Colon cancer constitutes 33% of all cancer cases in humans and the majority of patients with metastatic colon cancer still have poor prognosis. An important role in cancer development is the communication between cancer and normal cells. This may occur, among others, through extracellular vesicles (including microvesicles) (MVs), which are being released by both types of cells. MVs may regulate a diverse range of biological processes and are considered as useful cancer biomarkers. Herein, we show that similarity in the general chemical composition between colon cancer cells and their corresponding tumor-derived microvesicles (TMVs) does exist. These results have been confirmed by spectroscopic methods for four colon cancer cell lines: HCT116, LoVo, SW480, and SW620 differing in their aggressiveness/metastatic potential. Our results show that Raman and Fourier Transform InfraRed (FTIR) analysis of the cell lines and their corresponding TMVs did not differ significantly in the characterization of their chemical composition. However, hierarchical cluster analysis of the data obtained by both of the methods revealed that only Raman spectroscopy provides results that are in line with the molecular classification of colon cancer, thus having potential clinical relevance. Colon cancer is one of the most common cancers worldwide and in the last 15 years, the number of patients with this type of cancer has increased by 60% ,3. In caIn this study, by using Raman and FTIR spectroscopy, we determined the general chemical composition of four colon cancer cell lines: HCT116, LoVo, SW480, and SW620, which differ in their aggressiveness/metastatic potential ,24. In pTMVs released by four colon cancer cell lines HCT116, LoVo, SW480, and SW620 were isolated from culture supernatants by differential centrifugation. Cell lines differed in malignancy potential, as HCT116 was described as fast growing, SW480-having low metastatic ability, SW620-high metastatic ability, and LoVo-undifferentiated . The isoFor the size distribution measurements, nanoparticle tracking analysis (NTA) was used. As shown in By using two complementary spectroscopic techniques, FTIR and FT-Raman, the general chemical composition of the four colon cancer cell lines and their corresponding TMVs was determined. In the obtained FTIR  and Rama−1 to 3000 cm−1. In the case of TMVSW480 and SW480 cells  and between 3000 cm−1 and 3500 cm−1, were noticed when we compared the TMVsHCT116 and the HCT116 cell lines. When comparing LoVo . Moreover, in the Raman spectra of SW480 cells and TMVSW480, differences in the shape of the spectra and peak shifts originating from DNA, phospholipids, and protein vibrations (800–1100 cm−1) were visible. Furthermore, in the Raman spectra of TMVHCT116 and TMVSW620, absence of the peak corresponding to the C–H groups from cholesterol and cholesterol ester were noticed in comparison to their respective cell lines. The peaks originating from the stretching vibrations of the CH, NH, and OH groups were not observed in the Raman spectrum of the LoVo cell line in comparison with the Raman spectrum of the TMVLoVo. The identified peaks are described in detail in The acquired Raman spectra showed differences in the intensity values as well as in the shape of the spectra between the respective cell lines and TMVs released by them. In the spectra obtained for the HCT116 cells, the Raman intensities in the analyzed range were higher than for their TMVs a. Moreoving LoVo b and SW6ing LoVo d cells weir TMVs c, higher−1), proteins (~1250–1700 cm−1), and lipid (~2700–3000 cm−1) vibrations, respectively.To obtain information about the differences in the structure between cell lines and the corresponding TMVs released from them, the second derivative of the FTIR spectra was calculated . HoweverHCT116, TMVsSW480, and TMVsSW620 were compared with those of their respective cell lines. Furthermore, structural changes in the IR region corresponding to DNA and phospholipid functional group vibrations were observed when we compared the second derivative of the FTIR spectra obtained for the SW480 cell line and its respective TMVs. Indeed, it is well known that in the IR region between 1700–1600 cm−1, there is a vibration of the C=O group, which participates in the formation of intramolecular and intermolecular hydrogen bonds. In this case, the stretching vibrations of the C=O bands in the IR spectra are very sensitive to the formation and changes in the hydrogen bonds between peptide groups [−1; for structures of the β-form, the frequencies are in the range between 1640–1620 cm−1, while for β-sheet structures, it was 1670–1690 cm−1 [−1). This analysis enabled the determination of the protein secondary structure by quantifying the percentage of α-helix and β-sheet structures.While comparing the respective cell lines and their TMVs, the most visible, however very small, structural changes occurred in the IR region corresponding to lipids. Changes in the protein structure were also noticed, when the second derivatives of the FTIR spectra of the TMVse groups ,45. Intr690 cm−1 . To deteHCT116, one component band less was detected than in the HCT116 cells. Moreover, when comparing LoVo cells and the TMVs released by these cells, the TMVLoVo showed one component band less than the parental LoVo cells. Additionally, the component band areas corresponding to the α and β structures were different when comparing cells with their TMVs. The peak area values, percentage, and ratio of α and β secondary protein structures obtained for the analyzed cell lines and their TMVs are described in Similarity in the value of α and β secondary protein ratio structures in tumor cell lines and corresponding TMVs is presented. High similarities in the ratio of α-helix/β-harmonica in HCT116, LoVo, and SW480 cells and their corresponding TMVs have been detected. A similar trend was also observed for the SW620 cells and corresponding TMVs, however, it was less significant.To determine the similarities in the general chemical composition between the analyzed cell lines and their TMVs, we performed hierarchical cluster analysis (HCA) .LoVo (Euclidean distance around 0.25). When we compared Euclidean distance in HCA obtained for Raman and FTIR data, the lower value of this parameter was in FTIR HCA analysis , which showed that the samples included both exosomes and MVs. This heterogenic composition was further confirmed by western blot analysis and the expression of proteins characteristic for both exosomes and MVs. The FTIR and Raman spectra of the HCT116  confirme solvent . The sol2), we cannot observe any IR absorption lines since the molecule cannot change its dipole moment. It has been observed that molecules with a strong dipole moment are typically hard to polarize [HCT116, which could result in the observed distance between the cells and TMVs in the HCA analysis.Interestingly, in the HCA analysis , we founpolarize ,51. AddiMoreover, in the obtained spectra, some vibration peaks were absent while the peaks corresponding to functional groups within the measured samples have been identified. Therefore, using these two complementary methods, a whole chemical structure of analyzed samples was obtained. Differences in the peak-shifts as well as the absence of some peaks were observed in The two isogenic colorectal cancer SW480 and SW620 cell lines were derived from the same patient, the first originating from the primary tumor, whereas the latter was from a metastatic lesion to the lymph node, therefore similarities between these cells and their corresponding TMVs could be expected, as evidenced by several papers ,53,54,55On the other hand, Pablo et al. used Raman spectroscopy to distinguish different colon cancer cell types including SW480 and SW620. They showed that these two cell lines could be distinguished by ratio of α:β secondary protein structure. Furthermore, the PCA-LDA analysis showed that by using this approach, it is possible to distinguish different types of colon cancer cell lines with an accuracy of 92.4 ± 0.4% . These rTo summarize, in this report, through the use of FTIR and Raman spectroscopy, we showed that the chemical composition of TMVs is similar to the composition of the cells from which they are released. We suggest that Raman as well as FTIR spectroscopies could find a potential application for the first rapid chemical identification of circulating TMVs, reflecting the composition of their parental cancer cells. It seems however, that only the data obtained from the Raman analysis were in line with the molecular classification of colon cancer, having thus a potential clinical relevance. Further studies involving other tumor cell lines and their respective TMVs using Raman and FTIR spectroscopies would provide additional information on the usefulness of these methods, which could potentially translate into the clinical setting .TM, Corning, NY, USA) in a 37 °C humidified atmosphere with 5% CO2. All media were supplemented with 10% fetal bovine serum  and gentamicin (50 µg/mL), . The cells were cultured by bi-weekly passages and regularly tested for Mycoplasma sp. contamination with the PCR-ELISA kit , according to the manufacturers’ instructions.Colon cancer cell lines  were obtained courtesy of Prof. Caroline Dive, Paterson Institute for Cancer Research, University of Manchester. Human colon carcinoma cell line HCT116 was obtained from the American Type Culture Collection  and maintained according to the ATCC’s instructions. Briefly, HCT116 cells were cultured in McCoy’s 5A medium , while LoVo, SW480, and SW620 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose content  for 1 h at 4 °C. Pellets were washed twice in PBS to remove FBS and finally re-suspended in serum-free medium. Quantification of TMVs proteins was evaluated by the Bradford method . TMVs were tested for endotoxin contamination by the Limulus test, according to the manufacturer’s instruction  and stored at −20 °C until use. To simplify, TMVs were named according to their cell line of origin  containing the protease inhibitor cocktail (Roche). The concentration of samples was measured using the Bradford kit (Bio-Rad) as per the manufacturer’s instructions. A total of 20 μg of suspended TMVs was mixed with NuPAGE LDS Sample Buffer (4×) and NuPAGE Sample Reducing Agent (10×) . Samples were heated  and electrophoresed in 12% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Next, electrophoresed samples were transferred onto the polyvinylidene fluoride membrane . Then, after blocking for 1 h at room temperature in Tris buffered saline (TBS) with 0.1% Tween-20  and 1% bovine serum albumin , the membranes were incubated overnight at 4 °C with mouse mAb anti-Alix (3A9), rabbit mAb anti-CD9 (D801A), and anti-Flotillin-1 (D2V7J)  diluted 1:1000 as well as rabbit polyclonal Ab anti-CD63 (SIGMA) diluted 1:2000. As a loading control, rabbit anti-GAPDH antibodies  diluted 1:5000 were used. After incubation, the membranes were washed in TBS supplemented with BSA and Tween-20 and incubated for 1 h in room temperature with appropriate secondary antibody: goat anti-mouse