The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable  As pancreatic cancer is notoriously asymptomatic at an early stage, 80% of patients are diagnosed after the cancer has metastasised, making them ineligible for resection, which is the only curative treatment. The number of cases of pancreatic cancer has been steadily rising since 20043. It is currently the fourth most common cause of cancer death in the US, and by 2030 it is estimated that it will surpass breast and colorectal cancer to become the second most common cause of death by cancer4. The majority of pancreatic cancers are in the exocrine pancreas (95%) known as pancreatic ductal adenocarcinoma (PDAC), and 5% are in the endocrine pancreas5. The leading epidemiological factors include smoking, obesity, type II diabetes mellitus and acute pancreatitis, which account for approximately 25% of PDAC cases9.With a rapid progression and fatal outcome, pancreatic cancer is one of the deadliest of all cancers. Long-term survivors are limited to those with resected early stage tumours; however, overall survival rate is a dismal 9%, with a median survival time of 7–11 monthsin vitro patient tumour representative models. Established 2D cell lines are the most widely used model for the development and testing of chemotherapeutics for over 50 years10. The ability of cell lines to grow indefinitely makes them a low-cost, repeatable model, easy to manipulate and are an important base for discovery and proof-of-concept studies. Their importance in cancer research is indisputable, however, their use as a robust clinical model is questionable11. During passaging, cell lines undergo genetic modifications, such as copy number variation and point mutations12. Cell lines also have a high level of homogeneity, which does not represent the heterogenetic nature of PDAC tumours, and not all cancer subtypes are well represented as they are usually developed from aggressive and metastatic tumours, therefore tumour progression cannot be studied13. Deer et al.14 highlighted the genetic diversity and differences in adhesion, migration and tumorgenicity between the different PDAC cell lines in a detailed review of phenotypes and genotypes of PDAC cell lines. Primary tumour cultures derived from patient tumour or biopsy and grown in vitro in 2D tend to be heterogeneous, and are a better representation of the tumour of origin tissue as the cells are at an early passage number15. These models allow for the development of personalised cancer therapy as demonstrated through functional screening of chemotherapeutic drugs, modelling individual tumour response16. However, although an important tool in cancer research, there are limitations associated with primary cell culture, including the difficulty of obtaining tumour samples. Primary cell cultures are often difficult to establish as the initial sample may lack tumour cells; the outgrowth of stromal cells such as fibroblasts may overrun the culture, and the cells may only grow for a finite number of passages16.A limitation in the understanding of the disease progression and development of effective treatments in PDAC may be due the lack of in vitro models can address the limitations of growing cancer in 2D. Unlike 2D cultures, 3D cell cultures are not grown attached to plastic, so they adopt a more physiologically relevant morphology and signalling pathways similar to in vivo conditions17. Like solid tumours, 3D cell cultures are exposed to complex physiological and heterogeneous environments, resulting in varied exposure to oxygen, nutrients and stresses. This allows for the study of cell-to-cell interaction, drug penetration, response and resistance19. The 3D culture model contains proliferating, quiescent, hypoxic and necrotic cells, whereas in 2D cell models, all the cells are in the same growth phase20. Organoids are 3D cultures derived from organ specific stem cells, allowing for the self-organisation of cells to resemble structures from within an organ or tumour21. These 3D system models the physiology, shape, dynamics and cell make-up of the cancer and produces a relevant and highly adaptable model system22. Organoids can be derived from embryonic stem cells, induced pluripotent stem cells, and both tumour and normal organ restricted adult stem cells (aSCs)21. Organoids can be maintained in a 3D matrix which supports cell growth, such as Matrigel or hydrogels which are laminin rich, mimicking the pancreatic microenvironment in vivo23. Organoid culture requires special media containing a cocktail of growth factors that mimic the microenvironments of organ stem cell niches24. Unlike established and primary cell lines, organoids display cell heterogeneity after several passages and organoids which have been developed from the pancreas can be expanded, managed and cryopreserved and PDAC organoids can proliferate indefinitely22. Boj et al.25 developed a method for the establishment of both normal and neoplastic organoids from human and murine pancreatic tissues. Orthotopic transplant of both the PDAC and normal organoids resulted in full tumour and ductal development respectively. These methods are useful for the further development of PDAC research, including the study of the tumour microenvironment, personalised medicine, furthering the understanding of the genetics and for testing novel therapeutics in PDAC.Three-dimensional (3D) 26.Patient derived xenografts (PDX) are widely used animal models to study cancer. The patient tumour is grown subcutaneously or orthotopically in an immunodeficient mouse, and sub-cultured into new mice when the tumour reaches a sufficient size. PDX models allow for tumours to retain their cell-to-cell interactions, and orthotopic implantation allows for the tumour to grow in the same micro-environment as the original tumourin vivo and ex vivo drug testing, which is invaluable in PDAC research. However, disadvantages to the use of PDX models include: expensive to develop, time consuming, dependent on the use of animals, require ethics approval and are subject to strict regulations27. PDX models also have a slow take rate, and can take months to develop tumours. Moreover, as the tumour is sub-cultured, the tumour-associated stroma is replaced by murine stromal cells, such as blood vessels and fibroblasts28. Finally, SCID-mice which are used for PDX biobanks lack immune systems, and this limits the testing of drugs such as immune modulators, which are increasingly being used in cancer treatments.There are several advantages to using PDX tumours for the study of PDAC. PDX tumours are established from small amount of patient tumour; the tumours retain the original tumour heterogeneity, which can be lost in established cell lines. The PDX tumours also preserve the original tumours’ genetics and histological characteristics during passaging. These models are used as an unlimited resource for in vitro live material models. However, limitations for both include considerable amount of time required and cost to expand cultures in vitro, which makes it difficult to scale-up and perform downstream high‐throughput analysis. Here, we report methodologies for the establishment of PDX-tumour organoids, isogenetically matched 2D primary cell line and recapitulated primary cell line into organoids (CLOs). We show that organoids and CLOs maintain the same molecular and transcriptomic signatures compared to the 2D primary cell line, indicating the robustness and usefulness of CLOs in preclinical studies of pancreatic cancer.PDX models and 3D cancer organoid cultures are important preclinical 30 and cancer organoid models32 to bridge the in vivo/in vitro gap in PDAC research. Therefore, we developed a protocol to established organoids from PDX tumour with modified approaches from Boj et al.  gel derived from Engelbreth-Holm-Swarm murine sarcoma on adherent wells, and allowing the cells to attach and proliferate. This method allows for the rapid development of primary cell lines, which can be scaled up and manipulated. The 2D primary cells are cultured for two passages then recapitulated as 3D cultures  revealed PT291 organoids and CLOs clustered together; while PT127 PDX tumour and CLOs clustered together, separate from their corresponding isogenetically matched 2D primary cell line Fig. . HierarcFOSB , CAV1 (log fold change −5.7652 p-adj 4.89E-75) and AHNAK2  as the three top down regulated genes in the grouped CLOs compared to the grouped 2D primary cell lines. TFF3 , PLEKHB1  and LTB  were the top up regulated genes.In Fig. KIF12 had the highest log2 fold change  and FOSB  had the lowest fold change. The 10 most significant (p-adj ≤ 5.79E-14) up and down-regulated genes in PT291 organoids and CLOs compared to the isogenetically matched 2D cell line are outlined in Fig. SERPINA5  had the highest log2 fold change, and AHNAK2  displayed the lowest fold change.To further identify a specific transcriptomic signature common among the PDX tumour/organoid and CLOs we assessed the transcriptomic signature of the PT127 PDX-derived tumour and CLO compared to the 2D primary cell line, and the PT291 organoid and CLO versus the 2D primary cell line. We observed 144 genes up regulated and 130 genes down regulated  in both PT127 PDX tumour/CLO compared to the isogenetically matched cell line in 2D , and was assessed for the levels for protein using the TOPFlash dual luciferase assay34.The method for the establishment of PDAC PDX organoids and isogenetically matched cell lines was modified from Boj PDAC primary tumour tissue specimens were obtained from Saint Vincent’s University Hospital Ethics and Medical Research Committee, Dublin, Ireland between 2013 and 2017. All patients underwent pancreatic adenocarcinoma resection. All donors agreed by written informed consent to donate tissue and for study participation. The specimen donor’s personal information was confidential and protected except the date of birth. All samples and methods used in this study were approved by Saint Vincent’s University Hospital Ethics and Medical Research Committee and conducted in accordance with the relevant guidelines and regulations in compliance with the Saint Vincent’s University Hospital Ethics and Medical Research Committee. PDAC PDX tissues were generated by subcutaneous seeding in severe combined immunodeficiency disease (SCID) mice. Primary patient samples were confirmed as PDAC by a pathologist (N.S.) in Saint Vincent’s University Hospital, and PDX samples were also confirmed by pathology examination to maintain the human tumour content and morphology of the original tumour.All animal work has received ethical approval from the DCU Research Ethics Committee (DCUREC/2012/202) and was licensed by Department of Health (B100–4501) all methods were performed in accordance with the relevant guidelines and regulations in compliance with the DCU Research Ethics Committee and the Department of Health.After initial macroscopic pathological confirmation, material remaining after diagnostic sampling was cold transferred in RPMI media containing 1% Pen Step, 1% fungazone to DCU.scid/lcrCrl mice  were implanted subcutaneously with fresh patient tumour material. Under anaesthesia  a small incision was made in the skin of the left flank of the animal. The tumour piece was placed in the pocket under the skin and the wound sealed with a single staple. The animals were monitored post-surgery, and staple removal was within 10 days. Animals were monitored weekly for body weight and tumour development. Mice were monitored for tumour development for up to 1 year post implantation. Animal welfare monitoring criteria included tumour volume, tumour axis, body weight and condition, where maximum tumour volume is <2000 mm3, and maximum tumour axis is <20 mm. A decrease in body weight of >10% resulted in increased monitoring, with body weight decrease of 20% resulting in humane euthanasia. Tumour measurements were by calliper measurement and tumour volume was calculated as (HxDxW)/2, where H indicated height, D indicated depth and W indicated width. At experiment end, the animals were humanely euthanized and the tumour was harvested. PDX samples were confirmed by pathology examination to maintain the human tumour content and morphology of the original tumour.The tumour was cut into implant sized pieces (<2 mm) and rinsed with fresh serum-free RPMI media following transport. Severe combined immunodeficiency (SCID), CB17/lcr-PrkdcThe media for organoid establishment and culture is represented in Supplementary Tables 6 cells was centrifuged at 1500 RPM for 10 minutes at RT, and resuspended in 80 μL PB buffer, and 20 μL of the mouse cell depletion kit antibody cocktail . This suspension was incubated at 4 °C for 15 minutes. Following incubation, 400 μL of PB buffer was added to the cell suspension and mixed thoroughly. An LS column  was placed in the MidiMacs Separator  and rinsed with 3 mL PB buffer. The cell suspension was pipetted into the column, and the flow-through was collected in a 15 mL tube. The column was washed twice with 1 mL of PB buffer, and the flow-through was collected in the same tube. The cells were centrifuged at 1000 RPM for 5 minutes at 4 °C. All media was removed, using a P200 to avoid disrupting the pellet.Following centrifugation, the media was aspirated, and the pellet was resuspended in 5 mL PB buffer, making a single cell suspension. Using Trypan blue , the cells were counted and a cell suspension of 2 × 105 cells. 