Auger emitters (AEs) are radionuclides that can cause DNA damage when delivered close to the DNA. Due to the nuclear location of PARP1, radiolabelling of PARPi with AEs provide an efficient nuclear delivery mechanism. This study shows the radiosynthesis of an AE radiolabelled PARPi ([125I]-PARPi-01) and its therapeutic effect as monotherapy or in combination with chemotherapeutics in a panel of TNBC cell lines. We found that [125I]-PARPi-01 efficiently induces DNA damage with therapeutic effect irrespective of BRCA mutation. All responsive cell lines have homologous recombination deficiency. Short pretreatment with doxorubicin significantly reduces clonogenic survival of both responsive and resistant cell lines.Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer, with a high recurrence rate. Since treatment of BRCAmut metastatic breast cancer, but they have shown limited response in triple negative breast cancer (TNBC) patients. Combination of an Auger emitter with PARPis enables PARP inhibition and DNA strand break induction simultaneously. This will enhance cytotoxicity and additionally allow a theranostic approach. This study presents the radiosynthesis of the Auger emitter [125I] coupled olaparib derivative: [125I]-PARPi-01, and its therapeutic evaluation in a panel of TNBC cell lines. Specificity was tested by a blocking assay. DNA strand break induction was analysed by γH2AX immunofluorescence staining. Cell cycle analysis and apoptosis assays were studied using flow cytometry in TNBC cell lines (BRCAwt/mut). Anchorage independent growth potential was evaluated using soft agar assay. [125I]-PARPi-01 showed PARP1-specificity and higher cytotoxicity than olaparib in TNBC cell lines irrespective of BRCA their status. Cell lines harbouring DNA repair deficiency showed response to [125I]-PARPi-01 monotherapy. Combined treatment with Dox-NP further enhanced therapeutic efficiency in metastatic resistant BRCAwt cell lines. The clonogenic survival was significantly reduced after treatment with [125I]-PARPi-01 in all TNBC lines investigated. Therapeutic efficacy was further enhanced after combined treatment with chemotherapeutics. [125I]-PARPi-01 is a promising radiotherapeutic agent for low radiation dosages, and mono/combined therapies of TNBC.PARP1 inhibitors (PARPi) are currently approved for BRCA Triple negative breast cancer (TNBC) is the most aggressive and heterogenous subtype of breast cancer , accounting for 15–20% of all breast cancers -PARPi-01) derived from the structure of the clinical PARPi olaparib. This is the first study investigating the therapeutic effects of [125I]-PARPi-01 as mono/combined therapies across a panel of TNBC cell lines comprising BRCA1wt and BRCAmut subtypes. We found that, irrespective of the BRCA status, TNBC cell lines with mutations in PI3K pathway respond to [125I]-PARPi-01 monotherapy at a considerably lower dosage than a pharmacological dosage used in clinic. A short pretreatment with Dox-NP (100 nM) significantly enhanced mitotic arrest in resistant metastatic cell lines and reduced the clonogenic survival.PARP1 is an attractive target for AE delivery proximal to DNA, because of its intranuclear localisation. Using PARPi as a vehicle provides a mechanism of AE delivery to DNA, a strategy that might avoid the need for additional usage of DNA damaging chemotherapeutic agents. In this study, we report the radiosynthesis of an Auger emitting PARPi . The delivered [125I] iodide in dilute NaOH was concentrated by evaporation and redissolution in the needed volume but did not exceed a concentration of 0.5 M NaOH. Moreover, 5 µL of a PARP-01 precursor solution (15 mM in DCM) was evaporated in a microcentrifuge tube by a N2 gas stream and redissolved in 20 µL of HOAc. The identity of the PARP-01 precursor was 4-{[4-fluoro-3-({4-[4-(tributylstannyl)benzoyl]-1-piperazinyl}carbonyl)phenyl]methyl}-1(2H)-phthalazinone. Moreover, 10 µL of the concentrated radioiodide solution was added to the tube, and labelling was started by adding 20 µL of a fresh chloramine-T solution (1.0 mM in MeCN). The reaction was maintained for 10 min at room temperature (24 °C) and the crude reaction mixture was purified using an analytical HPLC method . The product fraction  was diluted with 10 mL of water and was solid phase extracted by a preconditioned Sep-Pak light C18 cartridge . The [125I]-PARP-01 was eluted using 1 mL of CH3CN and evaporated to dryness at 85 °C in a N2 gas stream. The residue was taken up in 60 µL of EtOH and diluted by isotonic saline until the EtOH concentration was below 8%.The radionuclide wt , TNBC BRCAmut , non-TNBC BRCAwt cell lines  and a mammary epithelial cell line MCF10A as control. All cell lines were confirmed for the absence of mycoplasma. The cell lines HCC1937 and HCC1395 were obtained from ATCC and SUM1315M02 from BioIVT. The cell lines BT20, MDA-MB-231, MDA-MB-468, MCF7, Hs578T, MCF10A, SUM149PT, and SKBR3 were obtained from the Department for Gynaecology, University Hospital of Aachen. The cell lines MDA-MB-231, MDA-MB-468, SKBR3, BT20, and MCF7 were cultured in DMEM, supplemented with 5% FBS and 1% penicillin/streptomycin. Hs578T was cultured in DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and 10 µg/mL insulin. HCC1937 and HCC1395 were cultured in RPMI supplemented with 5% FBS and 1% penicillin/streptomycin. SUM149PT was cultured in F12(Hams), supplemented with 5% FBS, 10 mM HEPES, 1 µg/mL hydrocortisone, and 5 µg/mL insulin. SUM1315M02 was cultured in F12(Hams), supplemented with 5% FBS, 10 ng/mL EGF, 10 mM HEPES, 1 µg/mL hydrocortisone, and 5 µg/mL insulin. MCF10A, a human mammary epithelial cell line was cultured in DMEM:F12, supplemented with 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 µg/mL insulin, and 1% penicillin/streptomycin. All cell lines were maintained in their culture conditions at 37 °C at 5% CO2 during culture and experiments.The cell-line panel consists of 11 cell lines: TNBC BRCA®, Alabaster, AL, USA), followed by treatment for 60 h with 1 μM olaparib. For temozolomide (TMZ)  and olaparib co-treatment, the cells were pre-treated for 48 h with 50 μM temozolomide before co-treatment with 1 μM olaparib for 24 h. Single treatments with [125I]-PARPi-01 (~1 MBq/106 cells), olaparib (1 μM), dinaciclib , and talazoparib  were done for 72 h, dinaciclib (5 μM), and 1 μM olaparib co-treatment was done for 72 h. For Dox-NP and [125I]-PARPi-01 combination, the cells were pre-treated with 100 nM Dox-NP 6 h prior to incubation with [125I]-PARPi-01 (~1MBq/106 cells) for 72 h. Untreated cells were incubated in growth medium supplemented with DMSO (≤0.2%) for 72 h.Different co-treatments or pre-treatment strategies were followed for each combined therapy. For liposomal doxorubicin and olaparib treatment, the cells were pre-treated for 6 h with 100 nM Dox-NP . Cells were collected and washed after treatment; they were incubated with cytoplasmic, membrane, and nuclear extraction buffers, subsequent to one another; the fractions were collected, and protein measured using a BCA assay. Western blot was performed using the fractions (30 µg/lane) and, subsequently, the radioactivity in the blot was detected via a phosphorimager by incubating the blot with a Fujifilm imaging plate overnight followed by detection using Typhoon FLA.SDS-PAGE was carried out using premade Any KD Mini-Protean TGX gels at 90 V for 1.5 h. After adequate protein separation, the proteins were transferred to PVDF membranes using a wet blotting  technique. The blots were then blocked with 1% Milk/TBST and incubated with a primary antibody (anti-PARP1 (1:1000); Abcam ab227244; anti-GAPDH (1:4000); cell signalling 14C10). For the phosphorimager analysis, after blotting, the blots were sandwiched with a Fujifilm Imaging plate for Bioanalyzer (BAS-MS 2040), and incubated overnight, and the protein bound activity was imaged using a phosphorimager (Typhoon FLA 7000 version 1.2).5 cells were seeded in 12-well plates and allowed to adhere overnight, and were treated with different mono/combined drugs, as described above. After treatment, the media was removed and the cells were incubated with [125I]-PARPi-01 (~50 KBq/well) in serum free media for 1, 4, and 24 h. Subsequently, the cells were washed, trypsinised, and collected. The cell-incorporated activity was detected using a Wizard2 Gamma Counter . The cells were then lysed and the protein concentration was measured by a standard Bradford assay to calculate the uptake per mg of protein. The experiments were independently carried out thrice.1 × 10Annexin V apoptosis assay was performed using the Roche Annexin-V-FLUOS staining Kit. Cells were collected after treatment and pelleted by centrifugation. After a PBS wash, the cells were resuspended in Annexin-V-FLUOS labelling solution with Annexin V-FITC and propidium iodide, and incubated in room temperature for 10 min. The cells were then analysed using flow cytometry . Similarly, Nicoletti assay for cell cycle analysis was performed by permeabilising cells with a 50:50 mixture of 1% sodium citrate and 1% Triton X. Subsequently the cells were supplemented with 100 ng/mL of propidium iodide and RNase A, incubated overnight, followed by flow cytometry and analysis .5 cells were seeded on glass cover slips prior to treatment in a 24-well plate. After treatment, cells were fixed with 4% paraformaldehyde for 20 min. They were subsequently washed with PBS and blocked with 5% FBS/PBS with 0.3% Triton-X. The cells were incubated overnight at 4 °C with primary antibody ; Rabbit Anti-pH2AX, 1:500 ) dissolved in blocking solution. After washing with PBS, the cells were incubated for 1 h with secondary antibodies  at room temperature. After the PBS wash steps, the cells were mounted with Roti Mount® Vectashield containing DAPI and analysed using fluorescence microscopy . Quantification was performed using CellProfiler 3.0 software.1 × 10A soft agar colony formation assay was done to investigate the colony formation of cells post-treatment. The cells were trypsinised, collected after treatment, and counted using a haemocytometer. Moreover, 2500 cells/well were seeded in 0.3% soft agar  in media on a 6-well plate, over a 0.5% soft agar underlay. They were grown for about 21–22 days before staining with 4-NBT  and colony counting under a microscope.Data were analysed using GraphPad Prism 9 version and statistical analyses were performed using ANOVA with appropriate post-test algorithms.125I]-PARPi-01 was synthesised by a straightforward 125I-iododestannylation of tributylstannyl-benzoylated derivative of olaparib, using chloramine T as an oxidant  and low molar activity (11.8 ± 4.8 GBq/µmol) [125I]-PARPi-01 was evaluated in vitro (in MDA-MB-231 cells) by a blocking assay upon co-treatment