and goat anti-rabbit  conjugated with horseradish peroxidase (Santa Cruz Biotechnology). The protein bands were visualized with the SuperSignal West Pico Chemiluminescence Substrate kit (Pierce), according to the manufacturer’s protocol and analyzed with ChemiDoc system (Bio-Rad).Average, modal size, and size distribution of the TMVs were obtained using the NANOSIGHT LM10-HS488FT14 Nanoparticle Characterization System . Briefly, 1 μL of the TMV suspension was diluted 1000× in filtered (0.22 μm) PBS to obtain the total sample volume of 1 mL. Next, approximately 700 μL of the sample was loaded manually into the measuring chamber using an insulin-type syringe, after which the syringe was mounted onto the pump and the sample was delivered at a constant flow rate of 80 units. Next, three one-minute videos were recorded by the sCMOS camera for each sample and used later for analysis using the NanoSight NTA 3.0 analytical software .8 cell/mL were centrifuged for 5 min at 3000 rpm. Subsequently, the cells were washed three times in isotonic solution  to ensure complete removal of trypsin and culture medium. Next, dense cell suspensions were placed onto an attenuated total reflection (ATR) crystal (FTIR spectroscopy) or CaF2 slides (Raman spectroscopy).For FTIR and FT-Raman spectrum acquisition, cells in the concentration 10−1. Each spectrum was obtained by averaging 64 scans recorded at a resolution of 4 cm−1. For statistical validation, three samples for each of the cell lines were measured. Moreover, each sample was measured three times. Baseline correction and vector normalization of the obtained spectra were performed using OPUS 7.0 software .All measurements were carried out on an EXCALIBUR FTS-3000 spectrometer  at room temperature. All spectra were recorded by attenuated total reflection (ATR) with a ZnSn crystal. A total of 0.5 mL of cell containing solution was deposited on the ATR ZnSn crystal. FTIR spectra were recorded between 4000 and 500 cm−1 with a laser power of 1 W. Unfocused laser beam was used with a diameter of approximately 100 μm and a spectral resolution of 8 cm−1. Raman spectra were processed by the Omnic/Thermo Scientific software based on 128 scans. The obtained spectra were normalized using vector normalization in OPUS 7.0 software . Moreover, each spectrum was smoothed using the Savitzky–Golay algorithm. The number of smoothing points was nine.FT-Raman spectra were recorded using a Nicolet NXR 9650 FT-Raman spectrometer  equipped with an Nd:YAG laser (1064 nm) and a germanium detector. Measurements were performed in the range from 150 to 3.700 cmThe secondary protein structure was analyzed by means of curve fitting using MagicPlot software . First, the second derivative spectra were calculated based on the ATR-FTIR spectra to determine the initial peak positioning for the curve fitting. The peaks were fitted using a Gaussian function and the area under the curve was considered at 100%. Each component was expressed as its percentage after fitting.−1–1700 cm−1 and between 2700 cm−1–3000 cm−1. PCA-LDA results had been verified by the leave-one-sample out cross-validated method, which involves leaving all spectra from a single sample out of the model once before assessing performance. This analysis was done using the OriginPro 2015 software . Moreover, to determine similarities between HCT116, LoVo, SW480, SW620 cell lines, hierarchical cluster analysis (HCA) with Euclidean distance was performed in Past software (developed by Oyvind Hammer). HCA analysis was done for the fingerprint region (between 800 cm−1 and 1800 cm−1) for both the Raman and FTIR spectra of the cells and TMVs. For this purpose, 518 points from the FTIR spectra and 259 points from the Raman spectra were analyzed using the paired group (UPGMA) algorithm.For all obtained spectra, vector normalization and baseline correction were applied. These operations were performed using OPUS 7.0 software. Moreover, in each FTIR as well as Raman spectrum, vibrations corresponding to nucleic acid, phospholipids, proteins, and lipids were analyzed. The number of obtained data from FTIR and Raman spectra was very large, therefore, to determine the similarity between the cancer and control cells, principal component analysis-linear discriminate analysis (PCA-LDA) was performed. First, PCA was undertaken, which reduced the dimensionality and the number of data variables, while maintaining as much variance as possible. Next, with the use of the LDA method, a suitable model for grouping data based on independent variables (wavelengths) was developed. Then, the PCA-LDA procedure was performed based on the selected spectral regions: between 1250 cm


Treatment of uveal melanoma (UM) is generally successful, with local primary tumour control being at 90–95%. Localized radiotherapy in the form of plaque brachytherapy or proton beam radiotherapy is the most common treatment modality in the UK. However, the basic mechanisms of radiation response, DNA repair and tissue reactions in UM have not been well documented previously. We have investigated the comparative radiosensitivity of four UM cell lines in response to exogenous radiation sources (both X-rays and protons), and correlated this with DNA repair protein expression and repair efficiency. We observed a broad range of radiosensitivity of different UM cell lines to X-rays and protons, with increased radioresistance correlating with elevated protein expression of ataxia telangiectasia mutated (ATM), a protein kinase involved in the signaling and repair of DNA double strand breaks. The use of an ATM inhibitor in UM cell lines enhanced radiosensitivity following both X-ray and proton irradiation, particularly in cells that contained high levels of ATM protein which are otherwise comparatively radioresistant. In proton-irradiated compared with non-irradiated primary enucleated UM patient samples, there was no significant difference in ATM protein expression. Our study therefore suggests that ATM is a potential target for increasing the radiosensitivity of more resistant UM subgroups. In the adiation ,10. ConsPhoton (X-ray) and proton radiotherapy are effective at inducing the formation of DNA damage, such as DNA double strand breaks (DSBs), which contribute to their therapeutic effect in cancer cell killing. Despite this, there is still a significant biological uncertainty with protons, particularly due to increases in LET at and around the Bragg peak, where the majority of the radiation dose is deposited . Cells aIn this study, we examined the radiosensitivity of UM cell lines following photon (X-ray) and proton irradiation, and identified that the most radioresistant cell lines contain increased ATM protein levels. These UM cell lines can be radiosensitized using an inhibitor targeting ATM. These results suggest a potential therapeutic strategy employing ATM inhibitors, which could be exploited for more effective treatment of UM by radiotherapy.β between the cell lines  polymerase-1, PARP-1; X-ray repair cross-complementing protein 1, XRCC1, polynucleotide kinase phosphatase, PNKP and AP endonuclease 1, APE1) and DNA DSB repair  in extracts from four UM cell lines derived from primary (Mel270 and 92.1) and metastatic (OMM1 and OMM2.5) tumors. Variability was observed particularly in the levels of ATM, XRCC1, PNKP and Pol ll lines . HoweverWe subsequently analyzed the comparative radiosensitivity of the cells to photon (X-ray) and proton irradiation by clonogenic survival assays. OMM2.5 and Mel270 were ~3.5–6.2-fold (across the dose response) more radioresistant to X-ray irradiation than OMM1 and 92.1 B,C. The We analyzed the comparative kinetics of DNA DSB repair in UM cell lines in response to photon and proton irradiation using neutral comet assays. We observed in response to photons that, surprisingly, all the four UM cell lines analysed  were able to largely repair DSBs within a 4 h period post-irradiation A,C. The n = 32) or non-irradiated . Examples of the degree of staining (from negative up to 65% staining) observed in cells from UM tissues are shown (p = 0.57).Given the range (~1.7–6.0-fold) of different expression of ATM protein in UM cell lines, and apparent links with modulating cellular radiosensitivity to photons and protons A–D, we are shown . There wre shown . Additiore shown . ATM stare shown . Finallyp = 0.33–0.72; one-sample t-test). We consequently analysed the impact of targeting ATM on the radiosensitivity of UM cell lines in response to both photon (up to 4 Gy) and proton (up to 8 Gy) irradiation. We observed that ATM inhibition resulted in increased radiosensitivity of both the OMM2.5 cells (by ~1.7-fold across the dose response) and, more dramatically, the Mel270 cell lines (by ~16-fold), but not the 92.1 cells, to photon irradiation  and proton beam therapy for UM, we have utilized both X-rays and protons to investigate the comparative radiobiology of four UM cell lines.The radiobiology of UM has only been subject to limited investigation. Early studies using single UM cell lines suggested that these are generally radioresistant ,4,5, butAs demonstrated in previous studies, our data support the variability in radiosensitivity between UM cell lines, and that there is no correlation with the UM cell line derivation, be it from a primary or a metastatic tumor. A novel finding of our study is that this degree of variability between UM cell lines is observed following both photon (X-ray) and proton irradiation. In fact, the OMM2.5 and Mel270 cell lines were consistently the most radioresistant, whereas the OMM1 and 92.1 were the most radiosensitive following both X-rays and protons. We also showed that cellular radioresistance appears to correlate largely with the levels of ATM that co-ordinates the signaling and repair of DNA DSBs. Indeed, an ATM inhibitor was able to enhance the effects of photons and protons in reducing clonogenic cell survival in a range of UM cell lines. This suggests a potential therapeutic strategy to enhance the impact of radiotherapy (photons and protons) in UM cell killing. Targeting ATM is currently being investigated to enhance the radiosensitivity of other tumor types, particularly glioblastoma ,19,20, bWe analyzed the expression of ATM in UM tissues, but did not observe any difference in immunohistochemical staining between proton-irradiated and treatment-naive UM, nor any correlation of ATM staining with known poor prognostic parameters or clinical outcomes. Given the small sample size, it is possible that our study was not powered sufficiently to identify any statistical difference. The phosphorylation of ATM and the relocation of the protein into the nucleus have been suggested to act as predictive biomarkers of radiotherapy response ,24,25 an106 brachytherapy but which have subsequently been successfully treated with protons.Interestingly, our analyses found no differences in the kinetics of DSB repair through neutral comet assay analysis in UM cell lines following X-rays, suggesting that there is no direct correlation with DSB repair efficiency and radiosensitivity. This could indicate that the