20 μL of organoid:12 mg/ml ECM solution was pipetted into poly-HEMA coated 48-well plate. The plate was placed in an incubator upside down at 37 °C for 20 minutes for ECM to polymerise. 350 μL of the Complete Human Feeding Media (CHFM)  per 1.5 × 105 cells were mixed in 100 μL of ECM diluted to 1 mg/mL and 50 μL was plated per well in a non-poly-HEMA coated 24 well plate. Cells were fed 500 μL of the CHFM with RhoK inhibitor  was added to the pellet and placed at 37 °C for 15 minutes. The pellet disrupted using “hard pipetting” . DMEM with 10% serum was added to stop trypsinisation. The organoids established as previously outlined above, and fed with RhoK inhibitor supplemented in CHFM for two days after trypsinisation, and then subsequently fed using CHFM without RhoK inhibitor.4 cells per well to be seeded was made, and centrifuged at 1500 RPM for 10 minutes at 4 °C. Cells were resuspended in 20 μL ECM per well, and plated and fed as outlined above. Two weeks after establishment, cells were passaged as organoids, as outlined above.Using early passage 2D primary cell lines, which had been established from organoids, cells were trypsinised, and a cell suspension of 5 × 10DNA was extracted from organoids and primary cell lines using DNeasy Blood & Tissue Kit . PCRs were performed using MyTaq™ PCR Kit. The first PCR is performed using a universal primer pair complementary to conserved sequences in cytochrome B and 16S rRNA genes.2O) up to 25 µl. Amplification was carried out in G-Storm, at 94 °C for 5 minutes, 59 °C for 5 minutes, followed by 35 cycles of 72 °C for 2.5 minutes, 94 °C for 30 seconds, 59 °C for 45 seconds, and followed by a final annealing step of 72 °C for 10 minutes. Samples were stored at 4 °C.100 ng DNA, 10 pmol Primers  up to 25 µL. Amplification was carried out in G-Storm, 94 °C for 5 minutes, 60 °C for 5 minutes, followed by 30 cycles of 72 °C for 1.5 minutes, 94 °C for 30 seconds, 60 °C for 30 seconds, followed by a final annealing step of 72 °C for 10 minutes. Samples were stored at 4 °C. 5 μL of second PCR product was run on a 2% agarose (Sigma), stained with SafeView , visualized under UV light, and imaged. A 100 bp DNA Ladder  was applied as a size marker.The amplified product from the first PCR was diluted 1:10 with ddHBrightfield imaging of PT127 CLOs and PT291 CLOs and matched organoids was performed on days 0, 3, 7, and 10 to determine the morphological changes of the cells. Pictures were taken using a Canon EOS 550D camera  through a Leica DM IL LED inverted microscope . Scale bars were added using ImageJ.4 in 500 μL was seeded in a glass bottomed 8-well plate , and grown overnight. Media was removed from the cells, and washed three times in a PBS 0.1% Tween 20  and 2% BSA  solution. Cells were fixed in ice-cold methanol for 5 minutes at −20 °C, and washed in the PBS-T-BSA solution.A cell suspension of 5 × 10Four wells of organoids at day 7 were combined and centrifuged at 1500 RPM for 10 minutes in a 1.5 mL Eppendorf tube. All media was removed from the cell pellet, and iPGell  was used according to the manufacturer’s protocol. Gel plugs organoids were placed in optimal cutting temperature (OCT) embedding matrix gel  in a disposable histology mold  and placed at −80 °C overnight. Organoids were cut into 10 μm sections using a cryostat  which was cooled to −25 °C prior to use. OCT embedded organoids were mounted onto the freezing block using OCT, and faced off by cutting 60 μm sections until the organoids were exposed. The organoids were cut into 10 μm sections and placed onto SuperFrost Plus slides , and stored at 4 °C until further use.Cells were blocked for 1 hour 30 minutes in 10% Goat Serum , the primary antibody , TaqMan Fast Advanced Master Mix , RT-PCR Grade Water  and cDNA template were prepared as described in the assay protocol using 20 ng of cDNA, in MicroAmp Fast Optical 96 well reaction plates . The plate was sealed using Adhesive PCR Plate Seals  and centrifuged briefly. Using an Applied Biosystems 7900 Real-Time PCR System qRT-PCR was performed using 50 °C for 2 minutes, 95 °C for 20 seconds, and 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds.A High Capacity cDNA Reverse Transcription Kit (Applied Biosystems 4368814) was used to synthesis cDNA from RNA. Master mix was prepared as described in the protocol. A G-Storm Thermocycler  was used to perform cDNA synthesis at 25 °C for 10 minutes for annealing, 37 °C for 2 hours for cDNA synthesis, 85 °C for 5 minutes for enzyme inactivation, and storage at 4 °C. Following synthesis, cDNA was stored at −20 °C. TaqMan qRT-PCR was used to assess Organoids and CLOs were grown as described for 14 days. TRI-reagent® (Sigma) was used to lyse cells, and RNA isolation was performed using Direct-zol RNA Miniprep Plus Kit . RNA samples were quality controlled with an RNA Screen Tape (Agilent) and quantified with Qubit RNA HS Assay kit. Sequencing libraries were prepared with 200 ng of total RNA for all samples. The Nugen Universal Plus mRNA protocol was followed as recommended by the manufacturer. Libraries were amplified by 15 cycles of PCR. Libraries were sequenced in one NextSeq550 run with the NextSeq500/550 High Output Kit v2.5 (300 cycles), sequencing was done with Paired End 150 nt reads and 8 nt indexes. Libraries were loaded at 1.4 pM, 5% PhiX control was used.Homo sapiens Ch37 genome (hg19) with hisat2 using default parameters. The resulting sam file was piped into samtools for bam conversion and sorting. The resulting featureCounts object can easily be manipulated to generate a counts matrix for DESeq2. This counts generation is written to counts.tsv for further analysis. Up/down regulated genes: differential expression genes (p < 0.001 and log2 fold-change ≧ 2) are detected using DESeq235 after filtering out reads count less than 10. Compare CLOs with CLs; compare PT127 PDX, CLO with CL; compare PT291 organoids, CLO with CL. The correlation of paired groups - for each sample, reads count is normalised to TPM. Median is applied if there is more than one sample in a group. Linear regression was performed to identify the relationship of paired groups: PT291 ORG and PT127 PDX; PT127 PDX and PT127 CLO; PT291 ORG and PT291 CLO, (p < 0.05). Raw and processed RNA-seq files are available at GEO (accession number GSE144737).Raw sequencing files were merged for each sample to generate a single fastq file per sample. Cutadapt was used to remove adapter sequences. Trimmed reads were aligned against the 36, with up to 90% of the cells in a PDX tumour being murine stroma28. When establishing primary cell lines from PDX samples, these murine fibroblasts can often quickly out-grow and outnumber the PDAC cancer cells in vitro, resulting in a completely unrepresentative model for PDAC37. In order to overcome this issue, we used mouse cell depletion kit, which results in the removal of infiltrating mouse cells from the tumour sample33. Utilisation of the mouse cell depletion kit to develop organoids and 2D primary cell lines from PDX tumours ensures the starting material is pure tumour tissue.In this study, we present a method for the establishment of organoids, and isogenetically matched 2D primary cell lines from PDAC PDX tumour samples and recapitulation of the cell lines to CLOs, which we show are morphologically, molecularly and transcriptomicly similar to their PDX derived tumour organoids. This method can be utilised for all cancer types, allowing laboratories to utilise PDX samples already available for the development of organoids and primary cell lines. While PDX tumours remain stable during passaging, tumour-associated stroma is replaced with murine stromal cellsex vivo, they are expensive to culture, require time-consuming maintenance and can be difficult to scale up in short periods. In order to overcome these issues, we highlight the establishment of cell line organoids (CLOs). The CLOs are developed from 2D primary cell lines within two passages, grown under the same conditions as PDX organoids. These CLOs were imaged, and found to be morphologically similar to the matched organoids. To validate CLOs as representative organoid models, we molecularly profiled the expression of tumour initiating markers in our cells. Expression of ALDH1A1 and CXCR4 was low in both the PT291 and PT127 2D primary cell lines, but consistently expressed in organoid and CLOs. ALDH activity selectively defines an enhanced tumour-initiating cells38. ALDH1A1 is involved in retinoic acid metabolism, and has a role in proliferation and differentiation39. The expression of the transmembrane glycoprotein, EpCAM was retained in all models, and is known to be highly expressed in PDAC and established cell lines41.While organoids are a more representative model of PDAC 42. The over expression of PDX1 in PDAC highlighted its role as an oncogene43. However, PDX1 expression may be linked to the changing stages during cancer development and progression. PDX1 loss or downregulation is associated with a more aggressive phenotype, and loss of PDX1 observed in subset of cells undergoing EMT45. Furthermore, upregulation of key stem cell markers, transcription factors NANOG, OCT4 and SOX2 in CLOs compared to 2D primary cultures indicates a pluripotent stem cell state and suppression of lineage-specific genes in these models46.Interestingly, PDX1 expression was low in CLOs compared to 2D primary cell lines. PDX1 is a transcription factor which is essential for pancreatic development and differentiation of all pancreatic cell lineagesTFF3, PLEKHB1, LTB and FOSB, CAV1, AHNAK2, respectively. Interestingly, FOSB is a proto-oncogene, and CAV1 expression has been linked with mammary tumorigenesis and increased lung metastasis48. AHNAK2 has also previously been found to be upregulated in PDAC49. The up regulated signature also contains increased expression of genes associated with cilia 52. Cilia have been found to play an important role in modulating signalling cascades including the WNT signalling pathway, and are essential in pancreatic tissue organisation54. The upregulation of stem cell markers ALDH1A1, ALDH1B1 and LRIG1 may indicate restored embryonic programs and dedifferentiation pattern among our CLOs56. This dedifferentiation theory is supported by a previous study which has shown that when implanted orthotopically, organoids recapitulate tumour progression from precursor PanIn lesions to metastatic carcinoma25. Interestingly, genes down regulated in PDX tumour/organoids and CLOs include genes which have increased expression in cancers, including potential PDAC biomarkers 62. There is also an increased expression of tumour suppressor genes LRIG1 and SEMA3F63. This gene signature also highlights the “phenotypic switch” of the 2D the primary cell lines as the cells invade through ECM, attach and proliferate, resulting in increased motility, invasion and proliferation of cells by up-regulation of oncogenic pathways.RNA-seq analysis highlighted by PCA that the PT291 organoids and CLOs clustered together as did the PT127 PDX-tumour and CLOs compared to their respective 2D primary cell lines. Additionally, hierarchical clustering comparing the dysregulated genes between PT291 and PT127 CLOs versus the 2D primary cell lines, revealed a clear signature pattern as shown by heat map Fig. . The thr65 include the lack of a relevant microenvironment integrating the immune and stromal components, as tumour derived organoids exclusive express ductal cells, but not of other pancreatic cell lines46. Tsai et al.66 constructed complex organotypic models containing tumour, stromal and immune components of the tumour microenvironment. This study showed spheroids derived from established PDAC cell line, Panc-1, a mouse xenograft cell line, and the A549 lung cancer cell line are phenotypically distinct from primary organoids as they formed homogenous, non-lumen forming spheres. However, the cell line models used by Tsai et al.66 are not primary cultures, and were not compared to their isogenetically matched organoid or PDX tumour, therefore are not directly comparable and tumour to tumour bias may exist.A limitation of our study was the use of non-metastatic surgical samples which were implanted as subcutaneous PDX tumours which did not metastasise in the mouse. In order to establish CLOs as an invasive disease model for PDAC, future work will include the establishment of preclinical models from biopsies of metastatic sites. Further limitations of our study and others studying PDAC organoidsin vitro organoid-similar model to study cancer. CLOs have the molecular and transcriptomic phenotype resembling organoids, however offer more flexibility, expandability and utility for downstream translational research and for the development of personalised treatment of PDAC patients.As thirty years of cancer research have done little to improve patient outcomes, additional disease modelling strategies are required to complement existing techniques and advance PDAC research. Here, we demonstrate that CLOs established from 2D primary cell lines are a physiologically relevant Supplementary tables.


