Lavandula angustifolia is a flowering plant that possesses anti-inflammatory and anti-depressive medicinal properties. Cancer is a deadly disorder affecting millions of people globally. It affects several human organs, including liver, stomach, and lungs. Several researchers are doing efforts to eliminate the disease around the globe. In this study, Chang and Huh-7 liver cell lines were utilized as human normal hepatocyte model and innovation to mimic the liver environment. Cytotoxicity of L. angustifolia seed extracts was investigated at two different concentrations (50% and 100%) against Chang and Huh-7 liver cell lines by colorimetric assay which is used to assess cell metabolic activities. The Chang and Huh-7 liver cell lines were treated with L. angustifolia seeds extracts (50% and 100%) and incubated for 24 and 48 hours under standard conditions . The 3--2, 5-diphenyltetrazolium bromide assay was employed to quantify cell survival. Seed extracts of L. angustifolia exerted varied cytotoxic effects depending on the concentration and treatment duration. The results indicated that L. angustifolia seed extracts with 100% concentration exhibited the highest cytotoxicity against Huh-7 and Chang liver cell lines. In conclusion, L. angustifolia seed extracts exhibited cytotoxic activity which can be enhanced based on the concentration and treatment duration. The findings of the current study are critical for the development of novel herbal-based therapies for fatal disorders such as liver cancer. However, more investigations are required to reveal cytotoxicity mechanisms of L. angustifolia seed extracts.Flowering plants are valuable in numerous ways, including food/feed supply for living organisms, fuel production, and medicinal uses. Several plant extracts/products are used to treat variety of serious ailments in human and animals.  Several diseases pose significant threats to human life. Although medical science has witnessed significant progress in the twenty-first century, there are still several diseases that are incurable. Therefore, cures for these diseases must be investigated. Cancer is a fatal health issue that affects people all over the world. It can arise in almost any tissue/organ of the body when anomalous cells grow uncontrollably, migrate to other organs through metastasizing process and cause death . Cancer in vitro model. The HuH-7 cell line was developed in 1982 from a 57-year-old Japanese male liver tumor patient’s hepatocyte cell line. It was then further refined and now being used in vitro to produce hepatitis C virus (HCV) medicines. The unique characters of HuH-7 cell line reveal that it is made up of epithelial-like, tumorigenic cells that stick to the surface of flasks or plates and proliferate as 2D monolayers. According to detailed literature review, this is the first study investigating the impact of lavender seed extracts on liver cancer cells.Different types of plants have been used in herbalism in various parts of the world in all civilizations at local level. The plant-based products are being suggested by professionals for the treatment of a variety of disorders. Numerous people around the world use herbal medications for a variety of medical needs. Plant species are thought to be a rich source of compounds that can be utilized for making active pharmaceutical or manufactured medications. Plant-based medicines are usually free of adverse effects, which makes them more popular among the public. Plant extracts serve various purposes, including natural coloring, crop pest management, food, cosmetics, and beverage etc. Natural plant species have been used not only as sources of food, but also as remedies for many diseases in the ancient era. Different reports suggested that there are unidentified properties in different parts of plants that can be used in the treatment of certain diseases . The curLavandula angustifolia Mill (LV) ] belongs to the mint family that is native to Mediterranean, Middle Eastern Asian countries, Arabian Peninsula, and Russia, also growing in Europe, United States, and Australia  methanol extracts at concentrations from 20 to 2400 μg/ml 24 hours after incubation are shown in Stereological investigation of Huh-7 liver cells’ viability exposed with L. angustifolia methanol extracts. Cell viability was not affected by LV50%, and LV100% methanol extracts-treated Chang liver cells at concentrations <200μg/ml, whereas significantly decreased by the concentrations >200μg/ml after 24 h incubation compared with control cells.Researchers around the world are looking at the use of plant-derived medicines as a healthcare supplement . Herbal L. angustifolia seed extract by indicating its cytotoxic property against Hun7 cells, human hepatocellular carcinoma cells, and Chang liver cells, explaining the cell death mechanism in vitro.Hepatocellular carcinoma (HCC), a type of malignant tumors, is causing high mortality of cancer-related death worldwide. Compared to other forms of cancers, HCC arises from several stages and multifactorial progression. Different risk factors drive the progression of HCC malignancy, and treatments are not effective once the disease is initiated , 27. TheL. angustifolia seed extract has cytotoxic-inducing effects in Huh7 and Chang liver cells in a duration and concentration-dependent mode. The cytotoxic property could be due presence of different pharmacological active principles in LV50% and LV100% seed extract. Based on the result, we found that 100% methanol extract of L. angustifolia seed extract increased cytotoxicity at 48 h compared to other time. A similar pattern of results we observed that Tridham and its active principles showed cytotoxic property in the human cancer cell lines [L. angustifolia grown in Brazil against GM07492 human fibroblasts cells [L. angustifolia exhibited a promising cytotoxicity against HeLa and MCF-7 cancer cell lines. Another recent work investigated the antiproliferative activity of L. angustifolia essential oils that grown in Tuscany  showed a notable effect against SHSY5Y neuroblastoma cell line [L. angustifolia seed extract induces active growth inhibition in hepatoma cells. Presence of several phytochemicals such as carvacrol, spathulenol, p-cymene-8-ol, caryophyllene oxide, and terpinolene present in L. angustifolia seed extract are responsible for the obtained results. Salleem et al. [We found that ll lines . Our fints cells . In addits cells , who fouell line , and them et al.  reportedL. angustifolia seed extract may provide the possibility of cancer prevention and therapy, which is evidenced by the inhibition of cytotoxicity in cancer cell lines. Among the two tested doses, 100% methanol extract exhibited promising cytotoxic properties. Further, additional investigations are warranted to delineate proper molecular mechanisms involved in the anticancer activity.In conclusion,


Appropriate sample preparation is critical for the analysis of cell cultures with mass spectrometry. It is important not only to maintain clear and consistent morphological features, but also the local chemical composition of the sample. In this proof-of-concept study, we evaluated two different sample preparation procedures for proteomic analysis using imaging mass spectrometry (IMS) of formalin-fixed multiple myeloma (MM)-cultured cells. Cytospin preparation resulted in more peaks with a signal-to-noise ratio > 3 as compared to formalin fixation and paraffin embedding. Overall, IMS technology holds the potential to stratify different cell lines to address the identification of differentially expressed proteins. We propose this approach as an additional feasible method for proteomic investigation of MM cell lines, and potentially applicable to other tumor types.Sample processing of formalin-fixed specimens constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature, dehydration, and embedding media affect downstream analysis, generating data dependent on technical processing rather than disease state. In this study, we investigated two different sample processing methods, including the use of the cytospin sample preparation and automated sample processing apparatuses for proteomic analysis of multiple myeloma (MM) cell lines using imaging mass spectrometry (IMS). In addition, two sample-embedding instruments using different reagents and processing times were considered. Three MM cell lines fixed in 4% paraformaldehyde were either directly centrifuged onto glass slides using cytospin preparation techniques or processed to create paraffin-embedded specimens with an automatic tissue processor, and further cut onto glass slides for IMS analysis. The number of peaks obtained from paraffin-embedded samples was comparable between the two different sample processing instruments. Interestingly, spectra profiles showed enhanced ion yield in cytospin compared to paraffin-embedded samples along with high reproducibility compared to the sample replicate. Mass spectrometry (MS)-based proteomics is an emerging approach to generate protein signatures from clinical samples for biomarker discovery ,2,3,4. IIn order to use IMS-based proteomics for the analysis of cell cultures in routine research and possibly in the clinical setting, the variability associated with the sample preparation steps that precede IMS analysis must be considered. We used multiple myeloma (MM) as a model system to establish an IMS experimental procedure for cultured cells.Multiple myeloma (MM) is a B cell malignancy of clonal plasma cells (PCs) accumulating in the bone marrow (BM), characterized by an intricate genomic and epigenomic landscape. Despite the remarkable therapeutic achievements that have led to an increased extent