inherent photon (X-ray) radiosensitivity is largely determined by other factors, such as chromatin structure, cell cycle progression, and cell survival mechanisms including apoptosis, autophagy and senescence. This requires further investigation in UM. However, in response to protons, there was a correlation with UM cells displaying reduced DSB repair capacity post-irradiation (OMM1 and 92.1), and their increased radiosensitivity compared to the other cell lines examined . This could alternatively suggest a different spectrum of DNA damage induced by proton rather than photon irradiation, or that the DSB repair pathways utilized  are distinct in their response to the different radiation modalities. Therefore, a comprehensive analysis of the induction and repair of DNA base damage and single strand breaks , and DSBs  following photon and proton irradiation is necessary to further understand this. It was noticeable, though, that lower doses of X-ray radiation (0.5–2 Gy) appeared to have a greater impact on reducing cell survival than protons, when comparing the response between OMM2.5 and Mel270 with OMM1 and 92.1. Given the lack of correlation with DSB repair efficiency, the mechanism through which reduced cell survival  is triggered in response to X-rays at these lower doses should be investigated in more detail. It should be noted that our proton experiments utilized low-LET protons at the entrance dose of a pristine beam, and therefore we would not expect LET, and subsequently DNA damage complexity, to play a role in the differential cellular response compared to photons. Nevertheless, this was recently discussed in the context of head and neck cancer cells , and reqThe following antibodies for immunoblotting were used. APE1, Pol β and XRCC1 antibodies were kindly provided by Dr G. Dianov. PNKP antibodies were also kindly provided by Prof M. Weinfeld. ATM (sc-23921), DNA-Pk (sc-9051), Ku-86 (sc-9034), PARP-1 (sc-53643) and RAD51 (sc-8349) antibodies were from Santa Cruz Biotechnology ; ATR antibodies (ab2905) were from Abcam ; 53BP1 (A300-272A) antibodies were from Bethyl Laboratories ; and actin (A5441) antibodies were from Sigma-Aldrich . 2 at 37 °C, and were authenticated in our laboratory by STR profiling. Cells were irradiated using a CellRad X-ray irradiator , or at the entrance dose of a passive scattered horizontal proton beam line at 60 MeV maximal energy (~1 keV/µm), as previously described [UM cells  were routinely cultured as monolayers in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1× penicillin-streptomycin and 1× non-essential amino acids. All cells were cultured under standard conditions in 5% COescribed .Whole cell extracts were prepared as previously described . BrieflyCells were seeded in 35 mm dishes to ~70% confluence at the time of irradiation. Following irradiation, cells were trypsinized, counted and seeded into 6 well plates, and colonies were allowed to grow for 10–14 days, prior to fixing and staining with 6% glutaraldehyde and 0.5% crystal violet for 30 min. Plating efficiencies for the non-irradiated cells were as follows: OMM1 (~25%), OMM2.5 (~15%), Mel270 (~35%) and 92.1 (~40%). Dishes were washed, left to air dry overnight, and colonies were counted using the GelCount colony analyzer . Relative colony formation (surviving fraction) was expressed as colonies per treatment level versus colonies which appeared in the untreated control. For inhibition experiments, media containing 10 µM ATM inhibitor  or DMSO as a vehicle only control were added onto the monolayer cells for 1 h prior to irradiation. The irradiated cells were then trypsinized, counted and seeded into 6 well plates containing 10 µM ATM inhibitor for a further 24 h. Following this, fresh media only was added until the colonies formed. Statistical analysis was performed using the CFAssay for R package .5 cell/ml, and 250 µl aliquots of the cell suspension were placed in the wells of a 24 well plate. For inhibitor experiments, media containing 10 µM ATM inhibitor (KU-55933) or DMSO as a vehicle only control was added to the cells 1 h prior to trypsinization. Cells in suspension were irradiated with 4 Gy X-rays or protons, and then embedded on a microscope slide in 1% low melting point agarose  in PBS. Slides were incubated for up to 4 h in an humidified chamber to allow for DNA repair, and subsequently cell lysis was performed in buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl pH 10.5, 1% N-lauroylsarcosine, 1% DMSO and 1% (v/v) Triton X-100 for at least 1 h at 4 °C. DNA was allowed to unwind in cold electrophoresis buffer  for 30 min in the dark, before separation of the DNA DSBs by electrophoresis at 25 V, ~20 mA for 25 min. Slides were washed three times with 1 × PBS before being allowed to air dry overnight. The agarose was subsequently rehydrated for 30 min in water (pH 8.0), stained for 30 min with SYBR Gold  diluted 1:10,000 in water (pH 8.0), and air dried again overnight. Cells  were analyzed from the dried slides using the Komet 6.0 image analysis software , and three individual biological replicates were performed.Neutral single cell gel electrophoresis (comet) assays for the measurement of DNA DSBs were performed as previously described ,29,30. Et-test.Sections (4 µm thick) from FFPE UM were deparaffinized and underwent heat induced epitope retrieval at 96 °C, pH 9.0 for 20 min on the Leica Bond RXm, followed by staining using the Leica Bond polymer refine red detection kit, according to the standard manufacturer’s protocols. A mouse monoclonal anti-ATM antibody  was used at 1:100 dilution. Sections were counter-stained with hematoxylin, dehydrated and mounted using a solvent based mountant. Negative controls used mouse IgG1 at the same concentration as the primary antibody. Staining was assessed and scored by three individuals . Statistical analysis was performed using a one sample We have demonstrated that UM cell lines display a broad range of radiosensitivity to photon (X-ray) radiotherapy and proton beam therapy, with an apparent correlation of increased radioresistance with elevated levels of ATM. Whilst no correlation between ATM protein expression in UM tissue samples and an adverse patient outcome was found, inhibition of ATM in vitro was shown to be effective in potentiating the effects of photons and protons in reducing clonogenic survival. Our data therefore suggest that targeting ATM, in combination with radiotherapy, can be a more effective strategy for the treatment of UM.


The drug resistance of first-line crizotinib therapy for ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion non-small cell lung cancer (NSCLC) is inevitable. Whether the administration of immune checkpoint inhibitor (ICI) therapy is suitable for ROS 1 fusion NSCLCs or after the development of crizotinib resistance is still unknown. In this study, five different crizotinib resistant concentration cell lines (HCC78CR1-5) from primary sensitive HCC78 cells were cultured. Ba/F3 cells expressing crizotinib sensitive ROS1 fusion and crizotinib resistant ROS1-G2032R mutation were used to explore the relationship between ROS1 fusion, ROS1-G2032R mutation and programmed death-ligand 1 (PD-L1) expression and the clinical potential of anti-PD-L1 ICI therapy. The signaling pathway net was compared between HCC78 and HCC78CR1-5 cells using RNA sequencing. Anti- PD-L1 ICI therapy was performed on mouse xenograft models with Ba/F3 ROS1 fusion or ROS1-G2032R mutation. HCC78CR1-5 showed more immunogenicity than HCC78 in immune-related pathways. The PD-L1 expression level was remarkably higher in HCC78CR1-5 with ROS1 fusion upregulation than HCC78 primary cell. Furthermore, the expression of PD-L1 was down-regulated by RNA interference with ROS1 siRNAs and up-regulated lower in Ba/F3 ROS1-G2032R resistant mutation than ROS1 fusion. Western blotting analysis showed the ROS1–SHP2 signaling pathway activation in HCC78CR1-5 cells, Ba/F3 ROS1 fusion and ROS1-G2032R resistant mutation. Mouse xenograft models with Ba/F3 ROS1 fusion showed more CD3+PD-1+ T cells both in blood and tissue, and more sensitivity than the cells with Ba/F3 ROS1-G2032R resistant mutation after anti-PD-L1 therapy. Our findings indicate that PD-L1 upregulation depends on ROS1 fusion more than ROS1-G2032R mutation. We share our insights of NSCLCs treatment management into the use of anti-PD-L1 ICI therapy in ROS1 fusion and not in ROS1-G2032R resistant mutation. At present, molecular targeted therapy is an important strategy for the treatment of non-small cell lung cancer (NSCLC) , 2. DrivROS1 fusion NSCLCs are sensitive to crizotinib, but their development of drug resistance is inevitable. As a new drug class, immunology checkpoint inhibitor (ICI) therapies, such as those involving anti-CTLA4 , anti-prCurrently, the pembrolizumab (anti-PD-1) is approved for use as first- and second- line therapy in patients with advanced NSCLC whose tumors express PD-L1 in immunohistochemistry analysis , 13. Nivin vitro and in vivo experiments.The mechanism of the regulation of PD-L1 expression in ROS1 fusion primary tumor and acquired resistance to crizotinib is still unknown. Although many options are available for molecular targeted therapies of primary sensitive or secondary mutation of ROS1 tyrosine kinase domain drug resistance mutation, it remains unclear about the clinical potential of anti-PD-L1/PD-1 ICI therapy on ROS1 fusion and ROS1-G2032R crizotinib resistant mutation. Our aims of this study were 1) to explore the relationship between ROS1 fusion, ROS1 G2032R crizotinib resistant mutation and PD-L1, and 2) the clinical potential of anti-PD-L1 ICI therapy in ROS1-driven and drug-resistant ROS1 fusion G2032R mutation cell lines and mouse models. In this research, we share our insights related to ICI therapy by examining the role of ROS1 fusion and ROS1 G2032R resistance mutation on PD-L1 expression in NSCLCs by http://www.cobioer.com/). The cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  with 10% fetal bovine serum (FBS). Crizotinib (Cat NO.: S1086), Lorlatinib (Cat NO.: S7536), TPX-0005 (Cat NO.: S8583) and SHP099 (Cat NO.: S8278) purchased from Selleckchem. The HCC78 cell line was maintained in RPMI-1640 medium that contained Crizotinib at a starting concentration of 100 nM until a ﬁnal concentration of 2 mM over 10 months. Then, the resulting resistant cells were maintained in RPMI-1640 medium with 0.5 mM crizotinib and were designated as HCC78CR1, -2, -3, -4, and -5 cell lines. HCC78 and HCC78CR1-5 cell lines were authenticated by detecting the SLC34A2–ROS1 fusion using next-generation RNA sequencing. All cells were maintained under a humidified atmosphere of 5% CO2 at 37°C. For in vitro studies, crizotinib was dissolved in dimethyl sulfoxide (Sigma Aldrich) at 10 mM and stored at -80°C. Ba/F3, Ba/F3 ROS1 fusion harboring SLC34A2-ROS1 (Cat NO.: CBP73191) and Ba/F3 ROS1-G2032R harboring both ROS1 fusion and G2032R mutation (Cat NO.: CBP73192) were also obtained from Cobier Company. The Ba/F3 ROS1 fusion, Ba/F3 ROS1-G2032R harboring SLC34A2-ROS1 with G2032R crizotinib resistant mutation and IL-3 producing