Advancements in thyroid cancer are dependent on the availability of experimental models allowing for investigation in the laboratory setting. Here, we detail the development and characterization of three cell-based models of follicular thyroid cancer that closely mimic the genetic and pathological progression of the disease seen in patients, including progression to poorly differentiated and anaplastic thyroid cancer in some cases. We anticipate that these newly developed models will allow for the discovery of novel mechanisms of disease progression and the testing of therapeutic interventions.Recent developments in thyroid cancer research have been hindered by a lack of validated in vitro models, allowing for preclinical experimentation and the screening of prospective therapeutics. The goal of this work is to develop and characterize three novel follicular thyroid cancer (FTC) cell lines developed from relevant animal models. These cell lines recapitulate the genetics and histopathological features of FTC, as well as progression to a poorly differentiated state. We demonstrate that these cell lines can be used for a variety of in vitro applications and maintain the potential for in vivo transplantation into immunocompetent hosts. Further, cell lines exhibit differing degrees of dysregulated growth and invasive behavior that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We believe these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and lead to the development of more personalized diagnostic and treatment strategies. Thyroid cancer is estimated to have affected 52,890 individuals in the US in the year 2020, making it the most prevalent endocrine malignancy and the most commonly diagnosed cancer among individuals aged 15–29 years . More thWith more conservative diagnostic and treatment recommendations being implemented for well-differentiated disease, the focus has shifted to developing treatments for the more severe PDTC and ATC. Although recent treatment strategies have focused on targeted pathway inhibition ,9, the sIt has become imperative to develop in vitro and in vivo pre-clinical models that recapitulate patient disease to screen prospective treatments for efficacy and decipher molecular mechanisms that govern therapeutic resistance. Although cell lines derived from human thyroid tumors provide a powerful tool to study novel therapies, progress in the field has been halted by the lack of validated human thyroid cancer lines. In 2008, Schweppe et al. reported that roughly 50% of commonly used thyroid cancer cells lines were misidentified, resulting in cell line redundancy and contamination with cells of other tissue types . SimilarRAS of the mitogen-activated protein kinase (MAPK) signaling pathway. Further, genetic alterations leading to activation in both the MAPK and phosphoinositide 3-kinase (PI3K)/Akt pathway become increasingly more prevalent as disease progresses, with some estimates indicating that up to 80% of ATCs possess genetic alterations in both pathways [G12VHras and homozygous Pten inactivation in mice to achieve concomitant MAPK and PI3K/Akt pathway activation. These mice develop FTCs that progress to PDTC [G12V/Ptenhom/TPO-CreHras mice]  . This moG12V/Pten−/−/TPO-CreHras mice of a pure 129/svJ genetic background. Thyroid tumors and wild-type thyroid glands were dissected and minced, followed by digestion in a solution of 1 mg/mL collagenase Type I  and 1 mg/mL dispase  in Hank’s Balanced Salt Solution at 37 °C with gentle shaking for 1.5 h. Following digestion, samples were centrifuged at 1200 rpm for 3 min and resuspended in Ham’s F12 medium  supplemented with 10% fetal bovine serum , 2 mM L-Glutamine (Gibco), and Penicillin/Streptomycin/Fungizone (Sigma). The samples were then plated into tissue culture flasks and maintained at 37° Celsius in 5% CO2. To ensure removal of contaminating stromal cells and outgrowth of the pure tumor cell lines, all cell lines were passaged at least 5 times after plating and then genotyped using primers specific for Pten and Pten recombination [Cell lines were isolated as previously described . Hras1, bination . Cell liCells were seeded into 8-chamber culture slides  and allowed to attach overnight. The following day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature followed by permeabilization with 0.5% Triton X-100. The cells were treated with 10% goat serum for 1 h prior to antibody staining to block any non-specific binding, and then incubated with anti-EpCAM antibody . The cells were then washed with cold PBS three times for 3 min each and incubated with AlexaFluor 594-labeled secondary antibody  at room temperature for 1 h. Slides were mounted in SlowFade mounting medium containing 4′,6-diamidino-2-phenylindole (Invitrogen) and imaged using the EVOS FL Auto Cell Imaging System.Pax8, Ttf1, Ttf2, Tg, and Tshr was measured using pre-designed primers . Equal amounts of RNA template were reverse transcribed using the Verso cDNA synthesis kit . The differential mRNA expression of software  was usedCells were homogenized on ice in RIPA buffer  supplemented with protease and phosphatase inhibitors (Thermo Scientific). Western blot analysis was performed on 30 µg of total protein separated by SDS-10% PAGE. The proteins were transferred to PVDF membranes and then probed with antibodies (1:1000) specific for PTEN (Millipore), P-AKTS473 (Abcam) pan-AKT, P-ERK1/2, ERK1/2, P-MEK, MEK, and β-actin . Specific immunoreaction was detected using HRP-conjugated secondary antibodies and Luminata Forte western chemiluminescence substrate (Millipore).Cells were plated in quadruplicate into 96-well plates. Pharmacologic inhibitors of PI3K  and MAPK/extracellular signal-related kinase (ERK) kinase (MEK)  or vehicle (DMSO) were added 24 h after plating. At designated time points, cell growth was assessed using the CellTiter-Glo luminescent cell viability assay . Results of cell proliferation in control media were confirmed by plating cells in triplicate in 6-well plates and determining raw cell number via automated trypan blue dye exclusion and the Via XR Cell Viability Analyzer .Cells were seeded in triplicate in 96-well dishes. As in proliferation assays, LY294002 (10 µmol/L), AZD6244 (250 nmol/L), or vehicle (DMSO) were added 24 h after plating. At designated time points, media were collected from cells and lactate dehydrogenase release was determined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega).Cells were seeded in triplicate in 6-well plates. LY294002 (10 µmol/L), AZD6244 (250 nmol/L), or vehicle (DMSO) were added 24 h after plating. At designated time points, cells and growth media were collected together for analysis. Samples were centrifuged at 2500 rpm followed by washing with PBS. Fixation was carried out with ice-cold 70% ethanol added dropwise while vortexing. Samples were stored in ethanol at −20 °C until staining. On the day of FACS analysis, cells were centrifuged to remove ethanol and stained with 300 µL of propidium iodide/RNAse staining buffer (BD Pharmingen) for 1 h at 4 °C. Samples were analyzed on the LRSFortessa flow cytometer (BD). Data were analyzed using Flow Jo v10  and the included Watson model of cell cycle .Cells were seeded in triplicate into 16-well chamber slides (Millipore) in a 1:1 mixture of Matrigel (Corning) and growth medium at a concentration of 250 cells/well. Thyrospheres were grown for 7 days. Subsequently, samples were rinsed with warmed PBS and fixed with 2% paraformaldehyde for 10 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 and blocked with 1% BSA at room temperature for 1 h. Samples were stained with anti-β-actin primary antibody  for two hours at room temperature or anti-phalloidin antibody conjugated with AlexaFluor 488  for one hour at room temperature. β-actin-stained samples were subsequently incubated with AlexaFluor 594 secondary antibody  and mounted in SlowFade mounting medium containing 4′,6-diamidino-2- phenylindole (Invitrogen). Phalloidin-stained samples were mounted in SlowFade mounting medium without DAPI (Invitrogen). All samples were imaged using the EVOS FL Auto Cell Imaging System.Cell suspensions were prepared at a concentration of 2000 cells per 20 µL of growth medium. Cells were pipetted into multiple 20 µL droplets onto the underside of a 10 cm tissue culture dish lid. The lids were carefully inverted and placed onto tissue culture dishes. Culture dishes contained 5 mL of growth medium to prevent the evaporation of droplets. After 7 days, the droplets were removed from the lids by gently washing with PBS and collected in a 6-well culture plate. Viability was assessed via staining with 1 µM of Calcein AM  for 30 min at 37 °C. Spheroids were visualized with the EVOS FL Auto Cell Imaging System.5G12V/Pten−/−/TPO-CreHras tumor cells  were pelleted by centrifugation, resuspended in 200 μL of a 1:1 mixture of PBS and matrigel (Corning), and injected subcutaneously into the right hind flanks of 8–12-week-old male wild-type 129/SvJ recipients. Tumor development was monitored weekly for 15 weeks, at which point the mice were sacrificed due to tumor burden.All animal experiments were performed at the University of Arkansas for Medical Sciences and approved by the IACUC. Briefly, 5.0 × 10G12V/Pten−/−/TPO-CreHras mice were dissected, minced and digested in a solution of serum-free DMEM (Gibco) containing 1 mg/mL Collagenase I (Sigma) and 1 mg/mL Dispase (Sigma) for 1.5 h at 37 °Celsius with gentle shaking. The digested samples were then filtered through 70μm nylon strainers  into 10 cm tissue culture dishes containing 10 mL of PBS. The samples were treated with 100 ug/mL DNAse I (Sigma) for 5 min at room temperature, centrifuged for 5 min at 1400 rpm, and resuspended in 1 mL red-cell lysis buffer to remove residual red blood cells. After 5 min of incubation