and frequency of response with unprecedented outcome improvement, MM patients may eventually evolve towards a drug-resistant phase ,23,24. MS-based methodologies have proven to be a valuable diagnostic tool for minimal residual disease monitoring in plasma cell dyscrasias, including MM . AdditioIn order to further assess the clinical utility of MALDI MS, we optimized the sample preparation workflow for the proteomic investigation of MM cell cultures by comparing two different sample processing methods, including the use of the cytospin sample preparation, formalin fixation and paraffin embedding, and automated sample processing apparatuses. Herein, we present an optimized methodological approach, comparing two sample-embedding instruments using different reagents and with different processing times. We evaluated the peptide molecular extraction and the quality of mass spectral data obtained from fixed cell specimens with both procedures.MM cell lines were cultured in RPMI-1640 medium  supplemented with 10% FBS and 1% P/S (Gibco) . The AMOFor each cell line, the supernatant liquid was decanted in a separate tube and cell pellets were removed with a disposable Pasteur pipette and placed into a Tissue-Tek Paraform biopsy centralized cassette . One third of the cell pellets was resuspended in 70% ethanol and stored at 4 °C until further use. The remaining cell pellets were then introduced in the automated tissue-processing machine , where they were transferred from container to container to be processed. The pellet of the cell line “AMO BZB (8425)” was processed with a different automatic machine  employing a similar solvent composition but a longer reagent incubation time. To create paraffin-embedded specimens, the tissue processors employ raised temperatures, a low-wattage microwave assistance (60 watts), and a vacuum infiltration technique to enhance processing and reduce the processing time from 8 h on a conventional processing platform to only 2.5 h when using Sakura Tissue-TEK-VIP, and to about 1 h for the Sakura Tissue-TEK-Xpress. FFPE cell sections (3 µm) were mounted onto indium-tin oxide-coated (ITO) glass slides  and dried at 37 °C overnight. Deparaffination occurred by heating the slides at 80 °C for 15 min, followed by incubation in xylene (2 × 5 min). Slides were dehydrated in 100% isopropanol, followed by graded ethanol  and deionized water washes, for 3 min each. Antigen retrieval was performed by heating the slides at 95 °C for 20 min in 10 mM of Tris buffer pH 9 using a decloaking chamber , followed by enzymatic retrieval using trypsin. Trypsin solution (0.025 µg/µL in 20 mM of ammonium bicarbonate) was applied on the samples with an automatic reagent sprayer  in 16 passes at 30 °C, with a 0.015 mL/min flow rate and a 750 mm/min velocity. Samples were then placed in a digestion chamber, prepared with saturated potassium sulphate solution, and incubated at 50 °C for 2 h.The resuspended cell pellets were mixed by pipetting, and 150 µL of the cell suspensions was cytocentrifuged onto ITO-coated glass slides  in a cytospin  at 1200 rpm for 10 min. Slides were then incubated in 10 mM of Tris buffer pH 9, together with the dewaxed sections described above for the antigen retrieval process, and air-dried trypsin solution (0.025 µg/µL) deposition onto cytospin slides, and the digestion process occurred using the same method as described for the FFPE cell sections .Digested samples were sprayed with a matrix solution  using the same sprayer apparatus in 4 passes at 75 °C, with a 0.120 mL/min flow rate and a 1200 mm/min velocity. MALDI MS data were acquired using a rapifleX MALDI Tissue-typer mass spectrometer  in positive-ion reflector mode in a mass range of 500–3200. Each mass spectrum was obtained by averaging signals from 200 laser pulses. External calibration was performed using the peptide calibration standard  . All mass spectra were baseline-subtracted during acquisition with the TopHat algorithm. Following mass spectra acquisition, cells were stained with hematoxylin and eosin.Spectral data were pre-processed using FlexAnalysis 4.2, ScilS Lab software, version 2022b Pro , and R-statistical software, version 3.5.3 and 4.0.3, for total ion count (TIC) normalization, mass alignment, peak counting, and feature detection. Statistical analysis using Wilcoxon/Kruskal–Wallis tests and receiver operating characteristic curve (ROC) analysis was performed to measure the discrimination quality of the ion peptide features between the cell lines.Examination of the stained cells by light microscopy revealed a nice balance of nuclear and cytoplasmic stains in both types of cell sample processing methods, however cytospin-stained cells showed better cellular architecture and resolution of nucleoli .m/z range 500–3000). The median total peak count and signal-to-noise ratios (S/N > 3) were used as quality parameters to compare all experiments. For each sample procedure, the overall amount of the peaks detected in the same cell line and for each individual replicate was similar . Nonetheless, the highest peak counts and lowest variance were achieved in all cytospin samples  and FFPE (m/z with log2 intensity cytospin/FFPE < 0 = 145) was obtained, indicating that the higher number of peaks for cytospin is not related to a higher general peptide ion intensity (m/z < 800) were likely to have a higher intensity in cytospin than in FFPE, and conversely peaks in the high mass range (m/z > 800) had a higher intensity in FFPE than in cytospin (m/z < 800). In order to improve data quality and to prevent characteristic discrepancies between data collected from different samples, it Is recommended to extract spectra from regions of interest sufficiently far from areas with few cells. This would reduce the inclusion of the background interference signal in the dataset, thus further improving the quality of the analysis. High-quality MS signals were obtained from both FFPE and cytospin sample processing. A qualitative evaluation based on visual inspection and comparison of spectral data and molecular images showed better MS data quality with respect to cells prepared by cytospin with a higher ion yield  was similar to the one obtained from cells embedded with the Sakura Vip apparatus . The number of peaks per single spectrum was high in the AMO WT cells, with a median peak count of 132 in replicate 1 . Peptide profiles of AMO WT and AMO BZB shared many peaks compared to the cytospin H929 GFP/LUC, whose MS data showed a different molecular profile trend A. An ovep-values . Sixteen m/z features were overexpressed in AMO WT, from which two features  showed more than a two-fold intensity difference between the two cell lines. Ten features were instead overexpressed in AMO BZB cells, where three features  exhibited more than a four-fold intensity difference. The identification of the m/z species making these specific signatures is beyond the scope of this study because they need validation in a larger dataset. Nevertheless, these results could be reproduced on separate experiments and on a different day. Nearly identical spectra as well as equivalent peak expression were obtained in the replicate experiments, where segregation of the two different cell lines, AMO WT and AMO BZB, following AUC analysis was also obtained. Such a highly reproducible technique could be applied to other types of cells and has the potential to be a useful tool for the stratification of cell lines based on their peptidomic profile, with the goal of rapidly identify novel potential tumor molecular signatures. Comparison of the average relative intensities of the This proof-of-concept study indicates that fixation in formalin followed by centrifugation in cytospin resulted in the recovery of higher numbers of peptides compared with fixation combined with paraffin embedding. It was also shown that IMS technology holds the potential to stratify cell lines of MM, including those with a similar genetic background but different drug sensitivity. We propose this approach as an additional method for proteomic analysis of MM samples, potentially applicable to other tumor types. The continued development of proteomics and its integration with genomic studies is expected to provide valuable information for the detection of molecular mechanisms and target proteins involved in MM onset and progression.


