on our results, we suggest that trials use a combination of MYCi975 and either docetaxel or doxorubicin and include MYC as a putative therapy predictive biomarker.The online version contains supplementary material available at 10.1007/s10549-022-06673-6. Although the treatment of patients with breast cancer has greatly improved in recent years , the forThe c-MYC oncogene is one of the best studied cancer driver genes, promoting tumorigenesis via diverse mechanisms such as stimulating cell proliferation, blocking apoptosis, altering metabolism and depressing host immunity –7. OveraIn breast cancer, MYC is the most frequently amplified gene , 10, witTargeting MYC for cancer treatment, however, has proved difficult as the protein lacks a suitable site for high-affinity binding of low-molecular-weight inhibitors , 16. BecThe origin and maintenance of the breast cancer cell lines used was as previously described , 20. The5 per well and incubated overnight at 37 °C. Cells were then treated with dimethyl sulfoxide (DMSO) or 10 μM MYCi975. Following 48 h of incubation, they were harvested and stained with annexin-V and propidium iodide using the Annexin-V-FITC Apoptosis Detection Kit (Invitrogen). FACS analysis was performed using a BD FACSCanto ™.Cell growth was assessed using 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as previously described , 20. ForFollowing treatment with MYCi975 or MYCMI6, cells were lysed in RIPA buffer (Sigma-Aldrich) containing 0.1% of Triton X-100 (Sigma-Aldrich) as well as protease and phosphatase inhibitors (Roche). Following 20 min incubation on ice, cell lysates were centrifuged at 21,000 g for 20 min at 4 °C. The resultant supernatants were collected and stored at − 80 °C. Extracted proteins (70 or 100 μg) were then separated on a 10% or 12% handmade SDS-PAGE or precast Bolt Bis–Tris gels (Invitrogen) and transferred onto a PVDF membrane (Cytiva) [For determining proteasome inhibition, cells were incubated with 10 µM MG132 (Sigma-Aldrich) for 22 h, followed by another 2 h incubation with 10 µM MYCi975 or DMSO. The 2 h incubation period with MYCi975 was chosen based on the results shown in Suppl Fig. 1, where degradation of survivin was clearly found after this time. For determining protein half-life, cells were treated with 10 µM MYCi975 or DMSO for 6 h and then incubated with cycloheximide (100 µg/ml). Cells were then harvested at the indicated time points and the relative levels of MYC were determined by western blotting.c-MYC protein was quantified by ELISA using a c-Myc  Human ELISA Kit, according to the manufacturer’s instructions . Absorbance was measured on a plate reader at 450 nm .RNA was extracted from cell lines using Trizol reagent (Sigma-Aldrich). One μg of RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Survivin primers were supplied by Sino Biological Inc. GAPDH (Sigma-Aldrich) was used as a housekeeping-gene control. The amplification process was performed with the Roche Light Cycler 480, according to the manufacturer’s instructions. Changes in gene expression were calculated using the ddCt method.All the raw data were analyzed using Microsoft Excel 2016. Graph Pad Prism 5 was used to graph the calculated data points and calculate statistical values. The significance of data were evaluated using the Student’s unpaired, two-tailed t-test. Spearman’s rank correlation was used to assess the correlation between two groups. Combination Index  was calculated using CalcuSyn software (Biosoft). CI < 1 at 50% or 90% inhibition was used to indicate synergism .p = 0.041).The effect of MYCi975 on cell proliferation was investigated using the MTT assay in a panel of 14 breast cancer cell lines, representative of the major molecular subtypes of this disease. Following 5 days incubation with increasing concentrations of MYCi975, the IC50 values for inhibition of growth varied from 2.49 to 7.73 µM  of apoptosis induced by MYCi975, we first tested its effect on 43 apoptosis-associated proteins using apoptosis antibody arrays . Figures Theoretically, the downregulation of survivin could have occurred at an mRNA or protein level. To establish the mechanism of downregulation, we performed cycloheximide chase experiments . As showAs MYCi975 appears capable of altering survivn levels, we wanted to assess if combined inhibition of MYC and survivin could produce synergistic effects. We treated cell lines with MYCI975 and the survivin inhibitor, YM-155 and evaluated the potential for enhanced interaction between the two drugs using the Chou–Talalay method . As showTo enhance the anti-proliferative effects of MYCi975, we combined it with two drugs widely used to treat breast cancer, i.e., docetaxel or doxorubicin. As shown in Fig. Similar with MYCi975, MYCMI-6 was previously reported to block the interaction between MYC and MAX and inhibit cell proliferation , 25. Of In this study, using a panel of 14 breast cell lines, we showed that the MYC inhibitor, MYCi975 reduced cell proliferation and promoted apoptosis in a cell line-dependent manner. In particular, the effect of MYCi975 on apoptosis was highly variable across the panel of cell lines investigated, ranging from effectively no induction of apoptosis in some cell lines to approximately 80% induction in others. Previously, Han et al.  reportedA potential mechanism by which MYCi975 mediated apoptosis in our work was by reducing expression of survivin. Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins which is believed to decrease apoptosis by inhibition of caspase activity, although the precise mechanism of this inhibition is unclear . By decrFurther evidence for a possible interaction between MYC and survivin was our finding of enhanced inhibition of cell proliferation when MYCi975 was combined with the survivin inhibitor, YM-155. We should state however, that our work does not exclude mechanisms other than a reduction in survivin levels by which MYCi975 might induce tumor cell apoptosis.Furthermore, although YM155 has been clearly shown to reduce survivin expression, it may possess other activities , which mPreviously, the MYC inhibitor, MYCMI-6 was also found to reduce cell proliferation in a diverse range of cancer cell lines , 24. DesAs previously reported with MYCi975 , we alsor = − 0.23, p = 0. 086).From a clinical point of view, a potentially important observation from our work was that the effects of MYCi975 on both proliferation reduction and apoptosis promotion was greater in TNBC cell lines than in non-TNBC cell lines. Furthermore, we found a significant inverse relation between cellular MYC levels and IC50 values for MYCi975, suggesting that MYC protein levels might be used as a predictive biomarker for response, assuming MYCi975 will enter clinical trials. In an earlier study, the MYCi975 analog, MYCi361 was found to exhibit a non-significant trend for an inverse relationship with MYC mRNA in the NCI60 panel of cells Below is the link to the electronic supplementary material.


Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) form a rare and remarkably heterogeneous group of tumors. Therefore, establishing personalized therapies is eminently challenging. To achieve progress in preclinical drug development, there is an urgent need for relevant tumor models.We successfully established three gastroenteropancreatic neuroendocrine tumor (GEP-NET) cell lines  and two gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) cell lines (NT-32 and NT-38). We performed a comprehensive characterization of morphology, NET differentiation, proliferation and intracellular signaling pathways of these five cell lines and, in addition, of the NT-3 GEP-NET cell line. Additionally, we conducted panel sequencing to identify genomic alterations suitable for mutation-based targeted therapy.Death Domain Associated Protein (DAXX), sensitizing NT-18LM to the Ataxia telangiectasia and Rad3 related (ATR) inhibitor Berzosertib, and a mutation in AT-Rich Interaction Domain 1A (ARID1A), sensitizing NT-38 to the Aurora kinase A inhibitor Alisertib. Small interfering RNA-mediated knock down of DAXX in the DAXX wild type cell line NT-3 sensitized these cells to Berzosertib.We found that the GEP-NEN cell lines exhibit a stable neuroendocrine phenotype. Functional kinome profiling revealed a higher activity of serine/threonine kinases (STK) as well as protein tyrosine kinases (PTK) in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38. Panel sequencing revealed a mutation in The newly established GEP-NET and GEP-NEC cell lines represent comprehensive preclinical in vitro models suitable to decipher GEP-NEN biology and pathogenesis. Additionally, we present the first results of a GEP-NEN-specific mutation-based targeted therapy. These findings open up new potentialities for personalized therapies in GEP-NEN.The online version contains supplementary material available at 10.1007/s13402-022-00727-z. Neuroendocrine neoplasms (NEN) form a heterogeneous group of malignancies comprising well differentiated neuroendocrine tumors (NET) and poorly differentiated neuroendocrine carcinomas (NEC). NEN originate from neuroendocrine cells ubiquitously dispersed throughout the body, in which the gastroenteropancreatic (GEP) tract represents the major site of primary tumor development , 2. AlthDeath Domain Associated Protein (DAXX), ATP-dependent helicase ATRX, X-linked helicase II (ATRX) and Multiple endocrine neoplasia type 1 (MEN1) genes have been reported. Additionally, germline mutations have been detected in the MEN1, CDKN1B, Von Hippel–Lindau (VHL), Neurofibromin 1 (NF1) and Tuberous Sclerosis Complex 2 (TSC2) genes [Tumor protein P53 (TP53), Retinoblastoma protein 1 (RB1) and Murine sarcoma viral oncogene homolog B (BRAF) genes [Several recent genomic investigations revealed main genes being involved in GEP-NET pathogenesis. Specifically, high occurrences of somatic mutations in the 2) genes , 6. The 2) genes . In contF) genes , 9. DespUnfortunately, clinical options for systemic GEP-NEN therapy are limited. So far, mammalian target of rapamycin (mTOR) inhibitors, multi-targeted receptor tyrosine kinase inhibitors, somatostatin analogues (SSA), and peptide receptor radiotherapy (PRRT) have been approved as targeted anti proliferative therapies in GEP-NET . RegardiAberrant activation of mTOR signaling has been implicated in GEP-NEN tumorigenesis, proliferation and angiogenesis , 13. TheSSA are well-established for the treatment of SSTR-positive GEP-NET patients, achieving prolonged intervals of disease stabilization –23. AddiRegarding GEP-NEC, most patients do initially respond to first line chemotherapy. However, the response duration is limited to a few months and a high rate of treatment failure to second line options is observed . Thus, dA major challenge in developing targeted therapies in GEP-NEN is the paucity of preclinical tumor models resembling the unique genetic and phenotypic tumor features. So far, only few GEP-NEN cell lines are available. Of these BON and QGP-1 are the most widely used cell lines. While BON is commonly regarded as a GEP-NET cell line, it is currently under discussion whether QGP-1 falls under the GEP-NET or GEP-NEC classification. Although NET marker expression is present in both the BON and QGP-1 cell lines, their genomic alterations, including mutations in tumor suppressor and oncogenes , do not normally occur in GEP-NET. Additionally, both cell lines exhibit an extremely high proliferation rate, hardly resembling a GEP-NET phenotype , 28. ThuDAXX and MEN1 genes in the GEP-NET cell lines and in RB1 and TP53 in the GEP-NEC cell lines. Furthermore, we show the feasibility of a mutation-based targeted therapy. Overall, our models provide a valuable research resource for both GEP-NET and GEP-NEC.To overcome this limitation, we have generated three GEP-NET and two GEP-NEC cell lines. Panel sequencing not only identified typical genomic alterations in the Newly established GEP-NET cell lines NT-18P, NT-18LM and NT-36 were generated from a G3 pancreatic NET (PanNET) primary tumor (NT-18P), a liver metastasis (NT-18LM) and a local recurrence 12 months after initial surgery and chemotherapy treatment (NT-36) of a 57-year-old male patient. Besides the confirmed chromogranin A (CgA) and synaptophysin (SYP) expression, histopathological examination revealed Ki-67-based proliferative indexes of up to 70%. The newly established cell line NT-32 was generated from a large cell neuroendocrine carcinoma of the pancreas of a 70-year-old female patient. Histopathology revealed the presence of CgA and SYP and a Ki-67-based proliferative index of 70%. Finally, cell line NT-38 was established from a duodenal small cell neuroendocrine carcinoma of a 78-year-old female patient. Here, histopathological examination revealed expression of SYP, while CgA expression was absent. The Ki-67-based proliferative index was 70%. The use of human tissue for the generation of permanent cell cultures was approved by the local ethical review board and informed consent from the patients was obtained prior to surgery . The present study was conducted in accordance with the Declaration of Helsinki.Primary cell culture was performed as previously described . BrieflyGrowth curve analysis was performed by trypan blue staining and manual cell counting. Briefly, cells were seeded in 24-well-plates in medium supplemented with EGF (20 ng/ml) and FGF2 (10 ng/ml). Cells were counted in a Neubauer counting chamber on Days 1, 3, 7, 14 and 24 and doubling times were determined by exponential growth curve analysis.Transfection and small interfering RNA (siRNA)-mediated knockdown of DAXX expression was performed using Lipofectamine RNAiMAX Transfection Reagent  according to the manufacturer’s instructions. Briefly, 5 pmol of pre-validated, quality controlled and DAXX-specific siRNA (DAXXHSS102654 Sequence (5’ – 3’) (RNA) – GGA GUU GGA UCU CUC AGA AUU GGA U), Stealth, Thermo Fisher Scientific, Massachusetts, USA) was used for transfection. An equal concentration of scrambled RNA (scrRNA)  was used as a control.Immunofluorescence (IFC) was used to detect CgA and SYP in GEP-NET and GEP-NEC cell lines. Additionally, IFC was performed to determine the Ki67-based proliferative index. In general, GEP-NEC and GEP-NET cells were cultured on collagen IV coated glass coverslips and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). Subsequent permeabilization was performed in 0.3% Tween 20 for 5 min at RT followed by blocking in 10% BSA 0.1% Tween 20 in PBS for 1 h. Next, the coverslips were incubated with 1:100 diluted primary antibodies  in a humified chamber overnight at 4 °C. Incubation of 1:400 diluted secondary antibody  was performed for 60 min at RT. After subsequent Hoechst 33,342  incubation for 5 min at RT, the coverslips were mounted on an object slide with a drop of fluorescent mounting medium . Finally, the cells were analyzed by fluorescence microscopy using a BioRevo BZ-9000 apparatus  and the proliferative Ki67 index was determined by counting Ki67 positive cells.MTT analysis was performed to quantify viable tumor cells in culture and to determine IC50 values after treatment with Adavivint, Alisertib, Berzosertib, Olaparib and Trametinib . For dose response analyses, cells were seeded in a 96-well plate under standard culture conditions. After overnight culture, the cells were treated with a dilution series of the various inhibitors. 