at room temperature, the red-cell lysis buffer reaction was neutralized with 20 mL of PBS. The cell suspensions were filtered again through 70 μM nylon strainers, centrifuged, and resuspended in FACS buffer . Cell suspensions were treated with Fc receptor blocking antibody (BD biosciences) for 20 min on ice. After Fc receptor blocking, antibodies specific to the following immune cell populations were added to the cell suspensions and incubated for 25 min at 4 °C in the dark: CD45-PECy7 , CD11b-PE , Gr-1-APC , F4/80-FITC . The samples were then washed with FACS buffer, centrifuged, and resuspended in FACS buffer containing Zombie Aqua viability dye (Biolegend). Samples were immediately analyzed on the LRSFortessa flow cytometer (BD). The data were analyzed using Flow Jo V.10 (BD). Samples were gated for immune cells of the myeloid  or lymphoid lineage . Myeloid lineage cells were further divided into macrophages  and neutrophils .Tumors from G12V/Pten−/−/TPO-CreHras cells  were plated at 300,000 cells/well in 6-well dishes in triplicate. In addition, wildtype thyrocyte cultures were generated as described above. When cells reached confluency, complete media was replaced by serum-free Ham’s F12 medium (Corning). Conditioned media were collected after 18 h, filtered through 40μm nylon strainers (Fisher Scientific) to remove debris, and stored at −20 °C until analysis.m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0. Proteins were identified by database search using MaxQuant  with a parent ion tolerance of 3 ppm and a fragment ion tolerance of 0.5 Da. Scaffold Q+S  was used to verify MS/MS-based peptide and protein identifications. Protein identifications were accepted if they could be established with less than 1.0% false discovery and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm ). Protein intensities were normalized using iBAQ (intensity-based absolute quantification) algorithm and differences between groups were calculated with assistance from the UAMS Bioinformatics Core. All differences between cell lines  and wildtype secretions with a fold change of greater than 2 and a multiplicity-adjusted p-value of ≤0.05 were considered for analysis. REACTOME pathway analysis software was used to identify known pathways containing protein targets. Pathways were ranked by the false discovery rate (FDR) value obtained from over-representation analysis.Conditioned media samples were processed and analyzed by the UAMS Proteomics Core, as follows: Purified proteins were reduced, alkylated, and digested using filter-aided sample preparation with sequencing grade modified porcine trypsin (Promega). Tryptic peptides were then separated by reverse phase XSelect CSH C18 2.5 um resin (Waters) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 60 min gradient from 97:3 to 60:40 buffer A:B ratio . Eluted peptides were ionized by electrospray (2.15 kV), followed by mass spectrometric analysis on an Orbitrap Fusion Lumos mass spectrometer (Thermo). MS data were acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375 to 1500 p-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).Excluding proteomics data, all other data were analyzed using Prism 6 software . Differences with G12V/Pten−/−Hras/TPO-Cre mice were dissected and enzymatically dissociated as previously described . Hemotoxylin and eosin staining of G12V/Pten−/−/TPO-CreHras tumors confirmed the model from which these cell lines were derived exhibit FTC-like histological characteristics, including enlarged follicular cells with mostly solid growth patterns, some intervening follicular patterns and pleomorphic nuclei  primary thyrocytes, while an increase in expression occurred in Hras1. The detection of Pax-8 transcripts confirms the thyroid origin of these cell lines. All three G12V/Pten−/−/TPO-CreHras thyroid cell lines  had detectable expression levels of Ttf-1. In contrast, all lines lacked the expression of Ttf-2, providing additional evidence of de-differentiation of the tumors from which these cell lines were derived. Collectively, Pax-8, Ttf-1, and Ttf-2 regulate the expression of Tg and Tshr [Tg and Tshr expression levels were undetectable in all cell lines. Similar to thyroid transcription factors, loss of Tg and Tshr expression has been associated with disease progression [G12V/Pten−/−TPO-Cre tumors demonstrate that the Hras-driven cell lines represent a true model of follicular thyroid cancer and its progression to a dedifferentiated state.Next, we sought to validate the thyroid origin of these cells and their differentiation status by measuring the mRNA levels of thyroid-relevant genes, including and Tshr C. Pax-8,sgenesis ,25. Losssgenesis ,26. Pax-gression ,28. Shwegression ,15. OverWe next determined the growth rates of the cell lines in culture A using tG12V/Pten−/−/TPO-CreHras cell lines by treating cells with LY294002 (PI3K inhibitor) and AZD6244 (MEK inhibitor) alone or in combination for 72 h. In all cell lines, the inhibition of PI3K or MEK alone resulted in a significant reduction growth  in the supernatant of cells treated with PI3K and MEK inhibitors using a colorimetric assay. In all cell lines, the inhibition of PI3K and MEK alone or in combination caused no increase in cell death relative to vehicle B. Cell cG12V/Pten−/−/TPO-CreHras cell lines offers the potential for rapid screening of therapeutics and more in-depth mechanistic studies in environments that better mimic the in vivo tumor environment.Although the two-dimensional growth of cells on plastic dishes remains the most popular method of in vitro experimentation, three-dimensional (3D) models of tumor growth are gaining favorability as they better recapitulate the environment of solid tumors and cellular responses to therapeutics. As a result, we sought to develop more physiologically relevant methods of growing our cell lines using previously established 3D culture methods. When grown embedded in Matrigel matrix, staining for actin filaments revealed that Hras1 grew as asymmetric multicellular aggregates with loose cytoskeletal arrangement, while H245T and H340T grew in dense, circular formations. B, left. G12V/Pten−/−/TPO-Crede novo Hras murine model [G12V/Pten−/−/TPO-CreHras cell lines in immunocompetent hosts by injecting cells subcutaneously into the flank of wildtype 129SvJ mice. Mice were monitored weekly for 15 weeks for the development of tumors. Upon sacrifice, 7 of the 11 mice were injected with Hras1 developed tumors, resulting in a tumor penetrance of 64%. Conversely, 5 of the 12 mice (42%) injected with H340 exhibited tumor growth. Mice injected with H245 were observed and monitored for 15 weeks, at which time they were euthanized, and no evidence of any tumor formation was found in these animals  and compared it to secretions from cultured wildtype thyrocytes  led to the development of FTC in mice [G12V/Pten−/−/TPO-CreHras mice and maintain many of the same characteristics as the de novo murine model. We have shown that these cells lines can be used for a variety of in vitro applications including mechanistic studies of targeted inhibitors and 3D culturing, while also demonstrating successful transplantation into syngeneic hosts. For these experiments, we chose to highlight a subcutaneous model of in vivo tumor growth as it represents a method of exploring tumorigenesis and evaluating the potential effects of anti-cancer agents that is readily accessible to researchers of many backgrounds without extensive training. However, we anticipate these cell lines could be used in more advanced in vivo modeling methods, including orthotopic implantation directly into the thyroid of wildtype recipients. Although beyond the scope of our present study, it would be of interest in future experiments to determine whether tumors developed from orthotopic implantation of the described cells would maintain a more well-differentiated phenotype in the endogenous thyroid environment. Additionally, although these cells were all derived from mice of the same genetic background, our results demonstrate heterogeneity within these cell lines that may better represent differences in clinical behavior observed in the patient population.Cancer cell lines derived from tumors of genetically engineered mouse models represent an invaluable tool that can be used for in vitro and in vivo research and pre-clinical studies for thyroid cancer. Here, we sought to generate cell lines specifically targeted at understanding the pathogenesis of follicular thyroid cancer (FTC). nly seen ,30. In a in mice . Aside fTg and Tshr are undetectable in all cell lines. This observation is likely reflective of the transition of the original endogenous tumors to a poorly differentiated state in G12V/Pten−/−/TPO-CreHras mice. As a result, these cell lines will allow for the investigation of the molecular basis by which thyroid tumors progress to poorly differentiated disease and for the testing of targeted signaling-pathway inhibitors that may potentially restore thyroid function.Over the last five to ten years, the focus of therapeutics has been heavily directed towards the treatment of radioiodine-refractory, poorly differentiated and anaplastic thyroid cancer. All of the Hras-driven lines described here partially express the transcriptional machinery necessary for normal thyroid differentiation; however, the expression of other markers of thyroid function including In sum, the establishment of three independent cell lines modeling follicular thyroid cancer represents a step towards developing much needed validated in vitro models for thyroid cancer research. We anticipate that these cell lines will offer powerful tools for in vitro and in vivo studies that will lead to the discovery of more personalized diagnostic and treatment strategies for patients with refractory thyroid cancer.