WEHI-3 cell lines were cultured in RPMI 1640 medium  with 10% FBS. The IL-3-dependent mouse pro-B cell line, Ba/F3, was cultured in 10% FBS and 10% WEHI-3-conditioned medium as a source of IL-3. To obtain the WEHI-3-conditioned medium, WEHI-3 cells were seeded into culture medium (107/100 mL) in the T75 culture flask and cultured for about 4 days until the color of medium turned almost yellow. The IL-3 containing medium was harvested, centrifuged, filtered with a microfilter system (0.2 μm), and kept frozen at –80°C.HCC78 (Cat NO.: CBP60100) and IL-3 producing WEHI-3 (Cat NO.: CBP60532) cell lines were obtained from Cobioer Company (Homo sapiens genome and transcriptome (gencode v19) using RNA STAR software (v2.4.0). The RNAseq datasets presented in this study can be found in online repositories. The names in the National Omics Data Encyclopedia and Project ID OEP000975 can be reached.Libraries for transcriptome sequencing (RNA sequencing) were constructed with NEB Next Ultra RNA Library Prep Kit for Illumina (NEB). Poly(A) tailed mRNA molecules were enriched from 1 μg total RNA with NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit. The mRNA was fragmented into approximately 200 base pair pieces. The first-strand cDNA was synthesized from the mRNA fragments reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized using DNA polymerase I and RNase H. The end of the cDNA fragment was subjected to an end repair process that included the addition of a single ‘A’ base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit  and an Agilent 2100 Bioanalyzer. After quantitative reverse transcription-polymerase chain reaction (RT-qPCR) validation, libraries were subjected to paired-end sequencing with pair end 150-base pair reading length on an Illumina HiSeq sequencer (Illumina). First, the sequenced reads in raw fastq format data were mapped to the hg19 The HCC78 cell lines were plated at 40% to 50% confluence in 12-well plates and incubated for 24 h before transient transfection for 72 h with siRNAs mixed with Lipofectamine reagent (Invitrogen). The siRNAs specific for ROS1 mRNA  and a nonspecific siRNA (5′-GUUGAGAGAUAUUAGAGUU-3′) were obtained from RUIBIO Inc.Cells were stained with biotinylated monoclonal antibodies against human PD-L1 (Biolegend) and with phycoerythrin-labeled streptavidin (eBiosciences) for flow cytometric analysis with Cytoflex instrument (Beckman coulter). The data were analyzed using FlowJo software.Western blotting analysis was performed as most described. Protein estimation was performed by using Thermo Pierce™ BCA Protein Assay Kit (cat NO.: 23225). Rabbit polyclonal antibodies against human phosphorylated ROS1 (Y1604), phosphorylated EGFR (Y1068), phosphorylated ERK, phosphorylated signal transducer and activator of transcription 3 (STAT3), and glyceraldehyde-3-phosphate dehydrogenase were obtained from Cell Signaling Technology. All antibodies were used at a 1:1000 dilution. Horseradish peroxidase-conjugated goat antibodies against rabbit immunoglobulin G were obtained from Abcam. Immune complexes were detected with the use of Pierce Western Blotting Substrate Plus (Thermo Scientific) and Las-mini 4000 system (GE Inc.).2×0.5 was used to calculate tumor volume (mm3). For the analysis of the effect of ICI therapy on ROS1 fusion or ROS1 G2032R resistance mutation, tumor-bearing animals were treated with a single dose of 100 µg per mouse by intratumoral injection.Ba/F3 Tumor Model: Ba/F3 ROS1 fusion and Ba/F3 ROS1-G2032R resistant mutation cell lines were implanted subcutaneously into the right flank of female C3H mouse  buffer). Mice were treated once daily by oral gavage with vehicle (PBS buffer) or crizotinib (25 mg/kg) was administered orally to tumor-bearing mice daily for 28 consecutive days, and tumor volumes were monitored every 2 days for 4 weeks. L×WIn this study, HCC78 cell line was selected for its SLC34A2–ROS1 fusion gene, whereas the other lung cancer cell lines are wild type for ROS1. To investigate the effect of ROS1 fusion on PD-L1 expression, we conducted two cell experiments by transfecting HCC78 cells with siRNA specific for SLC34A2–ROS1 fusion or treating HCC78 cells with crizotinib. Immunoblot analysis with antibodies of the total forms of ROS1 confirmed the downregulation of ROS1 and PD-L1 expression after using siRNA specific for SLC34A2–ROS1 . PD-L1 expression in HCC78 and HCC78CR1–5 was analyzed by flow cytometry  and immunoblotting  to confirm the cell surface and total expression, respectively. The HCC78CR1–5 sublines showed remarkably higher levels of PD-L1 than their parental HCC78 cell line. HCC78CR1–5 cell lines showed higher levels of PD-L1 protein compared with the HCC78 cell line, as shown by Western blotting, along with the upregulated expression of ROS1 fusion. These data overall suggest that the increased expression of PD-L1 is associated with ROS1 fusion.To further explore the relationship of PD-L1 expression and ROS1 fusion in crizotinib resistance, we cultured five HCC78 crizotinib resistant cell lines (HCC78CR1–5). Crizotinib resistance of HCC78CR1–5 was detected by Cell Counting Kit-8 with half maximal inhibitory concentration (CCK8 IC50) . HCC78CR1 harbored the most genes, of which 234 were upregulated, and 466 were downregulated. HCC78CR1–5 shared 145 genes, which were speculated to be related to the expression of PD-L1 in acquired drug resistance without resistant mutation. Enrichment analysis of the pathway showed that the HCC78CR1–5 cell lines are more immune sensitive to PD-1 signaling, cytokine signaling in immune system, and immune system compared with the HCC78 cells . Western blotting analysis showed that after the ROS1 fusion group was treated with crizotinib and anti-PD-L1, the expression of PD-L1 in the tumor immune micro-environment decreased, whereas the expression was up-regulated in the G2032R group , and T-cell immunoglobulin mucin-3 (TIM3) on Th and Tc cells was performed in blood and tissue  is the standard ; combinavia oncogenic signaling pathways, such as RAS/RAF/MEK/mitogen-activated protein kinase-ERK , which is hosted by the Chinese Academy of Sciences (CAS), under the Project ID OEP000975.Data Access: All the RNA-sequencing data of HCC78 and HCC78CR1-5 have been deposited at the National Omics Data Encyclopedia , the National Natural Sciences Foundation Key Program (81630071), the National Natural Science Foundation of China (81771689 and 81900194), the Ministry of Education Innovation Team Development Project (IRT-17R10), the CAMS Key Lab of Translational Research on Lung Cancer (2018PT31035), the CAMS Innovation Fund for Medical Sciences (CIFMS 2016-I2M-3-008), the Aiyou Foundation(KY201701), and the Wujieping Foundation(320675018477).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since the pioneering NCI-60 panel of the late'80's, several major screenings of genetic profiling and drug testing in cancer cell lines have been conducted to investigate how genetic backgrounds and transcriptional patterns shape cancer's response to therapy and to identify disease-specific genes associated with drug response. Historically, pharmacogenomics screenings have been largely heterogeneous in terms of investigated cell lines, assay technologies, number of compounds, type and quality of genomic data, and methods for their computational analysis. The analysis of this enormous and heterogeneous amount of data required the development of computational methods for the integration of genomic profiles with drug responses across multiple screenings. Here, we will review the computational tools that have been developed to integrate cancer cell lines' genomic profiles and sensitivity to small molecule perturbations obtained from different screenings. Builtissues) . All canolecules –11. Convstration –14. AlthInspired by the NCI-60 project, several collaborative efforts scaled up the number of cancer cell lines investigated in pharmacogenomics studies from the original 60 to more than 1,400, planning to reach over 10,000 publicly available cancer models in the near future . The mashttps://discover.nci.nih.gov/cellminercdb/) expands the analysis power of CellMiner, the original NCI-60 analysis tool, with the integration of the cancer cell line data from the Sanger/Massachusetts General Hospital GDSC, the Broad/Novartis CCLE, and the Broad CTRP  and the response to several DNA-targeted anticancer drugs as platinum derivatives, topoisomerase inhibitors, and poly (ADP-ribose) polymerase (PARP) inhibitors  is a web-based tool designed for the integrative analysis of drug response, mutations, and gene expression profiles derived from the NCI-60 consortium and the CCLE , gene mutations associated to drug responses (from drug to gene), and drugs associated to active gene signatures (from signature to drug). Starting from a drug correlated to gene mutations, gene expression profiles can be used to identify genes differentially expressed in cell lines sensitive to the selected compound. The statistics behind GDA is based on drug response data. Basically, all pairs of cell lines and drugs are defined as responsive if the relative sensitivity is smaller than two standard deviations of the left tail of the distribution of all relative sensitivities, and non-responsive otherwise. Based on genomic data, cell lines are classified as mutant if treated with the compound and carrying the selected set of mutations and as wild type if treated with the compound but without the specific set of mutations. Given these classifications, compounds are ranked using a score defined by the fraction of responsive in mutant multiplied by the fraction of non-responders in wild type. This score ranks each drug based on the enrichment of responsive in the mutant group. The statistical significance of this ranking is computed using a one-tailed Fisher's exact test for the enrichment of responsive in mutant as compared to non-responsive in wild type, given the number of non-responsive in mutant and responsive in wild type. Results are accessible through interactive graphical representations and tables and can be directly fed to external tools as Enrichr for functional annotation  was one of the first computational resources developed for the investigation of connections between transcriptomics and drug-induced perturbations (https://clue.io). In CLUE, the Query tool allows to input a gene signature  and search for perturbagens  that induce a similar (or opposite) expression profile in the treated cells. The statistical significance of the association is assessed through a connectivity score that takes into account the strength of the similarity between the query and the induced signature as compared to the enrichment of all other signatures in the database (CMap (rbations . As exterbations , the goarbations , 32. Therbations . This nedatabase . This apdatabase . From itEfforts to decipher the molecular mechanisms of cancer stimulated scientists to explore the interconnection between the genomic landscape of cancer models and their response to drug treatments. This resulted in large pharmacogenomics screenings that, with the advent of high-throughput technologies, generated large amounts of genomics and pharmacological data. However, the integration of these precious information is still challenging due to the variable type and number of drugs and cancer cell lines that have been screened by the various projects and the heterogeneous assays used for drug testing in the different studies , 33–35. JC and SB conceived the project. JC, MD, and SB wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors . Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression. Cutaneous melanoma is an aggressive disease representing one of the leading causes of mortality related to human cancers worldwide.In the recent years, several molecular alterations have been identified as occurring during melanoma initiation and progression , 2. The While the effects of the V600E mutation of BRAF have been extensively studied in experimental systems, several aspects of BRAF function remain poorly understood. Very few studies have investigated the role of BRAF expression in melanoma.Major advances have been made in the clinical management of melanoma patients carrying BRAF mutations with the adoption of two BRAF specific inhibitors, namely vemurafenib and dabrafenib, approved by the Food and Drug Administration (FDA) in 2011 and 2013, respectively. The correct identification of cancer driving mutations is of paramount importance in cancer diagnostics as this allows the appropriate selection of target treatments and the implementation of personalized therapies . Of noteWhen performing RNA-seq and Sanger sequencing on 15 melanoma cell lines, we found discordance between the results obtained by the two methods for three of the cell lines analyzed, specifically, these three cell lines showed a BRAF V600E mutation by Sanger sequencing but the mutation was not detected by RNA-seq. This prompted us to investigate whether this was due to a differential expression of BRAF WT and V600E alleles and whether these cell lines showed a different responsiveness to BRAF inhibitors as compared to the concordant WT and V600E mutant cell lines. We have tested three additional cell lines to confirm the data.2 with RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Gemini Bioproducts), 0.01% GPS , and 0.01% fungizone .Melanoma cell lines were derived from metastatic melanoma lesions from patients treated at the Surgery Branch, National Cancer Institute (NCI), Bethesda, MD and kindly donated by Dr Steven A. Rosenberg. All patients signed an informed consent approved by the Institutional Review Board of the National Cancer Institute. Two cell lines were purchased from ATCC, namely A375 and SKMEL28; one additional cell line, PIG1, immortalized from human melanocytes was kindly donated by Dr. Caroline Le Poole from Northwestern University, Chicago, Illinois, USA. These three cell lines were tested for validation purposes. Cells were cultured at 37 °C, in 5% COGenomic DNA was isolated using QIAamp DNA Mini Kit  according to the standard protocol. DNA quality and quantity were estimated using Nanodrop .http://frodo.wi.mit.edu/):Each sample was screened for mutations in exons 11 and 15 of BRAF gene and exon 1 and 2 of NRAS gene. PCR was performed in a 20 µl final volume, containing 50 ng of genomic DNA, 10 µl of Qiagen HotStarTaq Master Mix Kit  and 500 nM of forward and reverse primers with the following cycling conditions: initial denaturation at 95 °C for 10 min; 35 cycles at 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s; final step 72 °C for 10 min. Primers were selected using Primer3 software  and labeled using Big Dye terminator kit v3.1 . Excess dye terminators were removed using DyeEx 96 Kit columns following the manufacturer’s instructions (Qiagen). Sequencing was performed using Biosystems 3730 Genetic Analyzer  and analyzed by Sequencher software .® Poly (A) Purist™ MAG Kit). After the mRNA was fragmented in short fragments (approximately 200 bp), cDNA was synthesized by random hexamer primers (Illumina TruSeq Stranded mRNA Library Prep Kit). The double-stranded cDNA was purified by QiaQuick PCR extraction kit (Qiagen) and went through an end repair process with the addition of a single ‘A’ base, and then ligation of the adapters. These products were then purified by agarose gel electrophoresis and enriched with PCR to create the final cDNA library. The library products were sequenced and 300 bp sequences were generated via the GAIIx Illumina sequencing platform. Raw reads were imported on a commercial data analysis platform CLC Genomics Workbench . An average of 20 million reads were generated for each sample run as per Illumina recommendations. Quality control checks on raw sequence data from each sample were performed using the QC analysis application tool. Adapter trimming was done to remove ligated adapter from 3′ end of the sequenced reads with only one mismatch allowed. After reads have been processed to meet a quality standard, they were aligned to the Human reference genome UCSC-Hg19, using the ultra high-throughput short aligner provided by CLC bio software. A Transcript Discovery analysis was performed to generate a transcript annotation file with an estimation of the relative abundances of each transcript by counting the number of reads that mapped to the genomic location of that transcript. Transcription level assessment has been obtained by the number of fragments per kilobase of transcript per million fragments mapped (RPKM).Total RNA was isolated from the cell lines using miRNeasy minikit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity were estimated using Nanodrop (Thermo Scientific) and Agilent 2100 Bioanalyzer . A A260/A280 ratio of ~ 2.0 was considered indicative of RNA of good purity. A RIN  of 7 was set as cut off for RNA quality. Only samples with RIN > 7 were used for further experiments. Enrichment in mRNA molecules was obtained by using oligo (dT) magnetic beads  and the control WT (MEL-2805) and V600E (MEL-2492) cell lines were treated with BRAF inhibitors prior to running the proliferation assay and DNA and RNA mutational testing.6 cells were seeded in complete medium the day before treatment and incubated overnight at 37 ℃ with 5% CO2. Two BRAF-specific (PLX4720 and Vemurafenib) and one BRAF nonspecific inhibitor (Sorafenib) were purchased from Selleckchem, Houston TX and used for the inhibition treatment. Final concentration of PLX4720, Vemurafenib and Sorafenib were respectively 1 μM, 2 μM and 5 μM. Upon treatment, cells were used for DNA and RNA isolation (see below). Each experiment was done in triplicate. The same experiments were repeated on three additional cell lines for reproducibility, namely A375, SKMEL28 and PIG1. A375 and SKMEL28 are commercially available cell lines carrying the BRAF V600E mutation. PIG1 is a cell line transformed from normal epidermal melanocytes.About 5 x 103 cells in 100 µl of complete medium were seeded into each well of a 96-well tissue culture plate and incubated overnight at 37 ℃ with 5% CO2. Cells were treated with indicated inhibitors in serum-free medium for a period of 24 h, 48 h and 72 h. Each treatment condition was done in triplicate. Then 20 µl of MTT (5 mg/ml) were added to each well and plates were further incubated for 4 h at 37 ℃. The formed crystals were solubilized by addition of 150 µl of DMSO in each well and cells agitated on an orbital shaker for 20 min. The optical density was measured using a spectrophotometer at a wavelength of 570 nm. The BRAF inhibition experiment was repeated at least three times, in which each cell line was assessed in triplicate.The MTT assay is a colorimetric test used to assess the cell metabolic activity . The NADTotal RNA from the 15 cell lines was extracted using miRNeasy minikit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity were estimated using Nanodrop (Thermo Scientific) and Agilent 2100 Bioanalyzer . A RIN of 7 was set as cut off for RNA quality. Only samples with RIN > 7 were used for further experiments. First- and second-strand cDNA were synthesized from 300 ng of total RNA according to manufacturer’s instructions (Ambion WT Expression Kit).Genomic DNA was isolated using QIAamp DNA Mini Kit  according to the standard protocol. DNA quality and quantity were estimated using Nanodrop . Only DNA of A260/A280 of ~ 1.8 were processed for downstream analyses.Allele specific PCR was performed to detect BRAF V600E mutation and wild type BRAF sequence. Two PCR reactions  were performed for each sample. For the RNA allele-specific PCR, the reverse primer was used for both the PCR reactions and designed spanning exon 15 and exon 16. The forward primers were designed to possess two bases substitution at 3′-end compared to wild-type sequences. These primers were, respectively:BRAF15_R: 5′-GATGACTTCTGGTGCCATCC-3′BRAF15_WT_F: 5′-TAGGTGATTTTGGTCTAGCTACAGT-3′BRAF15_V600E_F: 5′-GGTGATTTTGGTCTAGCTACAAA-3′For the DNA PCR, primers were designed as previously reported .-(delta Ct) method and GAPDH as housekeeping gene.The PCR reaction was performed in a 25 μL final volume, containing 200 nM of forward and reverse primers, 2 μL of cDNA, 12.5 μL of 2× GoTaq MasterMix , which included BRYT Green as intercalating dye. In each reaction 0.3% HiDi formamide  was added to increase primer-annealing specificity. Cycling conditions were as following: initial denaturation at 95 °C for 10 min; 40 cycles at 95 °C for 15 s, 60 °C for 1 min. Reactions were run on a 7500 Fast Real-Time machine . To verify primers specificities, melting curves were generated at the end of PCR reaction. Fluorescent data were acquired during the extension phase. After 40 cycles, a melting curve for each gene was generated by increasing the temperature from 60 °C to 95 °C (1 °C for each step), while the fluorescence was measured. For each experiment a no-template reaction was included as a negative control. The qPCR data was analyzed using the 2Cells were harvested in a cell lysis buffer containing 20 mM Tris, pH 7.4, 1% Triton X-100, 40 mM NaF, 2 mM EDTA, 1 mM EGTA, 1 µg each of pepstatin, leupeptinand aprotinin, 1 mM PMSF, 1 mM NaVO4, 50 mM β-glycerophosphate, 40 mM p-nitrophenyl phosphate, and 1 mM DTT. The cell lysates were cleared of insoluble material by centrifugation at 14,000 g for 10 min at 4 ℃. The protein quantity was estimated by BCA Protein Assay Kit . Approximately 50 µg of protein was used in Western blotting. The Western blotting protocol was similar as described previously . Equal lt-test was used to determine differences of cell lines’ responsiveness to the inhibitors at day 3. ANOVA  test was used to evaluate the effects of BRAF inhibitors. All the analyses were performed using Statgraphics Centurion .Paired two-tailed Student’s Eight out of fifteen (53%) cell lines harbored the BRAF V600E mutation. Among the seven cell lines that did not display BRAF mutation, six cell lines harbored activating mutation in NRAS gene . BRAF and NRAS mutations were mutually exclusive in the cell lines tested.RNA isolated from the 15 cell lines was tested by RNA-seq. Discordance between Sanger sequencing and RNA-seq was observed for BRAF but