48 h after the initial treatment, all inhibitors were replenished and after 120 h MTT (5 mg/ml) was added to each well, followed by an incubation for 90 min at 37 °C. Subsequently, supernatants were aspirated, and precipitated formazan dye was solubilized with DMSO. Absorbance was measured at 540 nm using a Tecan infinite F50 spectrophotometer and viable tumor cells were calculated by the mean absorbance relative to DMSO control.CHGA: Hs00900370_m1, DAXX: Hs00985566_g1, ISL1: HS00158126_m1, NEUROD1: HS01922995_s1, NEUROG3: HS01875204_s1, PDX: HS00236830_m1, SSTR1: HS00265617_s1, SSTR2: HS00265624_s1, SSTR3: HS00265633_s1, SSTR5: HS00990407, Thermo Fisher Scientific, Massachusetts, USA) were used to assess gene expression signatures of neuroendocrine differentiation. Samples were analyzed as duplicates in a Step One Plus Real-Time PCR System . Using comparative Ct-analyses, gene expression values were normalized to that of the housekeeping gene GAPDH and were expressed as fold changes.Total RNA was isolated from cell lines using a Nucleospin RNA isolation kit  according to manufacturer’s instructions. The RNA quality was checked by spectrophotometric absorbance measurements (260/280 ratio). Subsequently, pure RNA was converted into cDNA by reversed transcription using a first strand cDNA synthesis kit according to manufacturer’s instructions . Pre-validated and quality controlled TaqMan® probes  using the Burrows Wheeler Aligner (BWA-mem Version 0.7.2-cegat) [Coding and flanking intronic regions were enriched using a SureSelectXT Library Prep Kit (Agilent) and a CeGaT tumor panel (Agilent), and were sequenced using an Illumina NovaSeq6000 system (CeGaT GmbH Tübingen)2-cegat) . Local r2-cegat)  to achie2-cegat) . The rem6 cells per 100 μl M-PER Mammalian Extraction Buffer containing Halt Phosphatase Inhibitor and EDTA-free Halt Protease Inhibitor Cocktail . Protein quantification was performed using a bicinchoninic acid assay according to the manufacturer´s instructions . Per array 1 µg (STK) or 5 µg (PTK) protein and 400 µM ATP were applied. Technical triplicates were analysed, and sequence-specific peptide phosphorylation was detected either in two steps  or in one step (PTK) using fluorescein–labelled antibody PY20 . Signals were recorded using a CCD camera and Evolve software . After quality control the final signal intensities were log2-transformed and used for further data analysis with BioNavigator software version 5.1 . Unless otherwise stated, data are expressed as the average signal intensity (± s. d.) of 144 (STK)/196 (PTK) peptide spots based on end levels of the phosphorylation curve. Significant differences were determined using One-way ANOVA comparisons and Dunnett’s test.To profile kinase activity a PamStation®12  was used as described previously . To analPreparation of whole cells lysates, electrophoresis and immunoblotting were performed as previously described . BrieflyP-values < 0.05 were considered as statistically significant * p-value < 0.05, ** p-value < 0.01. *** p-value = 0.001. **** p-value < 0.0001.) Data are presented as mean ± SD and 95% confidence interval of at least three replicates.Statistical analyses were conducted using GraphPad Prism 8. Shapiro–Wilk test was used to test for normal distribution. Comparisons of parametric data of two or more groups were done using two tailed unpaired t-test or one-way ANOVA, respectively. IC50 values were determined using an inhibitor vs. normalized response (variable slope) algorithm.  PDX and NEUROG3 were exclusively expressed by the well-differentiated NT-3 cell line, whereas NT-18P and NT18LM cells exhibited the highest NEUROD1 expression among all GEP-NEN cell lines tested. ISL1 expression was detected in all GEP-NET cell lines, whereas the GEP-NEC NT-32 and NT-38 cell lines hardly showed any ISL1 expression  updated GEP-NEN classification according to the grade of differentiation and Ki67 positivity. Well-differentiated GEP-NET can be subdivided into GEP-NET grade 1 (G1), grade 2 (G2) and grade 3 (G3) according to a Ki67 index (%) of < 3%, 3%—20% and > 20%, respectively. Poorly differentiated GEP-NEC exhibit a Ki67 index of > 20% and can be further subdivided into a small cell type and a large cell type. To assess the proliferative activity of our GEP-NEN cell lines, we performed immunofluorescent Ki67 staining and growth curve analyses. We found that GEP-NEC cell lines N-32 and NT-38 showed the highest Ki67 indexes with 50.40% ± 4.34% and 44.92% ± 2.65% positivity, respectively. Among the GEP-NET cell lines, NT-36 exhibited the highest Ki67 index with 27.71% ± 2.34% positivity followed by NT-18P and NT-18LM with 25.18 ± 2.35% and 25.57 ± 1.59% positivity, respectively. Consistent with a slowly proliferative G2 tumor, the well-differentiated NT-3 cell line showed the lowest Ki67 labeling index  and protein tyrosine kinase (PTK) activities in NT-3, NT-18LM, NT-32 and NT-38 cells were assessed using microarray-based functional kinomics. We observed a significantly higher number of strongly phosphorylated peptides in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38, i.e., the STK as well as the PTK array revealed increased overall STK and PTK activity in GEP-NET cells Fig. , B. AnalMEN1, DAXX and ATRX as the main mutated genes being apparently involved in the pathogenesis of GEP-NEN. However, further genetic modifications, especially gain of function and druggable mutations, are very rare and have not yet been elucidated. Thus, a mutation-based targeted therapy could so far not be established. Therefore, and to further characterize our established GEP-NEN cell lines, we performed sequencing of a 700 gene panel. We found that NT-3 cells harbored mutations in LRP1B (c.12010C > G), NSD1 (c.2875G > A) and PARP1 (c.826G > T). The GEP-NET cell lines NT-18P, NT-18LM and NT-36 harbored mutations in DAXX (c.1884dupC), MEN1 (c.85 > T), MSH6 (c.1095G > A), MECOM (c.1839delA), PIK3C2A (c.4694 T > A) and TCF3 (c.302A > G). Additionally, NT-36 cells exhibited a frameshift mutation in the RAD50 (c.2165dupA) gene. Among all GEP-NET cell lines tested, NT-18P carried a TP53 (c.472C > A) mutation exclusively. The GEP-NEC cell line NT-32 showed a BRAF gain of function mutation (c.1457_1471del), whereas a frameshift mutation was detected in the RB1 gene (c.1696-8_1699del) and stop codon mutation was detected in the TP53 gene (c.586C > T). NT-38 cells harbored loss of function mutations in the APC (c.4037C > A) and ARID1A (c.1681C > T) genes, and a gain of function mutation was detected in the RAF1 (c.776C > T) gene  /lymphoid enhancer-binding factor (LEF) [nM) Fig. .ARID1A wild type cell line NT-32 showed an IC50 of 9.6 µM   Fig. . AdditioµM) Fig. .DAXX mutated GEP-NET cell lines NT-18P, NT-18LM and NT-36 were exposed to the ATR inhibitor Berzosertib , the IC50 of the DAXX deficient cell line NT-18LM was 338.2 nM   to overcome replicative mortality . Interestib Fig. . We founnM) Fig. .RAF1 and BRAF was detected in NT-38 and NT-32 cells, respectively. Concordantly, administration of the MEK1/2 inhibitor Trametinib led to a significant reduction in viable cells with IC50 values of 76.2 nM  for NT-32 cells and 16.6 nM  for NT-38 cells, respectively  and 74.8% (p = 0.0001), respectively   and 68.90% (p = 0.0077), respectively   Fig. , B. Like5%) Fig. , F. In a5%) Fig. , G.Fig. Additionally, PARP cleavage was investigated to assess the effect of Berzosertib and Alisertib on apoptosis. We found that administration of 250 nM and 500 nM Berzosertib induced PARP cleavage after 24 h and 48 h, being particularly strong after 48 h. Specifically, we observed an 11.1-fold higher PARP cleavage in 500 nM Berzosertib treated NT-18LM cells compared to that in DMSO control cells Fig. . For AliDAXX wild type NET cell line NT-3. Quantitative RT-PCR revealed a siRNA-mediated reduction in DAXX expression of 30.89%  in proliferating cells   Fig. , K, L. H77) Fig. .Fig. 6ATGEP-NEN represents a minority among cancers of the digestive system and their rarity impedes the development of novel treatment regimens through large clinical studies. In addition, there is still a scarcity of reliable preclinical in vitro tumor models. Especially, to unravel molecular mechanisms underlying the unique tumor biology of GEP-NEN, well-characterized GEP-NET and GEP-NEC cell lines are urgently needed. To address this problem, we generated five novel GEP-NEN cell lines and showed that personalized molecular targeted therapies based on genetic profiling are feasible.ISL1 and NEUROD1. Additionally, these cell lines express SSTR2 and SSTR5, further indicating their neuroendocrine origin. Regarding the GEP-NEC cell lines, NT-38 cells lack expression of CgA, whereas both NT-32 and NT-38 cells express SYP. Importantly, all GEP-NEN cell lines maintained their neuroendocrine differentiation and morphology over 30 passages, rendering them into stable and continuous cell lines. Although NT-32 cells exhibit a heterogenous morphology, this morphology was consistent over the passages. Based on intra-tumoral heterogeneity, it is conceivable that some cells in culture respond differently to growth factors present in the culture medium. With a Ki67 proliferation index of 20%—30%, the newly established GEP-NET cell lines exhibit an unambiguously lower proliferation rate than that of the most commonly used PanNET cell lines BON and QGP-1. Despite the expression of SYP as a marker of neuroendocrine differentiation in both cell lines, they exhibit atypical GEP-NET mutations . Additionally, BON and QGP-1 exhibit a Ki-67-based proliferation index of > 90%, which hardly resembles the clinical nature of GEP-NET. Consequently, our cell lines represent a better model to investigate the role of GEP-NET specific mutations, thereby achieving a better understanding of GEP-NET biology. While there are additional GEP-NET cell lines  available, recent studies have shown that only GOT1 and P-STS exhibit a true neuroendocrine phenotype. A lymphoblastoid origin was postulated for the other three cell lines, further underpinning the challenging intricacy of GEP-NET cell line generation [Establishing reliable GEP-NEN cell lines has remained exceptionally difficult, mainly due to their low proliferative behavior and unresolved issues regarding optimal culture conditions. We successfully generated three GEP-NET and two GEP-NEC cell lines. All GEP-NET cell lines exhibited a neuroendocrine phenotype as confirmed by the presence of CgA and SYP, and expression of the NET-specific transcription factors neration . More reneration . DUE1 an. observed a diminished adhesion and migration of PanNET cells after pharmacological