Stichodactyla haddoni. Anticancer and hemolytic activity of crude venom was evaluated and followed by fractionation using RP-HPLC. Breast, Brain, and Colon cancer cell lines were selected to assessment of anticancer activity and toxicity. IC50 of crude venom on the abovementioned cancer cell lines was as 4.13, 6.58, and 31.54 µg, respectively. According to the results obtained by paired sample t-test and comparison of toxicity of the fractions in normal cell line, F10, designated as hadonin, was determined as the candidate anti-cancer fraction. The non-toxic dose of F10 was 20 ng in which showed respectively 66, 29, and 7 anticancer activities on breast, brain, and colon cancer cell lines. According to results, anticancer activity of hadonin is of high pharmaceutical value to follow its therapeutic potency in animal model. In conclusion, the venom of the Persian Gulf sea anemone contains a potential anticancer agent with reasonable activity at nanogram level against three kinds of cancer cells with no toxicity on normal cells. Chemotherapy is still one of the main therapeutic regimens in cancer patients but its toxicity is a hard challenge for every patient yet. One of the available solutions is tracing for non-toxic anticancer agents from natural resources. Numerous proteins and peptides in the venom of sea anemones are potentially useful agents with pharmacological properties. Concerning to significance of this issue, the current study was aimed to finding a non-toxic anticancer fraction from the venom of the Persian Gulf sea anemone,  Cancer is globally still a main public health concern. According to the data collected by the International Agency for Research on Cancer (IARC) and World Health Organization, 8.2 million People (13%) die each year from cancer worldwide.Breast, colon, and brain cancers are of high significance regarding the clinical signs, prevalence, complications of anticancer drugs, and treatment failures , 2. AlthRegarding this vital condition, discovery or design of a non-toxic anticancer drug is being necessitated. One of the available solutions is tracing for non-toxic agents from natural resources.Numerous proteins, peptides, and chemical agents in the venom of venomous marine animals are potentially useful biologically active molecules with pharmacological properties. During the past decade, many studies have been focused on tracing of novel drugs from marine animals -15. ®)   , trabect®) , cytarabepocyt®) , 19, andepocyt®) , 20 are A series of drug candidates derived from marine invertebrate and vertebrate animals are in different phases of clinical studies including Plitidepsin, bryostatin, hemiasterlin, and elisidepsin against lymphoma, leukemia, colon, breast, and various cancers -24.H. crispa, H. magnifica, E. quadricolor, C. adhaesivum, and H. malu against three human cancer cell lines , Glioblastoma (U87mg), Colon cancer (SW948), as well as study of their toxicity. Regarding the significance of searching for a non-toxic anticancer agent, the goal of the current study was aimed to determine the anticancer activities of all HPLC purified fractions from the Persian Gulf sea anemone, Reagents and media3--2,diphenyltetrazolium bromide (MTT) [Sigma], bicinchoninic acid solution (BCA) [iNtRON Biotechnology Co. South Korea], Triton purchased [Sigma–Aldrich Co], Dimethyl Sulfoxide (DMSO) . RPMI-1640 medium , DMEM medium , Fetal bovine serum (FBS) , Penicillin /Streptomycin , Trypsin–EDTA , Trypan blue [Sigma], Methanol .Sample collection Stichodactyla haddoni weighted 85.82 gram was collected manually at depth of 30m from coastal waters of Lark island, Persian Gulf (the south of Iran) in August 2015. The specimens were kept in -20 °C and transferred to Venom and Biotherapeutics Molecules Laboratory (VBML) at Pasteur Institute of Iran, Tehran. One sample of The algorithm of study design Our final objective was reaching a non-hemolytic non-toxic anticancer fraction against examined cancer cell lines. The following chart is described as classified goals in each step . Venom extraction and preparationThe frozen specimens thawed in room temperature and surface mucus layer was cleaned. Tentacles were removed from base by a sterile scalpel, trimmed to small pieces, and divided to two equal parts weighted 8.96 grams for venom extraction. Macroscopic and microscopic view of tentacles is showed in Water extractionSixty nine mL sterile deionized water added to 8.96 grams of fragmented tentacles, mixed in a vortex and incubated at 4 °C for 24 h. The solution was centrifuged  at 10625 g for 10 min. Supernatant lyophilized in a freeze dryer system . The powder resuspended in sterile water for injection and stock solution maintained at -70 °C. Protein estimation and SDS-PAGEConcentration of crude venom was determined by bicinchoninic acid (BCA) protein assay method according to manufacturer instructions (iNtRON Biotechnology Co. South Korea). Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried out according to standard method . The fraHemolysis assay for water extract of crude venomThis assay was performed as described before . Fresh hSerial amounts of venom prepared in a microplate ranged from 100 to 0.78 µg in 100 µL normal saline and 2% RBC suspension (100 µL) added to each well and incubated for 1 h at 37 °C. The samples were than centrifuged for 10 min at 1664 g (Sigma 3-18k), and the absorbance of the supernatant was determined at 540nm in a microplate spectrophotometer . Normal saline and triton X100 were used as negative and positive control respectively. The percent of hemolysis was calculated as follows:  Equ.1Anticancer activity of crude extract against cancer and normal cell linesCell CultureThe breast cancer cell line (MDA- MB-231), colon cancer cell line (SW948), brain cancer cell line (U87mg) and Normal Human Dermal Fibroblast (NHDF) line were purchased from Pasture Institute of Iran. MDA- MB-231 and SW948 cell lines were cultured in RPMI 1640  whereas U87mg and Human fibroblast cell lines in DMEM . The cells were seeded in tissue culture flasks with 10% FBS and 100U/microgram penicillin/streptomycin and incubated at 37 °C, 5% CO2, and 80% humidity. Breast Cancer, Brain Cancer, Colon Cancer, and Normal Human Fibroblast were abbreviated as “BC”, “BrC”, “CC”, and “n” respectively. Cytotoxicity assay4 cells/well in culture medium (100µL) supplemented with 10% FBS. After 24 h incubation , cells were washed with PBS. Then the fresh media with 10% FBS (100 µL) was added to each well. The cells exposed to serial amounts of crude venom ranged from 100 to 0.78 µg and incubated for 24 h at 37 °C. The cells with culture medium alone were considered as negative control. The cytotoxicity effects of crude extract were determined using MTT assay . The celMTT (5 mg/mL in PBS) was added to each well (10 μL MTT/90 μL medium) and the plates were incubated at 37 °C for 4 h and then 100 μL DMSO was added to each well. The plates incubated at room temperature in dark room for 10 min with shaking and finally the absorbance was measured on a microplate spectrophotometer  at 570 nm. The percentage of cell death was calculated according to the below formula: Equ. 2Fractionation of crude venom by RP-HPLC The lyophilized venom was dissolved in distill water (100 µL) and insoluble material discarded by centrifugation at 10625 g for 10 min. The protein fractions were separated using a HPLC instrument (Knauer-Germany). Fifty µL of the prepared venom (8 µg/µL) manually injected to C18 column  and eluted in a linear gradient of acetonitrile containing 0.5% TFA (solution B) and 0.5% TFA in water (solution D) at flow rate 1 mL/min. The resultant peaks were detected at 214nm and 280nm, and were collected manually. The obtained fractions were lyophilized in a freeze dryer system . The powder resuspended in sterile water for injection and stock solution maintained at -70 °C. Concentration of the fractions was estimated as detailed above. Hemolysis assay for HPLC collected fractionsHemolytic activity was performed as detailed above with some modification. Briefly, erythrocytes were prepared as above and a suspension of 0.5% RBC was prepared in normal saline. Serial amounts of fractions prepared in a micro plate ranged from 1.56 to 0.01 µg in 100 µL normal saline and 0.5% RBC suspension (100 µL) added to each well and incubated for 1 h at 37 °C. The other steps were done as outlined before and the percent of hemolysis was calculated according to above formula. Anticancer evaluation of hemolytic and non-hemolytic fractions The breast cancer cell line (MDA- MB-231), colon cancer cell line (SW948), brain cancer cell line (U87mg), and NHDF cell line were cultured and incubated as described before. The cytotoxicity effects of HPLC fractions were determined using MTT assay . All conStatistical Analysist-test. For estimation of correlation between hemolytic activity of crude venom and doses, linear regression test was performed.Variance of hemolytic activity for examined doses was determined using one sample t-test was carried out. Paired sample student t-test was performed to cross comparison of anticancer activities of crude venom between BC and BrC, BC and CC, BrC and CC cell lines. Linear regression test was done to evaluate the correlation between anticancer activity and examined doses between cancer and normal cells as well as Regression between cancer cells.To control the variance of examined doses in anticancer assay of the crude, one sample t-test. Linear regression test was done to evaluate correlation between hemolysis activity of the fractions and examined doses. Variance of examined doses in hemolysis assay of the collected fractions was checked by one sample t-test. Furthermore, Comparison of toxicity of non-hemolytic fractions on normal cell line was evaluated by paired sample t-test.  Difference of anticancer activity between the fractions, against each cancer cell line, was estimated by paired sample t-test. To evaluate the similarity of anticancer activity of candidate fraction on breast, brain and colon cancer cell lines, linear regression test was done. In order to determine the candidate anticancer dose, toxicity of examined doses on cancerous and normal cell lines was compared point to point together. Significance between anti-cancer activity of the candidate fraction on Brain, breast, and colon cancer cell lines and NHDF cell line was compared by paired sample student Assessment of the purity for purified candidate fraction by SDS-PAGETo control the purity of eluted candidate fraction, the resultant peak was collected and freeze dried in a freeze dryer system . The powder resuspended in HPLC grade water and its concentration was determined by BCA assay. The fraction was loaded onto a 12% polyacrylamide gel and stained with Coomassie brilliant blue as noted above.Protein profile of crude venomSDS-PAGE results showed about 12 separate bands and molecular weight of observed proteins ranged approximately from 8 to 250 kDa . Hemolysis assay for water extract of crude venom and statistical analysis2= 0.299)  . P value < 0.001). According to linear regression assay no significant correlation was observed between hemolytic activity and doses (R2 = 0.3).Analysis of variance for hemolysis activity showed that in almost all doses, the activities were similar (As showed in 2= 0.978). Slope of hemolysis activity gradually raised from 83.6 to 100% that was corresponded to the amounts ranged from 0.78 to 25 µg. Slope of hemolysis activity intensely increased from 3.13 to 77.2% that this raising trend corresponded to amounts ranged from 0.023 µg (23 ng) up to 0.39µg (390 ng) . Cross comparison of anticancer activities of crude venom between BC and BrC, BC and CC, BrC and CC cell lines showed significant difference of anticancer activity by Paired sample student t-test respectively . IC50 of crude venom against BC, BrC, CC, and normal cell lines was observed at 4.13, 6.58, 31.54, and 93.45 µg respectively. Analysis of variance for anticancer activity of crude venom on BC, BrC, and CC cell lines showed that activity was similar together in almost all doses  and also toxicity on all three cancer cell lines were very greater than normal cells. Based on linear regression analysis, cross comparison of anticancer activity of crude venom between BC and BrC, BC and CC, BrC and CC cell lines showed significant correlation respectively . Comparing the induced toxicity of crude venom between BC, BrC, and CC cell lines and NHDF cell line (n) showed a significant difference . Linear regression analysis showed significant correlation between anticancer activity of crude venom on BC, BrC, and CC with normal cell lines by increasing examined doses . The highest activities of the first six fractions on human RBCs were equal to 30, 31, 28, 8, 4, and 6% at 1.56 µg, respectively. The fractions from F7 to F11 showed no hemolytic activity at all amounts . RegressThe highest activities of the first six fractions on human RBCs were equal to 30, 31, 28, 8, 4, and 6% at 1.56 µg respectively. The fractions from F7 to F11 showed no hemolytic activity at all amounts. Regression analysis was showed significant correlation between hemolysis activity of each fraction and examined doses .Correlation of hemolytic activity and hydrophobicityBased on the order of elution time in which the fraction eluted from the column, hemolysis activity was descended significantly . 