of [125I]-PARPi-01 (1 nM) with 100× higher molar concentration of olaparib (100 nM). One-hour post incubation, co-treatment with olaparib reduced uptake (%) significantly (p < 0.05) to 1.15 ± 4.8% as opposed to control uptake (14.5 ± 4.8%) thereby proving target specificity ([ oxidant A. The ra oxidant . No deio oxidant . During Bq/µmol) . The tarcificity B.125I]-PARPi-01 treatment in vitro in triple negative breast cancer (TNBC), a panel of cell lines with BRCA deficient (mutant) or proficient (wild type) genotypes was chosen. Amongst the BRCAwt panel, two TNBC cell lines representing the basal A subtype  and two cell lines belonging to the aggressive basal B subtype  were chosen. The panel of TNBC BRCAmut cell lines consisted of representative cells from Basal A (HCC1937) and B subtypes . Additionally, two hormone receptor positive breast cancer cell lines  were included to evaluate their responsiveness, along with a non-malignant mammary breast epithelial cell line (MCF10A). In western blot analysis, TNBC cell lines did not display statistically significant differences in cellular PARP1 expression, whereas benign breast epithelial cells showed enhanced expression (To determine therapeutic effects of [pression .125I]-PARPi-01 uptake upon pretreatment in their previously reported dosages. The cells were pre-treated with the cyclin-dependent kinase (CDK) inhibitor dinaciclib , DNA alkylating temozolomide  prior to incubation with [125I]-PARPi-01. Pre-treatment with the DNA intercalating liposomal doxorubicin  was done for 6 h prior to treatment with [125I]-PARPi-01. Liposomal formulation of doxorubicin was chosen due to the prevalent clinical use of this type of formulation as opposed to Doxorubicin in solution. The cellular uptake and retention were evaluated at 1, 4, and 24 h later. Additionally, [125I]-PARPi-01 uptake upon long term exposure to olaparib  and talazoparib  were studied. This was performed to assess if a long-term exposure (72 h pre-treatment) has a blocking effect on 125I-PARPi-01 uptake or leads to a possible uptake increase due to enhanced PARP1 expression. Due to the enhanced cytotoxicity of talazoparib as compared to olaparib, they were treated at 10 nM and 1 µM, respectively. Remarkably, while a time dependent increase in uptake was observed in almost all cancer cell lines irrespective of pre-treatments (except SKBR3), the mammary epithelial cell line MCF10A showed time dependent decrease despite of detected high cellular PARP1 expression  to the PARPi olaparib (1 µM) was effective in blocking uptake (0.8 − 0.5x) up to 24 h in almost all cell lines, except Hs578T and SUM1315M02. Interestingly, a pre-treatment with the highly affine PARPi talazoparib (10 nM) did not block as effectively as olaparib . Exposure to chemotherapeutic drugs did not show any significant impact on cellular -PARPi-01 monotherapy. The DNA damage level was further increase upon combination with Dox-NP pre-treatment . Notably, this effect was not observed uniformly in all cell lines, indicating differences in DNA repair pathways due to their varying underlying genotypes. Amongst the BRCAmut cohort, only the epithelial SUM149PT showed to be responsive with significantly higher dsDNA damage upon [125I]-PARPi-01 monotherapy. The epithelial HCC1937 showed significant dsDNA damage induction upon combination treatment. In HCC1395 and SUM1315M02 cell lines, the dsDNA damage level was not significant. The breast cancer cell lines SKBR3 remained unaffected whereas MCF7 did show higher DNA damage upon each treatment. Importantly, even if MCF10A showed significant H2AX formations upon [125I]-PARPi-01 monotherapy, the foci amount was lower when compared to that detected in the responsive malignant breast cells.Amongst the BRCAreatment . Howeverreatment . [125I]-125I]-PARPi-01 mono-treatment  three Basal A epithelial TNBC cell lines BT20, MDA-MB-468 and HCC1937 showed changes in the cell cycle distribution with a significant increase in the most radiosensitive G2-M phase (p < 0.001)  . This re< 0.001) B.mut) also showed a decrease in the G0–G1 population despite a lack in significant mitotic arrest. The TNBC SUM1315M02 (BRCAmut), HER2+/hormone receptor positive (HR+) breast cancer cell lines SKBR3 and MCF7 (BRCAwt) did not show significant changes upon treatment with [125I]-PARPi-01 all one and in combination with Dox-NP. Additionally, changes in the cell cycle distribution was evaluated for other combination chemotherapy strategies and compared to that induced by an Auger emitter treatment  as monotherapy and further completely inhibited after combined pre-treatment with Dox-NP (mut) and mutant HRas (HRasmut) genes in MDA-MB-231 and Hs578T, respectively, which are known to drive the epithelial–mesenchymal invasion mediated tumourigenicity  with colony diameters >250 µm . This hih Dox-NP . Similargenicity  35,36,3,36,3125Iormation , possiblmut TNBC patients, several BRCAmut patients are still unresponsive warranting PARPi sensitivity based stratification -MAPi  [123/125I]-KX1 -MAPi via local intratumoral injection or delivery. Similarly, 125I-KX1 was tested in vitro in ovarian cancer cell lines and showed reduction in cell survival.Auger emitter (AE) based therapy strategies have gained recognition and are studied in several cancers ,43,44,45r probe)  and Ruca25I]-KX1 . During 125I]-PARPi-01. Our radiosynthesis yielded up to 2.5 times higher molar activity and significantly higher radiochemical yield than reported by Wilson et al. [125I]PARPi-01, at molar concentrations of 0.01–0.04 µM. Importantly, it is 250–1000 times lower than minimal cytotoxic concentration of olaparib proving amplified therapeutic efficiency in the clinic -PARPi-01 can be of therapeutic value in TNBC. Nevertheless, TNBC is highly heterogenous and can show varied response. Due to this, a tailored approach based on clear understanding of subtype-specific response is crucial [125I]-PARPi-01 monotherapy. The most radiosensitive cell-cycle phase is the G2-M phase and enhanced cell cycle arrest in this phase shows that the Auger emitter functions by being recruited to the PARP1 mediated DNA repair machinery possibly in the S-phase followed by further DNA damage and arrest in the G2-M phase.This AE-based PARPi: -PARPi-01 induced dsDNA damage, significant cell cycle arrest and reduction in clonogenic survival. As shown in other studies, these cell lines are PARPi sensitive, which resulted in olaparib mediated enhanced γH2AX formation and mitotic phase arrest upon PARPi mono  and combination therapies as presented here [125I]-PARPi-01 sensitivity in BT20 [125I]-PARPi-01 based PARP inhibition coupled with DNA damaging approach. Thus, HRD positive patients harbouring somatic/germline PTEN deletion/loss-of-function can clearly benefit from targeted [125I]-PARPi-01 therapy.Among the cell lines investigated in this study, BRCAted here . This ex in BT20 . Similar in BT20 ,49. Howe in BT20 . TAI als in BT20 ,52. In s in BT20 ,39. In owt), Hs578T (BRCAwt) and HCC1395 (BRCAmut)), MDA-MB-231, and HCC1395 showed talazoparib sensitivity, accordingly to previous studies [125I]-PARPi-01 observed in uptake assay. However, their MGMT deficiency and response to TMZ predisposes these subtypes for TMZ combination with [125I]-PARPi-01 treatment -PARPi-01 mono-therapy significantly induces γH2AX formation, mitotic phase arrest higher than olaparib and blocks the tumorigenicity in TNBC cells regardless of BRCA status, PI3K mutations and RAS family mutations. In resistant subtypes, therapy combining [125I]-PARPi-01 with doxorubicin pre-treatment showed enhanced killing efficacy with a complete inhibition of colony formation. The general therapeutic efficacy is remarkable, considering the apoptosis rates detected in some cell lines. This underlies the unique therapeutic potential of Auger electron emitters and of the presented approach combining the endogenous irradiation and PARP inhibition. Therefore, Auger emitter-based [125I]-PARPi-01 therapy provides a promising and attractive alternative to conventional chemotherapy for patients stratified to have better prognosis with PARP1 monotherapy or in combination with DNA damaging agents. Owing to its high efficacy and low required dosage, [125I]-PARPi-01 is expected to induce less systemic toxicity.Taken together these results show that [125I]-PARPi-01, derived from the clinical PARP inhibitor olaparib, and assessed its therapeutic value in different TNBC subtypes. Apart from the ability of [125I]-PARPi-01 to cause DNA damage at (up to) significantly lower concentrations than olaparib, we identified responsive subtypes of TNBC. Responsive subtypes with underlying impaired DNA repair mechanism are forced towards apoptosis upon treatment with [125I]-PARPi-01, while the benign mammary epithelial cell line is not affected significantly. For the non-responsive TNBC subtypes, a combination therapy strategy involving Dox-NP as conditioning for treatment with [125I]-PARPi-01 significantly increased the therapeutic response, resulting in a complete inhibition of the clonogenic growth. Thus, an Auger-emitting inhibitor of PARP is a promising therapeutic strategy that can be  fitted by combined pre-treatment, accordingly, to the unique phenotype of each single TNBC subtype.In this study, we developed and evaluated an Auger emitter-based therapeutic PARP inhibitor [


Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI  showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI  promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI  promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis. Abnormalities in bone formation are characterized in bone diseases such as osteoporosis and periodontitis . The numOsteoblasts play important roles in controlling skeletal integrity by forming new bone and remodeling old bone, and the dysfunction of osteoblasts in proliferation, migration, and differentiation contributes to the pathogenesis of bone diseases ,8,9,10. Paeonia suffruticosa +, molecular formula C20H28O12; 1H-NMR  δ: 7.75 , 6.81 , 6.66 , 5.03 , 4.97 , 2.64 , 4.04 , 3.97 , 3.89 , 3.86 , 3.77 , 3.49-3.66 , 3.38 ; 13C-NMR  δ: 200.7 (C-7), 166.3 (C-4), 160.5 (C-2), 133.2 (C-6), 122.7 (C-1), 111.0 (C-5), 109.0 (C-1″), 103.3 (C-3), 102.6 (C-1′), 80.6 (C-3′), 78.2 (C-5′), 78.1 (C-3″), 77.1 (C-2′), 75.1 (C-4′), 74.9 (C-2″), 71.5 (C-6′), 68.9 (C-4″), 65.7 (C-5″), 56.4 (OCH3), 32.3 (C-8).Light yellow powder; EI-MS 2 and 95% air as previously described [MC3T3-E1 pre-osteoblasts (CRL-2593) purchased from the American Type Culture Collection (ATCC)  were cultured in α-minimum essential medium (α-MEM)  without L-ascorbic acid  supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere of 5% COescribed .Osteoblast differentiation was induced using osteogenic supplement medium (OS) containing 50 μg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP). The medium was replaced every two days during the incubation period as previously described .Cell viability and proliferation were assessed using a 3--2,5-diphenyl tetrazolium bromide (MTT) solution to detect NADH-dependent dehydrogenase activity, as previously described . Formaza2 and 95% air. Cell migration was also assessed with some modifications using the Boyden chamber assay as previously described [Cell migration was assessed using a wound healing assay as previously described . Brieflyescribed . BrieflyOsteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with PALI  for seven days. ALP staining assay was performed as previously described . Cells wOsteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with PALI  for seven days. ALP activity assay was performed as previously described .Osteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with PALI  for 14 days. ARS staining was performed as previously described . BrieflyWestern blot analysis was performed as previously described . BrieflyImmunofluorescence was performed as previously described . Cells wwww.graphpad.com, accessed on 4 January 2021). Data are presented as mean ± standard error of the mean (SEM). Statistical differences between the groups were analyzed using a Student’s unpaired t-test. Statistical significance was set at p < 0.05.Statistical analysis was performed using GraphPad Prism software version 5.0.0 for Windows (GraphPad Software Inc., San Diego, CA, USA;


ST3GAL1/2/3/5/6, ST6GAL1/2, ST6GALNAC2/6, and ST8SIA1/2), which are known to play a role in tumor progression. We could show that glycation of BEN-MEN-1 cells led to decreased expression of ST3Gal5. This resulted in decreased synthesis of the ganglioside GM3, the product of ST3Gal5. In the malignant meningioma cell line, we observed changes in expression of sialyltransferases  after glycation, which correlates with less aggressive behavior.Meningiomas are the most common non-malignant intracranial tumors and prefer, like most tumors, anaerobic glycolysis for energy production (Warburg effect). This anaerobic glycolysis leads to an increased synthesis of the metabolite methylglyoxal (MGO) or glyoxal (GO), which is known to react with amino groups of proteins. This reaction is called glycation, thereby building advanced glycation end products (AGEs). In this study, we investigated the influence of glycation on sialylation in two meningioma cell lines, representing the WHO grade I (BEN-MEN-1) and the WHO grade III (IOMM-Lee). In the benign meningioma cell line, glycation led to differences in expression of sialyltransferases ( Meningiomas arise from the arachnoid and are the most common non-malignant intracranial tumor ,2,3,4,5.Like many other tumors, meningiomas use glucose as a primary energy source (Warburg effect), which is considered as one of the “hallmarks of cancer” ,12,13.During glycolysis, up to 0.4% of the glucose is converted into methylglyoxal (MGO). MGO is a typical side product of glyceraldehyde-3-phosphate, which is generated by the aldolase reaction from fructose-1,6-bisphosphate. Please note that MGO is more than 20,000 times more reactive than glucose . PreviouThe dicarbonyl MGO reacts primarily with proteins  or to a small extent also with DNA and lipids. This non-enzymatic reaction between the carbonyl groups of dicarbonyls  or monosaccharides  and the amino groups of proteins is called glycation ,24. AnotAnother common posttranslational modification is glycosylation. In contrast to glycation, glycosylation is an enzymatic addition of carbohydrates, glycans to a non-carbohydrate-structure, commonly a lipid or protein in the endoplasmatic reticulum (ER)/Golgi. Sialylation is of deep interest and describes the addition of sialic acids (Sia) to lipids  or proteins ) through sialyltransferases (ST) .N-acetyl neuraminic acid (Neu5Ac) represents the major Sia of mammals. It is synthesized from UDP-N-acetyl glucosamine (UDP-GlcNAc) in the cytosol [N-acetyl glucosamine 2-epimerase/N-acetyl mannosamine kinase (GNE) [N-acetyl galactosamine  alpha 2,6-sialyltransferases  and alpha 2,8-sialyltransferases (ST8Sia1-6) [N-glycans [N-glycosylation sites such as transforming growth factor β (TGFβ) or glucose transporter 4 (GLUT4) show rapid response to increasing GlcNAc concentrations causing complex glycan formation and branching [ cytosol . The keyse (GNE) . Sialyla8Sia1-6) ,37. The 8Sia1-6) ,37. High-glycans . Especiaranching . Ganglioranching . This isranching ,42,43.In this study, we compared the expression of STs in benign and malignant meningioma cells and found significant differences between these two. Furthermore, we investigated the role of the glycating metabolite MGO on the expression of STs in both benign and malignant meningioma cells. Thereby, we could show that glycation has a dramatic effect on the expression of STs and consequently on the GM3 expression. As a result, this could change sialylation-dependent tumor progression in meningioma.® CRL-3370™) was obtained from American Type Culture Collection . Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 100 µg/mL of streptomycin, 100 U/mL of penicillin, 4 mM of glutamine, and 10% fetal bovine serum  at 37 °C in a 5% CO2 incubator. The cell lines were split every 2–3 days with 0.1% Trypsin-EDTA (Ethylenediaminetetraacetic acid) solution for 2 min.The human benign meningioma cell line BEN-MEN-1 was obtained from Leibniz-Institute DSMZ  and the human malignant meningioma cell line IOMM-Lee  were cells  without MGO or GO treatment. The cell lines were cultivated for 24 h. RNA was isolated using the Quick-RNA™ MiniPrep Kit  according to the manufacturer’s instructions. The quality and concentration of the RNA were analyzed using the NanoDrop 1000 Spectrophotometer . RNA (2 μg) was transcribed into cDNA using SuperScript™ II Reverse Transcriptase according to the manufacturer’s instructions. PCR reactions were performed using DreamTaq DNA polymerase (Thermo Fisher Scientific), and products were separated on a 1.5% agarose gel. The following conditions were used: initial denaturation for 2 min at 95 °C, 35 cycles , final elongation for 5 min at 72 °C. We use for all sialyltransferases the same primer pairs which were used in a previous study [Cells were seeded in 12-well plates at a density of 3.95 × 10us study .−ΔΔCT). All reactions were performed in triplicate.The sialyltransferase expression of untreated meningioma cell lines  and after 24 h of glycation with 0.3 mM MGO were measured via quantitative real-time PCR (qPCR) using the iQ™ 5 Multicolor Real-Time PCR Detection System  and qPCR GreenMaster  with the same primer pairs used for normal PCR. The following conditions were used for qPCR: initial denaturation for 1:30 min at 95 °C, 40 cycles , final elongation for 1 min at 72 °C, followed by a melting curve analysis. The expression level of sialyltransferases in control and glycated cell lines was determined relative to GAPDH , calculated as ΔCT. The control was relatively computed to glycated cell line  was added to the samples followed by 15 min sonication. Methanol (350 μL) was added to the cell pellets and incubated for 4 h with shaking at room temperature. The upper phase containing GSLs was collected after centrifugation at 2700× g for 20 min. Then, 400 μL of chloroform/methanol  was added, followed by adding 400 μL of methanol/water . After sonication and centrifugation, the upper phase was collected and pooled to the previous sample. The process of adding methanol/water , sonication, centrifugation and removing the upper phase was repeated another two times. In each replicate, the upper phase was collected and replaced by the same volume of methanol/water . The combined upper phases were dried under vacuum in an Eppendorf Concentrator 5301  at 30 °C.Extraction of GSLs and preparation of GSL-glycan alditols from cells were performed in triplicate as previously described . The celv/v), 2 mL of methanol followed by equilibration with 2 mL methanol/water . The extracted GSLs were loaded to the cartridge and washed with 2 mL methanol/water . The GSLs were eluted from the column with 2 mL methanol and 2 mL chloroform/methanol . The samples were dried under vacuum in an Eppendorf Concentrator at 30 °C.Before the purification of the GSLs using reverse-phase (RP) SPE, the samples were dissolved in 100 μL methanol followed by the addition of 100 μL water. TC18-RP-cartridges were prewashed with 2 mL of chloroform/methanol , EGCase I buffer (4 μL) and water (34 μL) (pH 5.2) was added to each sample and incubated for 36 h at 37 °C. The released glycans were collected and loaded on TC18-RP-cartridges, which had been preconditioned with 2 mL of methanol and 2 mL of water. The samples were washed with 200 μL of water and residual glycans were loaded to the cartridge. Then, 500 μL of water were added to the cartridge to wash the glycans from the column. The flow-through and wash fractions were pooled and dried in an Eppendorf Concentrator at 30 °C.The reduction was carried out with slight modifications following the same procedure as described in previous work ,46. In bv/v) acetonitrile/10 mM ABC. The trap column was packed with 5 μm particle size PGC stationary phase from Hypercarb PGC analytical column , while the PGC nano-column was packed with 3 μm particle size PGC stationary phase from Hypercarb PGC analytical column .The analysis of glycan alditols was performed using PGC nano-LC-ESI-MS/MS following a method described previously ,46. MeasTo analyze glycans, 2 μL injections were performed and trapping was achieved on the trap column using a 6 μL/min loading flow in 1% solvent B for 5 min. Separation was achieved with a linear gradient from 1% to 50% solvent B over 73 min, applied followed by a 10 min wash step using 95% of B at a 0.6 μL/min flow rate. The column was held at a constant temperature of 35 °C.m/z range of 340–1850 in enhanced mode using negative ion mode, smart parameter setting was set to m/z 900. MS/MS spectra were recorded using the top 3 highest intensity peaks.Ionization was achieved using the nanoBooster source (Bruker) with a capillary voltage of 1000 V applied and a dry gas temperature of 280 °C at 3 L/min and isopropanol enriched nitrogen at 3 psi. MS spectra were acquired within an Structures of detected glycans were studied by MS/MS in negative mode. Glycan structures were assigned based on the known MS/MS fragmentation patterns in negative-ion mode ,48, elutN-acetylglucosamine; yellow square is N-acetylgalactosamine; blue circle is glucose; yellow circle is galactose; red triangle is fucose; purple diamond is N-acetylneuraminic acid, grey diamond