inhibition of Src kinase family members [Using kinomics analyses, we found a significantly higher frequency of tyrosine-phosphorylated peptides in the GEP-NET cells NT-3 and NT-18LM compared to that in the GEP-NEC cells NT-32 and NT-38. In particular, we found that members of the SrcB subfamily  and the ABL kinase were strongly activated in the GEP-NET cells NT-3 and NT-18LM. Recently, upregulation of LCK was reported in primary and metastatic PanNET samples, while LCK expression was absent in normal islet cells . Intrigu members . Additio members . Regardi members . However members . CollectDAXX mutations. DAXX was originally identified as a Fas death receptor binding protein mediating JNK-induced apoptosis [DAXX mutated cell line NT-18LM with the ATR inhibitor Berzosertib showed a reduction in viable cells, a reduction in proliferating cells and a significant induction of apoptosis. SiRNA-mediated knockdown of DAXX sensitized the DAXX wild type cell line NT-3 to ATR inhibition. However, apoptosis was not induced by Berzosertib in siDAXX transfected NT-3 cells. It is conceivable that transfected NT-3 cells develop only a partial ALT phenotype accompanied by a lower responsiveness to Berzozertib compared to DAXX mutated tumor cells, since siRNA-mediated DAXX knockdown resulted in only a partial reduction in DAXX expression. Even though a recently published study showed that ALT positive cells do not exhibit a general sensitivity to ATR inhibitors, we were able to demonstrate a response of DAXX mutated/knockdown cells to ATR inhibition [DAXX mutation this could be clinically developed into the first personalized therapy for GEP-NET patients.Genomic alterations resulting in an aberrantly activated growth of tumor cells, have been widely used as therapeutic targets in many cancers. Whereas, GEP-NET lack a classical oncogene addiction, GEP-NEC oncogene addiction has so far not been well studied. Thus, establishing targeted treatments remains challenging. Using our newly established GEP-NEN cell lines, we identified potentially druggable genomic modifications paving the way for the design of novel treatment strategies for GEP-NET and GEP-NEC patients. Specifically, we found that NT-18P, NT-18LM and NT-36 cells harbor deactivating poptosis , 47. Howpoptosis , 49. A rpoptosis . Althougpoptosis –52. Intepoptosis , 53. Berpoptosis –56. TreaARID1A. ARID1A represents a subunit of the SWI/SNF chromatin remodeling complex which is involved in regulating the accessibility of transcription factors, DNA polymerases and DNA damage repair proteins to specific regions within the genome. As a tumor suppressor, ARID1A was already identified in various cancers as being able to modulate cellular proliferation, invasion and metastasis [. revealed synthetic lethality in ARID1A deficient colorectal cancer cells to Aurora kinase A inhibitors. Alisertib, a specific Aurora kinase A antagonist has already been found to induce a potent reduction in tumor growth in solid as well as hematologic neoplasms [. in breast and colon cancer cells [A potential druggable genomic alteration was also found in the GEP-NEC cell line NT-38. Here, we detected a loss of function mutation in tastasis . In a prtastasis . Intrigueoplasms , 60. Coner cells . UnfortuBRAF and RAF1 mutations in NT-32 and NT-38 cells, respectively. Both cell lines showed a response to the MEK inhibitor Trametinib, but not to the BRAFV600E-specific inhibitor RAF709. In GEP-NEC, the incidence of BRAF mutations is estimated to be 5%—25%. Two recent case reports of high-grade colorectal NEC showed an initial response to combined BRAF/MEK inhibition, but both experienced disease progression after a short treatment period [BRAF mutations in GEP-NEC proved to be more complex than expected. Our GEP-NEC cell lines represent an ideal tool to investigate MEK/RAF inhibitor treatment response.We also identified t period –64. HencAPC mutation accompanied by increased activation β-catenin. Treatment of NT-38 cells with the Wnt inhibitor Adavivint led to a strong reduction in cellular viability in our study. Comprehensive molecular profiling of GEP-NEC identified APC mutations, particularly in colorectal NEC, with similar frequencies to those in colorectal adenocarcinomas. Consequently, it can be assumed that the primary site of GEP-NEC development is a determinant factor in the occurrence of this specific mutation. Whether APC mutations represent potential therapeutic targets is questionable as, so far, effective Wnt inhibitors have been shown to impair healthy tissue homeostasis and regeneration [Deregulated Wnt/β-catenin signaling has been implicated in neuroendocrine tumorigenesis, and prior studies showed antitumor properties of Wnt inhibitors in GEP-NEN cells –68. Indeneration . TherefoObviously, our study has limitations. First, we presented only a small number of cell lines. Thus, given the well-known heterogeneity of GEP-NEN, these cell lines do hardly capture the full spectrum of GEP-NEN biology. Secondly, we did not study how the identified mutations affect tumor biology, therapeutic response to established therapies and tumor progression. Particularly, in vivo studies of xenografted cells will enhance our knowledge in this regard. Nevertheless, establishing continuous cell lines will maintain a crucial role in cancer research, especially for mechanistic and therapeutic studies .DAXX- and ARID1A-mutated in vivo xenograft models and investigate how ATR and Aurora kinase A inhibition reduces growth in DAXX- and ARID1A-mutated tumors on a molecular basis.Future studies will include molecular and functional genomics to identify genes and expression profiles associated with malignant progression and therapy resistance. Another great opportunity of the new cell lines is to perform unbiased drug screening aiming to identify novel treatment concepts for GEP-NEN. Additionally, we will verify the treatment efficacy of Berzosertib and Alisertib in We successfully generated three GEP-NET and two GEP-NEC cell lines proving them as a valuable resource to further investigate GEP-NET and GEP-NEC. These models allowed us to assess novel therapeutic strategies. In particular, pharmacological inhibition of ATR and Aurora kinase A in DAXX and ARID1A deficient cells may represent a promising therapeutic strategy in GEP-NEN patients.A) PTK array and (B) STK array. Upstream STK kinase analysis of pooled NT-32 + NT-38 vs NT-3 (C) or NT-18LM (D)  > 0: higher kinase activity in NT-3 or NT-18LM; specificity score (log2) > 1.3; white to red bars: statistically significant changes. (DOCX 395 kb)Supplementary Figure 1 Box plots summarizing the log2-transformed signal intensities of (Below is the link to the electronic supplementary material.


Cancer is a complex disease, and its treatment is a big challenge, with variable efficacy of conventional anticancer drugs. A two-drug cocktail hybrid approach is a potential strategy in recent drug discovery that involves the combination of two drug pharmacophores into a single molecule. The hybrid molecule acts through distinct modes of action on several targets at a given time with more efficacy and less susceptibility to resistance. Thus, there is a huge scope for using hybrid compounds to tackle the present difficulties in cancer medicine. Recent work has applied this technique to uncover some interesting molecules with substantial anticancer properties. In this study, we report data on numerous promising hybrid anti-proliferative/anti-tumor agents developed over the previous 10 years (2011–2021). It includes quinazoline, indole, carbazole, pyrimidine, quinoline, quinone, imidazole, selenium, platinum, hydroxamic acid, ferrocene, curcumin, triazole, benzimidazole, isatin, pyrrolo benzodiazepine (PBD), chalcone, coumarin, nitrogen mustard, pyrazole, and pyridine-based anticancer hybrids produced via molecular hybridization techniques. Overall, this review offers a clear indication of the potential benefits of merging pharmacophoric subunits from multiple different known chemical prototypes to produce more potent and precise hybrid compounds. This provides valuable knowledge for researchers working on complex diseases such as cancer. Cancer is a complex group of multiple diseases characterized by inappropriately controlled cell proliferation and replication eventually resulting in disruption of normal physiology, metabolism, or structure. Benign tumors are self-limited and do not invade or metastasize, but in complex stages groups of cells display uncontrolled growth, invasion and metastasis . In the Cancer is a major global health care problem that continues to remain a leading cause of morbidity and mortality, with >277 different cancer types. According to estimates from the World Health Organization (WHO) in 2019, in 112 of 183 countries, cancer is the first or second major cause of death before the age of 70 and ranks third or fourth in a further 23 countries. In 2020, for all cancers, 19,292,789 new cases were estimated globally, with a total of 9,958,133 cancer deaths . AccordiOver the past 20 years, cancer treatments have improved significantly, and more potent medications have better safety profiles and more precise molecular targeting. Drug resistance is a major challenge with cancer treatment. During clinical usage, almost all targeted anticancer medications encounter resistance. Numerous processes have been related to drug resistance, including genetic and/or epigenetic mutation, amplification, cancer stem cells (CSCs), efflux transporters, apoptotic dysregulation, and autophagy, among others ,7,8,9.