2 = 0.789). The subsequent fractions (F7-F11) eluted by acetonitrile ranged from 9.22% to 15.07 and had no hemolytic activity  and was recorded at 1.6 µg. The highest anticancer activity induced by F4 on both brain and colon cancer cells were showed in 87 and 69%, respectively at 1.6 µg. The lowest activity against breast, brain, and colon cancer cells were induced by F9, F5, and F10, respectively  and 12. The slope of anticancer activity was intensely raised up to 0.4 µg for almost all fractions against all three cancer cell lines and trend of activity was dose dependent. This trend was gradually tended to rising or to be constant while the doses are being increased  and 12. 2BC= 0.529 - 0.95, R2BrC= 0.679 - 0.868, R2CC= 0.528 - 0.945). Anticancer activity of examined doses of all fractions against breast, brain, and colon cancer cells were correlated significantly together by linear regression analysis . It means that toxicity of higher and lower doses are totally the same but it is still a chance to finding a non-toxic dose. Analysis of variance for toxicity of non-hemolytic fractions on normal cells  showed that activity of each dose of each fraction was totally the same as the other one  while F7 and F8 had similar toxicity with F10 and F11, and F9, respectively . Paired sample student t-test . Comparing the mean toxicity between these fractions showed that F10 had the least toxicity (Mean: 11.75% ± 3.7)  . 2F7 = 0.691, R2F8 = 0.736, R2F9 = 0.772, R2F10 = 0.759, R2F11 = 0.736)  . p value <0.001) and also comparison of toxicity of F10 between cancer and normal cell lines showed significant difference  according to paired sample t-test analysis. F10 induced significant different toxicities when used against three cancer cell lines  compared to normal cell line (R2n = 0.759) was completely dose dependent  (2 high linearity that was seen at the range of 0.023 µg (23 ng) up to 0.39 µg (390 ng) (R2= 0.978) indicated strange linear trend of activity. Although there is a lack of correlation between hemolytic activity and doses (R2 = 0.3), a relative meaningful correlation about 81% is considerable to describe dose dependent trend of activity . In reference to calculated R2, high linearity was seen at examined range of doses (R2= 0.914). This result demonstrates that hemolytic activity strangely was linear at the range of 0.78 to 25 µg.Hemolysis activity of the crude was generally not dose dependent at estimated range (R= 0.299) . Accordi2=0.299) but at two distinct ranges of 0.023-0.39 µg (R2=0.978) and 0.78-25 µg (R2=0.914) activity was dose dependent and linearity was significant. Similarly, Subraminan et al. in 2011 showed the hemolytic activity of P. indicus and P. sinensis against chicken erythrocytes and also H. magnifica and S. hadonii against chicken and goat erythrocytes , not only may give us a chance to finding an anticancer fraction but may boost our chance to apply lower doses of anticancer agent and avoiding toxicity in normal cell line. The results obtained from Cross comparison of anticancer activities of crude venom between BC and BrC, BC and CC, BrC and CC cell lines showed that each examined cancer cell line has different sensitivity when exposed to the same crude venom. Crude venom induced more lethality on BC cells than the other cancer cell lines. Significant difference in toxicity of crude venom against examined cancer cell line compared to normal cell line  is an interesting phenomenon pointed out to existence of a non-toxic anticancer fraction in crude venom. According to significant cross-correlations between anticancer activity of crude venom on BC and BrC, BC and CC, BrC and on CC cell lines, similar mechanism of toxicity would be contemplated. In feference to significant correlation between anticancer activity of crude venom on BC, BrC, and CC with NHDF cell lines by increasing examined doses, this similarity would suggest a common dose dependent trend of toxicity on all three cancer cell lines and normal cells as well.Amazing Similar activity of crude venom on BC, BrC, and CC cell lines based on variance analysis , mouse lymphocyte leukemia cells (L1210), and human embryonic lung diploid cells (HEL299) were inhibited at 6.54, 10.07, and 2.34 mg/mL, respectively . Comparing the mean activity of all fractions together by paired sample t-test showed significant difference between them  except for the following pairs including F1-F2, F1-F3, F2-F4, F2-F5, and F5-F6 . According to analysis of variance for all hemolytic fractions, similar activity at all doses of each fraction was showed (2 = 0.74-0.999). Analysis of correlation between hemolysis activity and examined doses by linear regression test indicated that all fractions had dose dependent manner. Cross comparison of trend of hemolysis in all hemolytic fractions was performed by linear regression test and the results showed similar trend of activity in all fractions .Analysis of variance for anticancer activity of isolated fractions on cancer cells showed that activity of each dose of each fraction was generally similar together  in exception of some fractions. In reference to the difference of hydrophobicity in isolated fractions (Cross comparison of the mean of anticancer activity of each fraction with the other fractions by Paired sample ractions  and diff2= 0.528 - 0.95).Anticancer activity of examined doses of all fractions against breast, brain, and colon cancer cells were correlated significantly together by linear regression analysis , mouse lymphocyte leukemia cells (L1210), human embryonic lung diploid cells (HEL299) were inhibited at 40.32, 29.99, and 29.74 mg/mL respectively  (3.5 μg/mL), Non-Small Cell Lung Cancer Carcinoma (NSCLC) (2.8 μg/mL), liver carcinoma (BEL-7402) (3.6μg/mL), stomach adenocarcinoma (BGC-823) (7.4 μg/mL), NIH Swiss mouse embryo (NIH/3T3g) (3.4 μg/mL) (In reference to Jiang 4 μg/mL) . Src I aet al. in 1988 isolated a toxin from the sea anemone Stoichactis helianthus that exhibited toxicity on HL-60 human myelocytic leukemia cells  in a concentration-dependent manner (EqTX-I cytolysis toxin from the sea anemone t manner  while haActinia equina was studied for cytotoxicity against human glioblastoma U87 and A172 cell lines. After 24 h of treatment, 10 mg/mL EqTx-II showed remarkably cytotoxic and reduced the viability of U87 and A172 cells to 60 and 48%, respectively, but was toxic on normal cells at 10 mg/mL and decreased the viability to 80% (EqTx-II from y to 80%  while haAccording to obtained results, anticancer activity of hadonin is of high pharmaceutical value to follow its therapeutic potency in animal models. In conclusion, the venom of the Persian Gulf sea anemone contains a potential anticancer agent with reasonable activity against three kinds of cancer cell lines with no toxicity at nanogram level on normal cells.


Emerging evidence has shown the oncogenic roles of leptin in modulating cancer progression in addition to its original roles. Analyses of transcriptomic data and patients’ clinical information have revealed leptin’s prognostic significance in renal cell carcinoma (RCC). However, its biological effects on RCC progression have not yet been explored. Clinical and transcriptomic data of a RCC cohort of 603 patients were retrieved from The Cancer Genome Atlas (TCGA) and analyzed to reveal the correlation of leptin with clinical outcomes and the hierarchical clustering of gene signatures based on leptin levels. In addition, cox univariate and multivariate regression analyses, cell migration upon leptin treatment, identification of putative leptin-regulated canonical pathways via ingenuity pathway analysis (IPA), and the investigation of induction of Wnt5a, ROR2, and Jun N-terminal Kinases (JNK) phosphorylation activation were performed. We first observed a correlation of high leptin levels and poor outcomes in RCC patients. Knowledge-based analysis by IPA indicated the induction of cancer cell migration by leptin, which was manifested via direct leptin treatment in the RCC cell lines. In RCC patients with high leptin levels, the planar cell polarity (PCP)/JNK signaling pathway was shown to be activated, and genes in the axis, including CTHRC1, FZD2, FZD10, ROR2, WNT2, WNT4, WNT10B, WNT5A, WNT5B, and WNT7B, were upregulated. All of these genes were associated with unfavorable clinical outcomes. WNT5A and ROR2 are pivotal upstream regulators of PCP/JNK signaling, and their correlations with leptin expression levels were displayed by a Pearson correlation analysis. The inhibition of signal transduction by SP600125 reversed leptin-mediated cell migration properties in RCC cell lines. The results indicate the prognostic impact of leptin on RCC patients and uncover its ability to promote cell migration via PCP/JNK signaling. Ob (LEP) gene, which was cloned in 1994 by Friedman and colleagues. It was noted that the gene was also called leptin after the Greek “leptos”, meaning thin [LEPR), which regulates energy expenditure and hunger [Emerging evidence has demonstrated leptin’s roles in regulating cancer progression, including tumor proliferation, metastasis, angiogenesis, and drug resistance . Leptin ing thin . Leptin d hunger ,4. Recend hunger ,6,7,8. Fd hunger . Howeverd hunger ,12. Critd hunger . Howeverp = 0.013, p = 0.006, multivariate, We first explored leptin’s clinical significance in ccRCC. Clinical information and transcriptomic data of a ccRCC cohort of 603 patients were retrieved from The Cancer Genome Atlas (TCGA) and investigated . PatientTo further explore the potential phenotypes that might be regulated upon leptin upregulation leading to poor clinical outcomes in cancer patients, those RNA-Seq data from the ccRCC cohort from the TCGA were investigated . A totalp < 0.05–0.01, Cancer cell migration ability was then examined by transwell assays. ccRCC cell lines were treated with leptin at concentrations of 500 or 1000 ng/mL prior to performing the assay. The treatments with dosages lower than 500 ng/mL revealed the nonsignificant effects in RCC cell lines. Caki-1, ACHN, and A498 cells exhibited a significant increase in cell migration upon leptin addition, though the effect was not observed in 786-O cells . The expression profiles and clinical follow-up data were investigated and retrieved in the Human Protein Atlas/The Pathology Atlas database ,22,23,24The noncanonical WNT/PCP pathway is known to play a pivotal role in promoting cell movement associated with JNK phosphorylation activation ,25,26. AVHL mutation [LEPR) than other cell lines which might in part explain the difference upon the same dose of leptin treatment  was performed according to the pipeline which was previous described [® Pathway Analysis platform according to the instructions.Ingenuityescribed . BrieflyAll human renal adenocarcinoma cell lines were obtained from the American Type Culture Collection  and were gifts from Dr. Michael Hsiao of the Genomics Research Center at Academia Sinica in Taiwan. Culture methods and experimental conditions were according to our previous publication .5 cells in serum-free culture medium were studied in each transwell experiment. A498, ACHN, and Caki-1 cells were allowed to migrate for 3.5 h, and migration time for 786-O cells was 0.5 h.The method followed the precedent research article . In vitrThe method and experimental conditions were previously described . After bp values at the following levels were considered significant: * p < 0.05, ** p < 0.01, and *** p < 0.001. For estimates of the survival rates, clinical follow-up data were calculated by the Kaplan–Meier method, and were compared using the log-rank test. In addition, student’s t-test was applied for other statistical analyses.All data are presented as mean ± S.D. The The results indicate the prognostic significance of leptin in predicting the unfavored overall survival in RCC patients, and uncover its ability to promote cell migration of RCC cells via the activation of the PCP/JNK signaling pathway.