not for NRAS mutation assessment. Table Out of the fifteen melanoma cell lines tested, twelve cell lines showed concordant results between Sanger sequencing and RNA-seq. Three cell lines, namely MEL-2523, MEL-3025 and MEL-3104, were mutated by Sanger sequencing but wild type by RNA-seq. Of note, there were no cell lines in which the mutation was detected only by RNA-seq but not by Sanger sequencing. Thus, we hypothesized that in these three “discordant” cell lines the wild type allele was the only one to be expressed at the RNA level.To test this hypothesis, we assessed the BRAF mutational status at the RNA level by allele specific qPCR. The three discordant cell lines resulted mutated by RNA allele specific qPCR, consistent with the Sanger sequencing data. This suggested that the mutated allele was also expressed at the RNA level in contrast to our hypothesis.A closer look to the RNA-seq data Table  showed tt-test. ANOVA test was also performed to evaluate the combined effect of the BRAF inhibitors  whose mutation assignment was concordant between RNA-seq and Sanger sequencing Fig. . We haveWe have further explored the responsiveness to BRAF inhibitors in three additional cell lines, namely A375, SKMEL28 and PIG1. The mutation analysis revealed V600E mutation for both A375 and SKMEL28 while PIG1 was homozygous V600 wild-type. Consistent with the assumption that BRAF mutated cell lines respond to BRAF-specific inhibitors, A375 and SKMEL28 showed responsiveness to PLX4720 and vemurafenib but they did not respond to the non-specific inhibitor. Conversely, PIG1 proliferation curves did not show differences across all the inhibitor conditions, suggesting that the BRAF specific inhibitors had no effect on PIG1  as compared to the control WT and V600E cell line Fig. . The WT We have tested the expression of phosphorylated-ERK (p-ERK) protein as marker of BRAF activation, and consequently of MAPK pathway activation. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors Fig. . OverallThe assessment of the sensitivity and specificity of mutation detection of different methods is particularly relevant in the diagnostic setting. As an example, the identification of BRAF V600E mutation is important to indicate which melanoma patients will likely respond effectively to vemurafenib treatment , 19. OthSince the discovery of BRAF mutations in melanoma by Davies and colleagues in 2002 , severalHere we aimed at assessing whether a lower overall BRAF mRNA expression may correlate with a lower responsiveness to BRAF inhibitors. To our knowledge this question has not been previously investigated. We found here that three cell lines whose mutation assignment was discordant from the DNA and RNA levels and that express very low level of BRAF mRNA respond less to BRAF V600E specific inhibitors.The response to BRAF inhibitors can be very different from patient to patient , 22. A mTwo studies have assessed BRAF protein expression in melanoma , 24. TheAdditionally, the issue of intratumoral heterogeneity has not been assessed in this study, we are planning to investigate whether the presence of different clones in the same cell line may account for the discordant mutation assignment.We show here that a low BRAF mRNA expression may explain a lower responsiveness to BRAF inhibitors in melanoma cell lines. These finding should be validated in additional studies with cohorts of a bigger size and employing tumor tissues rather than cell lines.Additional file 1: Table S1. ANOVA test between treatments at time point 3d (day 3).


Based on recent advances in organoid research as well as the need to find more accurate models for drug screening in cancer research, patient-derived organoids have emerged as an effective in vitro model system to study cancer. Showing numerous advantages over 2D cell lines, 3D cell lines, and primary cell culture, organoids have been applied in drug screening to demonstrate the correlation between genetic mutations and sensitivity to targeted therapy. Organoids have also been used in co-clinical trials to compare drug responses in organoids to clinical responses in the corresponding patients. Numerous studies have reported the successful use of organoids to predict therapy response in cancer patients. Recently, organoids have been adopted to predict treatment response to radiotherapy and immunotherapy. The development of high throughput drug screening and organoids-on-a-chip technology can advance the use of patient-derived organoids in clinical practice and facilitate therapeutic decision-making. Organoid research has been an emerging field in the past decade since organoids have the potential to provide better cancer drug screening. While cell culture is important for determining whether or not a drug should advance to clinical trials , 2, a stSeveral reviews detail the advantages of using PDOs. In a review from 2017, the researchers describe how PDOs allow us to study the cells’ stem-like properties since they can maintain the chemoresistance and genetic mutations observed in the original tissue . Dutta eMany primary research articles have also been published on this topic. Lie et al.  publisheCompared with other reviews, the current review focuses on the use of organoids in clinical application. Specifically, it focuses on PDO drug screening reported in previous studies and presents a detailed table of the specific drugs and organoids that have been tested. The authors hope that such tables can provide clinicians and scientists with an overview of important PDO models for drug screening. Furthermore, compared to Rossi et al.’s  discussiThere are four commonly used in vitro tumor models Fig. . Each tu5 to 107 cells that are cultured from tissue form these immortal cell lines [Cell lines are derived from cells that have acquired oncogenic mutations that permit them to grow indefinitely . It is ell lines .In 2012, Barretina et al.  publisheSeveral alternatives to 2D methods of cell culture have been established. One of these is the 3D culture of cell lines to form spheroids. Studies have shown that 3D cell cultures show improved cell morphology, mechanical properties, differentiation, and viability compared with those of 2D culture , 2. DrugThe development of primary cell cultures provides a more personalized way of culturing cells, as it allows scientists to use cells that correspond to individual patients, rather than using one patient sample to represent all patients with that particular disease. Culturing primary cells involves growing cells from fresh patient tissues and using those that grow successfully. Unfortunately, culturing primary cells presents many problems. Many of the cells stop growing shortly after culturing because they undergo a process called ‘senescence’ , which is characterized by growth arrest, preserved chromosome integrity, and gradual death, or ‘crisis’ , which is characterized by chromosome instability and high rates of cell death . FibroblThe establishment of PDOs has offered great potential in cancer research, since the year 2013. A PubMed search shows that the term “patient-derived organoids” in published studies begin to appear (n > 4) in 2013, during which five papers had been published on the subject. The number of papers is steadily increasing over the years, and by 2019, about 260 papers had been published. PDOs can provide the advantages found in both 3D spheroids (the improved morphology), primary cell lines (each culture can represent the patient it came from), and many more.Briefly, the culture of PDOs begins with mincing up the patient tissue and plating the cells in drops of a solid jelly-like substance that is usually Matrigel or Basement Membrane Extract. These substances are two kinds of solubilized basement membrane extracts derived from Engelbreth-Holm Swarm mouse sarcoma consisting of laminin, collagen IV, entactin, and heparan sulfate proteoglycans , 11. TheTo begin with, gene expression profiles tend to remain more stable in PDOs. To illustrate this, seven prostate organoid lines showed identical mutational landscapes to those of their corresponding tumor tissues after three months of in vitro culturing, harboring similar disease-specific genomic alterations as the tumor tissue . In anotThe reproducibility of PDOs is also promising. Sachs et al.  generateIn cancer treatments, drugs are used before and after surgical removal of the tumor. These drugs are mainly used in chemotherapy, targeted therapy, radiotherapy, and recently, immunotherapy . IndividResearchers are now further confirming the usefulness of organoids by showing their in vitro to in vivo correlations. This has been done by performing co-clinical trials in which parallel studies looking at drug responses in patients are compared to the drug responses in the corresponding pre-clinical models . The proUsing mice, Broutier et al.  showed aNumerous studies in large-scale drug screens have been published, testing the efficacy of different drugs by looking at the IC50 of the drugs. A list of these drug screens is presented in Table With the recent development of drug screens with PDOs, other cancer treatments, such as radiotherapy, have also been investigated. Yao et al. studied the ability of organoids to predict drug response to chemoradiation in rectal cancer. They found that chemoradiation responses in patients were highly matched (85%) to the organoid responses, with the sensitivity data of 68 organoids matching their corresponding patient outcomes while 12 organoids did not . AnotherUsing organoids to study immunotherapy has been an emerging technique. In a study published in December 2019, Cattaneo et al.  describeAn important factor supporting the clinical applicability of organoids is efficiency and reproducibility. Efficiency is important since it allows for a quick turnaround time from the time of the biopsy to know the type of therapy that is most suitable for the patient. As described in previous studies, organoid culturing involves individually digesting, plating, and passaging each organoid for each patient , 27, 28.High throughput screening methods have allowed for rapid drug screens. For example, liquid handling robotics have been used to generate a dilution series for each compound for PDO drug screening . These rOrganoids-on-a-chip technologies have also been developed, in which organoids are grown in microfabricated cell culture environments. Briefly, an organ-on-a-chip is a recreation of an organ by putting together its basic elements and functional units. This includes the different cell types, structural organization, and microenvironment . An orgaThe emergence of organoid technology provides new and promising clinical applications. PDOs can provide a more accurate platform for drug screening to help clinicians decide the most suitable treatment regimen for their cancer patients. Numerous studies have shown promising correlations between organoid responses and patient responses to cancer treatments. High throughput screening and organoids-on-a-chip are new technologies that facilitate the widespread use and clinical application of organoids.