(a) two or more medicines that operate on distinct sites simultaneously or concurrently; (b) multi-targeting or promiscuous drugs treatment; and (c) hybridization drugs ..36 . Mechanistically, compound 36(a) induced appototic cell death via caspase activation with an IC50 of 7.8 µM quinoline (DIMIQ) at IC50 values 1.14, 2.19, 1.50, 9.70, and 5.70 µM, respectively. Furthermore, compound 38a showed excellent topo II inhibitory activity at concentration of 0.05 µM with control DIMIQ (Conc. 0.5 µM), m-AMSA (Conc. 0.05 µM), and daunorubicind (Conc. 0.5 µM) and cell cycle inhibitory activity at a concentration of 0.10 µM with control DIMIQ (Conc. 1.02 µM). Additionally, it showed positive inhibitory results in the G2M phase of the cell cycle. The structure of quinoline-indole based hybrid derivatives is given in 38(b–e) against human cancer cell lines is shown in Luniewski et al. (2012) synthesized quinoline based indole hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 39(a) showed excellent activity against MDA-MB-231, HeLa, SMMC-7721, and QSG-7701 (hepatocyte cell line) at IC50 0.12, 0.08, 0.34, and 10.76 µM with control Etoposide with IC50 values of 5.26, 2.98, 3.48, and 28.75 µM, respectively. Furthermore, compound 39(a) showed excellent activity apoptosis-inducing activity in on HeLa cell lines (for 48 h). The structure of quinolinebased ursolic acid hybrids derivatives is shown in 39(b–e) against human cancer cell lines is shown in Jin et al. (2019) synthesized quinoline-based ursolic acid hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 40(a) showed excellent activity against MB231, MB468, MCF7, 184B5 , and MCF10A cell lines, having GI50 values of 3.4, 0.7, 2.3, 9.0, and 12.3 µM. Control chloroquine, had GI50 values of 22.5, 28.6, 38.4, 76.1, and 81.26 µM and Cisplatin having GI50 value 23.7, 31.0, 25.8, 25.5, and 51.51µM, respectively. Furthermore, compound 40(a) showed cell cycle interruption at the meta phase, and also increased the liposamal volume in cancerous cells which led to cell death. The structure of quinoline based piperazine hybrids is given in 40(b–e) against human cancer cell lines is shown in Solomon et al. (2019) synthesized quinoline based sulphonamide/piperazine hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 41 showed potent activity against HCT-116, Caco-2 (human colon cancer cell), HT-29, and CCD-18C (colonic fibroblasts), having IC50 values of 14.26, 19.56, 19.66, and 28.28 µM, respectively. Control etoposide had comparative values of 38.10, 32.90, 35.10, and 58.90 µM, respectively. Furthermore, hybrid 41 was evaluated for anti-malarial activity, showing more potent activity than lumefantrine on the 3D7 strain of Plasmodium falciparum. A quinoline-based gallium(III) hybrid is depicted in Kumar et al. (2014) synthesized quinoline-based gallium(III) hybrids and evaluated their anticancer activity. Synthesized hybrid Quinoline containing drugs that have received FDA approval include lenvatinib, cabozantinib, and bosutinib which are protein kinase inhibitors and are used to cure medullary thyroid cancer and chronic myelogenous leukemia accordingly. FDA approved drugs with quinoline hybrids are depicted in Quinones are found in all living beings; particularly animals, plant and microbes. Through serving as crucial links in the cell nucleus respiratory cycle, they play a vital role in the power generation of such species. Numerous distinct medicinal uses of quinones have been noted, such as antiviral, antithrombotic, antiplatelet, antibacterial, antifungal, anti-inflammatory, and antiallergic properties ,102.42(a) showed excellent activity against HeLa, LS174 , A549, and MRC-5 (multipotent stem cells) with IC50 values of 2.73, 6.44, 28.84, and 48.76, respectively. Furthermore, compound 42(a) showed caspase based apoptosis in G2/M and S phase of cell division. The structure of quinone-based chalcone hybrids derivatives is given in 42(b–e) against human cancer cell lines is shown in Markovic et al. (2015) synthesized quinone based chalcone hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 43(a) showed excellent activity against KB (throat cancer cells), KB/VCR (colon cancer cells), A549, and HL60, with IC50 values 4.05, 1.28, 0.62, and 1.73 µg/mL. A control vincristine had IC50 values of 0.46, 0.26, and 12.09 µg/mL , respectively, and adriamycin had an IC50 value 0.02 µg/mL (HL60). The structure of quinone-based pyran hybrid derivatives is given in 43(b–e) against human cancer cell lines is shown in Jiang et al. (2015) synthesized quinone based pyran hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound Doxorubicin, daunorubicin, and mitoxantrone drugs are used to treat several types of cancer. These drugs are shown in The imidazole derivatives have recently gained a lot of interest and are already present in many existing treatments. Imidazole was first synthesized in 1858 using glyoxal and formaldehyde by Heinrich Debus . Various44(a–e)  are listed in Xiao-Dong Yang et al. (2012) synthesized different derivatives of novel hybrid compounds between 2-phenylbenzofuran and imidazole. Myeloid liver carcinoma (SMMC-7721), colon carcinoma (SW480), breast carcinoma (MCF-7), lung carcinoma (A549) and leukemia (HL-60) cell lines were used for in vitro study of the cytotoxic effects of synthesized hybrids with cisplatin (DPP) as the standard drug. Five derivatives showed more potent cytotoxic activity than standard DPP. The structure of imidazole based benzofuran hybrid derivatives are given in 2-phenyl-3-alkylbenzofuran and imidazole compounds. Myeloid liver carcinoma (SMMC-7721), colon carcinoma (SW480), breast carcinoma (MCF-7), lung carcinoma (A549) and leukemia (HL-60) cell lines were used for in vitro cytotoxic testing of synthesized hybrids, with DPP as the standard drug. They found that five derivatives showed more potent cytotoxic activity than standard DPP. The structure of imidazole based benzofuran hybrid derivatives is given in 45(a–e)  are listed in Wen Chen et al. (2013) synthesized numerous derivatives of novel hybrid 46(a–e) are listed in Al-blewi et al. (2013) synthesized numerous derivatives of novel imidazole-1,2,3-triazole hybrids. The synthesized compounds were screened for their anticancer activity against three different types of cancer, namely human colon carcinoma (Caco2 and HCT116), human cervical carcinoma (HeLa) and human breast adenocarcinoma (MCF-7) cancer cells-, using doxorubicin as a standard drug. They found that these derivatives showed potent more cytotoxic activity than standard doxorubicin. The structure of imidazole based triazole hybrid derivatives is given in 47(a–e) are listed in Yanping Hu et al. (2019) designed and synthesized a series of artemisinin-imidazole hybrids derivatives with multidrug resistance (MDR) reversal activity. All hybrids were screened in vitro for anticancer activities against four human cancer cell lines, human breast cancer (MCF-7), human non-small-cell lung (A549), (HEPG-2), breast cancer (MDA-MB-231) and normal human hepatic cells (L02). Adriamycin was used as reference drug. They found that most of the synthesized compounds showed higher anticancer activities than artemisinin. The structure of imidazole based artemisinin hybrid derivatives is given in 48(a–e) are listed in Wen-Jian Song et al. (2012) designed and synthesized a series of novel hybrid compounds of 2-substituted benzofuran and imidazole. In vitro anticancer activity of synthesized hybrids against a panel of human tumor cell lines i.e., ovarian carcinoma cell line (Skov-3), leukemia (HL-60) and breast carcinoma (MCF-7) was evaluated. They found that the hybrid compounds were more selective than standard DPP. The structure of imidazole based benzofuran hybrid derivatives is given in Different imidazole-based anticancer hybrid drugs that are FDA approved/or under clinical trials are shown in , and theSelenium (Se) is a unique trace element. Se directly or indirectly exerts antioxidant functions in the human body. However, during recent years, researchers reported that Se containing compounds exhibit superior anticancer effects, with high efficacy and selectivity . It has Organic Se compounds have lower systemic effects, fewer side effects, and strong anti-tumor action. They also have a greater ability to inhibit metastasis. Several novel organic Se compounds have been synthesized in order to further improve the selectivity, specificity and efficacy and to lower the toxicity .3) derivatives as potential anticancer agents. The anticancer activity of the synthesized compounds was assessed using human cancer cell lines, human colon adenocarcinoma cells (SW480), human cervical cancer cells (HeLa), human lung carcinoma cells (A549), human breast adenocarcinoma cells (MCF-7). The in vitro biological evaluation was conducted at 24, 48 and 72 h intervals, and it was found that the organoselenium hybrid compounds were more selective than standard Fluorouracil (5FU). The structure of organoselenium hybrid derivatives is given in 49(a–c) are listed in Xianran He et al. (2020) synthesized organoselenium (SeCF50(a–e) are listed in Guilherme A. Jardim et al. (2017) synthesized selenium-containing quinone-based 1,2,3-triazoles with potential antitumor activity via rhodium-catalyzed C-H bond activation and click reactions. All compounds were evaluated against five types of cancer cell lines: human promyelocytic leukemia cells (HL-60), human colon carcinoma cells (HCT-116), human glioblastoma cells (SF295), human lung cells (NCIH-460) and human prostate cancer cells (PC3), using paclitaxel as a positive control. L929 cells were also used to test the cytotoxic potential of the naphthoquinoidal derivatives in non-tumor cells. Overall, these compounds represented promising new lead derivatives with potential antitumor activity. The structure of selenium based quinone triazole hybrids is given in 51(a–e) are listed in An B et al. (2018) synthesized selenium-containing 4-anilinoquinazoline hybrids and evaluated them as tubulin polymerization inhibitors. All the synthesized compounds were screened against a panel of six human tumor cell lines, human colon cancer cells (RKO), HEPG2, breast adenocarcinoma (MCF-7), human epithelial cervical cancer cells (HeLa), human colon cancer cells (HCT116), and human gastric cancer cells (MGC803). An antiproliferative activity assay showed that most of compounds inhibited human cancer cells at low nano-molar concentrations. These compounds disturbed microtubule dynamics, lowered mitochondrial membrane potential, and stopped Hela cells in the G2/M phase, ultimately leading to apoptosis. The structure of selenium based anilino quinazoline hybrids is given in 51(a–d) are listed in Hairong Tang et al. (2021) synthesized novel selenium-containing chiral 1,4-diarylazetidin-2-ones and biologically evaluated for antitumor activities. All the newly synthesized selenium-containing compounds were screened for their antiproliferative activity against four human cancer cell lines, human epithelial cervical cancer cells (HeLa), human hepatoma cells (HUH-7), ovarian carcinoma cells (Skov-3) and human ovarian cancer cells (A2780), using paclitaxel as the positive control. The structure of Selenium based arylazetidin hybrids is shown in 53 displayed more than two-fold higher cytotoxicity to A2780 cells than to normal ovarian epithelial cells (IOSE80). Further research demonstrated that these inhibitors generated reactive oxygen species (ROS), harmed mitochondrial membrane potential (MMP), stopped cells from entering G0/G1 phase, and ultimately promoted apoptosis in the A2780 cells. The structure of selenium-based diarylimidazole hybrids is shown in 53(a–e) are listed in Sheng Huang et al. (2021) designed and synthesized fourteen novel selenium N-heterocyclic carbene (Se-NHC) compounds derived from 4,5-diarylimidazole and evaluated their antiproliferative activity towards ovarian cancer cells (A2780) and normal ovarian epithelial cells (IOSE80). Most of them were more effective towards ovarian cancer cells (A2780) than HepG2 hepatocellular carcinoma (HCC) cells. In addition, compound Different selenium-based potent anticancer drugs their chemical structure , mode ofPlatinum (Pt) medicines are still among the most often used anticancer treatments after more than 40 years of use. It is not unexpected that new research into changes in DNA repair pathways provides a reasonable explanation for Pt medicines’ efficacy since they primarily target DNA . The fir54(a–e) are listed in A. Graham et al. (2012) synthesized platinum−acridine hybrid anticancer agents. In vitro cytotoxicity activity was evaluated on different cell lines, ovarian cancer (OVCAR-3), breast cancer , pancreatic (PANC1) and non-small cell lung cancer cells (NCI-H460) using cisplatin as the positive control. The structure of Platinum−acridine hybrids is given in 55–60) are listed in Jian Zhao et al. (2012) designed and synthesized six novel platinum (II) complexes 1−6 bearing different furoxan moieties as nitric oxide (NO) donors. The furoxan groups were introduced to the platinum complexes to release NO, which may have synergisticaction with the platinum-based moieties on the tumor cells. It was found that all compounds exhibited higher cytotoxicity against human cancer cell lines HCT-116 and SGC-7901 compared to standard carboplatin, and oxaliplatin. The structure of platinum hybrids is shown in 50 values. The structure of Platinum (iv) dihydro-2-quinolone hybrids is given in 61(a–e) are listed in Liu Z et al. (2021) prepared dihydro-2-quinolone (DHQLO) platinum (IV) compounds. Cytotoxic profiles of DHQLO platinum (IV) complexes were tested against five carcinoma cell lines including, ovarian cancer (SKOV-3), human cervical cancer cell (HeLa), human lung cancer (A549), murine colon cancer (CT-26), a cisplatin resistant cell line (A549R), and one normal human embryonic kidney cell line (293T). The antitumor activities of DHQLO platinum (IV) compounds were tested using the MTT assay with cisplatin and oxaliplatin as reference drugs. Cells were treated with drugs at different concentrations for 48 h, and the results were given as IC62−66, 62−66) were tested against four tumor cell lines including human lung cancer (A549), human ovarian cancer (SKOV-3), murine colon cancer (CT-26) and a cisplatin resistant cell line (A549R).A human normal liver cell LO-2 was also evaluated. The results are given in 62–66 displayed moderate to effective antitumor activities against the tested tumor cell lines   benzodiazepine-3,11-dione (PBD) derivatives and evaluated their HDAC6 inhibitory effect. When R = H, the activity of the linker with the benzene ring was moderate. However, when the linker was extended by one methylene, the compound’s activity drastically dropped (107a vs. 107b). The structure of pyrrolo-benzodiazepine-dione hybrids is given in 50 of compounds  against HDAC6 enzyme is shown in Li et al. (2021) synthesized new pyrrolo  against cancer cell lines is shown in Chen et al. (2013) synthesized a new series of PBD-triazole hybrids and their cytotoxicity was investigated on various mouse and human cells, taking tubastatin A as a positive control. Cis-hybrids , has been shown to have strong anticancer effects on a variety of cancer types.One of the most significant bioactive substances with a chalcone structure (isoliquiritigenin) was isolated from liquorice roots. Butein, a physiologically active flavonoid found in Chalcone-based hybrid compounds have the potential to improve selectivity and anticancer activity while also overcoming drug resistance. Therefore, combining the chalcone moiety with additional anticancer pharmacophores is a promising strategy for creating new anticancer drugs. In recent times, a number of chalcone hybrids have been prepared and evaluated for their cytotoxic activity; some of them are found to have remarkable activity both in vitro and in vivo .111(a) and 111(b) were the most effective compounds with IC50 values of 7.14 µg/mL and 7.6 g/mL, respectively. The structure of chalcone based phenothiazine hybrids is shown in 111(a–d) against cancer cell lines is shown in Zahrani et al. (2020) synthesized chalcone-based phenothiazine derivatives and evaluated their cytotoxic activity against two carcinoma cell lines (human breast cancer cell line MCF-7 and human hepatocellular carcinoma HEPG-2 cells) compared with anticancer standard drugs cisplatin and doxorubicin under the similar conditions following the MTT (methylthiazol-tetrazolium) colorimetric assay. 112(a–d) against cancer cell lines is shown in Shivapriya et al. (2021) produced chalcone benzoxadiazole hybrids and evaluated their cytotoxic activity against the human epidermal carcinoma (KB) cell line using MTT assay. Chalcone-benzoxadiazole hybrids were prepared through the Claisen–Schmidt condensation reaction. The structure of chalcone-based benzoxadiazole hybrids is given in 113a–d, and evaluated their cytotoxic activity against three target cell lines: human breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HEPG-2) using the MTT assay method with doxorubicin as a reference drug. The initial results showed that some of the chalcones exhibited significant antiproliferative effects against most of the cell lines, with selective or non-selective behavior, with IC50 values found to be in the 1.65 to 34.28 µM range. Compound 113(a) showed maximum cytotoxic activity towards given cell lines. The structure of chalcone-based triazolo-quinoxaline hybrids is given in Alswah (2017) et al., designed and synthesized a series of triazolo-quinoxaline-chalcone derivatives 114(a) maximum activity. The structure of chalcone based melatonin hybrids is given in 114(a–d) against cancer cell lines is shown in Yepes et al. (2021) synthesized a new series of hybrid molecules by inclusion of two scaffolds: chalcone and melatonin. To achieve this goal, biologically active chalcone was attached via a non-hydrolizable thioalkyloxy linker to the corresponding melatonin bioisostere scaffold. The anticancer activity of the synthesized hybrids was evaluated against an in vitro model of colorectal cancer. In this study, two cell lines were used, i.e., the non-malignant (CHO-K1) and human colon adenocarcinoma cells (SW480). The reference drug used was 5-FU. Compound 115a–e) were tested in vitro for their anticancer activities against, PC12, BPH-1 and MCF-7 cells using the MTT assay. With IC50 values in the micro molar range (9.1–98.7 mM) against all tested cancer cells, all synthesized compounds demonstrated moderate to good antiproliferative activity and compound 115(e) showed maximum cytotoxic activity. The structure of chalcone-based quinoxalin hybrids is given in 115(a–e) against cancer cell lines is shown in Ma et al. (2021) synthesized chalcone-based quinoxalin as anti-cancer hybrids. All of the synthesized compounds (The coumarin scaffold (2H-1-benzopyran-2-one) is extensive in nature, and it’s derivatives exhibit various antibacterial, antifungal, antimalarial, and anticancer