Drimane and coloratane sesquiterpenoids are present in several plants, microorganisms, and marine life. Because of their cytotoxic activity, these sesquiterpenoids have received increasing attention as a source for new anticancer drugs and pharmacophores. Natural drimanes and coloratanes, as well as their semi-synthetic derivatives, showed promising results against cancer cell lines with in vitro activities in the low micro- and nanomolar range. Despite their high potential as novel anticancer agents, the mode of action and structure–activity relationships of drimanes and coloratanes have not been completely enlightened nor systematically reviewed. Our review aims to give an overview of known structures and derivatizations of this class of sesquiterpenoids, as well as their activity against cancer cells and potential modes-of-action. The cytotoxic activities of about 40 natural and 25 semi-synthetic drimanes and coloratanes are discussed. In addition to that, we give a summary about the clinical significance of drimane and coloratane sesquiterpenoids. According to the World Health Organization, cancer is the most common cause of death in the 21st century and one of the world’s major health challenges . ParticuNatural products have proven to be a versatile source of new drugs and pharmacophores in recent decades ,10,11. F4), which was first isolated in 1948 from the stem bark of Drimys winterii , wCanellaceae, Polygonaceae, and Winteraceae species . Th. ThDrimyll lines . Compoun17, 20–22, and polygodial (2) from the bark and leaves of Zygogynum pancheri and Zygogynum acsmithii and tested their cytotoxic activity against KB adenocarcinoma, HL-60 leukemia, and HCT116 colon cancer cell lines . On. OnWarbu2), warburganal (24), and muzigadial (26) from the stem bark of Warburgia ugandensis and tested their cytotoxic activity against leukemia (MV4-11 and THP-1), melanoma (Sk-Mel29), and glioblastoma (LN-229) cell lines (26) exhibited three-to-five-fold higher activities than polygodial (2). The activity of warburganal (24) was also increased compared to polygodial (2) as previously observed by Xu et al. [26) increases its cytotoxicity.Kitte and Tretbar et al. obtained the sesquiterpenoids polygodial (ll lines  53]. Mu. Mu2), wu et al. . The res27–33 from Dendrodoris carbunculosa  P388 cells with IC50 values of 4–17 µg/mL. Lactone 33 showed increased cytotoxicity compared to 27–32. Simultaneous incubation of P388 cells with drimanes 27–33 and adriamycin or vincristine led to no reversal effects.Sakio et al. obtained drimane lactones and acetals unculosa  54]. Dr. Dr27–3334–36 were isolated from Perenniporia maackiae by J. Kwon et al. and tested against ACHN renal, HCT-15 colon, MDA-MB-231 breast, NCI-H23 lung, NUGC-3 stomach, and PC-3 prostate cancer cell lines -configuration or a keto group at the C6 position proved to be cytotoxic during their investigations.Drimane acetals ll lines  55]. Co. Co34–3637–39 from the fungus Aspergillus ustus and investigated their impact on a murine lymphoma cell line (L5178Y) on PC-12 and HeLa cells  turned out to be a suitable target because it can be isolated in sufficient quantities from different sources and possesses a less complex structure with reactive aldehyde groups . By. By49), 2) . A possible reason is also the enhanced membrane mobility of the bis-acetals because of the less polar structure compared to polygodial (2).Dasari et al. proposed that the reversible formation of bis-acetals might lead to a controlled release of reactive dialdehydes inside the cell and an increased cytotoxicity . Therefo2) . Bis-ace2) (2. 64 against MCF-7 cancer (GI50 = 7.0 µM) being 10-fold more active than polygodial (2). In general, GI50 values of all Wittig derivatives 64–71 were about 21–42 μM for A549-, Sk-Mel-28-, U373-, and Hs683 cancer cells. Additionally, it was demonstrated that 64 forms an isolable pyrrole product with benzylamine. No Wittig derivatives of 9-epipolygodial (49) were synthesized.In further works, Dasari et al. focused on the Wittig derivatization of the C12 aldehyde group of polygodial (2)  66]. Th. Th2) (649) and the Wittig derivative 64 were investigated more intensively . An. An2) (62) leading to a loss or an increase in cytotoxicity (2) are strategies to increase its cytotoxic effects ..75 (147]75 inhibits DNA binding of the transcription factors NF-kB and Stat-3 to the survivin promoter. DNA binding of NF-κB and Stat-3 to the CMV promoter was not impaired. Interestingly, structurally related eudasmane sesquiterpenoids and meroterpenoids lead to the attenuation of NF-κB dependent pathways [In further experiments, it was shown that pathways ,149. Tanpathways . In futuAround half of the small molecules that have been approved for cancer chemotherapy since 1940 are natural products . All of Drimane and coloratane sesquiterpenoids were shown to exert promising anticancer activity in vitro. However, no preclinical studies evaluating their in vivo cytotoxic activity nor clinical trials were accomplished until now. In vivo studies are only available for antidiabetic and antimalarial activities of some drimane sesquiterpenoids and plant extracts ,154. In 50 values in the low nanomolar range were only observed for a few drimanes and coloratanes against certain cancer cell lines [Possible reasons are the lack of detailed knowledge about a specific target or modes-of-action, cancer cell specificity, or insufficient anticancer activities of drimane and coloratane sesquiterpenoids. Until now, ICll lines ,50,52,53ll lines ,156,157.In this review, we gave an overview of the cytotoxic activity of drimane and coloratane sesquiterpenoids against cancer cells. Over the past decades, natural drimanes and coloratanes were isolated and their cytotoxic potential was evaluated. Additionally, several semi-synthetic derivatives were obtained from polygodial to increase its cytotoxic activity. It is demonstrated that the drimane and coloratane scaffold holds great potential as a lead structure for anticancer drug development. However, none of the components are used as a therapeutic agent or in a clinical trial so far. Furthermore, several modes-of-action of drimane and coloratane sesquiterpenoids were discussed. Detergent effects on subcellular membranes, DNA damage, and ROS production are commonly observed for drimanes. It was also shown that cancer cell exposure to drimane-containing plant extracts can cause cell cycle arrest. Additionally, dialdehyde drimanes activate the TRPA1 ion channel. The related ion channel TRPV1 was often supposed to be addressed by drimanes, but current data show that TRPV1 only plays a minor role in the cytotoxic action of this class of sesquiterpenoids. However, whether there is a relation between these actions or they are caused independently needs to be further evaluated. Therefore, a detailed analysis of differentially expressed genes using microarrays or next-generation sequencing methods could be key methods to reveal targets or mode-of-action of drimane and coloratane type sesquiterpenoids in anticancer activity. Proper target identification will lay a solid basis for future lead compound discovery and candidate selection for preclinical and clinical development.


MYC expression and impairs the DNA damage response. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent DNA damage sensing and repair. We hypothesized that JQ1 would promote a DNA repair-deficient phenotype that sensitizes neuroblastoma cells to PARP inhibition.JQ1 is a bromo- and extraterminal (BET) domain inhibitor that downregulates MYCN-amplified (BE(2)-C and IMR-32), and two non-MYCN-amplified (SK-N-SH and SH-SY5Y). Cells were treated with JQ1 (BET inhibitor), Olaparib (PARP inhibitor), or in combination to assess for therapeutic synergy of JQ1 and Olaparib. Treated cells were harvested and analyzed. Quantitative assessment of combination treatment synergy was performed using the median effect principle of Chou and Talalay.Four human neuroblastoma cell lines were examined: two 50 of JQ1 by 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C, IMR-32, SK-N-SH, and SH-SY5Y cell lines, respectively. In the MYCN-amplified cell lines, BE(2)-C and IMR-32, combination treatment decreased gene expression of MYCN relative to single-drug treatment alone or control. Combination treatment decreased protein expression of DNA repair proteins Ku80 and RAD51, led to accumulation of DNA damage marker phospho-histone H2A.X, and increased caspase activity. In the non-MYCN-amplified cell lines, SK-N-SH and SH-SY5Y, combination treatment induced G0/G1 cell cycle arrest.Combination treatment with Olaparib decreased the ICMYCN-amplified neuroblastoma cells, this effect may be induced by downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest.Combination BET and PARP inhibition synergistically inhibited neuroblastoma tumorigenesis in vitro. In The online version contains supplementary material available at 10.1007/s12672-022-00563-5. Despite advances in treatment, high-risk neuroblastoma (NB) accounts for approximately 15% of cancer-related mortality in children with a five-year survival rate of less than 40% . TherefoMYCN amplification, a feature of high-risk NB due to its role in promoting NB tumorigenesis and proliferation, has made transcription factors of oncoproteins such as N-myc attractive potential therapeutic targets. However, these oncoproteins have historically been resistant to conventional treatment strategies due to a paucity of high-affinity binding sites [MYC expression by displacing BET bromodomains from chromatin after selectively recognizing and competitively binding histone acetylated lysine residues [ng sites . Moreoveng sites . This hang sites , 6. BET ng sites –9. BET ing sites . In precresidues , 6. JQ1 residues , 11. Desresidues , 12.MYCN-amplified NB cells in vitro [Poly(ADP-ribose) polymerase (PARP) proteins play a vital role in numerous cell processes including DNA repair, cell signaling, and cell death . PARP inin vitro . Tumors in vitro . This hain vitro .MYCN amplification status may affect the efficacy of this combination therapy.A combination of BET and PARP inhibition has demonstrated efficacy in inhibiting tumorigenesis in preclinical models of multiple cancer types, including pancreatic, breast, ovarian, prostate, and lung cancers , 17, 22.Primary antibodies against Neurofilament-M (NF-M)(#2838), caspase3 , pAKT (#4060), AKT (#4685), p53 (#2524), N-myc (#94055), C-myc (#5605), Ku80 (#2180), RAD51 (#8875), phospho-histone H2A.X (Ser139) (#2577), and PTEN (#9118) were purchased from Cell Signaling Technology . Primary antibody against neuron-specific enolase (NSE) (ab53025) was obtained from Abcam . Primary antibody against p21 (#610234) was obtained from BD Bioscience . Primary antibodies against β-actin and Ponceau S solution were obtained from Sigma-Aldrich . Anti-GAPDH antibody (#20357) and secondary anti-mouse, anti-rabbit, and anti-goat antibodies were obtained from Santa Cruz Biotechnology, Inc .2 and 95% air.The human NB cell lines SK-N-SH, SH-SY5Y, BE(2)-C