is N-glycolylneuraminic acid.Structures are depicted according to the Consortium of Functional Glycomics (CFG). Blue square is t-test against the control group, both cell lines of a theoretical value of 1 , were executed. Figures show the average mean with standard deviation (SD) and levels of significance are represented within the figures.All analyses and visualizations were performed using OriginPro 2019 software . Paired Student ST3GAL1–6 in BEN-MEN-1 and IOMM-Lee. ST3GAL1–ST3GAL3 and ST3GAL5–ST3GAL6 were detected in both cell lines. The band intensity of ST3GAL2 was higher in the malignant cell line compared with the benign cell line, whereas the ST3GAL3, ST3GAL5–6 band intensities were higher in BEN-MEN-1 compared to the malignant cell line. Agarose gel of ST6GAL1–2 is shown in ST6GAL2 was only expressed in the benign cell line. In contrast, no differences could be found in terms of band intensity of ST6GAL1 in both cell lines. The expression of ST6GALNAC1–6 for both cell lines is shown in ST6GALNAC2 was detectable in BEN-MEN-1. Expression of ST6GALNAC4–6 was detectable in both cell lines. The band intensities of ST6GALNAC5 and ST6GALNAC6 were higher in IOMM-Lee compared to BEN-MEN-1. The expression of ST8SIA1–6 in both cell lines is shown in ST8SIA1–2 and ST8SIA5–6, the expression has been detected in both meningioma cell lines. In BEN-MEN-1, we observed a higher expression of ST8SIA2 and ST8SIA6 compared to the malignant cell line. Again, the band intensity of ST8SIA5 was stronger in IOMM-Lee compared to the benign cell line.Since there is evidence that sialyltransferases have an impact on tumorigenesis, we analyzed benign (BEN-MEN-1) and malignant (IOMM-Lee) meningioma cell lines regarding differences in expression of sialyltransferases , Table 1ST3GAL1–6 in BEN-MEN-1 (ST3GAL1 (1.4812 ± 0.115 fold change), ST3GAL2 (3.143 ± 0.476 fold change), and ST3GAL3 (1.28 ± 0.189 fold change) expression were increased in contrast to non-glycated cells in BEN-MEN-1. Furthermore, the relative expression of ST3GAL5 (0.7863 ± 0.0933 fold change) and ST3GAL6 (0.572 ± 0.126 fold change) were decreased in contrast to non-glycated cells. The relative expression of ST3GAL1 (0.601 ± 0.223 fold change), ST3GAL2 (0.288 ± 0.0535 fold change), ST3GAL3 (0.6175 ± 0.217 fold change), ST3GAL5 (0.4561 ± 0.1271 fold change), ST3GAL6 (0.502 ± 0.1325 fold change) were decreased in contrast to non-glycated cells in the malignant cell line.Since the expression of sialyltransferases is different in the benign and malignant meningioma cell lines, we quantified the sialyltransferase mRNA expression level after 24 h of glycation of the cells to verify the influence of glycation on sialylation. EN-MEN-1 A and IOMEN-MEN-1 B. GlycatST6GAL-family in BEN-MEN-1 (ST6GAL2 was only detected in BEN-MEN-1 and increased after treatment with MGO (1.624 ± 0.188 fold change).EN-MEN-1 A and IOMEN-MEN-1 B. GlycatST6GALNAC1–6 in BEN-MEN-1 (ST6GALNAC2 expression decreased after glycation in contrast to the untreated benign cell line (0.6807 ± 0.1106 fold change). ST6GALNAC4 expression is not affected in BEN-MEN-1 (2.556 ± 1.232 fold change) and IOMM-Lee (1.005 ± 0.2552 fold change), but glycation decreased the mRNA expression of ST6GALNAC5 in both cell lines . Glycation led to a higher expression of ST6GALNAC6 in the benign cell line (1.5141 ± 0.1999). ST6GALNAC6 expression is not influenced by glycation in the malignant cell line (0.839 ± 0.203).Moreover, the mRNA expression of EN-MEN-1 A and IOMEN-MEN-1 B is alsoST8SIA 1–6. Glycation influenced more strongly the expression level of these sialyltransferases in BEN-MEN-1 cells compared to the malignant IOMM-Lee cell line. The expression of ST8SIA1 was highly increased (2.696 ± 0.627 fold change) after glycation in the benign cell line (ST8SIA2 (3.2171 ± 0.6837 fold change) and ST8SIA5 (1.696 ± 0.3475 fold change) were both increased in BEN-MEN-1 , which is in line with the decreasing expression of ST3GAL5 (see: 8 ± 9.15 × 106) were decreased by 235% compared to the glycated cells (5.61 × 107 ± 3.89 × 106).To prove that MGO-induced reduction of ST3GAL5 expression has an impact on BEN-MEN-1 cells, we quantified ganglioside GM3 by PGC nano-LC-ESI-MS/MS. AL5 see: A. The noST3GAL5 expression as MGO. We could show by Western blot analysis that 0.3 mM glyoxal (GO) leads to glycation in both cell lines (data not shown). Using qPCR of cDNA of BEN-MEN-1 cells -β1-induced epithelial-mesenchymal-transition, migration, and invasion, and a knockdown resulted in the opposite [ST3GAL2 and ST6GAL1 were significantly upregulated in tumors with positive perineural invasion status [ST3GAL3 was increased in the glycated BEN-MEN-1 cell line but decreased in glycated IOMM-Lee cells. Expression of ST3GAL3 is important for the regulation of biosynthesis of brain disialoganglioside (GD)1a and trisialoganglioside (GT)1b [ST3GAL3 has an impact on cell adhesion and invasion. Glycation of meningioma cell lines resulted in decreased ST3GAL5 expression in both cell lines. This sialyltransferase is also known as monosialoganglioside (GM3) synthase [ST3GAL5) as MGO. However, in IOMM-Lee cells, we observed no effect after glycation with GO, which could be explained by higher glyoxalase 1 activity, which degrades dicarbonyls and has been described in many studies on cancer and glycation [ST3GAL6 was reduced in both glycated meningioma cell lines, which is known to play a key role in the generation of functional Sialyl Lewis X [ST3GAL6 can lead to decreasing migration and invasion in 5637 and J82 UBC cells as well as decreasing adhesion and migration in multiple myeloma cells [There are many reports of changes in ST expression in cancer. Overexpression of opposite . Anothern status , which we (GT)1b . In sevesynthase  and suppsynthase  and the synthase . The totsynthase . Other ssynthase ,42,66,67lycation ,69,70. T Lewis X . Decreasma cells ,73.ST6GAL1 expression, as we have shown in glycated BEN-MEN-1 cells, was also found in lung, colon, glioma, prostate, cervical, and breast cancer tissues [ST6GAL1 decreased metalloproteinases (MMPs) expression and suppressed invasive potential of A549 and H1299 cells in vitro [ST6GAL1 upregulation, a tumor-suppressive role [ST6GAL2 was found in different types of cancer and was associated with breast cancer with higher expression of intracellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, CD24, MMP2, MMP9 and C-X-C motif chemokine receptor (CXCR)4 [Increased  tissues ,78,79,80ive role . The upr (CXCR)4 ,83.ST6GALNAC2 is known as a metastasis suppressor in breast cancer and a low expression of it, as we observed in glycated BEN-MEN-1 cells, is associated with a bad prognosis [ST6GALNAC2 correlates with metastases to the lymph system [ST6GALNAC4 expression was elevated in glycated BEN-MEN-1. High expression of ST6GALNAC4 leads to the prevention of O-glycan chain elongation [ST6GALNAC4 gene led to an inhibition of invasive behavior in vitro and in vivo [ST6GALNAC5 in both glycated meningioma cell lines in our study could be a sign of transformation, because it is restricted to the brain and synthesizes GD1alpha in the nervous tissues [ST6GALNAC6 in glycated BEN-MEN-1 cells. A study in colon cancer has shown that ST6GALNAC6 is responsible for the synthesis of sialyl Lewis (a), which is a significant inductive mechanism in cancer progression [rognosis ,85. In Crognosis . In conth system . The expongation . In anot in vivo . The low tissues ,91. We hgression ,93.ST8SIA1 in glycated BEN-MEN-1 cells could lead to a weak prognosis for patients. In contrast, we observed decreased expression of ST8SIA1 after glycation in IOMM-Lee cells. In melanoma brain metastases, it was shown that ST8SIA1 (GD3 synthases) is upregulated and the GD3 expression is increased, which was associated with a bad prognosis [ST8SIA1 is one of the key drivers for malignancy in glioblastoma [ST8SIA2, as we observed in glycated BEN-MEN-1 cells, plays a role in the invasive behavior and was significantly associated with the risk of relapse in non-small-cell lung carcinoma [The increased expression of rognosis . In gliorognosis . In addiblastoma . Mennel blastoma . A studyblastoma . The incarcinoma . The siaarcinoma .The glycation of meningioma cell lines has opposite effects in benign or malignant meningioma cells. Overall, glycated BEN-MEN-1 cells express more sialyltransferases than unglycated, whereas glycation of IOMM-Lee cells leads to a downregulation of the sialyltransferase expression. These observations support our recent observations that glycation of BEN-MEN-1 cells lead to increased invasive potential .ST3GAL1/2/3/5/6; ST6GAL1/2; ST6GALNAC2/6 and ST8SIA1/2. These STs have a direct or indirect impact on tumor progression. The decreased expression of ST3GAL5 after glycation results in a decreasing expression of GM3 in benign meningioma cells. The expression levels of some sialyltransferases  of the glycated IOMM-Lee cell line were inhibited, which indicates less aggressive behavior.To sum up, glycation of meningioma cell lines has cell line-specific effects. The glycated BEN-MEN-1 cell line is affected in a different expression of