pharmacological properties.1R2) were the most potent with inhibition of most of the cell lines. The compound with ethanolamine as a substituent (NR1R2) at position 7 of the coumarin-benzimidazole scaffold, was the most potent synthesized compound. The structure of coumarin-benzimidazole hybrids is given in 116(a–e) against cancer cell lines is shown in Kamaldeep Paul et al. (2013) synthesized coumarin-benzimidazole hybrids. The synthesized compounds showed potent activity against leukemia cancer cells , K-562, RPMI-8226), colon cancer cells , melanoma cancer cells  and breast cancer cells . The compounds containing substitution (NR50 of compounds 117 (a–e) against cancer cell lines are shown in R. An et al. synthesized amide containing 1,2,3-triazole hybrids, which showed moderate to excellent activity against MDB-MB 231 cell lines under both normoxic and hypoxic conditions. The structure of coumarin-triazole hybrids is given in 118(a) was more potent than 3,4-dimethoxyphenyl chalcone 118(b), which showed higher activity than chalcone with 3,4,5-trimethoxyphenyl moiety 118(c).H.A. Elshemy et al., (2017) synthesized coumarin-chalcone hybrids having anticancer activity against HEPG2, leukemia, and WI-38 cell lines. All coumarin-chalcone hybrids had potent activity against HEPG2 and K562, and weak activity against WI-38 cell. Among the coumarin-chalcone hybrids, 4-methoxyphenyl chalcone 119(c) showed the highest activity against a leukemia cell line (k562) while compound (119a and 119b) showed potent activity against the HEPG2 cell line and moderate activity against the WI-38 cell line. The structure of coumarin containing chalcone hybrids is shown in 50 (µM) of compounds  against cancer cell lines is shown in In the coumarin-acrylohydrazide series, compound 50 = 1.55µM) contained A with fluorine atom as R with two carbon chain lengths between triazole and coumarin moieties. The structure of coumarin containing uracil hybrids is given in 50 (µM) of compounds 120(a–e) against cancer cell lines is shown in Mohit Sanduja et al. (2020) synthesized uracil-coumarin-based compounds as potent anticancer hybrid compounds. The uracil-coumarin hybrid compounds inhibited the MCF-7 cancer cell proliferation more effectively compared to standard 5-FU. The most potent compound 120b (GI121(e) had the highest antiproliferative activity and was more potent than standard, doxorubicin. The structure of coumarin containing furoxin hybrids is given in 50 of compounds 121(a–e) against cancer cell lines is shown in Zhuo zing et al., (2018) synthesized novel coumarin-based furoxin hybrids which had potent activity against Hela cell proliferation. The compound 122(b) induced apoptosis and affected cell cycle progression in human hepatoma (Bel-7402) cells. The structure of nitrogen mustard contain oridonin hybrids is given in 50 of compounds 122(a–d) against the cancer cell line (Bel-7402) is shown in Shengtao Xu et al. (2014) synthesized natural oridonin bearing nitrogen mustard hybrid as potential anticancer compound. The hybrid showed activity against the multidrug resistant cell lines SW620/AD300 and NCL-H460/MX20. Compound 123(a–e) was most potent and exhibited activity against human leukemia (MV4-11) cells. The compounds 123(b) and 123(e) had potent activity against MCF-7 and HCT116. The structure of nitrogen mustard contain thiazole hybrids is given in 50 of compounds 123(a–e) against cancer cell lines is shown in Laczkowski et al. (2016) synthesized nitrogen mustard-based thiazole hybrid drugs that showed antiproliferative activity against human cancer cell lines MV4-11, A549, MCF-7 and HCT-116. Among the all derivatives, b) and 124(d) were the most potent. The structure of nitrogen mustard-containing triazine hybrids is shown in 50 of compounds 124(a–d) against cancer cell lines is shown in Kolesinska et al. (2012) synthesized hybrids containing a nitrogen mustard-triazine scaffold as potent anticancer compounds. These were prepared by rearrangement of mono, bis and tris--1,4-diazacyclo [2.2.2] octanium chlorides leading to the formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three piperazine, respectively. Their cytotoxicity and alkylating activity depended on substituents on the triazine ring and nitrogen mustard. Among the above mentioned compounds, 124(125(a) showed the highest growth inhibition on IGROV (ovarian cancer cells) with a GI50 of 0.937 µg/mL. The structure of androstene oxime-nitrogen mustard hybrids is shown in 50 (µg/mL) of compounds (125(a-b),126(a-b)) against cancer cell lines is shown in Acharya et al. (2017) synthesized androstene oxime-nitrogen mustard hybrids and nitrogen mustard conjugates of various steroidal oximes. The conjugation was achieved by oxime-ester linkage. The 17-E-steroidal oxime benzoic acid mustard ester, 3β acetoxy-17E-[p-amino]benzoyloxiamino-androst-5ene 126a and 126b showed excellent anticancer activity against the HepG2 cancer cell line with an IC50 of 6.1 and 7.9 µM, respectively. The structure of the pyrazole-based indole hybrid is given in 50 of compounds 127(a–e) against cancer cell lines are shown in Hassan et al. (2021) synthesized indole-pyrazole hybrids as anticancer agents. The newly synthesized hybrids were screened for their cytotoxicity activities in vitro against four human cancer types, i.e., colorectal (HCT-116), breast carcinoma (MCF-7), liver carcinoma (HepG2) and lung carcinoma (A549). Among all indole-pyrazole hybrids, 128c showed good src inhibition activity at 10 µM and was found to be most potent compound. The structure of benzofuran-pyrazole hybrids is given in 50 of compounds 128(a–d) against cancer cell lines is shown in Somaia et al. (2015) synthesized benzofuran-pyrazole-based hybrids as anticancer drugs. The newly synthesized compounds showed remarkable growth inhibitory activity against leukemia (CCRF-CEM), A549 (lung carcinoma), and HCT-116  cells. Compound 50 of compounds (129–133) against cancer cell lines is shown in Abdelaal et al. (2021) synthesized quinazoline-pyrazole hybrids as potential antiproliferative agents. The newly synthesized compounds showed activity against hepatocellular carcinoma (liver) HEPG2, mammary gland (breast) MCF-7 and colon cancer HCT-116 cells. The structure of quinazoline-pyrazole hybrids is given in 134 (b) was found to active against A549, SiHa, COLO205 and HePG2 cell lines with IC50 values of 4.94, 4.54, 4.98 and 2.09 µM, respectively, and was non-toxic against normal cells. The structure of pyrazoline-pyrazole hybrids is given in 50 of compounds 134(a–e) against cancer cell lines is shown in Washim Akhtar et al. (2021) synthesized 15 novel pyrazoline-pyrazole hybrids, all of which showed activity in the MTT growth inhibition assay against five cancer cells lines: MCF-7, A549, SiHa, COLO205 and HePG2 cells. Compound a–c) demonstrated promising results against all tested cell lines. The non-cyclized compounds (amide derivatives) favored anticancer activity. The structure of pyrazole acrylic acid based oxadiazole hybrids is shown in 50 of compounds 135(a–c) against cancer cell lines is shown in Garima Verma et al. (2018) synthesized pyrazole acrylic acid-based oxadiazole and amide hybrids as anticancer and antimalarial agents. The anticancer activity of newly synthesized compounds was measured using the sulforhodamine B assay. Compounds 135(136(d) was found to be most potent compound. The structure of biquinoline-pyridine hybrids is given in 50 of compounds 136(a–e) against cancer cell lines is shown in Chetan B. Sangani et al. (2014) synthesized biquinoline-pyridine hybrids as potential EGFR and HER-2 kinase inhibitors by base catalyzed cyclocondensation through one potent multicomponent reaction. All the synthesized compounds were tested against A549  and Hep G2 (liver cancer) cell lines. Enzyme inhibitory activity was measured against HER-2. Compound 137 showed the most potent activity against HEPG2 with an IC50 of 2.5 µM, while compound 138(c) was the most potent compound against A549 and MCF-7 cell line with IC50 values of 10.8 and 6.3 µM, respectively. The structure of isatin-pyridine hybrids is shown in 50 of compounds  against cancer cell lines is shown in W.M. Eldehna et al. (2015) synthesized isatin-pyridine hybrids as potential antiproliferative agents. They showed antiproliferative activity against hepatocellular carcinoma (HEPG2), lung cancer (A549) and breast cancer (MCF-7) cell lines. Compound 139(a–d) showed activity against HCT116 and compounds 140(a–d) showed activity against MCF-7 cancer cell lines. The structure of pyrazolo  against cancer cell lines is shown in E.K. Hamza et al. (2020) synthesized pyrazolo ,4 pyridiFor a very long time, medicinal chemists from all over the world have been trying to develop new and effective cancer treatments. Combination therapy and hybrid chemotherapeutics haves become more common, because a complex disease such as cancer cannot be properly treated with a single drug. This study presents rational approaches behind the design of anticancer agents employing molecular hybridization. This method has potential because it combines two moieties to create new molecular scaffolds. Molecular hybridization offers a wide range of applications since it can produce compounds with distinct and/or multiple modes of action and minimal side effects. The few examples included in this article are not intended to be an exhaustive collection of anticancer hybrids, but to provide a quick explanation of the idea and its potential uses for researchers working in this field.


Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood–brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC The solute carrier (SLC) superfamily constitutes more than 65 families and over 400 genes responsible for the influx and efflux of a wide range of molecules such as organic and inorganic ions, sugars, and amino acids across membranes . These tAmong motor neuron diseases, ALS is considered to be the most prevalent disorder resulting in muscle paralysis . ALS casG93A, a mutant cell line (MT), has an overexpression of human SOD1 gene mutation due to the substitution at the 93 position of glycine with alanine d-serine was markedly higher in the MT cells, whereas [3H]l-serine was higher in the WT cells. The kinetics parameters also elucidate the altered affinity and capacity. In case of [3H]d-serine, the affinity was higher in the MT cells as compared to WT; however, the affinity for [3H]l-serine in WT cells were two times lower in MT cells as compared to WT cells  gene . For a cWT cells . From thd-serine . The altd-serine .3H]taurine is transported by TauT/Slc6a6 in rat brain capillary endothelial cells (TR-BBB13) [3H]taurine uptake has shown the activity of transporters at both membranes luminal and antiluminal of BCECs. Saturable taurine transport showed high affinity and low capacity systems, Michaelis-Menten constant ; 12.1 ± 0.5 μM and velocity (Vmax); 4.32 ± 0.05 nmol/30 min/mg protein, for the luminal uptake, whereas, Km; 13.6 ± 2.4 μM and Vmax; 2.81 ± 0.02 nmol/30 min/mg protein, for antiluminal uptake of [3H]taurine taurine .3H]taurine, where the uptake was markedly higher in the MT cell line as compared to the WT cell  expressing H1 high strain mice) in a pattern similar to that of the motor neurons of ALS patients . Due to  WT cell . FurtherHeat shock factor-1 (HSF1) mediated expression of TauT showed a compensatory effect against oxidative stress, which is considered the key factor in ALS pathogenesis. It has been concluded that TauT is one of the key markers for diagnosing stress in motor neurons, and the regulation of Slc6a6 may slow the process of neurodegeneration . Additiol-arginine [14C]citrulline through the BBB was carried by Slc7a5/LAT1 [14C]citrulline was mediated by Slc7a5/LAT1 transporter In a similar manner, the roles of essential amino acids such as tryptophan in both the BBB and ALS model cell lines have been studied, and the findings suggested that LAT1 was also involved in the transport of [3H]tryptophan citrulline transport in the BBB showed that two saturable processes are involved in the transport, and the results showed that at high affinity site, higher affinity and capacity, whereas at a low affinity site, there was a lower affinity and capacity in the TR-BBB cell lines [m values 1–10 μM for high affinity and 10–100 μM for low affinity [14C]citrulline in ALS have also been studied. The data showed that the high affinity and low capacity transport systems were involved in the MT compared to the WT [A previous study on [ll lines . Anotheraffinity . Similaro the WT . These r14C]l-citrulline in a concentration-dependent manner. A previous study reported no inhibition at 0.2 mM, whereas a significant inhibition was observed at 0.5 mM in both NSC-34 cell lines l-arginine was found to be concentration-dependent in ALS model cell lines, and the kinetics revealed that in the MT cell line, the affinity was lower and capacity was higher at a high affinity site, whereas at a low affinity site there was no significant difference between WT and MT cell lines, as shown in 3H]arginine in ALS cell lines [The SLC7 family is subdivided as LATs and cationic amino acid transporters (CATs) . For theell line  and alsoell line . L-Argins of ALS  and has s of ALS . The uptll lines . Similarll lines . Furtherll lines .14C]PBA transport to the brain across the BBB [14C]PBA transport characteristics and transporters involved have been studied in ALS cell lines, and the results indicated that sodium-coupled MCT1 (SMCT1/Slc5a8) and MCT1 both help [14C]PBA transport to NSC-34 cell lines [MCTs play a vital role in cellular metabolism and energy pathways in several tissues . The SLC the BBB . Additioll lines .14C]PBA transport by TR-BBB showed that the transport was concentration-dependent, and the Michaelis–Menten constant demonstrated that the carrier-mediated transport of PBA was pH-dependent, with the Km four times higher at pH 7.4 than the Km at pH 6.0. In contrast, Vmax was five times lower at pH 7.4 than pH 6.0  analysis in MT revealed that high edaravone concentration is required to reach 50% inhibition [3H]VPA uptake.Conversely, our previous study on valproic acid (VPA) has shown the neuroprotective effects of VPA in the ALS disease model. The study data suggested that the transporter SMCT1 was commonly involved in mediating the transport of VPA in NSC-34 cell lines . Transpo6–0.8 mM , that isll lines . IbuprofT and MT . In addill lines  (Table 2hibition . These rl-carnitine (ALCAR) in the BBB [3H]ALCAR transport in TR-BBB cells was carried by OCTN2. Expression of OCTN2 in the cells confirm the involvement of OCTN2 in TR-BBB cells. Another study has reported that OCTN2 in the brain and astrocytes are responsible for the transport of ALCAR and l-carnitine [l-carnitine has shown the involvement of both OCTN1 and OCTN2 in the transport of carnitine in motor neuron NSC-34 cell lines [l-carnitine to neuronal cells in human has increased neuronal mitochondrial functions and thus has a role in preventing the pathological conditions related to ALS disease [3H]l-carnitine in ALS model cell lines exhibits saturable processes and two affinity sites. The data shown in l-carnitine possesses two affinity sites in ALS model cell lines. Additionally, various pharmacological compounds such as quinidine, pyrilamine, diphenhydramine (DPH) and metformin have shown the significant inhibitory effect of the uptake of carnitine in ALS model cell lines. The organic cationic compounds showed competitive inhibition with l-carnitine, showing they compete for the same binding sites [l-carnitine showed significant inhibition on the uptake of [3H]paeonol in ALS model cell lines. The OCTN1 and PMAT transporters showed altered behavior in the disease model of ALS in the uptake of paeonol ..93].Further, in our previous study on TR-BBB cell lines, CHT1 was expressed in the brain, whereas OCT2 was expressed in the brain and TR-BBB B 50]. C. C50]. C3H]choline transport in the BBB were retrieved from our previously published article. The data presented in 3H]choline uptake was concentration-dependent, and the Eadie–Hofstee plot showed a straight line, indicating a single saturable process with Km (26.2 µM) and Vmax (397 pmol/mg protein/min) choline uptake by half in the ALS disease model cell lines. Amiloride was the most sensitive drug in the ALS model cell line to achieve the maximal half concentration, whereas choline inhibition showed the most sensitivity in the TR-BBB cell line. A previous study on muscle cell lines involving the H1 receptor antagonist, DPH, showed the inhibition of histamine receptors, and the estimated IC50 value, 1.01 µM, helped control the intracellular calcium taurine was higher in the disease model as compared to the control. It was concluded that increase in the levels of taurine might be due to increase in the TauT levels in the disease model cell line. TauT modulation might delay the neurodegeneration and is considered to be a novel biomarker for ALS. PBA was transported across the BBB by MCT1; however, in addition to MCT1, PBA transport in ALS cell lines was mediated by SMCT. L-Carnitine was transported by OCTN1/2 in ALS cell lines whereas its acetylated form ALCAR transport was mediated by OCTN2 in TR-BBB cells. CHT1 was the main transport for choline across the BBB; however, no CHT1 expression was observed in ALS cell lines. CTL1 is responsible for choline transport in NSC-34 cell lines. In addition, IC50 evaluation of basic drugs showed that choline in BBB and amiloride were the most sensitive in ALS cell lines. Additionally, it is shown that the therapeutics for ALS such as riluzole and edaravone can be transported by LAT1 or SMCT1, respectively, in ALS cell lines. In conclusion, ALS is a devastating neuronal disease and need of hour is to find the possibilities to cure and alleviate the symptoms related to ALS. Hence, adequate knowledge of transporter involvement will be beneficial in delivering novel drugs in ALS. Comprehending the concept of SLC transporters involved in the transport of compounds will aid the development of new drugs and their delivery in brain and motor neuron diseases.Overall, in this review, the representative transporters, which are related with the transport of amino acids including small amino acid, large neutral amino acids, basic amino acids, as well as acidic drugs including PBA and VPA and essential nutrients such as choline and carnitine in BBB and ALS model cell lines. Affinity and capacity plays important role in understanding the transport ability of compounds into the cells. Therefore, we compared the kinetic parameters obtained in BBB and ALS cell lines. In the BBB, affinity of LAT1 is very high, but capacity is very lower than ALS cell lines.