Moreover, when compared with SAL used alone, our experiments proved for the first time increased selectivity of SAL-based dual therapy with 5-fluorouracil or gemcitabine, especially towards A2780 cell line. Looking closer at the results, SAL acted synergistically with 5-fluorouracil towards the drug-resistant A2780 cell line. Our results suggest that combinations of SAL with other antineoplastics may become a new therapeutic option for patients with ovarian cancer.Polyether ionophore salinomycin (SAL) and its semi-synthetic derivatives are recognized as very promising anticancer drug candidates due to their activity against various types of cancer cells, including multidrug-resistant populations. Ovarian cancer is the deadliest among gynecologic malignancies, which is connected with the development of chemoresistant forms of the disease in over 70% of patients after initial treatment regimen. Thus, we decided to examine the anticancer properties of SAL and selected SAL derivatives against a series of drug-sensitive  and derived drug-resistant  ovarian cancer cell lines. Although SAL analogs showed less promising IC Despite developing new therapeutic strategies and extensive knowledge about tumor biology, ovarian cancer (OvCa) remains a leading cause of mortality among gynecologic malignancies; asymptomatic early stages and lack of ambiguous biomarkers enabling detection of the disease lead to late diagnosis, mostly at stage III and IV ,2. Five-1, A few theories have been proposed to explain the development of chemoresistance in human malignancies. The most likely one is related to cancer stem cell theory . AccordiStreptomyces albus (strain no. 80614) [This is a monocarboxylic polyether antibiotic naturally synthesized by . 80614) . SAL was. 80614) . Since t. 80614) . SAL act. 80614) ,16,17.50 against cisplatin as compared with their parental population  and tested their activity towards OvCa cell lines  and reference anticancer drug—CDDP  or gemcitabine (GEM) towards OvCa cells. Therefore, we decided to check if SAL shows desired results when combined with these commonly used anticancer drugs. For this purpose, we prepared 1:1 molar mixtures of SAL and 5FU/GEM  towards normal cells. Judging by ICfied SAL .50 of derived cell lines , based on the ICll lines A. The RIll lines A. Contraes of RI A.6 and 7) developed even mild resistance during our experiments in both OvCa cell lines (2–4) than the corresponding ester derivatives (compounds 6–7). The only exception to this rule was ester 5   A.Further, to establish therapeutic potential of the tested anticancer agents, we used the difference in the antiproliferative activity towards the OvCa cell lines and the corresponding normal cell line to calculate the values of the selectivity index (SI) B. The SI1 + GEM, 1 + GEM, Selectivity of SAL as well as its amide and ester analogs was very promising, especially in non-resistant and resistant to cisplatin A2780 cell lines B. Howeve1 + 5FU and 1 + GEM , Bcl2 associated X protein (Bax), and caspase-3 (CASP3) was performed .3–5) caused increased Bcl2 expression. It is worth mentioning that a combination of 5FU with SAL caused downregulation of Bcl2. In all tested compounds the expression of Bax was inhibited in comparison with the control. Among them, a combination of SAL with 5FU caused decreased expression of Bax compared to 5FU alone. A similar effect onto Bax expression was observed in the GEM and its combination with SAL. In A2780 CDDP cell line, CASP3 expression in SAL and 5FU was decreased, and their combination caused a similar effect in comparison to control. However, the expression of that protein in GEM, 1 + GEM, 3, 4, and 5 variants was enhanced in comparison with the control. The combination of SAL with 5FU caused the enhanced expression of the full form of CASP3, which confirmed synergistic action of these compounds. On the other hand, the antagonistic effect was observed in GEM and its combination with SAL, where the cytotoxic effect was enhanced in only GEM-treated cells.In A2780 cells, the Bcl2 decreased in all groups in comparison with the control A. The lo4, compared to control  treatment is development of chemoresistant residual cancer cells. Cancer stem cell (CSC)-targeted therapies should thus be developed . One of In this study, we derived cisplatin-resistant cell lines A2780 CDDP and SK-OV-3 CDDP and evaluated the antiproliferative activity of selected amides and esters of SAL, as well as SAL used alone or in combination with other commonly used cytostatic agents, such as 5-fluorouracil (5FU) and gemcitabine (GEM), against these OvCa cells. To confirm the observed effect, Western blot analysis of proteins related to apoptosis  was also performed.50 against cisplatin estimated by MTT assay. These variants also showed elevated expression of genes related to drug transporters. ABCB1, ABCB4, ABCG2, and ABCC2 are well-described markers of drug resistance development in many cancers [We obtained resistant variants of cancer cell lines, as confirmed by increased IC cancers ,33,34,35 cancers ,37.6, Our experiments provided interesting data regarding the activity of SAL and its derivatives against platinum resistance developed in OvCa cell lines in vitro. Creation of platinum-resistant OvCa cell line (SK-OV-3) did not induce the resistance of cancer cells to the action of SAL and its derivatives. This indicated that different molecular mechanisms are responsible for either platinum or SAL resistance, and additionally SAL could be identified as an effective agent in overcoming the platinum resistance of OvCa cells. However, A2780 CDDP variant exhibited moderate sensitivity to SAL and its 2,2,2-trifluoroethyl ester derivative (compound +CD117+), and inhibition of the NF-ĸB signaling pathway  [−1 applied in the pancreatic cancer cell lines [+-dependent, whereas in the case of ENT, they are mediated by facilitated diffusion [Our experiments did not confirm the high anticancer activity of SAL derivatives reported recently in primary acute lymphoblastic leukemia (ALL) cells ,40, whicin OvCa) ,42,43,44in OvCa) ,46,47. Oin OvCa) ,49. In cin OvCa) ,51. We tll lines ,53,54,55iffusion ,54,55.50 effect at distinct concentrations of studied compounds, SAL amide derivatives (3–5) caused enhanced expression of CASP3 in A2780 and A2780 CDDP cell lines, which could suggest different or more effective induction of apoptosis by these molecules. We did not observe a similar effect in SK-OV-3 cells, which could be related to intrinsic resistance mechanisms due to their metastatic origin. However, there is a lack of detailed mechanistic studies explaining the differences between SAL and amide derivatives against OvCa cell biology; thus, their further detailed characterization of action should be performed. On the other hand, for SAL used alone, we obtained similar findings as the Parajuli group, where the A2780 CDDP variant was tested, and a decrease of the Bcl2, Bax, and CASP3 was noted at similar doses that we used in our study; however, in their study, only after 1 µM of SAL, caspases were significantly increased [The effect of combination of SAL with GEM or 5FU was confirmed by Western blot analysis of proteins related to apoptosis . Besides the same ICncreased ,56. Theyncreased .In summary, all findings mentioned above corresponded well with our results. Potent anticancer activity of SAL, lack of SAL resistance in platinum-resistant OvCa cell lines, and reversible SAL resistance imply the possible application of SAL in overcoming either primary or acquired platinum resistance (in platinum-resistant cell lines/patients). Further analyses of the SAL treatment used alone or in combination with other anticancer drugs, such as 5FU and GEM, will require identification of the most effective combination of SAL and the cytotoxic agent, and the mode of their administration . Our results may contribute to the development of anticancer therapy based on SAL, which may give hope for heavily treated OvCa patients. Further studies should focus on discovering the mechanisms of action for SAL and its derivatives.® following acidic extraction, using the previously described procedure [2Cl2 and stirred vigorously with a layer of aqueous sulfuric acid (pH = 1.0). The organic layer containing salinomycin   and 1% penicillin-streptomycin . MRC-5 pd19 was cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine , 1% penicillin-streptomycin and 1% non-essential amino acids (NEAA) . To generate the cisplatin-resistant cell lines , increasing doses of cisplatin (CDDP)  were added to the culture medium, starting from the concentration of 100 ng mL−1. Then, the cells were exposed to CDDP (3 cycles of 3 days each). After that, the cell culture medium was replaced with the fresh one without drugs for the next 3 days or one week until the cells recovered. After the 3 cycles, the dose of cisplatin was doubled until the concentration of 1000 ng·mL−1 was achieved. Then, to maintain a resistant phenotype, 1000 ng·mL−1 of CDDP was added once per 2 weeks for 3 days.In this study, OvCa cell lines A2780 and SK-OV-3  and human fetal lung fibroblasts cell line  were used to evaluate the antiproliferative activity of the tested compounds. OvCa cell lines were cultivated in RPMI 1640 containing 25 mM HEPES and 5 mM 5) were washed twice in Dulbecco’s phosphate buffered saline  and suspended in TRI reagent . Next, RNA was isolated using Direct-zol RNA MiniPrep  according to the manufacturer’s instructions. Then 1 µg of RNA was collected to synthesize cDNA using iScript kit . The cDNA was diluted 20 times, and 2.5 µL was added to the reaction mix composed of FastStart Essential DNA Probes Mix and specific probes . The RT-qPCR reaction was performed as previously described [The cells  and left for 2 h at 37 °C. After that, the medium was replaced with DMSO  and left at 37 °C for 10 min until the crystals were dissolved. The measurement was performed with a plate reader, Multiskan FC  at 570 and 690 nm. Then, IC50 and mean 95% CI were determined using GraphPad Prism 6 . The values of resistance index (RI), selectivity index (SI), and combination index (CI) were calculated as described previously [For the evaluation of cell proliferation inhibition, we used the MTT -2,5-diphenyltetrazolium bromide) assay as previously described . Brieflyeviously ,60.50 concentration of selected compounds, the OvCa cell lines were lysed using RIPA lysing buffer . For analysis, 10 µg of protein was used, calculated using Pierce™ BCA Protein Assay Kit  according to the manufacturer’s instructions. The procedure was performed as previously described [After 72 h of exposure to ICescribed . Brieflyt-test) was performed using GraphPadPrism 6 package . The data were deemed significant at p < 0.05. All experiments were repeated at least three times.The statistical analysis of genes expression (Student’s


