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.


For many cancer types, including colorectal carcinoma (CRC), combined modality treatments have shown to improve outcome, but are frequently associated with significant toxicity, illustrating the need for new therapeutic approaches. Based on preclinical data, TRAIL receptor agonists appeared to be promising agents for cancer therapy especially in combination with DNA damaging regimens. Here, we present the combination of the second-generation TRAIL receptor agonist APG-880 with radiation in a new and clinically relevant 3D model system.To investigate the effect of APG-880 in combination with radiation we performed short-term cytotoxicity and long-term clonogenic survival assays in established CRC cell lines, and in tumor organoids derived from colon cancer patients.APG-880 is a potent inducer of apoptosis in CRC cell lines and in patient-derived CRC organoids. Furthermore, a supra-additive effect on cytotoxicity was found when APG-880 and radiation were combined simultaneously, with combination indices around 0.7. Lastly, in the long-term survival assays, we demonstrated a radiosensitizing effect of APG-880 with dose enhancement factors between 1.3 and 1.5.In a new, clinically relevant CRC-organoid model system we demonstrated a more than additive combined effect between the second-generation TRAIL receptor agonist APG-880 and radiation. The highest incidences occur in Western countries Radiosensitizers are agents that increase the lethal effect of ionizing radiation and, ideally, achieve greater tumor cytotoxicity than would have been expected from the additive effect of each modality. Some radiosensitizers are used in the clinic already, while others are being studied for new indications. For example, non-specific agents such as cisplatin or 5-FU have shown to improve local control and overall survival when added to radiation therapy and are integrated in standard treatment protocols Since apoptosis is an important mechanism of radiation induced cell death, modulation of apoptosis sensitivity has been shown to increase tumor cell kill and relatively spare normal tissues, thereby enhancing therapeutic outcome in vivo, which is independent of cross-linking to Fc receptors A novel, second-generation TRAIL-receptor agonist has been engineered that simultaneously binds up to six TRAIL receptors, thereby improving the ability to form receptor clustering on cancer cells to induce superior anti-tumor efficacy C release The rational for combining TRAIL and radiation in our study, is that these agents activate the two distinct pathways leading to apoptosis in vivo efficacy suggesting that this second generation molecule may be superior to previously tested TRAs Unfortunately, TRAIL-based therapies have not yet led to improved clinical responses mainly because the first-generation TRAIL receptor agonists (TRAs) failed efficacy and did not meet clinical expectations as tested in phase I and II trials Second, the model systems used over the past years to test TRAIL efficacy were suboptimal in their capacity to predict clinical activity. Indeed, 2D cell culturing techniques are now considered structurally and functionally inferior to mimic cancer and are of limited use to predict successful clinical translation 22.1APG-880 stock solution (10.6 mg/ml) was provided by AbbVie , aliquoted in 2 μl portions and stored at −80 °C. Thawed samples were only used once.2.2Colon cancer derived cell lines HCT116 and HT29 were purchased from the American Type Culture Collection (ATCC). Jurkat cell line J16, were kindly provided by prof. dr. J. Borst . All cell lines were grown according to ATCC protocols.2.3Surgical specimens (in case of organoid cultures ITO17 and ITO60), or core needle biopsy material (in case of organoid culture ITO77), collected within a clinical trial at the NKI (NL48824.031.14) were dissected, stored in the central biobank and used for the establishment of organoids. For our experiments we used three different biobank-stored organoids from three different patients. After institutional approval, the biobank stored organoids ITO17, ITO60 and ITO77 were thawed and cultured according to the protocol earlier described Regarding the organoids used in this study, organoid ITO77 was derived from a peritoneal metastasis and organoids ITO17 and ITO60 originated from a primary colorectal carcinoma. Their mismatch repair (MMR) status is known: ITO17 and ITO60 are MMR proficient, while ITO77 is MMR deficient.2.4Cells were exposed to gamma rays from a Gammacell® 40 Exactor  at a dose rate of approximately 1 Gy/min.2.5Apoptosis was determined by staining cells with bis-benzimide or propidium iodide to detect morphological nuclear changes as described earlier 2.6Western blot analysis was performed to detect DR4 and DR5 protein. Cells were treated with radiation and/or APG-880, washed and lysed in Triton lysis buffer as described earlier 2.7DR4 and DR5 epitope expression was determined by flow cytometry as described by Verbrugge et al. 2.8To determine the cytotoxic potential of APG-880 in organoids we dissociated the organoids with TriplE Express  at day 1. We expanded the cells