and IMR-32 were purchased from the American Type Culture Collection . Cells were maintained in Rockwell Park Memorial Institute (RPMI) culture medium 1640 with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere consisting of 5% CO3, 2 × 103, 1 × 103, and 3 × 103 cells per well, respectively, in RPMI culture medium with 10% FBS and allowed to attach overnight. Cells were subsequently treated with JQ1, Olaparib, or both. Cell viability with combination treatment was measured after same day treatment with both drugs for all four cell lines. The SK-N-SH, BE(2)-C, and IMR-32 cell lines also underwent a second experiment evaluating the effects of staggered treatment (JQ1 followed by Olaparib 24 h later). Cell viability was measured using Cell Counting Kit-8 (CCK-8) colorimetric assay  72 h after last treatment dose for each treatment group.SK-N-SH, SH-SY5Y, BE(2)-C, and IMR-32 cells were plated in 96-well plates at 3 × 10Each cell line was plated at two different cell densities in 6-well plates. SK-N-SH, SH-SY5Y, BE(2)-C, and IMR-32 cells were plated at 8000 and 16,000, 3000 and 10,000, 5000 and 10,000, and 100,000 and 150,000 cells per well, respectively. Cells were allowed to attach for 48 h prior to treatment with JQ1, Olaparib, or combination. JQ1 and Olaparib dosages were 300 nM for the SK-N-SH and BE(2)-C cell lines and 150 nM for the IMR-32 cell line. The SH-SY5Y cell line was treated with 200 nM of JQ1 and 500 nM of Olaparib. Cells were observed for five to seven days after treatment to evaluate for changes in treatment-induced morphology. Images were captured using a BioTek Cytation 5 Cell Imaging Multi-Mode Reader .SK-N-SH, BE(2)-C, and IMR-32 cells were plated at 3000, 1000, and 10,000 cells per well, respectively, on 12-well plates in triplicate. 48 h after plating, cells were treated with JQ1, Olaparib, or both. The JQ1 dose was 50 nM for the SK-N-SH and BE(2)-C cell lines and 125 nM for the IMR-32 cell line. The Olaparib dose was 100 nM for the SK-N-SH and IMR-32 cell lines and 1000 nM for the BE(2)-C cell line. Cells were allowed to grow for 6–10 days after treatment. SK-N-SH and BE(2)-C colonies were stained with 0.01–0.05% crystal violet dye, photographed, and counted using the Bio-Rad Gel Doc XR + Imager .Due to the IMR-32 cell line’s propensity to detach from wells with washing, cells were alternatively trypsinized, centrifuged, resuspended in fresh media, and placed on dual chamber slides with trypan blue dye for cell counting using the Bio-Rad TC10 Automated Cell Counter .In order to assess for a dose-dependent response to treatment using clonogenic assays, the SK-N-SH and BE(2)-C cell lines were also plated at 1500 and 500 cells per well, respectively, in 24-well plates in triplicate. Cells were treated with varying doses of JQ1, Olaparib, or combination treatment 48 h after plating. SK-N-SH and BE(2)-C cells were allowed to grow for 8 or 5 days after treatment, respectively, prior to staining with 0.02% crystal violet dye, photographing, and colony counting.ACTB. Amplification was performed for 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 40 s at 72 °C. Primers used to detect the expression of qPCR were the following: MYCN , PTEN forward: 5’-CGGGCTCAGGCGAGGGAGAT-3’, reverse: 5’-GCCCACGGCTCCACCTTCC-3’), ACTB .Total RNA was isolated and purified using a Trizol Reagent . The High-Capacity cDNA Reverse Transcription Kit  was used to synthesize complementary DNA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using iTaq Universal SYBR Green Supermix , and data were collected with a CFX96 instrument . Results were normalized to an endogenous control, https://imagej.nih.gov/ij/, 1997–2018).Cells were collected using cell lysis buffer and denatured samples were prepared for immunoblotting, as we have previously described , 24. EquSK-N-SH and SH-SY5Y cells were plated separately in 6-well plates at 180,000 cells per well and allowed to attach overnight. Cells in the treatment group received a combination of JQ1 (200 nM) and Olaparib (500 nM) at 0 and 24 h time points. Samples were collected 16, 24, and 48 h after initial treatment. Samples were trypsinized, centrifuged, washed and resuspended in phosphate-buffered saline (PBS), and fixed in 70% ethanol at 4 °C. Prior to cell staining, samples were centrifuged and resuspended in PBS. RNase, DNase-free was added to each sample and subsequently incubated at 37 °C for 30 min. Samples were chilled on ice prior to staining with propidium iodide. Stained cells were analyzed with the flow cytometer .3 cells per well and allowed to attach overnight. Cells were treated as part of one of four treatment groups , Olaparib (3000 nM), or combination) and cultured for 72 h. Cells were subsequently washed with PBS and fixed with 4% formaldehyde in 1× PBS at room temperature. They were then washed in 1 × PBS three times prior to being blocked in blocking buffer for one hour. Primary antibody was subsequently applied at a concentration of 1:400 and incubated at room temperature for 1.5 h. The samples were again washed three times in 1× PBS prior to incubation with Alexa Fluor 594 dye-tagged secondary antibody  at a concentration of 1:500 for 30 min. Samples were again washed in 1× PBS three times. Coverslips were mounted and slides were left to dry. 4’,6-diamidino-2-phenylindole (DAPI) was used for staining nuclei. Images were captured using a Biotek Cytation 5 Cell Imaging Multi-Mode Reader .The phospho-histone H2A.X (Ser139) antibody was obtained from Cell Signaling Technology . BE(2)-C cells were plated on coverslips in a 12-well plate at a density of 2–6 × 10Therapeutic synergy of combination BET and PARP inhibition was quantitatively assessed using the Chou-Talalay calculation and Compusyn software  .t-tests, Mann–Whitney U tests, and analyses of variance using GraphPad Prism . A p value ≤ 0.05 was considered significant.All experiments were repeated in triplicate. The scoring index and relative expression values were expressed as mean ± SEM or SD. Statistical analyses were performed using Student’s and Welch’s MYCN-amplifying NB cell lines, BE(2)-C and IMR-32, and two non-MYCN-amplifying NB cell lines, SK-N-SH and SH-SY5Y, were treated with JQ1 alone and Olaparib alone to identify the IC50 doses of each drug. Each cell line was subsequently treated using increasing doses of JQ1 in combination with each cell line’s respective IC50 dose of Olaparib. When treated on the same day in combination, the IC50 dose of JQ1 decreased 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C -C cell line and decreased 1.7-fold and 51.3-fold in the IMR-32 and SK-N-SH cell lines, respectively -C cell line may reflect the relatively more rapid doubling time of this cell line. Together, this data suggest that the optimal timing of combination treatment may vary by cell line.We subsequently tested whether treating each cell line with JQ1 24 h prior to treatment with Olaparib could sensitize NB cells and enhance this inhibitory effect in the BE(2)-C, IMR-32, and SK-N-SH cell lines. When staggering treatment with these two drugs by 24 h, the ICPrevious studies have shown that treatment with JQ1 induces neuroblastoma cell differentiation in BE(2)-C cells, demonstrated by morphologic changes including a polarized phenotype with longer neurites, as well as increased protein expression of neural differentiation markers ZNF423 and NF-M .MYCN-amplifying and non-MYCN-amplifying cells demonstrated changes in morphology after treatment with JQ1, as well as after combination treatment with JQ1 and Olaparib  protein expression in the BE(2)-C cell line and a 2.0-fold increase in neuron-specific enolase (NSE) protein expression in the SK-N-SH cell line with combination treatment at 24 h, demonstrated by immunoblotting with densitometry in Fig. p = 0.0097 and p = 0.0012, respectively)-C and SK-N-SH cell lines to quantitatively assess colony formation in cells treated with JQ1 alone, Olaparib alone, or both drugs in combination relative to control. Combination treatment significantly decreased colony formation in both the BE(2)-C and SK-N-SH cell lines with better efficacy than either drug treatment alone. Figure ely)Fig. B.Fig. 3Cp = 0.0019). Combination treatment of IMR-32 with JQ1 and Olaparib decreased the number of viable cells by 78.6% relative to control , as well as a dose 2.5 times higher in concentration . Each cell line was then treated with varying combinations of these doses . As expected, each cell line demonstrated a dose-dependent response to single drug and combination treatment. Fewer colonies were formed after high dose single drug treatment relative to low dose single drug treatment relative to low dose for each drug and the JQ1H + OlapH treatment group demonstrated the lowest number of colonies overall for each cell line. In the BE(2)-C cell line, the JQ1L + OlapL and JQ1H + OlapH treatment groups demonstrated a 16.5% and 47.5% decrease in colony formation relative to control. In the SK-N-SH cell line, the JQ1L + OlapL and JQ1H + OlapH treatment groups demonstrated a 31.8% and 71.4% decrease in colony formation relative to control.To assess for a possible dose-dependent response to treatment, colony counts were also performed in the BE(2)-C and SK-N-SH cell lines after treatment with increasing doses of JQ1, Olaparib, or combination therapy Fig. C. Each cMYCN promoter, leading to decreased transcription of MYCN [MYCN expression were therefore evaluated using RT-qPCR. We found that BE(2)-C and IMR-32 NB cells treated with JQ1 demonstrated decreased expression of MYCN with lowest expression seen in cells treated with both JQ1 and Olaparib . The IMR-32 cell line demonstrated a 2.25-fold and 4.51-fold decrease in MYCN expression