Protaetia brevitarsis larvae (PBL) and evaluated their fermentation levels. After pretreatment with the paste and sauce extracts, HepG2 cells were damaged with ethanol (EtOH), and then the effects of the paste and sauce were evaluated. As a result, we confirmed that the PBL paste and sauce extracts reduced the aspartate aminotransferase (AST) and alanine aminotransaminase (ALT) content in the medium as compared to soybean (Glycine max) sauce and paste extracts. In addition, the PBL paste and sauce extracts significantly lowered the level of tumor necrosis factor (TNF)-α and interleukin (IL)-6, which are biomarkers of inflammation, and significantly increased the inhibition rate of superoxide dismutase (SOD) and reduced glutathione (GSH), which are antioxidative indicators, in proportion to the amount of PBL added to the paste and sauce. These results suggest that an intake of PBL paste and sauce, a novel type of fermented food made from insects, may be effective for liver protection through anti-inflammatory and antioxidative effects against hepatocyte injury caused by EtOH. We made paste and sauce using protein-rich  Protaetia brevitarsis larvae (PBL), Tenebrio molitor larvae (TML), Allomyrina dichotoma larvae, Zophobas morio larvae, Gryllis bimaculatus, Bombyx mori larvae and pupae, and Oxya chinensis sinuosa can now be legally used as edible insects. Accordingly, studies have been actively conducted in Korea to develop various types of food using insects.Insects are emerging as a new protein-rich food source because of the many advantages they possess as a protein food . They arP. brevitarsis is an insect belonging to the Coleoptera Scarabaeidae family. It has already been proven as non-toxic through good laboratory practice (GLP) procedures [P. brevitarsis has been used for a long time in Korea as a traditional medicine for liver disease, as its efficacy on the liver had been recorded in the old Korean medical book entitled Donguibogam in 1613 [ocedures ,5 and reocedures . PBL is ocedures . P. brev in 1613 . On the  in 1613 .Glycine max) sauce (Ganjang) and paste (Doenjang) are made from soybean koji, which is fermented by inoculating microorganisms or inducing the natural attachment of microorganisms. Fermented soybean koji is aged in salt water for 2–3 months, and the liquid and solid are separated from each other. The liquid is Ganjang, and the solid is Doenjang [Soybean  PBL, which are registered as edible insects and a type of new food raw material in Korea [In this study, we evaluated the fermentation degree of PBL koji fermented by inoculation of in Korea . A. oryzae was bought from Chungmu Fermentation . The soybeans were immersed in distilled water for 12 h and braised for 1.5 h. For the preparation of koji, the steamed PBL and soybeans were used alone or mixed at a ratio of 3:7 or 1:1, mashed, and inoculated with 0.2%, 0.4%, and 0.8% A. oryzae. Afterward, the PBL and/or soybeans were molded into a square frame and fermented for 72 h at 30 °C, with a relative humidity of 70%. After fermentation, the samples were dehydrated in a dry oven for 24 h at 60 °C, completing the koji preparation. Subsequently, the koji was soaked in 20% salt water for 50 days. After 50 days, the paste (solid) and sauce (liquid) were separated, and then the sauce was sterilized at 110 °C. Depending on the proportion of PBL added into the koji, we made PBP100 (100% PBL paste), PBP50 (50% PBL/50% Glycine max (GM)), and PBP30 (30% PBL/70% GM) pastes and PBS100 (100% PBL sauce), PBS50 (50% PBL/50% GM), and PBS30 (30% PBL/70% GM) sauces for this study. The GMP100 (100% GM paste) and GMS100 (100% GM sauce) used in this study were purchased from SEMPIO, Seoul, Korea.The soybean  was cropped in Sunchang, Korea, and w/v). The diluted samples were mixed for 1 h using a shaker, centrifuged at 4000 rpm for 10 min, and then filtered using Whatman® No.1 filter paper for primary filtration and a 0.45 μm syringe filter for secondary filtration.The koji prepared in this study was milled and diluted with distilled water at a ratio of 1:10 (w/v), and extracted over 24 h. After centrifugation at 3000 rpm for 10 min, the supernatant was filtered using Whatman® No.1 filter paper for primary filtration and a 0.45 μm syringe filter for secondary filtration. Afterward, the supernatant was concentrated using a speed vacuum  for 48 h and dissolved in 20% DMSO. The sauce was centrifuged at 3000 rpm for 10 min and filtered using Whatman® No.1 filter paper for primary filtration and a 0.45 μm syringe filter for secondary filtration. Each koji, paste, and sauce extract was applied to all the experiments in this study.The paste made from koji was freeze-dried, milled, diluted with 95% EtOH at a ratio of 1:5 , and protease activity was determined using a Pierce™ Protease Assay Kit , according to each supplier’s protocol. The extracted samples were analyzed for free amino acids, according to the method of the Korean Food Standard Codex . The prepared samples were blended with 16% trichloroacetate and rocked for 15 min. After centrifugation at 3000 rpm for 15 min, the analysis of free amino acids by liquid chromatography  was performed with the supernatant. 6 cells per well of a 6-well plate, and incubated for 24 h. Afterward, the media were exchanged with 2 mL of DMEM containing 10% FBS, 1% AA, and the paste or sauce extract. After incubation for 4 h, the media were changed with 2 mL DMEM containing 10% FBS, 1% AA, and 300 mM EtOH. Each well was covered with parafilm to prevent EtOH vaporization and incubated for 24 h. After incubation, the cells and media were divided by centrifugation  and applied to all the experiments in this study. HepG2 cell viability was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS; Promega, Madison, WI, USA). HepG2 cells were incubated at 37 °C in a 5% carbon dioxide atmosphere with DMEM  containing 1000 mg/L glucose, 10% FBS, and 1% antibiotic–antimycotic (AA) . When the cell saturation reached 80% confluence, the cells were suspended in trypsin, plated at 1 × 10The media isolated in ® Reagent , and cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit . TNF-α and IL-6 levels were measured by the BrightGreen 2X qPCR MasterMix-No Dye , according to the supplier’s manual. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control, and relative gene expression levels were analyzed using the 2−ΔΔCt method. Primers for GAPDH , TNF-α , and IL-6  were used for cDNA amplification [The total RNA of the HepG2 cells was isolated using TRIzolfication .The cells isolated in t-test. SPSS version 18  was used for analysis. Statistical significance was defined at 0.05 probability.The results are indicated as the mean ± standard deviation (SD), and all measurements were repeated three times. Differences between groups were tested with the Student’s A. oryzae and fermentation time. The amino nitrogen content and protease activity increased in proportion to the quantity of A. oryzae inoculum and fermentation time. The greatest value of 0.64 mg/mL was measured when the PBL koji had been fermented for 72 h after inoculation with 0.4% A. oryzae , PBP100 (100% PBL paste), PBP50 (50% PBL/50% GM), and PBP30 (30% PBL/70% GM) as pastes and GMS100 (100% GM sauce), PBS100 (100% PBL sauce), PBS50 (50% PBL/50% GM), and PBS30 (30% PBL/70% GM) as sauces.Amino nitrogen content, protease activity, and free amino acid content were measured to evaluate the degree of PBL fermentation by the inoculation amount of . oryzae A. For th. oryzae A. As a r. oryzae B. TherefTo evaluate the degree of aging in the PBL pastes and sauces made with PBL koji, the amino nitrogen and protease activity were measured in each PBL paste and sauce . The amip < 0.001 was shown in the 300 mM EtOH concentration, 300 mM was determined as the concentration of EtOH that causes toxicity to hepatocytes . The ALT levels in the EtOH-damaged HepG2 cells after pretreatment with PBP30, 50, and 100 extracts were significantly decreased compared with only EtOH-treated HepG2 cells (34.46 IU/L). The ALT levels in the cells pretreated with GMP100, PBP30, PBP50, and PBP100 extracts were 24.59, 27.62, 21.27, and 19.75 IU/L, respectively. Likewise, for the pretreatment with GMS100, PBS30, PBS50, and PBS100 extracts, the ALT levels were 26.43, 25.66, 20.55, and 19.22 IU/L, respectively. These results showed that the AST and ALT levels in the EtOH-injured HepG2 cells after pretreatment with PBL paste and sauce were lower than those after treatment with GM paste and sauce, except PBP30. The ALT levels showed a decrease similar to that of silymarin (18.24 IU/L) when the larvae contents were 50% and 100%. As the content of insects increased in the paste and sauce, it was found that the AST and ALT levels decreased. In particular, it was confirmed that the reduction effect was highest in the paste and sauce made with 100% PBL. Although the crude extract of the paste and sauce was used, the hepatoprotective activity was similar to that of silymarin, a single substance. In addition, when the content of insects was more than 50%, the AST and ALT reduction effects were greater than those of the GM-based paste and sauce.The AST levels in the EtOH-injured HepG2 cells after pretreatment with GMP100, PBP30, PBP50, and PBP100 significantly decreased compared to only EtOH-treated HepG2 cells (56.45 IU/L). The AST levels in the cells pretreated with GMP100, PBP30, PBP50, and PBP100 extracts were 36.11, 37.35, 29.69, and 25.26 IU/L, respectively. For the pretreatment with GMS100, PBS30, PBS50, and PBS100 extracts, the AST levels were 36.78, 35.18, 33.47, and 28.23 IU/L, respectively. Accordingly, pretreatment with GMS100, PBP30, PBP50, PBP100, PBS30, PBS50, and PBS100 extracts significantly decreased the AST levels in EtOH-damaged HepG2 cells compared to only EtOH-treated HepG2 cells. In addition, compared to the single substance, silymarin (23.32 IU/L), a similar decrease was observed for all treatments  were prepared using PBL larvae composed of approximately 58% protein on a dry weight basis. The hepatocyte protective effects of PBL paste and sauce, which have long been reported to have liver protection efficacy, against ethanol damage were investigated . As a result of the analysis of 16 free amino acids in koji after fermentation, all the free amino acids except arginine and tryptophan significantly increased. However, according to previous reports, during the fermentation process of GM, only glutamic acid, glycine, aspartic acid, and proline increase rapidly . TherefoWhen the amino nitrogen level and relative protease activity was compared with pastes and sauces made of GM, it was found that the proteins were further fermented in the paste and sauce made with PBL50 and PBL100 than with GM. Furthermore, when the amino nitrogen level and relative protease activity were compared with the pastes and sauces made of 30% , 50% , and 100%  of TML , it was AST and ALT hepatic enzymes have been commonly used as indices to determine hepatocyte injury severity, because they tend to increase in the blood amid necrosis and tissue destruction of hepatocytes during hepatocellular injury ,31,32,33P. brevitarsis larvae extract to rats with liver damage induced by CCl4 [In EtOH-injured hepatocytes pretreated with the PBL paste and sauce extracts, the expression grades of TNF-α and IL-6 were inhibited at similar levels to those of the pretreatment with silymarin, which has been known to have hepatoprotective, anti-inflammatory, antioxidant, and antiapoptosis effects ,35,36,37 by CCl4 ,38,39.GSH activity and the SOD inhibition rate demonstrated that the pretreatment with the PBL paste and sauce extracts had a greater antioxidant effect on the EtOH-injured hepatocytes than GMP or GMS extracts. As the GSH enzyme in the liver plays a significant role in liver defense, protecting against liver damage by detoxifying many toxic compounds , we expeThe above results suggest that PBL fermented food will protect the liver by inhibiting inflammation through the suppression of cytokine expression, raising GSH activity and the SOD inhibition rate, which are biomarkers of an antioxidant effect. In addition, this activity is similar to that of silymarin, used as a positive control. In the future, it will be necessary to separate a single active substance with hepatoprotective activity from PBL paste and sauce to investigate the mechanisms for hepatoprotective effects.As a result of evaluating the efficacy of PBL after applying alcohol damage to hepatocytes pretreated with PBL paste and sauce extracts, it was found that PBL paste and sauce protects hepatocytes through antioxidant and anti-inflammatory effects. The degree of liver protection efficacy of the PBL paste and sauce was generally better than that of GM, similar to silymarin, and proportional to the amount of insect content included in the paste and sauce. ’Therefore, we suggest that PBL fermented food is a new food that not only has excellent taste and nutrition but also has the potential to assist in liver protection.