BTK is a dominant bioactive kinase expressed within both cancer and immune cells of GBM tissue. Complex cell co-cultures might better model the impact of kinase inhibitors as therapeutics in GBM. Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A “functional proteomics” screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival. Glioblastoma (GBM), a World Health Organization (WHO) grade IV astrocytoma, is the most common aggressive tumour originating in the brain . The incEGFR) aberrations (PDGFR) and vascular endothelial growth factor receptor (VEGFR) were found in 40% and 39% of examined GBMs, respectively, making them attractive potential targets for therapeutic inhibition . BTK inhibitors are currently being used in GBM clinical trials NCT03535350). Because BTK is known to be expressed in both cancer cells and immune cells 35350. Be, 37, we The functional status of protein kinases in GBM tissue and patient-matched tumour-derived cell lines was measured using a Pamstation12 kinomics platform . The plaIn patient G-222, the total kinase activity (specific activity) was substantially higher in lysates derived from GBM tissue relative to the patient-matched cell line  staNext, we performed a comparison between three replicates of G-222 control GBM stem-like cells and three replicates of G-222 tissue and we found that 142 peptides (142/194) were higher in G-222 tissue . A totalt test, using the three biological replicates of the G-222 tissue and of the G-222 cell line grown under standard conditions . These data were viewed in a volcano plot to assess the peptides that were differentially phosphorylated using lysates derived from G-222 GBM tissue or from the G-222 cell line  was analyzed by unsupervised hierarchical clustering with a nditions . A totalell line . These dell line  are propell line .P-value < 0.05) peptides (substrates) exhibited higher phosphorylation activity in G-222 GBM tissue compared with G-222 control cells.List of statistically significant  peptides (substrates) exhibited higher phosphorylation activity in G-222 control cells compared with GBM G-222 tissue.List of statistically significant . In addition, these same 18 peptides also exhibit higher phosphorylation states using the kinomics platform, when comparing G-221 tissue versus G-221 derived cell line  , 42, 43.ell line .P-value < 0.05) Bruton tyrosine kinase–related peptides (substrates) that exhibited higher phosphorylation activity in G-222 tissue compared with GBM G-222 control stem-like cell line. This table is taken from Table S1.List of statistically significant  , 10 GBM In GBM cell lines and mice xenografts, BTK silencing or inhibition results in reduced neurosphere forming ability, migration, and glioma cell proliferation, and induces apoptosis , 44, 45.The apparently discordant expression we observed between BTK protein expression in GBM-derived stem-like cell lines and tumour tissue is important to resolve because BTK is presumed to be expressed in GBM cancer cells, and as such, BTK inhibitors are being developed in clinical trials for GBM patients (NCT03535350). Therefore, we set out to define the cell type(s) in which BTK protein is expressed in GBM tumour tissue to better inform an interpretation of this emerging clinical trials data and better stratify patients.Cancer tissue lysate contains many cell types, including tumour cells, normal tissue architectural cells, and immune infiltrates. Our observation that BTK expression was lower in GBM cell lines compared to GBM tissue samples may reflect that its expression is in a non-cancer cell type or the GBM stem-like cells from a BTK-negative tumour niche. In addition, BTK expression is classically associated with cell lineages of the haematopoietic system, such as B-lymphocytes, monocytes, and macrophages , 47. HowWe first used immunohistochemistry from formalin-fixed paraffin-embedded (FFPE) material to assess BTK protein expression in a glioma tissue microarray (TMA) alongside immune cell markers, such as CD163. SOX2 was used as a marker of GBM cancer stem-like cells. The TMA consisted of 127 WHO grade II–IV glioma samples, including GBM grade IV, anaplastic astrocytoma (AA) grade III, anaplastic oligodendroglioma (AO) grade III, diffuse astrocytoma (DA) grade II, and oligodendroglioma (O) grade II. The clinical and molecular information for each of the 127 glioma tissue samples used to generate the TMA are summarised in We evaluated BTK protein expression by quantifying the percentage of BTK-positive staining in each core using QuPath, an open-source quantitative pathology and bioimaging software . We inclWe compared BTK protein expression in serial FFPE-derived sections against expression of immune cell markers in various glioma tissue samples using antibodies to macrophage cells (CD163) and against the glioma cancer stem-like cell marker (SOX2)  was therefore performed to further examine the extent of BTK, SOX2, and CD163 co-expression within individual cells in GBM and a low-grade glioma , and S9.We next assessed the extent of co-expression of BTK and CD163 antibodies in a high-grade glioma . We founBecause CD163 expression predicts poor prognosis in GBM , we nextP = 0.026), chemotherapy (TMZ) treatment  and KPS > 80  (P = 0.038)  . Whereas= 0.038) . TogetheThis study identifies a novel relevance of BTK towards GBM molecular biology. We have shown that: (1) BTK protein expression predicts good prognosis in GBM; (2) BTK is not expressed in state-of-the art GBM stem-like cell lines, but can be expressed in mixed cancer and immune cell populations within matched GBM tissue; and (3) because BTK inhibitors is already being planned for clinical trials, developing better ex vivo cell cultures that recapitulate BTK protein heterogeneity in tissue  will betKinase inhibitors represent one of the most dominant drug types currently available for use in human cancers . To exteSince the 1950s when the HeLa cell line was introduced , tumour-Studies that have compared pathway-specific gene expression of tumour cell lines, tumour tissue samples and normal tissues samples reported that most of the cell lines exhibited up-regulation of genes related to cell cycle progression and metabolic pathways , 63. GenThe differences in kinome activity have several potential explanations. First, in-vitro culture conditions may select for cells that are not representative of the tumour tissue cancer cell bulk. Second, the tumour tissue lysate is a homogenate of immune, endothelial, and stromal cells, in addition to tumour cells, and the observed kinase activity may relate to those non-tumour cells. The kinase activity of these non-tumour cells likely impacts on the druggable tumour cell proteome itself. Our observations using IHC suggest that some of the observed differences between the in-vitro and tissue analysis comes from the glioma microenvironment. This is not surprising because the microenvironment is not recapitulated in culture. For example, in-vivo oxygen concentration is reported to be less than 2% , not theThe dominating kinase target that we identified in tissue samples was BTK. BTK is a non-RTK belonging to the TEC kinase family . MutatioIn glioma, BTK is considered a cancer cell biomarker , althougFinally, across glioma grades, it was interesting to note that BTK protein expression was observed in different cellular compartments; the nucleus in lower grade and/or the cytoplasm in higher grades, and this is consistent with published studies , 71. BTKIn conclusion, our observations that GBM tissue samples have a higher, if not different, kinome compared with GBM-derived cell lines suggests that we can improve the ex vivo complexity of GBM cell models to maximize physiological relevance with respect to kinase signalling and kinase inhibitor drug screening. More sophisticated models are needed that better recapitulate the biochemical complexity of the human tumour tissue. Better understanding of tumoral heterogeneity in different cell types exhibiting kinase expression has important implications for future drug screening and development of targeted therapies.http://gcgr.org.uk/) and cultured according to a previously published method  condition (control kept in same media).GBM patient samples were acquired under informed consent according to local ethical approvals (LREC 115/ES/0094). Human neural samples were acquired under informed consent according to local ethical approvals (08/S1101/1). GBM stem-like cell lines  and human neural stem-like cell lines (GCGR-NS12ST_A and GCGR-NS17ST_A) were obtained from The Glioma Cellular Genetics Resource (GCGR)  tissue lysates and cancer cell lines lysates were performed using the Pamstation12 platform  and the PTK PamChip (PamGene) containing 196 phosphopeptides as previously described , 82. In 2 transformed to display signal in all peptides (https://www.pamgene.com/ (PamGene 2017).BioNavigator6 (Ver 6.3.67.0)  software was used to create kinetic activity post-wash curve slope using images captured in several exposure times , multiplied by 100 and logpeptides . Moreovepeptides . VSN is GBM surgical tissues and cancer cell lines were lysed in kinase lysis buffer or 8 M Urea buffer, 0.1 M Tris (pH 8.5). After protein concentration determined using the Bradford or BCA method, a total of 30–50 μg of total protein was loaded into SDS page acrylamide gels then transferred to nitrocellulose membrane. The membranes were blocked using 5% (wt/vol) non-fat milk powder in PBS + 0.1% (vol/vol) Tween 20 (PBST) for 1 h at room temperature then incubated with the mouse anti-BTK antibody , gently shaking overnight at 4°C followed by the mouse secondary mouse antibody (DAKO) for 1 h at room temperature. Loading antibody β-actin (Sigma-Aldrich) was incubated for 1 h at room temperature followed by the secondary antibody (DAKO). The immunoblots were washed three times each for 5 min with PBST between each step. Antibody binding was detected by enhanced chemiluminescence.Immunohistochemistry (IHC) staining was performed in the Pathology Department at the University of Edinburgh. Samples were sectioned from FFPE tissue blocks at a thickness of 5 μm and stained on positively charged slides (Thermo Fisher Scientific) to maximize tissue adherence. IHC was performed using a BOND III autostainer with Bond Polymer Refine Detection Kit  according to manufacturer instructions. Slides were blocked with Peroxide 3–4% (vol/vol) hydrogen peroxide for 5 min. Antigen retrieval was performed using Bond Epitope Retrieval Solution 1   for 20 min at 100°C. Slides were incubated at room temperature with a BTK antibody , SOX2 , or CD163  for 20 min. Visualisation was achieved using 3,3′-diaminobenzidine (DAB) staining and haematoxylin counterstaining (Leica Biosystems). Finally, slides were dehydrated and cleared in xylene before coverslips were applied. Three FFPE tissue blocks  were used to optimize the BTK antibody dilutions before applying on the TMA.FFPE GBM and Oligodendroglioma blocks were cut into 5 μm sections in the Pathology Department at the University of Edinburgh. Immunofluorescence staining was performed as described previously . BrieflyThe TMA was constructed as previously described . BrieflyTMA slides were scanned using a Hamamatsu NanoZoomer XR Digital Pathology slide scanner at ×40 magnification. Digital images of the slides were analyzed using QuPath, an open-source pathology and bioimaging software (version 0.2.0-m7) . We alteTo validate and confirm QuPath accuracy, we performed a comparison between QuPath results and manual counting. Manual counting is the gold standard for assessing protein expression in IHC. For this comparison, we calculated the relative error (RE) of the QuPath method as /manual. The threshold for a “real difference” between QuPath and manual count was set as RE > 0.200 (20%), based on a threshold previously described to assess interobserver variability P-value ≤ 0.05 was considered to be statistically significant.BTK immunohistochemistry data were analyzed by SPSS (version 25). A Kruskal–Wallis test was used to evaluate the differences between the grades. The survival curve was estimated by using the Kaplan–Meier method and differences were tested by the log-rank test. One patient was not included because of unavailability of survival data. A This study includes no data deposited in external repositories.