with JQ1 and combination treatment, respectively .BET inhibition with JQ1 has been demonstrated to displace BRD4 from the  of MYCN , 26. TheImmunoblotting was performed to assess the effects of treatment on N-myc protein expression at 24, 48, and 72 h -C and IMR-32 cell lines demonstrated increased caspase activity, expressed as a ratio of cleaved caspase-3 to full-length caspase-3 protein, with combination treatment relative to control or single drug treatment beginning 48 and 24 h after treatment, respectively -C NB cells are known to have a p53 mutation. Therefore, this accumulation of p53 may instead be a surrogate of another process, such as accumulation of DNA damage. These findings prompted the inclusion of the IMR-32 cell line for analysis in this study given its p53 wild type status.Given p53’s known role in inducing apoptosis in DNA damaged cells, the data suggest that combination treatment with JQ1 and Olaparib may cause an accumulation of p53 that contributes to apoptosis in MYCN-amplifying cell lines. After immunoblotting demonstrated SK-N-SH cells’ increase in protein expression of p21 -C cells using the phospho-histone H2A.X (Ser139) antibody to evaluate whether JQ1 enhances DNA damage induced by Olaparib treatment. The phosphorylated Ser139 variant of histone H2A.X is a known marker of DNA damage and signifies cellular response to DNA strand breaks [p < 0.0001). There was also a statistically significant difference in mean and median fluorescence between control and Olaparib treatment groups (p < 0.0001), as well as control and combination treatment groups (p < 0.0001).To further assess the hypothesis that combination treatment may be associated with apoptosis in the setting of DNA damage in H2A.X Ser9 antibodd breaks , 28. ConJQ1 inhibits and regulates BRD4, a member of the BET family of bromodomains and a transcriptional and epigenetic regulator. BRD4 serves as a chromatin platform responsible for gene expression and recruitment of components of DNA repair to sites of DNA-double strand breaks to facilitate DNA damage repair . We therIt is thought that cells that incur DNA damage marked by 5’-3’ resection of DNA ends will utilize HR mechanisms and prevent NHEJ . RAD51 iMYCN amplification status.With combination treatment, BE(2)-C cells demonstrated a sustained 2.08%, 2.58%, and 9.62% decrease in Ku80 protein expression at 24, 48, and 72 h, respectively, relative to control ’s known role in regulation of DNA repair via induction of RAD51 expression , we evalPTEN after drug treatment. The IMR-32 cell line demonstrated no significant difference in PTEN gene expression between treatment groups -C and non-MYCN amplifying SK-N-SH cell lines based on the median-effect principle of Chou and Talalay using Compusyn software  [However, the absence of a standardized definition of therapeutic synergy has historically allowed for additive effects of combination therapy to be mistaken for synergy . We therNJ, USA) . The resNJ, USA) . Fa rangNJ, USA) . TherefoNJ, USA) , 35.MYCN-amplifying BE(2)-C and non-MYCN amplifying SK-N-SH cell lines demonstrated therapeutic synergy at increasing Fa values as demonstrated by CIs less than 1. For instance, combination treatment of BE(2)-C cells with JQ1 and Olaparib at their approximate IC50 dosages led to an Fa = 0.509 and CI = 0.33, signifying therapeutic synergy. Furthermore, at these treatment doses and Fa = 0.509, dose-reduction index (DRI) data demonstrated that combination treatment required 3.9-fold less JQ1 plus 14.0-fold less Olaparib to achieve the same 50% inhibition in a 1:1 combination in comparison to the doses required of each drug alone. However, combination treatment at lower doses yielding lower Fa did not consistently demonstrate a CI less than 1. Similarly, combination treatment of SK-N-SH cells with lower doses of JQ1 and Olaparib yielding lower Fa (0.3–0.5) was associated with CIs of 0.95–1. However, higher doses yielding a higher Fa of 0.7 were associated with a substantial decrease in CI to 0.45. Furthermore, this was associated with a DRI of 2.2-fold less JQ1 plus 374.7-fold less Olaparib to achieve the same 70% inhibition in a 1:1 combination in comparison to the doses required of each drug alone. This data suggest that combination treatment of both MYCN-amplifying and non-MYCN-amplifying cells with doses of JQ1 and Olaparib sufficient to yield higher treatment effect are associated with true synergy and substantial dose-reduction indices compared to single drug treatment.Both the Patients with high-risk NB undergo multimodality treatment consisting of multi-agent chemotherapy induction, surgical resection, consolidation chemotherapy, autologous stem cell rescue, and radiation therapy followed by treatment with anti-ganglioside-2 (anti-GD2) immunotherapy, cytokines, and cis-retinoic acid . DespiteMYCN expression, induce differentiation toward a neural fate, prevent proliferation, and induce cell cycle arrest and apoptosis [MYCN-dependent NB cells, causing progression through the cell cycle despite DNA damage and ultimately mitotic catastrophe and apoptosis [MYCN, as well as those with 11q deletion and/or low ataxia-telangiectasia mutated (ATM) expression [In preclinical models of NB, BET inhibition has been shown to downregulate poptosis , 10, 11.poptosis , 37. Widpression , 37–42. MYCN amplification status, although the mechanism may vary.A combination of BET and PARP inhibition has demonstrated efficacy in inhibiting tumorigenesis in preclinical models of numerous malignancies, including pancreatic, breast, ovarian, prostate, and lung cancers , 17, 22.MYCN-amplified NB cell lines, combination treatment was associated with downregulation of MYCN gene expression and N-myc protein expression, downregulation of DNA repair proteins, accumulation of p53 and DNA damage, and induction of apoptosis. This proposed mechanism of downregulation of the DNA damage response contributing to synergistic cytotoxicity is consistent with preclinical studies of other cancer types, including pancreatic, ovarian, breast, prostate, and lung cancers [In  cancers , 17, 22.MYC amplification. Fiorentino et al. concluded that combination BET and PARP inhibition demonstrated preferential or selective inhibition on small cell lung cancer cells with an active MYC signaling pathway [MYC-amplified NB cells remain susceptible to combination treatment, albeit seemingly through a different mechanism of action. Lee et al. demonstrated that JQ1 alone was sufficient to halt proliferation in the SK-N-SH cell line [MYCN-amplifying SK-N-SH and SK-N-AS cell lines [MYCN-amplifying cell lines. Our results supported this hypothesis, with an 11.93% increase in G0/G1 cell cycle arrest at 16 h and a 6.78% increase in G0/G1 cell cycle arrest at 48 h in the SK-N-SH and SH-SY5Y cell lines, respectively, after combination treatment relative to control. This data suggest that combination treatment may affect cell cycle progression and alter checkpoint inhibition.Previous preclinical studies of BET and PARP inhibitors have largely focused on cancer cell lines with  pathway . Howeverell line . Wyce etll lines . Given tThe results of this study have substantial potential translational relevance. Several PARP inhibitors, including Olaparib, Rucaparib, Talazoparib, and Niraparib, have already been approved by the FDA for the treatment of other cancers , 44. NumBRCA-mutated ovarian cancers. However, Karakashev et al. demonstrated that combination treatment with BET inhibition suppressed TOPBP1 and WEE1 expression, resulting in accumulation of DNA damage, checkpoint defects, and apoptosis in preclinical models of BRCA-wild type ovarian cancer [The BET and PARP inhibition combination treatment strategy may also aid in overcoming other challenges in cancer treatment, such as treatment resistance. Our results demonstrated that BET inhibition sensitized neuroblastoma cells to PARP inhibition in vitro. These results are consistent with studies of other cancer types. PARP inhibition is a known clinical strategy against n cancer .BET inhibition may also work synergistically with other agents and treatment modalities. BET inhibition downregulates RAD51, whose overexpression has been associated with chemoresistance in multiple cancer types, including neuroblastoma –47. In aThis study is limited by its preclinical design. Further work is needed to test the safety and effectiveness of combination BET and PARP inhibition clinically. Another limitation is that BET inhibition likely affects the expression of other genes and proteins not tested in the current study that may affect or correlate with the observed synergistic effects of BET and PARP inhibition. Future directions include evaluating the effects of BET and PARP inhibition on other therapeutic agents and treatment modalities, such as radiation therapy, used in the management of NB.MYCN amplification status; however, the mechanism of action may vary. In MYCN-amplified cell lines, combination treatment was associated with downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest. A combination of BET and PARP inhibition is a potentially novel, efficacious treatment strategy in the management of NB.In conclusion, the combination of BET and PARP inhibition synergistically inhibited NB tumorigenesis in vitro. These results were seen regardless of Additional file 1: Figure S1. Staggering combination treatment with Olaparib 24 h after treatment with JQ1 has variable response on the IC50 of JQ1 by cell line. When staggering treatment of Olaparib 24 h after treatment of JQ1, the IC50 of JQ1 increased 6.4-fold in the BE(2)-C cell line  and decreased 1.7-fold and 51.3-fold in the IMR-32  and SK-N-SH  cell lines, respectively.


Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines. Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) mimic key features of human retinal development and form a laminated structure with time-dependent biogenesis of all major retinal cell types, including photoreceptors with rudimentary outer segment-like structures and partial functionality  formation and invagination to give rise to retinal pigment epithelium (RPE) and all the major cell types that make up the neural retina (NR). Stem cells cultured in vitro can be guided to follow a similar path. While many cell lines proceed through the process efficiently with current protocols, others show much lower yield for reasons that remain unclear. Therefore, identification of extrinsic factors that facilitate and increase the consistency of RO differentiation is still required. One such candidate is nicotinamide (NAM), the amide form of vitamin B3, which has been reported to favor neural and RPE differentiation from hPSC and display anti-apoptotic functions on hPSC-derived neural cells  of NAM in this process. We report that NAM treatment for the first 8 days of retinal differentiation significantly increased RO production across multiple cell lines, including the ones that proved to be previously intractable using multiple established protocols. ROs derived with NAM showed a morphology and retinal cell type biogenesis indistinguishable from the controls. Further analyses indicate that NAM treatment favored neural induction over non-neural ectoderm cell fate at the early stage of differentiation and suggest that this effect is mediated, at least in part, through the inhibition of bone morphogenesis protein (BMP) pathway, a well-known anti-neuralizing signal  line is the t-test and statistical significance was defined as p < 0.05.The number of ROs in 3 independent differentiation batches was quantified based on morphological criteria . Comparisons between non-treated and NAM-treated conditions were performed using an unpaired Student's t-test and statistical significance was defined as p < 0.05.Brightfield images were taken using an EVOS XL Core Cell Imaging System . Fluorescence images were acquired with LSM-700 or LSM-880 confocal microscope  with Zen software. Fiji and Photoshop CC 2019 software were used for image export, analysis and processing. Comparisons between non-treated and NAM-treated conditions were performed using an unpaired Student's Total RNAs were extracted using RNeasy Plus Mini kit . At least 3 ROs from different batches were used for each biological replicate. Quality of isolated RNAs was assessed using Bioanalyzer RNA 6000 nano assays  and only high-quality RNAs (RNA integrity number (RIN) > 8) were used for construction of mRNA sequencing library. One hundred nanogram of total RNA were used to construct the strand-specific libraries using TruSeq Stranded mRNA Library Prep Kit-v2  with slight modifications as previously described  . All hPSThe number of ROs generated with and without NAM treatment was quantified at D60 based on morphological criteria . Substantial variation in differentiation capacity was observed across hiPSC lines. While some lines displayed high efficiency in RO production without NAM treatment , others such as hiPSC3 produced <1 RO/batch on average . NAM treTo determine whether ROs obtained by NAM treatment differentiated similarly to non-treated ones, we assessed the biogenesis of major retinal cell types at different time points . As expeRobust expression of rhodopsin (RHO) and cone opsins (L/M and S-opsin) highlighted the emergence of rod and cone photoreceptors under both conditions at D150 . MoreoveWe then investigated NAM mechanism of action in hiPSC1 and hiPSC3 lines, which were selected as typical examples of a high and low yield line, respectively. We first compared the morphology of non-treated hiPSC1 and hiPSC3 . One dayTo confirm that NAM treatment favors neural and eye field commitment, we evaluated the expression of anterior neuroectoderm (SOX1), eye field (PAX6), NR (CHX10) and RPE (MITF) markers identified in previous study  in NAM-treated EBs , an essential cofactor involved in ATP production and DNA repair  Gene Expression Omnibus (GEO), FR and TL: conceptualization. FR, ZB, and TL: methodology. FR and ZB: data analysis. FR, HC, LG, and RK: investigation. FR and HC: writing—original draft. FR, HC, ZB, AS, and TL: writing—review and editing. TL and AS: funding acquisition and resources. TL: supervision. All authors contributed to the article and approved the submitted version.This research was supported by Intramural Research Programs of the National Eye Institute , and Mr. Yair Mendels through the USHER2020 Foundation.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Head and neck squamous cell carcinoma are characterized by a high degree of inter- and intratumoral heterogeneity. Well-characterized preclinical models represent the heterogeneity of this disease and enable the development of innovative therapeutic concepts. The present work dealt with the establishment of patient-individual tumor models to explore new treatment approaches for HNSCC patients and to identify suitable biomarkers for predicting treatment response. In this study, tumor specimens from advanced cancers of the oral cavity, hypopharynx, and larynx were used to establish individual tumor models. These novel cell lines were used to apply different combinations of strategies, preclinically, and to overcome intrinsic resistance mechanisms.CDKN2a deletion, which resulted from a R58* mutation. Moreover, this cell line demonstrated chromosome 12 polysomy, accompanied by an increase in CDK4-specific copy numbers. In HNSCC16 P1 M1, we likewise identified polysomy-associated CDK4-gains. Although not sensitive to abemaciclib per se, the cell line showed a G1-arrest, an increased number of acidic organelles, and a swollen structure. Notably, intrinsic resistance was conquered by Cisplatin because of cMYC and IDO-1 downregulation. Additionally, this Cisplatin-CDKI combination induced HLA-ABC and PD-L1 upregulation, which may enhance immunogenicity. Performing functional and molecular analysis on patient-individual HNSCC-models, we identified CDK4-gains as a biomarker for abemaciclib response prediction and describe an approach to conquer intrinsic CDKI resistance.This study aimed to refine combined targeted approaches on well-characterized, low-passage tumor models. Upon in vivo xenografting in immunodeficient mice, three cell lines from locally advanced or metastatic HNSCC were established. Following quality control and basic characterization, drug response was examined after therapy with 5-FU, Cisplatin, and cyclin-dependent kinase inhibitors . Our cell lines showed different in vitro growth kinetics, morphology, invasive potential, and radiosensitivity. All cell lines were sensitive to 5-FU, Cisplatin, and THZ1. One cell line (HNSCC48 P0 M1) was sensitive to abemaciclib. Here, Cyto-FISH revealed a partial  Representative preclinical models are important tools to improve knowledge on tumor biology and treatment response. An ideal model preserves the phenotype and molecular features of the tumor, but also the complex and heterogeneous tumor microenvironment (TME) including immune and stromal cells. However, no available model covers all these characteristics . Three eTP53-mutated and wild type), immune landscapes , and clinical prognosis (poor to moderate) , dinaciclib , THZ1 , and Cisplatin . A readout was conducted with crystal violet staining. The Bliss Independence model was used to analyze potential synergistic and additive effects between the substances.Cells were seeded in 96-well plates in three technical replicates and treated after 24 h incubation at 37 °C and 5% COAn apoptosis–necrosis assay was carried out after 2 × 72 h of treatment, supplemented by cell cycle analysis after 2 × 72 h treatment as described previously .Acidic compartments were visualized as described . TherefoGAPDH was self-designed and used as a housekeeping gene . MYC (Hs00153408_m1), PDK2 (Hs00176865_m1), SKP2 (Hs01021864_m1), and IDO-1 (Hs00984148_m1) were 6-FAM labeled and purchased from ThermoFisher Scientific .RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR was carried out as described . Reactio4 beads/mL; size 10 µm; Polyscience, Warrington, PE, USA) were added to the 200 µL of cell suspension in the respective FACS tubes. Measurement of the beads and residual tumor cells stopped when 5000 beads were counted.HNSCC48 P0 M1 was stained with 5 µM 5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester  before seeding. 