TNFRSF10B, TNFRSF10C, TP53TG5, MDM2, BAX, BCL-2 and CASPASE-8 genes and their participation in the resistance/susceptibility mechanism of these tumor cell lines.The objectives of this study were to determine the sensitivity of ovarian cancer (OC) cell lines (TOV-21G and SKOV-3) to cisplatin and to the recombinant human TRAIL (rhTRAIL), and to evaluate the expression profile of 50 values associated with Cisplatin and rhTRAIL, inhibition of cell growth was observed using MTT assays in two human OC cell lines (SKOV-3 and TOV-21G). The analysis of gene expression was performed using quantitative real-time polymerase chain reaction (qRT-PCR).To determine the IC50 values for cisplatin and for rhTRAIL were 270.83 ug/mL and 196.5 ng/mL, respectively. The same concentrations were used for TOV-21G. Different gene expression profiles were observed in each tested cell line. CASPASE-8 and TNFRSF10B expression levels could predict the response of both the cell lines to rhTRAIL alone or the response to a combination of rhTRAIL and cisplatin. In addition, we observed a relationship between BCL-2 and BAX expression that may be helpful in estimating the proliferation rate of the OC cell lines.Both cell lines had different susceptibility profiles to the tested drugs. In the SKOV-3 cell line, the ICCASPASE-8 and TNFRSF10B are good predictors of responses to these treatments.SKOV-3 and TOV-21G respond differently to cisplatin and rhTRAIL exposure, and expression of  In 20orldwide . In Brazas 6,150  with an The high mortality rates of OC is partially due to the inefficient detection of the disease in its initial stage, resulting in the diagnosis of 75% of the patients in the advanced stages . The majTNFRSF10B and TNFRSF10C), repair genes (TP53TG5 and MDM2), and apoptotic cascade genes  in these cell lines, and to analyze their participation in drug resistance and drug susceptibility.The TRAIL (TNF-related apoptosis-inducing ligand) receptor pathway has been a focus of studies that are aimed at developing a new strategy to combat OC resistance to cisplatin treatment . TRAIL c2.TOV-21G (Cat. #CRL-11730™) and SKOV-3 (Cat. #CRL-7566™) were acquired from the American Type Culture Collection . TOV-21G was cultivated in the Dulbecco's Modified Eagle's Medium - High Glucose, supplemented with 15% fetal bovine serum. The SKOV-3 line was cultivated in the McCoy's 5A (Modified) medium supplemented with 10% fetal bovine serum. Both cell lines were incubated at 37°C with 5% CO5 cells/well) in triplicate and incubated for 24h at 37°C with 5% CO2. After 24h, the wells were washed with 1X PBS and the cells were treated with varying concentrations of cisplatin (275 ug/mL-100 ug/mL) and rhTRAIL (600 ng/mL-0.78 ng/mL). Non-treated cells were used as experimental controls. After 24h, MTT solution was added to the plate , cells were incubated for 3h and the absorbance at 540 nm was measured using a Spectramax M5e. The concentration of each drug that inhibited the viability of 50% of the cells (IC50) from each cell line was calculated using an Origin 8.5.1 software.The TOV-21G and the SKOV-3 cell viabilities in the presence of cisplatin and rhTRAIL were determined using the 3--2,5-Diphenyltetrazolium Bromide (MTT) method. Cells were plated in 96-Well cell culture plates (1.0x1050) using TRIzol®  according to the manufacturer’s instructions. The total RNA concentration and the 260/280 absorbance ratio were measured using the NanovueTM Plus Spectrophotometer  microvolume spectrophotometer, and RNA integrity was evaluated by 1% agarose gel electrophoresis. Total RNA was then treated with RNase-free DNase Set® (Qiagen) and 2 μg of RNA was used for complementary DNA (cDNA) synthesis using the M-MLV Reverse Transcriptase® kit (Sigma), according to the manufacturer’s instructions.Total RNA was extracted from untreated control cells and cells treated with cisplatin, rhTRAIL or a combination of each drug , using the PowerUp™ SYBR® Green Master Mix, according to the manufacturer’s instructions. TBP and RPS26 genes were included as control genes for normalization. The primers used in this study have been shown in The expression of ® qPCR System (Stratagene). The 2(-Delta C(T)) method  method  was usedp<0.05. Clustering analysis was performed using the UPGMA method and the Euclidian distance was calculated using the BioNumerics software version 7.5 .Data were analyzed using the SPSS 18.0 software package . Differences in the gene expression were statistically evaluated using the non-parametric Kruskal-Wallis and the Levene tests. Differences were considered statistically significant when 50 being 275 ug/mL  on the viability of the SKOV-3 and the TOV-21G cell lines. All cisplatin concentrations tested reduced the viability of SKOV-3 cells, with the IC75 ug/mL . Concent00 ng/mL . Using te assays . Althouge assays . Thus, w50 were used to evaluate the effect of combining these drugs on the SKOV-3 cell viability. Upon treatment with a 1:1 ratio of cisplatin:rhTRAIL, the cell viability was found to be 28.49%. This corresponded to a strong synergistic effect, since the cell density was almost half of that observed when the drugs were used separately. The IC50 associated with combining the drugs could not be statistically calculated. Thus, in order to evaluate the effect of combined cisplatin-rhTRAIL treatment on gene expression, we opted to consider the IC50 of the SKOV-3 cells to correspond to a 2:2 cisplatin:rhTRAIL ratio, since this produced ∼50% cell viability (44.58%) (Different ratios of cisplatin and rhTRAIL at IC(44.58%) .TNFRSF10B, TNFRSF10C, TP53TG5, MDM2, BAX, BCL-2 and CASPASE-8) following treatment of each cell line with cisplatin or rhTRAIL alone, or treatment with a combination of cisplatin and rhTRAIL. The SKOV-3 cells exhibited down-regulation of all genes studied in response to each treatment, with a few exceptions: the rhTRAIL treatment resulted in the up-regulation of TNFRSF10B and CASPASE-8, while cisplatin and combined treatments resulted in the up-regulation of TP53TG5  for the characterization of Epithelial OC (EOC)  when diagnosed. They are characterized by rapid growth and are extremely aggressive, with high chromosomal instability and mutations in TP53 in >95% of the cases  . Type I he cases . TP53 isDespite the heterogeneity observed in OC, all cases are treated as a single disease ,21. The TP53, and is representative of a type I tumor. SKOV-3 originates from metastatic cells (ascites) of an ovary adenocarcinoma and is characterized by platinum-resistance, mutations in TP53, thus representing a type II tumor.In this study, we used the SKOV-3 and the TOV-21G cell lines as models of EOC in order to study the effects of cisplatin and rhTRAIL on the apoptosis-related gene expression. TOV-21G is derived from a primary clear cell carcinoma , contains the wild-type 50 values were calculated as 270.83 ug/mL for cisplatin and 196.5ng/mL for rhTRAIL. However, variation in cell viability was observed upon treatment of the TOV-21G cells with cisplatin or rhTRAIL, therefore the IC50 values could not be accurately determined. It was not possible to observe the dose-response effects, as increased drug concentration did not affect the cell viability proportionally. Karbownik et al. . These data suggest TP53 may have responded to DNA stress by transactivating its targets. It has been reported that TP53 has been involved in the induction of many genes related to apoptosis, including TRAIL death and defectives receptors (TP53 wild-type) and the EVSA-T (TP53-mutated) breast cancer cell lines before and after treatment with doxorubicin. Doxorubicin binds DNA and interrupts replication, a mechanism of action similar to that by cisplatin. In the MCF-7 cells, TNFRSF10A, TNFRSF10B and TNFRSF10C receptor expression was increased upon doxorubicin treatment, whereas this profile was not observed in the EVSA-T cells treated with doxorubicin. This suggests that TP53 may induce the expression of these receptors in the MCF-7 cells. In our study, increased expressions of TNFRSF10B and TNFRSF10C receptors after cisplatin treatment was not observed. Despite that the TOV-21G cells (TP53 wild-type) showed lower expressions of TNFRSF10B and TNFRSF10C receptors , other mechanisms may be involved in the control of expression of the TRAIL receptor in ovarian tumors.Analysis of the gene expression following cisplatin treatment demonstrated up-regulation of eceptors . Almodóveceptors  analyzedBCL-2 genes, which promote cell survival and the expression of the pro-apoptotic protein BAX  and the SKOV-3 (20.34 ratio) cells. In order to determine whether this synergism was due to the gene expression changes, a data clustering analysis was performed. However, genetic signatures were not revealed. Several studies have reported that increased transcription of the TNFRSF10A and TNFRSF10B receptors is related to the response to the DNA-damaging chemotherapeutic agents such as cisplatin, doxorubicin, and etoposide in several cell types (TNFRSF10A and TNFRSF10B in response to the combined treatment with TRAIL and cisplatin. Despite up-regulation of these receptor genes after combined treatment, protein expression was not significantly altered. Duiker et al. (CASPASE-8 expression rather than increased TNFRSF10B receptor expression. We observed up-regulation of CASPASE-8 and down-regulation of TNFRSF10B in both cell lines treated with a combination of both drugs, corroborating with the data described by Duiker et al. (A synergistic effect between cisplatin and rhTRAIL was observed in our cytotoxicity assays and confirmed by an increase in the ll types ,34. Usinll types  demonstrr et al.  also evar et al. .CASPASE-8 and TNFRSF10B expression may predict the response of OC cell lines to rhTRAIL alone or in combination with cisplatin. Our study suggests that BCL-2 and BAX expression may be helpful in the assessment of the proliferative rate potential of OC cell lines, and highlighted that the development of new therapeutic strategies for the OC should focus on the individual molecular and genetic characteristics of different tumor subtypes. In light of these findings, our future research will aim to explore the exact mechanism behind the synergy between cisplatin and rhTRAIL.Braga LC is the main researcher, responsible for the conceptualization, data collection and analysis, funding acquisition, project administration, literature review and manuscript revision.Gonçales NG was responsible for data analysis, resources, data collection and interpretation and for writing the manuscript original draft. Furtado RS was responsible for the literature review, manuscript revision and submission. Andrade WP was responsible for the data collection and interpretation. Silva LM was responsible for the conceptualization, data analysis, supervision and manuscript revision. Silva Filho AL is the research advisor responsible for the conceptualization, supervision and validation.