Choline kinase alpha (CHKA) is a promising target for the development of cancer therapeutics. We have previously reported ICL-CCIC-0019, a potent CHKA inhibitor with high cellular activity but with some unfavorable pharmacological properties. In this work, we present an active analogue of ICL-CCIC-0019 bearing a piperazine handle (CK146) to facilitate further structural elaboration of the pharmacophore and thus improve the biological profile. Two different strategies were evaluated in this study: (1) a prodrug approach whereby selective CHKA inhibition could be achieved through modulating the activity of CK146, via the incorporation of an ε-(Ac) Lys motif, cleavable by elevated levels of histone deacetylase (HDAC) and cathepsin L (CTSL) in tumour cells; (2) a prostate-specific membrane antigen (PSMA) receptor targeted delivery strategy. Prodrug (CK145) and PSMA-targeted (CK147) derivatives were successfully synthesized and evaluated in vitro. While the exploitation of CK146 in those two strategies did not deliver the expected results, important and informative structure-activity relationships were observed and have been reported. Abnormal lipid metabolism is a common feature observed in many cancers . EnhanceA number of small-molecule CHKA inhibitors have been developed as novel anti-cancer strategies for CHKA inhibition . HemichoN,N-dimethylaminopyridine (DMAP) derivatives bearing alkyl linkers of varying lengths were designed by Trousil et al. as CHKA inhibitors ; ICL-CCIC-0019 caused rapid and sustained lipid synthesis inhibition by acting on CDP-choline pathway and also showed potent in vivo anti-tumor activity. The compound, however, induced mitochondrial changes and slight reduction in body weight (not below the standard 15–20%) observed in the rodent model bearing HCT116 xenografts; implying adverse pharmacological effects which precluded clinical translation D4-FCH has been reported elsewhere D4-FCH (740 kBq) in presence of inhibitors in 1 mL for an additional hour at 37 °C. Cells were washed with ice-cold PBS three times and lysed in 1 mL RIPA buffer. The radioactivity of 800 µL lysate from each well was counted on a gamma counter  and normalized to protein content as determined by BCA assay.HCT-116 and C4-2B cells were seeded at a density of 5 × 10t-tests from GraphPad Prism 7.0 were used to determine the significance in the experiments. Statistic differences were significant at 0.01 < p < 0.05, very significant at 0.001 < p < 0.01 and extremely significant at 0.0001 ≤ p < 0.001 and p < 0.0001.Data were represented as mean values ± standard deviation (SD). Unpaired two-tailed The synthesis of CK146 from building blocks 1 and 2 is described in 3 PSMA-maleimide, Creative Chemistry Ltd., Hampton, UK). This produced 7 in a moderate yield of 56% which was deprotected to produce the final compound CK147 (8) ready for biological evaluation without further purification  was performed by contract synthesis  following a literature procedure . In brieThe conjugate stability of CK147 was assessed by HPLC, where ICL-CCIC-0019 served as an internal standard. At the early time points, CK147 remained stable in medium at 37 °C (93.0 ± 0.8% intact parent at 2 h and 89.7 ± 0.3% intact parent at 4 h). After 72 h, the concentration of CK147 in medium reduced to 60.0 ± 0.3% (21.7 ± 0.2 μg/mL) . Chemica50) of ICL-CCIC-0019, CK146 and CK145 were evaluated in these four cell lines by SRB assay. It was shown that all the three compounds potently inhibited the proliferation of cancer cells at low sub-micromolar concentrations (50 in four cancer cell lines: 0.5 ± 0.02 μM); CK146 still displayed potent antiproliferative activity (mean GI50 against four cancer cell lines: 2.5 ± 0.3 μM) with HepG2 being most sensitive to the treatment (0.21 ± 0.01 μM). The overall antiproliferative activity of CK145 was similar to CK146 (mean GI50 against four cancer cell lines: 5.0 ± 0.3 μM), but it resulted in less growth inhibition in HepG2 and Caco-2 cells. All three compounds displayed weaker antiproliferation in Caco-2 cells—a less tumorigenic colon cancer cell line—compared to HCT-116 cells.Appropriate cell lines were selected on the basis of mRNA levels of HDAC I class and CTSL using Cancer Cell Line Encyclopedia RNA sequencing database . The HDAtrations . ICL-CCI50 across eight cell lines of 0.6 ± 0.1 μM and 3.8 ± 0.9 μM respectively (50: 66.7 ± 7.2 μM) and the correlation of GI50 with PSMA expression was not observed.In addition, the antiproliferative activity of CK147 was assessed in PSMA-positive  and PSMA-negative cell lines . The expression of CHKA and PSMA in prostate cell lines was determined by immunoblotting a. ICL-CCectively b. HoweveUptake of ICL-CCIC-0019 and CK145 in HCT-116 cells increased with incubation time . The cel3-protected CK147) were included in the experiment for comparison. In both experiment groups, ICL-CCIC-0019 (tR: 7.36 min: sec), CK146 (tR: 6.30 min: sec) and CK148 (tR: 11.30 min: sec) were detected in the cell lysates by HPLC following 4 h incubation, while uptake of CK147 was not seen (−1 protein and 34.1 ± 0.6% μg−1 protein for FCS positive and negative groups respectively (−1 protein) compared to CK147  and the other was FCS-free medium (FCS negative group). ICL-CCIC-0019, CK146 and CK148 ((OtBu)not seen . ICL-CCIectively . Signifito CK147 . Drug upHPLC/MS analysis of cell lysates and the medium after 4 h incubation with CHKA inhibitors confirmed the stability of all the compounds in cells and medium. Decomposition or interconversion was not observed from the HPLC/MS .−6 cm/s). Notably, CK14 was the most subject to efflux with an efflux ratio of 87; the permeability of CK147 was too poor to permit calculation of the efflux ratio.The predicted in vitro pharmacokinetic characteristics of compounds were evaluated by four ADME assays, namely kinetic solubility assay, metabolic stability assay using human liver microsome S9, permeability and efflux study using the Caco-2 cell line, and matrix stability assay using fasted state simulated gastric fluid (FaSSGF). All tested CHKA inhibitors were highly soluble (>150 μM) in 0.1 M PBS (2% DMSO) . All CHKTM Kinase Assay. This assay measured generated ADP as a function of the kinase activity via a luciferase reaction  . T. T47]. T50 against recombinant CHKA2: ICL-CCIC-0019: 270 nM; CK14: 150 nM) D4-FCH was utilized in this work to predict the in vitro choline uptake inhibition by compounds of interest. Parallel to the lipid kinase screening results, CK145 inhibited the uptake of [18F]D4-FCH in a similar trend to CK146, which suggested that the intact CK145 harbored CHKA activity.A 50) of CK147, in 7 prostate cell lines with differential PSMA expression and the HCT-116 colon cell line, was unrelated to PSMA expression; furthermore, all the tested cell lines were not sensitive to CK147. The observed poor antiproliferative activity of CK147 relative to CK146 implied that the two mechanisms driving the cellular uptake of CK147, (1) PSMA-driven internalization and (2) choline transporter-driven internalization, were not as efficient as expected. This led to subsequent experiments to investigate cellular uptake efficiency and in vitro metabolism by HPLC analysis of cell lysates and [18F]D4-FCH competitive study in the whole cells.A small-molecule peptide (Glu-Urea-Lys) with high affinity towards PSMA was conjugated to CK146 via a non-degradable linker, which gave rise to CK147, a proposed ligand-drug conjugate (LDC) for targeted PCa treatment b 53]. C. C53]. C3-protected form of CK147. Electrostatic interactions may also explain the concentration reduction of CK147 observed in the stability study whilst no chemical decomposition was detected. The compound may have formed complexes with serum albumin over the time and then have been removed in the next protein precipitation step before MS analysis.At physiological pH, CK147 contains two positive charges from the quaternary ammonium moieties, and three negative charges from the carboxylates of the Glu-Urea-Lys peptide. Although not zwitterionic due to the charge imbalance, we propose a putative mechanism where the positive quaternary ammonium charges may be masked by inter- or intramolecular electrostatic interactions which neutralize the charges and form unfavorable structural configurations capable of inhibiting both PSMA-binding and CHKA activity  55]. Th. Th55]. A novel derivative of ICL-CCIC-0019 bearing a piperazine handle (CK146) was successfully synthesized and retained biological activity towards CHKA; our work opens up the possibility for further structural elaboration of CK146, to explore the chemical space around this moiety and build up SAR to modulate biological and pharmacokinetic properties. Interestingly, the introduction of the piperazine structure to the scaffold seems to offer CHKA inhibitors additional activities towards PIK4CB, a potential antimalarial and antiviral target. In this work, we presented the successful transformation of CK146 into two drug development programs to produce targeted cancer therapies via prodrug strategy (CK145) and drug delivery strategy (CK147), although conversion to active components or targeted delivery was not achieved. Regardless, we have demonstrated the potential for modulating CHKA activity via piperazine functionalization and work in our laboratory will continue, including molecular modelling studies to understand the interactions between these molecules and the binding site of interest, to develop a suitable candidate for clinical translation.


Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients’ care. Tremendous progress has been made.50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data (https://manticore.niehs.nih.gov/cancerRxTissue). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug.In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (ICWe demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.The online version contains supplementary material available at 10.1186/s12864-021-07581-7. Studies that characterize human cancer cell lines and evaluate their sensitivity to drugs provide valuable information about the therapeutic potential and the possible mechanisms of action of those drugs. Those studies allow the identification of genomic features that are predictive of drug responses and make it possible to relate findings from cell lines to tissue samples and, ultimately, to translate laboratory results into patients’ care.50 value . GDSC also quantified the basal level gene expression of many of the cancer cell lines using microarray  into 100 sets of 30 genes. We observed that many genes were being selected at frequencies more than 100 times above that expected by chance. The most frequently selected genes for each drug are potentially informative about that drug’s mechanism of action as well as about a cancer cell line’s sensitivity to the drugs. For some other drugs, multiple genes were selected with lower but distinctly higher-than-random frequencies, suggesting that multiple genes together are necessary for predicting cell-line sensitivity for those drugs. Many of the drug-gene interactions were also identified by others [For each of the top 272 predictable drugs, we counted how many times each gene was selected into the 100 sets of the C19orf33 (chromosome 19 open reading frame 33) was among the most frequently included predictive genes for the largest number of drugs, appearing in more than 20% of the predictive gene sets for 17 drugs  with the IC50 values of more than 100 drugs for those cell lines  inhibitors for the cell lines ; both were discovered and characterized in immortalized cells as being upregulated compared to senescent cells [C19orf33 expression may be a general biomarker for the sensitivity of cancer cell lines to many chemotherapeutic agents.nt cells . IMUP-1 nt cells –26. Overnt cells . Our datABCB1 appeared in more than 20% of the predictive gene sets. ABCB1 encodes ATP binding cassette subfamily B member 1, a member of the superfamily of ATP-binding cassette (ABC) transporters. ABCB1, also commonly known as MDR1, is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity [ABCB1 is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs [For 14 drugs of diverse mechanisms of action, cificity . Expresser drugs –30.MDM2 was selected in nearly 100% of gene sets predicting sensitivity to nutlin-3a. Other known p53 known target genes (CDKN1A and RPL22L1) were also selected in nearly 90% of those predictive gene sets, suggesting that high expression of these genes identifies tumors with a wild-type p53, and thus responsive to a further p53 activation by nutlin-3a.Many of the most frequently selected genes were also known targets of the drugs  is a HSP90 inhibitor, and NQO1 expression is inversely correlated with 17-AAG IC50s in cancer cell lines [NQO1 was selected in 100% in the predicted gene sets for tanespimycin sensitivity. ADK (adenosine kinase) was selected in 100% of gene sets predicting sensitivity to AICAR. AICAR is an analog of adenosine monophosphate (AMP) that is capable of stimulating AMP-dependent protein kinase (AMPK) activity [The drugs PD173074 and AZD4547 are two potent fibroblast growth factor receptor (FGFR) inhibitors ; FGFR2 w protein ; BCL2 wall lines . Similaractivity . AICAR pactivity .SPRY2  was among the most frequently selected genes for predicting sensitivity to all six MEK1/2 inhibitors in GDSC datasets  , indicating that cancer cell lines with higher SPRY2 expression are more sensitive to the MEK inhibitors. The other most frequently selected genes for MEK1/2, BRAF, and ERK1/2 kinase inhibitors were ETV4 (ETS variant transcription factor 4) and SPRY4  was selected in 100% of gene sets that predicted sensitivity to acetalax. On the other hand, TRPM4 was selected in fewer than 5% of the sets for all other drugs, suggesting that the TRPM4-acetalax interaction is specific. The mechanism of action for acetalax is unknown. Recent studies suggested that TRPM4 may be implicated in regulating cancer cell migration and in the epithelial-to-mesenchymal transition [TRPM4 in prostate cancer cell lines increased Snail protein expression and reduced expression of E-cadherin [TRPM4 expression in the CCLE cancer cell lines was inversely correlated with the IC50 of acetalax in those cell lines     as an ex50 values of trametinib for the ~ 11,000 TCGA RNA-seq samples. Our results indicated that the median predicted IC50 values of trametinib for melanoma  and intestine tumors  were much lower (showing higher sensitivity to trametinib) than those for all other tumor types  samples. The ability to predict drug sensitivity of individual tumors is important for personalized medicine.Although the median predicted ICBRAF mutations were not associated with the predicted sensitivity to trametinib  . For each gene, we divided the TCGA samples for each tumor type into two groups based on whether or not the sample carried mutations in that gene. We considered all genes in the mutation data. To conduct this analysis on a gene and tumor type combination, we required the tumor type to have at least five samples that carried the gene mutation. We then used the Wilcoxon rank-sum test to assess whether predicted sensitivity to trametinib differs between the two groups. Interestingly, we found that nib Fig. a. Howeve31) Fig. b. Similapes Fig. c. Specif pathway . Those r50 values) of all TCGA  < 0) to about 35 of the 272 drugs . Many of the drugs target DNA/protein synthesis, cell cycle, microtubules, and the mTOR pathway. Most of the drugs were also predicted to be similarly cytotoxic to normal samples from TCGA  with more than 20 normal samples.For each of the 272 drugs, we compared the median predicted IC50) value for tumor samples was more than one logarithmic unit lower than that for corresponding normal samples in at least one of the 14 tumor types  lower than the median of the medians from all tumor types. We identified 109 such drugs , most (96) of which were predicted to have lower IC50s for either diffuse large B cell lymphoma (DLBC), thymoma (THYM) or both. Interestingly, 12 of the remaining 13 drugs that were predicted not specific for DLBC, THYM or both are kinase inhibitors, consistent with the notion that kinase inhibitors target specific cellular pathways. Eighty-three drugs were predicted to have higher specificity for a unique tumor type; 74 for DLBC, 5 for SKCM (skin cutaneous melanoma), 2 for THYM, and 1 for HNSC (head-neck squamous cell carcinoma) and 1 for KIRP . Our analysis suggested that the B-Raf proto-oncogene (BRAF) inhibitors  are specific for melanoma  and breast invasive carcinoma (BRCA) Fig. b. Severa50 values of a drug for the samples of the subtype with the median of the medians of the predicted IC50 values for the five subtypes. We focused on drugs for which the difference in the medians exceeded 0.5 logarithmic units (corresponding to a 1.65-fold difference in IC50). Among the 270 drugs, seven drugs met this criterion  with the IC50 values of hundreds of chemotherapeutic drugs in those cell lines  for melanoma and colorectal cancer, ERBB/EGFR inhibitors (afatinib and sapitinib) for head-neck squamous cell carcinoma, and BCL2 inhibitors  for low grade glioma and glioblastoma multiforme. We further uncovered that OSI-027 was highly specific for breast cancer, especially the Her2-positive subtype breast cancer tumors. Furthermore, our result suggests that We also predicted that a few drugs not only were tumor-type specific but also induced higher sensitivity in tumor tissue than normal tissue for those tumor types. Those drugs may have better clinical efficacies. Our result suggested that paclitaxel (microtubule inhibitor) (commonly known as taxol) was more specific for breast, lung, and uterine tumors than other tumor types and that those tumors were overall more sensitive to paclitaxel than corresponding normal tissue. Similarly, we predicted that trametinib was specific for melanoma, colorectal tumors and rectal tumors; our analysis also found that those tumors were also more sensitive to trametinib than the normal tissues of the same origins. Sapitinib (ERBB inhibitor) was predicted to have the highest specificity for lung squamous cell carcinoma and was also predicted to be more cytotoxic to lung carcinoma than to normal lung tissue.50 values for all TCGA tumor samples  are more specific for basal-like breast cancer subtypes whereas ABT737 and navitoclax sapitinib and afatinib are more specific for the luminal subtypes especially luminal B. We predicted that OSI-027 (mTOR inhibitor) is specific for breast cancer among all 33 TCGA tumor types, especially the Her2-positive breast cancer subtype. Since we have provided the predicted IC50 values of OSI-027 for breast cancer cell lines from GDSC2 are the lowest among all 265 cancer cell lines; however, OSI-027 IC50s for breast cancer cell lines from GDSC1 were not among the lowest. Consequently, we predicted that OSI-027 with drug ID 1594, but not drug ID 299, is specific for breast cancer. GDSC recommends using assay data from GDSC2 when available, our results for OSI-027 were based GDSC2 data. However, additional replications are warranted.It is worth pointing out that OSI-027 was assayed twice  with two different drug IDs (299 for GDSC1 and 1594 for GDSC2). GDSC1 screened 906 cancer cell lines for the drug; whereas GDSC2 screened 265 cancer cell lines. The ICThere are many challenges associated with relating findings from cell lines to tumors and clinical applications. For example, in vitro assays do not capture organ responses. Although we used the largest collection of cancer cell lines in our predictive models, our models have limitations when applied to datasets that may not be represented by the training data. Nonetheless, translating findings from cell lines to tumors has had some success , 21, 22.k-nearest neighbor rule to predict drug response of an unknown sample. The predicted value of a sample is taken as the average of the values of its k-nearest neighbors. Because of the averaging, the most extreme predicted values, either high or low, usually cannot be as extreme as the corresponding observed values. Therefore, although the correlation between the predicted and observed values can be high, e.g., 0.8, the magnitude of the predicted values is generally pulled in from the extremes; the trend of the predicted values among the samples, however, is usually preserved  algorithm combines a genetic algorithm for feature selection and the k-nearest neighbor method for classification or prediction [d genes (see below) whose expression levels can accurately predict observed IC50 values using the k-nearest-neighbors prediction rule. The prediction rule is simple: the predicted IC50 value of a sample is defined as the average of the observed IC50 values of its k nearest neighbor samples (excluding itself) as determined by Euclidean distance in the d-dimensional space defined by a gene set. In making predictions for a testing-set sample (see below), we considered only samples within the corresponding training set as potential neighbors.The GA/KNN . The squared-error loss = N is the number of samples in the training set. The GA/KNN algorithm applied here sought a set of d genes to minimize this loss function.The GA/KNN algorithm is designed to optimize, either minimize or maximize, an objective function. For prediction, a typical objective function being minimized is the sum of the squared deviations between the observed and predicted IC). To identify the optimal number of nearest neighbors (k) and the “chromosome” length d, we systematically evaluated 16 combinations of k  and d   genes whose expression levels were best predictive of the IC50 values of samples in the training set using a leave-one-out cross-validation procedure [d genes was subsequently used to predict the IC50 values of the testing-set samples. The average IC50 value of the k-nearest (k = 3) training neighbors of a testing sample was taken as the predicted IC50 value for the testing sample. The above procedure was repeated 100 times independently, each started with a new random partition into training and testing sets. Over the 100 random training-testing partitions for a given drug, each sample would be expected to appear in about 90 training sets and about 10 testing sets. The final predicted value for a training-set sample was the average of the predicted IC50 values for that sample over the subset of the 100 independent training-testing partitions in which that sample appeared in a training set; analogously, the final predicted value for a testing-set sample was the average predicted IC50 value over the partitions where that sample appeared in a testing set.For high dimensional data, multiple sets of rocedure . For eacrocedure . That seBecause each training-testing partition provided a set of 30 genes as predictors, we used the frequency with which a gene was selected into the 100 sets of 30 predictor genes as a measure of the importance of that gene in prediction.