24 h later, PBMCs  were isolated by a density gradient centrifugation and added (tumor cell:PBMC ratio: 1:10). PBMCs were stimulated with 100 IU/mL of IL-2 . Abemaciclib [0.1 µM], Pembrolizumab [10 µg/mL] or the combination of both were added simultaneously with the PBMCs. After 1 × 72 h, cells were harvested and collected in FACS tubes. For the readout, 50 µL of 1:35 diluted beads , one-way ANOVA (Dunnett’s multiple comparison), two-way ANOVA (Tukey’s multiple comparison) or T-test was performed. If the normality test failed, the Kruskal–Wallis or U-Test was performed.All values are given as mean ± SD  or mean ± SEM (in vivo approach). Statistical evaluation was performed using GraphPad PRISM software, version 8.0.2 . The criterion for significance was set to Three novel HNSCC cell lines HNSCC16 M1 P1, HNSCC46 P0 M2, and HNSCC48 P0 M1 were generated after xenografting in NSG mice. Clinicopathological patients’ characteristics are summarized in TP53 mutations; while in the other cases, the p53-expression pattern is not mutation-specific. Because of tissue processing artifacts, the immunohistochemistry data  for HNSCC46 are not fully exploitable.The histological pattern of all three cases is that of a moderately differentiated squamous cell carcinoma A. HNSCC4+CD4+/CD3+CD8+ T cells and CD19+ B cells. CD16+CD56+ natural killer (NK) cells were found in all cases with the highest abundance in HNSCC46. This tumor case additionally showed the highest number of tumor-associated macrophages (TAM). The infiltrating CD3+ T cells showed a low level of CD274+ (no data available for HNSCC48). Roughly 15% of T cells were CD273+ (=PD-L2) and numbers comparable between the three HNSCC cases, while CD276+ was highest on HNSCC46/HNSCC48 (vs. HNSCC16).The TME was heterogeneous B. The stTP53 hotspot mutations (single or multiple) were detectable in all cases  of individual mutations revealed higher VAFs in the PDX and cell line compared to the patients’ tumors  and CDK4 were checked as potential biomarkers. All cell lines showed chromosome 9 polysomy with high copy numbers of the respective centromer , indicative for a gene specific CDKN2A deletion and thus confirming sequencing data.Gene-specific amplifications of entromer . In HNSCCDK4 is of high relevance for CDKI treatment. HNSCC16 P1 M1/HNSCC48 P0 M1 demonstrated polysomy of the chromosome 12, with the latter having 4–6 chromosomes/cell  with a molecular profile reflecting HPV-unrelated HNSCC. This included TP53 mutations in all cases [SMAD4 and CDKN2A mutations in HNSCC48 [CDKN2A cyto-FISH identified a partial gene deletion accompanied by chromosome 9 polysomy. In the other two cases, CDKN2A gains were equal to chromosome 9 copies  or remained unchanged with only chromosome 9 polysomy (HNSCC46). Chromosome polysomy is a common characteristic of advanced tumors and was described in p53 dysregulated HNSCC before [To address this issue, we initially tested HNSCC cell line establishment directly from patient tumor samples. Though this was described before ,21, we fll cases , along w HNSCC48 . CDKN2A C before .The direct comparison between patients’ tumors, PDX, and the corresponding cell line revealed preservation of the molecular profile—at least in early passages. This analysis is imperative to guarantee high-quality data generation and to correlate molecular features of the parental tumor with drug response. Rather unexpected were the VAFs of the PDX from HNSCC48. In HNSCC16 and HNSCC46, the VAFs remained stable or increased during transfer, likely because of increasing amounts of mutated cell clones and/or outgrowth of one dominating clone in culture. But in the HNSCC48 case, the VAF transiently decreased during passage, likely because of intratumoral heterogeneity. It can thus be assumed that a subclone in the tissue used for the PDX generation could not adapt to in vitro conditions. This phenomenon shows a limitation of our study and of cell lines in general and can only be vanquished by including different regions of one tumor. Still, this is laborious, time-consuming, and costly and thus hardly realizable. Solid cancers initially originate from one mutated cell clone with sustained proliferation ability and immune evasion capacity. But with disease progression, subclones develop and different genomic alterations occur, as a result of genomic instability. In HNSCC, up to six different subclones were found . Hence, Our cell lines exhibited various growth kinetics, radiation sensitivity, migration/invasion potential, and only HNSCC46 P0 M1 was positive for PD-1. Two of three cell lines formed solid spheroids in vitro and engrafted rapidly in vivo. HNSCC46 P0 M2 is the only exception, forming loose spheroids and showing slow engraftment rates, thus reflecting the reduced population doubling time in vitro. Still, the fact that two-thirds of cell lines formed spheroids allows further research to be a step closer to the in vivo setting. Such 3D models recapitulate interactions in the TME, transport properties, oxygen, nutrient, and proliferation gradients .CDKN2A due to a pathogenic R58* mutation may be the most likely molecular correlate for abemaciclib response [CDKN2A. We therefore checked for CDK4 gene amplifications via cyto-FISH because abemaciclib has a higher selectivity for CDK4 than for CDK6 [CDK4 gains. Such polysomy-associated CDK4 gains were also detectable in HNSCC16 P1 M1, but not HNSCC46 P0 M2 cells, which may explain our findings best. This is supported by the observation of morphological changes, such as an increased number of acidic organelles, a swollen cell structure and also the G1 phase arrest upon abemaciclib monotherapy in HNSCC16 P1 M1. We therefore propose CDK4 gains as a biomarker for response to abemaciclib, additionally to the known CDKN2A deletion. This allows us to conclude that polysomy and the associated increased copy number of the CDK4 gene increases the abemaciclib response . HNSCC46 P0 M2 was the most chemotherapy resistant and “special”. Though, eventually coincidentally, it is worth mentioning that the TME of the patients’ tumors harbored a high number of TAMs and NK cells as likely indicators for poor drug response. Such a link between the TME and cellular characteristics has been described for (intrinsic drug-resistant) cancer-stem cells only [Radiosensitivity was classified based on clinical doses . All celresponse ,32. Vicefor CDK6 . HNSCC48lls only  and demaCMYC is a prominent marker and was also higher in our cell lines than in adjacent normal mucosa [IDO-1 after abemaciclib treatment. Future research should address sequential pharmacological inhibition of IDO-1 (Indoximod or Epacadostat) [The challenge to improve the drug response is partially attributed to the upregulation of genes mediating drug resistance. l mucosa . Abemacil mucosa . We inteadostat)  to boostadostat) ,39. Giveadostat) . Using aadostat) , we routCDK4 gains as a new “surrogate” marker for abemaciclib response and describe a personalized approach to conquer intrinsic CDKI resistance.Summing up our findings, these newly established patient-individual tumor models guide the selection of drugs (combinations) and help in detecting intrinsic or acquired resistance mechanisms. With our complex set of analyses, we not only addressed morphological, molecular, and immunological aspects of advanced HNSCC, but also identified This work shows that well-characterized patient-individual tumor models are crucial for identification of effective therapy (combinations) and play an important role in detecting intrinsic resistance mechanisms. However, it also illustrates that in light of the intertumoral heterogeneity of HNSCC, a comprehensive characterization is necessary to draw general conclusions between the cell lines investigated or to explain different results and to identify potential new biomarkers.Here, we propose CDK4 gains as a biomarker for response to abemaciclib. Of note, abemaciclib resistance was reversible in one case (HNSCC16 P1 M1) by Cisplatin. This highlights the potential for combination therapies, especially in the context of intrinsic resistance towards targeted therapies.This study provides further evidence for the therapeutic potential of CDKIs and shows that they can be combined with classical chemotherapy. These findings contribute to our understanding of how the treatment of HNSCC can be improved in the future.


KRAS is mutated in approximately 25% of cancer patients and first KRAS G12C-specific inhibitors showed promising responses. Pancreatic cancer has the highest frequency of KRAS mutations but the prevailing KRAS G12D mutation is difficult to target. Inhibition of the GTP exchange factor (GEF) SOS1—KRAS interaction impairs oncogenic signaling independently of the specific KRAS mutations. In general, cell lines exhibiting KRAS mutations show specific alterations in respect to glucose utilization, signal transduction and stress survival. The aim of this investigation was to check the putative synergy of the SOS1 inhibitor BAY-293 with modulators targeting specific vulnerabilities of KRAS-mutated cell lines in vitro. The cytotoxicity of BAY-293 combinations was tested against MIA PaCa-2 (G12C), AsPC1 (G12D) and BxPC3 (KRAS wildtype) cell lines using MTT tests and calculation of the combination indices (CI) according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose utilization, inhibitors of the downstream MAPK pathway and several chemotherapeutics in dependence of the specific KRAS status of the cell lines. In particular, divergent responses for BAY-293 combinations between pancreatic and NSCLC cell lines were observed for linsitinib, superior inhibitory effects of trametinib and PD98059 in NSCLC, and lack of activity with doxorubicin in case of the pancreatic cell lines. Phosphoproteome analysis revealed inhibition of distinct signaling pathways by BAY-293 for MIA PaCa-2 on the one hand and for Aspc1 and BH1362 on the other hand. In conclusion, BAY-293 exhibits synergy with drugs in dependence of the tumor type and specific KRAS mutation. Around 85% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDACs) . PDAC isKRAS and inactivating mutations in CDKN2A, TP53, and SMAD4 with an incidence of approximately 85%, > 50%, 60–70%, and 50%, respectively [KRAS are G12D and G12V accounting for 80–90% of KRAS alterations in PDAC [KRAS G12C mutant pancreatic cancer patients [The most frequent genetic alterations in PDAC are activating mutations in ectively . Genes aectively , 12. Theectively . The mos in PDAC –16. Thus in PDAC –19. Howepatients .KRAS G12C mutations may be targeted by other inhibitors or in an indirect manner instead of covalent binding of the G12C inhibitors to the cysteine residue of the active center. RAS proteins exchange GDP for GTP for activation and the GEF SOS1 activates KRAS by increasing this