Lapatinib is a tyrosine kinase inhibitor widely used as a treatment for a Human Epidermal growth factor Receptor 2 (HER2) (+) breast cancer patients. However, when resistance is acquired through continued exposure, and it is associated with a poor prognosis for patients. In this study, we identified HSP90 as a common node for acquired resistance to lapatinib in two lapatinib resistant cell lines using proteomic analysis. Notably, in vitro and in vivo studies demonstrated synergy effect between lapatinib and an HSP90 inhibitor were observed in the estrogen receptor (+) HER2 (+) breast cancer cell only. These results could be a potential strategy for future clinical trials for HSP90 inhibitors in treatment—refractory HER2 (+) metastatic cancer patientsLapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials. Breast cancer is known as the most frequently diagnosed cancer in women worldwide and one of the main causes of death. In particular, 20 to 30% of breast cancers are classified as human epidermal growth factor 2 (HER2) positive and have aggressive biological characteristics compared to other breast cancer subtypes, reducing overall survival ,2. HER2/Now, HER2 (+) breast cancers are considered highly sensitive to chemotherapy and/or targeted agents. However, with continued exposure to therapeutics, cancer cells adaptively activate alternative survival pathways and reprogram tumor biology. However, efforts for new drug development other than HER2-directed agents are insufficient. Lapatinib is a dual inhibitor targeting both HER2 and EGFR that has an anti-cancer effect on HER2-positive breast cancer by inhibiting MAPK/Erk1/2 and PI3K/Akt downstream pathways . CurrentThis study identified the mechanism of resistance in lapatinib-resistant breast cancer cell lines using proteomics and examined whether the identified mechanisms overcome lapatinib resistance through in vitro and in vivo models. Our data suggest that HSP90 is a target for lapatinib-resistant cancers, but ER-positive subtypes may be a more suitable population in future clinical development.50 for SKBR3 and BT474 cells were 52 nM and 30 nM, respectively. In comparison, those of LR-SKBR3 and LR-BT474 were 4.5 μM and 0.7 μM, respectively  revealed that LR-SKBR3 cells were significantly enriched with “G2M checkpoint” and “E2F target” genes compared with the SKBR3 cell line A. ConverTo define acquired resistance to lapatinib in HER2 (+) breast cancer, SILAC-based quantitative phosphoproteomics were performed in lapatinib-sensitive and lapatinib- resistant cell lines A. A totaPathway and gene ontology (GO) analyses were performed with all proteins obtained from differentially upregulated phosphopeptides (>2-fold) using Enrichr. Spliceosome, mRNA surveillance pathway, and RNA transport were upregulated in LR-SKBR3, while RNA transport, and AMPK signaling pathway, mTOR signaling pathway were enriched in LR-BT474 B,C. ThesThe HSP90 chaperone complex regulates the stability, activation, and maturation of more than 200 oncogenic client proteins. So, targeting HSP90 offers the potential to simultaneously destroy many proteins involved in the lapatinib resistance mechanism ,20.50 values of lapatinib-resistant cell lines  were significantly higher than those of parent cells .To determine the effect of HSP90 inhibition, cell proliferation was evaluated after treatment with a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in HER2 (+), lapatinib-sensitive, and lapatinib-resistant cell lines. 17-DMAG treatment was effective in inhibiting cell proliferation of both lapatinib-resistant and parent cell lines in a dose-dependent manner. As shown in HSP90 inhibition still induced downregulation of HER2, EGFR, and Akt to the same extent in resistant cells compared to parent cells . Dephosp17-DMAG and lapatinib in each cell line were used to assess synergy of the two drugs in a dose-dependent manner. Synergy studies were done with the doses of lapatinib and 17-DMAG determined in the MTT assays using a single drug . Sub-IC53. In contrast, the mean tumor volume of groups treated with 17-DMAG only, lapatinib only, or a combination of 17-DMAG and lapatinib at day 53 were 727 ± 94 mm3, 738 ± 127 mm3, and 435 ± 112 mm3, respectively. A significant additive effect in tumor growth inhibition was observed with the combination of 17-DMAG and lapatinib.In vivo antitumor effects of 17-DMAG and lapatinib combinations were investigated in a xenograft model. Implanted tumors were established with parent cells and lapatinib-resistant cells on each flank. Tumor volume growth and inhibition after each treatment from 15 to 53 days after tumor implantation are plotted in 3 and it showed significant synergism with growth inhibition. After sacrifice of BT474 and LR-BT474 tumor-bearing mice, the expression level of HER2 and ER in tumors was examined by immunohistochemistry. In BT474 mice, HER2 expression (3+) and ER (Allred score 6–7) expression levels showed little difference in the group with combination 17-DMAG and lapatinib compared to vehicle (control). However, in LR-BT474 mice, HER2 expression (2–3 +) was slightly reduced in the group with combination 17-DMAG and lapatinib compared to vehicle (control), and ER (Allred score 6–8) expression was considerably reduced  breast cancer has evolved from a cytotoxic chemotherapy plus single antibody combination to a dual-targeted strategy and antibody-drug conjugates ,23. ReceIn this study, we identified that HER2 (+) breast cancer cells have different molecular mechanisms of acquired lapatinib resistance according to ER positivity at the transcriptional level. Further proteomic analysis confirmed HSP90 as an important node for acquired resistance; thus, HSP90 is a potential target for both LR-SKBR3 and LR-BT474 cells. In vitro data, however, demonstrated that combination of lapatinib and HSP90 inhibitor (17-DMAG) were synergistic in LR-BT474 only. A xenograft model showed clear synergy in suppressing LR-BT474 cells by combination lapatinib and 17-DMAG. Taken together, our results indicate that acquired resistance to lapatinib develops from different mechanisms according to ER positivity, and HSP90 can be targeted in ER (+) BT-474 cells only.The HSP90s are highly conserved molecular chaperone proteins as one of the sub-family of HSPs and have distinctive role in stability and function of conformationally labile proteins. In cancer cells, HSP90 expression is induced by microenvironmental stresses, including hypoxia, chemotherapy, and immunologic attack, rather than gene amplification or point mutation . It’s chn = 13) cohort  × (width [mm] 2)/2. Animal weights were also measured twice a week.All animal studies were approved by the Korea University Animal Care and Use Committee (# KOREA-2019-0129). First, 17-β-estradiol 60-day release pellets (Innovative Research of America) were transplanted into the left flank of athymic nude Foxn1nu female mice  1 day before tumor inoculation. After that, 5 × 10At the end of the study, tumors were fixed with 4% formalin provided by the Department of Pathology. In addition, Hematoxylin and Eosin (H&E) staining of tumor tissue and ER and HER2 staining were performed by a skilled pathology department. The HER2 and ER scores on completed slides were independently reviewed by pathologists.Graphs and statistical comparisons were performed in Graph-pad Prism v8.3 software. Multi T-test was used to compare lapatinib-resistant cell lines and parent cell lines for each drug. A two-way ANOVA with Bonferroni’s post-test was used to compare groups for mouse tumor growth. Additionally, a T-test was used to compare ER Allred scores between groups.To investigate the combination effect of lapatinib and 17-DMAG in lapatinib-resistant cells, isobologram was plotted and combination index (CI) was calculated using CompuSyn (version 1.0) software .We identified HSP90 as a common node for acquired resistance to lapatinib in both BT474 and SKBR3 cells using proteomic analysis. However, in vitro and in vivo studies demonstrated synergy between lapatinib and an HSP90 inhibitor, 17-DMAG, in LR-BT474 cells only. Further GSEA using transcriptional analysis revealed that the two HER2 (+) breast cancer cell lines have different resistance mechanisms after long-term exposure to lapatinib; estrogen response-related genes are major mechanisms in BT474 cells. The molecular mechanisms we delineated here may be a potential strategy for future clinical trials using HSP90 inhibitors in treatment for refractory HER2 (+) metastatic cancer patients.