to form small organoids of comparable sizes, and treated them with a defined concentration range at day 4, then performed metabolic activity assays with the CellTiter‐Glo® 3D reagent kit  at day 10.2.9Single cells were plated and allowed to attach before treatment. Cells were irradiated and treated with the indicated amount of APG-880, or sham-treated, immediately after irradiation and cultured for at least 14 days to allow colony formation. Colonies were fixed and stained with 0.2% crystal violet/2.5% glutaraldehyde. Colonies consisting of 50 cells or more were counted. The surviving fraction of cells was calculated by normalizing plating efficiency values of the treated samples to the untreated controls.2.10In order to determine the clonogenic potential of organoids we dissociated growing organoids with TriplE Express  at day 1. We plated the organoid derived single cells in Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix  and covered the matrix with growth medium as described above. Immediately after seeding the single cell suspension of the organoids, they were treated with the appropriate dose of radiation and/or concentration APG-880 and cultured for 14 days. Pictures of the colonies were taken with a cooled Hamamatsu ORCA R2 Black and White CCD-camera on a Zeiss AxioObserver microscope with a 10x/0.30 ECPlan-Neofluar Ph1 objective, run by ZEN2.3 Zeiss acquisition software. Analysis of the pictures and counting the number of organoids sized at least 50 cubic micrometer was performed with ImageJ. Organoids counts were done manually and double checked by an independent technician, in a blinded setting.2.111/(Dx)1 + (D)2/(Dx)2, and was previously applied by Zerp et al. The effects on cancer cells by ionizing radiation and APG-880 was characterized by calculating the Combination Index (CI) according to the classic isobologram equation described by Chou and Talalay 33.1First, we validated by Western blotting that DR4 and DR5 are expressed in HCT116 and HT-29 cell lines A. Patien3.2To confirm the apoptotic mode of cell death we first validated by Hoechst staining that APG-880 induces DNA condensation/fragmentation, a hallmark of apoptosis in HCT116 and HT29 cell lines and in CRC organoids . To quanDue to the 3D nature of the organoids we used a different quantification method that is presented with the results of the combination experiments in 3.3We next established the sensitivity of the colon carcinoma derived cell lines HCT116 and HT29 towards radiation and APG-880 in clonogenic survival assays, the gold standard for long-term cytotoxicity. HCT116 was more sensitive than HT29 for both treatment modalities A and B.F3.4When both treatment modalities were combined, we found a more than additive effect as quantified by calculating the combination index (CI) A and B. 3.5Because the 3D nature of the organoids hampered the Hoechst quantification method used in the cell lines, we applied a different quantification method for cell death/loss of viability, i.e. the cell titer glow 3D assay that is especially developed for measuring cell death in 3D cell culturing conditions C. The ce3.6In clonogenic survival assays in which APG-880 and radiation were combined, HT29 displayed the most effective decrease in clonogenicity at doses around 100 ng/ml. Above these concentrations APG-880 alone was too cytotoxic and at lower concentrations no reduction in radiation-induced clonogenicity was seen. In 37 of 1.5  and radiation in a clinically relevant organoid model system.We found that APG-880 and radiation show an enhanced combined effect in both short term and long term tumor cytotoxicity assays. The simultaneous activation of the intrinsic pathway by APG-880 and the extrinsic apoptotic pathway by radiation The CRC organoid model presented here was selected for its known high TRAIL receptor expression level. As expected, under control culturing conditions the expression levels of both DR4 and DR5 receptors were readily detectable. Furthermore, the CRC model is a relevant model for drug-radiation combinations as colorectal tumors are frequently treated with radiation alone, or in combination with conventional chemotherapy.in vivo setting was the insufficient clustering and inefficient DISC formation by TRAIL antibodies which likely prohibited effective pro-apoptotic signaling One of the main limitations of the first-generation TRAs in the The tumor organoid models that we use here mimic more closely the heterogeneity and structural similarities of growing tumors Taken together, we demonstrate here that in short-term as well as long-term assays, radiation combined with APG-880 causes an enhanced effect on tumor cell kill in CRC cell lines and in CRC patient-derived organoids. Importantly, this clinically relevant new CRC organoid model system can be leveraged to address mechanistic combination studies with TRAIL-R agonists to better define the role of the apoptotic pathway to radiation treatment sensitivity.This work was partially supported by a research grant from AbbVie Inc. The funding source had no role in the study design. AbbVie Inc. participated in the interpretation of data, review, and approval of this publication.