GTP turnover [. Besides non-G12C KRAS mutations, amplifications of native KRAS are frequently found in pancreatic cancer.Non-turnover , 22. Thuturnover , 24. Sevturnover , 26. In turnover , 27. Howturnover . BesidesKRAS-amplified cancer cells to MEK inhibition and the SOS1 inhibitor BI-3406 has been demonstrated to synergize with the MEK inhibitor trametinib [BI-3406 is a potent SOS1 inhibitor that is orally bioavailable and active in cells with KRAS mutations, but not on KRAS wild-type cells . BAY-293ametinib , 29. Amoametinib –32.KRAS wildtype [Targeting of KRAS in pancreatic cancer except for the G12C mutation may be achieved by using SOS1 inhibitors that as single drug are expected to possess limited anticancer activity. Therefore, combinations with compounds that hit specific vulnerabilities of KRAS-driven cells may provide sufficient clinical efficacy for significant tumor treatment. For the present investigation, the SOS1 inhibitor BAY-293 was used to check for synergism with a range of compounds targeting signaling pathways, metabolism and DNA damage. BAY-293 is not optimized for clinical use but can be employed as test compound that shares a similar chemical core structure with the BI-3406 SOS1 inhibitor. The results obtained for the G12C-mutated MIA PaCa2 and the G12D-mutated pancreatic cancer cell lines are compared to the BxPC3 cell line that exhibits an unusual wildtype . Promisiwildtype .Chemicals were obtained from Sigma-Aldrich  or from Selleck Chemicals . Dulbecco’s phosphate buffered saline (PBS) was purchased from Gibco/Invitrogen . Compounds were prepared as stock solutions of 2 mg/mL in either DMSO or 0.9% NaCl for cisplatin and aliquots stored at − 20 °C. Equivalent concentrations of DMSO were supplemented for medium controls.Permanent cell lines BxPC3, MIA PaCa-2 and AsPC-1 were obtained from the American Type Culture Collection . The BH1362 KRAS G12C NSCLC cell line was established from a pleural effusion according to the Ethics Committee EK-21-210-1221 of the Viennese Hospital Association. Aforesaid cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS  and Penicillin/Streptomycin (Sigma-Aldrich). Upon confluence cells were detached with trypsin/EDTA (Sigma-Aldrich) and counted with a LUNA cell counter .4 cells in 200 µL medium were treated for four days with twofold dilutions of the test compounds in 96-well microtiter plates in quadruplicate . The plates were incubated under tissue culture conditions and cell viability was measured using a modified MTT -2,5-diphenyltetrazolium bromide) assay . Optical density was measured using a microplate reader at 450 nm and values obtained from control wells containing cells and media alone were set to 100% proliferation. IC50 values were calculated using the Origin software . For the assessment of the interaction of the test compounds, tests were performed comprising the individual drugs alone and in combination, followed by analysis using the Chou-Talalay method with help of the Compusyn software . The combination index CI < 0.9 indicates synergism, CI > 1.1 indicates antagonism and 0.9 < CI < 1.1 indicates an additive effect.Aliquots of 1 × 10The phosphorylation status of the cancer cell proteins was assessed using the Proteome Profiler Human Phospho-Kinase Array Kit  recognizing 37 kinase phosphorylation sites and two related proteins. The kit was performed according to the manufacturer´s instructions and spots evaluated using the ImageJ and Origin software.Statistical analysis was performed using Student’s t test for normally distributed samples . Values are shown as mean ± standard deviation (SD). Statistical significance is marked by an asterisk (*).50 was 2.07 ± 0.62 µM for BxPC3, 2.90 ± 0.76 µM for MIA PaCa2 and 3.16 ± 0.78 µM for AsPC-1. BxPC3 is wildtype for KRAS while the other two cell lines have a KRAS G12C and a G12D mutation, respectively. After determination of the cytotoxic effects of BAY-293 a possible synergistic or antagonistic effect was tested using a range of compounds. Figure The activity of BAY-293 was assessed on all cell lines, namely BxPC3, MIA PaCa-2 and AsPC-1. The ICIn Fig. The CI values were calculated for all three cell lines and 14 different modulators. We grouped the modulators according to their targeted pathway. BAY-293 was tested for antiproliferative effects in combination with 2-DG, dichloroacetate (DCA), metformin (MET), linsitinib (Lins) that modulate glucose utilization and the signaling of insulin/insulin-like receptors as well as WZB117, a glucose transporter 1 (GLUT1) inhibitor, respectively. Initial concentrations were 5 mM for 2-DG, 10 mM for DCA, 5 mM for metformin, 5 µM for linsitinib and 2 µM for WZB117. In Fig. The next group of compounds tested were the MEK inhibitors, namely trametinib (TRAM) and PD98059, the mTOR inhibitor rapamycin (RAPA), the CDK4/6 inhibitor palbociclib (PALB) and the pan-CDK inhibitor flavopiridol (Flavo). Initial concentrations were 50 µg/ml trametinib, 5 µM PD98059, 5 µM rapamycin, 10 µM palbociclib and 1 µM flavopiridol. TRAM and PD98059 exhibited a synergistic effect in BxPC3 and AsPC-1 and an additive effect in MIA PaCa-2 when combined with BAY-293, respectively , pemetrexed (PEME), doxorubicin (DOXO) and SN-38 see Fig. . The forThe phosphorylation of selected kinases of MIA PaCa-2 and Aspc1 pancreatic cancer cell lines were compared to BH1362 NSCLC cell line carrying a KRAS G12C mutation for controls and cells pretreated with BAY-293 in Western Blot arrays. In MIA PaCa-2 cells Akt shows decreased phosphorylation at S473 (unchanged at T308) and CREB exhibited a minor decrease in phosphorylation  and accounts for 7% of all cancer deaths with increasing occurrence . Median r growth , 37–40. r growth . Howeverrognosis , 42.The advantage of inhibition of mutant KRAS is the avoidance of toxicity against normal tissues expressing wild-type KRAS. However, the RAF-MEK-ERK kinase cascade downstream of KRAS is indispensable for cells and recruitment of compensatory mechanisms cause resistance to monotherapy . KRAS G1KRAS wildtype patient-derived xenograft (PDX) models had a minor antitumor effect but metformin treatment inhibited KRAS G12D tumor growth significantly [KRAS-mutated cancers are distinguished by alterations in metabolic pathways including elevated glycolysis and glutaminolysis as well as increased breakdown of fatty acids and nucleotides , 52, 53.ficantly . This fiCancer cells show aerobic glycolysis, the well-known Warburg effect, and dichloroacetate (DCA) diverts the glucose utilization from aerobic glycolysis to respiration by inhibition of mitochondrial pyruvate dehydrogenase kinase 1 (PDK1) , 61. DCAMutations in RAS cause permanent overaction of this G-protein and result in increased cell proliferation and survival due to boosted signaling via RAS-RAF-MEK-ERK and other pathways . The firKRAS mutant cells, in particular drugs targeting DNA topoisomerases, such as camptothecin derivatives and anthracyclines such as doxorubicin [Other possible additions to wildtype KRAS inhibitors may comprise cytotoxic anticancer drugs. In wildtype EGFR NSCLCs, a platinum/pemetrexed regimen was inferior for KRAS-mutant versus wildtype KRAS patients and this type of chemoresistance may be due to mutated KRAS-dependent induction of NRF2-mediated cellular stress response , 77. Herorubicin . HoweverInhibition of KRAS by the SOS1 inhibitor BAY-293 is expected to disturb the several downstream signaling pathways via Akt, the MAPK pathway or via RAL in dependence of the specific KRAS mutation and the cellular background. According to the present results, MIA PaCa-2 KRAS G12C is affected by BAY-293 via regulation of AKT, p70 S6 kinase and kinases such as Pyk-2 and Src kinases Src and Yes. In contrast, the effects of BAY-293 in Aspc1 cells carrying a G12D KRAS mutation result in altered phosphorylation of CREB, EGFR, ERK1/2 and Src kinases. The response to BAY-293 in BH1362 NSCLC KRAS G12C cells was similar to the alterations found in Aspc1 cells, namely concerning CREB, EGFR and ERK1/2 with additional changes in beta-catenin and Src kinases.32 and Tyr64 alters the protein conformation that inhibit the function of KRAS [Akt controls cellular growth, proliferation, metabolism as well as survival and migration resulting in enhanced tumorigenesis and induction of chemoresistance in various tumor types –82. Akt  of KRAS .Active CREB was found overexpressed in lung adenocarcinoma in dependence of mutant KRAS signaling . Elevate50 410 nM) and Calu-1 cells . In a panel of 60 cancer cell lines BAY-293 displayed a relatively broad anticancer activity spectrum. Interestingly, all SOS1 inhibitors BAY-293, BI-3406 and RM-0331 target essentially the same binding pocket of this GEF, establishing BAY-293 as model compound for in vitro studies [Inhibition of mutated KRAS has concentrated on covalent inactivation of Cys12 of KRAS G12C, on blocking KRAS downstream signaling, or prevention of KRAS–GEF interactions , 91. In  studies , 92. BI- studies . SOS1 in studies . In part studies .KRAS G12C inhibitors for the respective tumors and inhibition of downstream signaling is actively pursued in clinical studies. Interference with glucose uptake and metabolization at different levels seems to synergize with SOS1 inhibition as well as a range of conventional chemotherapeutics. The efficacy of all combinations is critically dependent on tumor type and on specific KRAS mutations. Furthermore, the distinct signaling pathways inhibited by BAY-293 must be taken into account for considering kinase inhibitors.The present results demonstrate that special vulnerabilities of KRAS-triggered tumor cells can be exploited successfully in drug combinations targeting different pathways Fig. . That SO