ρP) and Spearman (ρs) correlation coefficients between the observed and predicted IC50 values for samples in the training and testing sets, respectively. We designated those drugs whose ρP and ρs values were both greater than or equal to 0.4 (ρP ≥ 0.4 and ρS ≥ 0.4) for the testing set samples as predictable drugs.We computed both the Pearson  and a testing set (10%), repeating the partitioning 100 times, as above. In addition, with each partition, we treated all TCGA samples in the tumor data and the GTEx samples in the GTEx data as additional “testing” samples for prediction.50) for each combination of cell line and compound as a measure of the sensitivity of cell viability in that cell line to the compound. We downloaded the ln (IC50) data from the GDSC website https://www.cancerrxgene.org/downloads/bulk_download. When combining data from both releases, if IC50s for the same cell line and compound were present in both, we kept only the one from GDSC2 as advised by GDSC. In total, 453 drugs were assayed, among which 397 were unique (56 had two different drug IDs). For those with two unique drug IDs, we did not combine them but rather treated each as it were a unique drug as GDSC did on their website. Like GDSC, we refer to the ln (IC50) values simply as IC50 throughout the manuscript, unless specified otherwise.GDSC screened 397 distinct compounds and 1000 distinct cell lines in two releases: GDSC1 screened 320 compounds in 987 cell lines whereas GDSC2 screened 175 compounds in 809 cell lines. GDSC reports the ln  (CCLE_RNAseq_rsem_genes_tpm_20180929.txt) and converted Ensembl gene IDs into official gene symbols using the annotation file (gencode.v19.genes.v7_model.patched_contigs.gtf). For 111 genes , multiple Ensembl entries corresponded to the same gene symbol; we used the average expression value for those genes. The distribution of the number of cancer cell lines per drug across the 573 drugs is summarized for each cancer type in Table CCLE measured gene expression profiles using RNA-seq for 1019 cancer cell lines. We downloaded the gene expression data from the CCLE website (https://gdac.broadinstitute.org/). We then log2-transformed those expression values after adding 1 to each.TCGA makes available RNA-seq gene expression profiles from 771 normal and 10,339 tumor samples encompassing 33 tumor types . We similarly transformed the data as above.GTEx has available RNA-seq gene expression data for normal tissue samples. We downloaded these data  from the GTEx website 50s for that drug. Denote the number of such cell lines for drug D by ND, CCLE and the number of genes in the expression profile by G . We created a gene expression data matrix  for each drug, with each row indexing a gene and each column indexing a cell line. We also created a corresponding drug-specific vector of IC50 values . Here ND, CCLE ranged from 38 to 579 with 25th, 50th, and 75th percentiles of 473, 538, and 553, respectively. For clarity, we refer to these data matrices as “cell-line data”.Among the cell lines used by GDSC, we identified all those for which CCLE provided gene expression profiles. Accordingly, for each of the 453 drugs from GDSC, we have CCLE gene expression profiles for a subset of the cell lines with ICG = 19,163). Thus, we created 400 new expression data matrices , one for each drug. Here ND, CCLE + TCGA ranged from 11,129 to 11,670 with 25th, 50th, and 75th percentiles of 11,563, 11,629, and 11,644, respectively. We refer to these data matrices as “tumor data”.We augmented each of the 453 matrices of cell-line data with columns of RNA-seq expression profiles for the tumor samples from the TCGA using the common genes between the two (G = 19163). We created an additional 400 expression data matrices , one for each drug. Here ND, CCLE + GTEx ranged from 5932 to 6473 with 25th, 50th, and 75th percentiles of 6367, 6970, and 6447, respectively. We refer to these data matrices as “GTEx data”.Similarly, we augmented each of the 453 matrices of cell-line data with columns of RNA-seq expression profiles for the normal tissue samples from GTEx using the common genes between the two (https://gdac.broadinstitute.org/) for all 31 TCGA tumor types .Hormone status of the TCGA breast invasive carcinoma (BRCA) tumor samples (file name: BRCA.clin.merged.txt) was downloaded from the Broad GDAC firehose . Thus, each sample has a mean expression of 0 and standard deviation of 1. Let i = 1, ⋯ND, CCLE + TCGA or ND, CCLE + GTEx (depending on the data set) and j = i, ⋯, G and si are the mean and standard deviation of the expression values for sample i.where Additional file 1: Table S1. The 10 most predictable drugs. Table S7. TCGA tumor types and sample size (A) and summary statistics for the distribution of the number of cancer cell lines per drug across the 573 drugs for each cancer type separately (B). Table S8. Main parameters of the GA/KNN algorithm used for the analyses of all datasets. Table S9. Training and testing performances for various combinations of k and d. Table S10. Comparison of training and testing performances between two independent runs with 100 and 1000 runs, respectivelyAdditional file 2: Table S2. Drugs for which predictive genes were selected in more than 20 of 100 predictive gene setsAdditional file 3: Table S3. The expression level of C19orf33 in cancer cell lines was positively correlated with the ln (IC50) values of more than 100 drugs for those cell linesAdditional file 4: Figure S1. Inverse correlation between SPRY2 expression (Z score) in cancer cell lines and the observed ln (IC50) of the six MEK inhibitors for those cell lines. Figure S2.TRPM4 expression (Z score) in cancer cell lines is inversely correlated with observed ln (IC50) values of acetalax for the cell lines. Figure S3. Drugs that were predicted to have high tumor-to-normal sensitivity for some tumor types. Figure S4. Scatter plot of the counts of genes selected into the sets of 30 chromosomes from two independent runs with 100 runs and 1000 runs, respectivelyAdditional file 5: Table S4. Predicted pan-tumor median ln (IC50) values for tumor and normal samplesAdditional file 6: Table S5. Predicted ln (IC50) values for all TCGA RNA-seq samplesAdditional file 7: Table S6. Drugs that were predicted to be specific to tumor type(s)


Mollugo nudicaulis Lam.(M.nudicaulis) extract by MTT assay and by monitoring the CXCR4 and HER2 expression through RT-PCR analysis.Results shown that the n-hexane extract of M.nudicaulis have anticancer activity IC50 values of 32.46±0.92 µg/mL on A2780 cell lines. It is further found that the CXCR4 and HER2 mRNA and proteinexpression were significantly reduced in M.nudicaulis treated A2780 cell lines. Thus, the n-hexane extract of M.nudicaulis is a natural source of bioactive compounds as potential anticancer agents.It is of interest to document the inhibition of A2780 cell proliferation using  In the current scenario, research and development (R&D) in pharmaceutical industries emphasize finding the superordinate actions intended to deal with the intervention of cancer and therapeutic agent counteracting force .Cancer,Mollugo nudicaulisLam. (M.nudicaulis) is one of the Indian medicinal plants which belongs to Molluginaceae family . Unlike a synthetic drug, natural products possess significant activities in the controlling and preventing various diseasesand disorders without causing unwanted side effects . Hence, e family . Traditie family . Ethanole family . Data onM. nudicaulis was gathered from Keeranur, Pudukkottai District, Tamil Nadu, India, and it was established as genuine by Dr. G.V.S. Murthy, Botanical Survey of India, Tamilnadu Agricultural University Campus,Coimbatore, Tamil Nadu (Reference: BSI/SRC/5/23/10-11/Tech 420), then the plant materials were dried under the shade condition, pulverized and stocked in air tight jar at 4°C for future analysis [The whole fresh plant of analysis .M. nudicaulis was obtained by the method of exhaustive extraction [13 - check with author]. Briefly, 300g of the plant material was soaked in 1500 mL of n-hexane contained flask and placed on a rotating shaker for 72 hrsat 25°C (average room temperature). Finally, the collected infusion was concentrated through a rotary evaporator (RE-2A evaporator) set at 40°C. Further, it was stored at 4°C for future studies.The plant material of 2. Towards theend of incubation, the medium was secluded and 10 µL of MTT was added proceeded by addition of 100 µL of DMSO to each well to solubilize the formazan crystals. It was then left in murky place for four hrs at room temprature. The absorbence wasassessed at the wavelength of 595 nm using a mircotitre plate reader and results were analyzed in triplicate and the percentage was calculated.Cell growth suppression was found by MTT assay . BrieflyTotal RNA isolation, cDNA conversion and real-time PCRThe mRNA expression levels of CXCR4, HER2 were examined using real-time PCR. The sum of RNA was detached by using using a TRIR kit  and assessed spectrometrically by the method of Fourney et al. (1988) .The RNAThe sample (50 µg) was subjected to heat denaturation at 96°C for 5 min with Laemmli buffer. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels and then transferred to PVDFmembrane . The membrane was blocked with 5% blocking buffer  in TBS-T  for anhour at room temperature succeeded by incubation with primary antibody to CXCR4 and HER2 at adilution of 1:1000. The membrane was washed thrice repeatedly with TBS- T and then incubated for an hour in horseradish peroxidase (HRP)-conjugated mouse/rabbit secondary antibody by 1:7500 dilutions in TBS-T. The membrane was again subjected to repeated washfor three times with TBS and TBS-T. Protein bands were visualized in chemidoc using enhanced chemiluminescence reagents . The detected bands were quantified by Quantity Software (Bio-Rad). Later, the membranes were incubated instripping buffer  at 55°C for 40 min. Following this, the membranes were re-probed using β-actin antibody (1:5000). In this study, a β-actin wasused as loading control.The obtained results from the assays were showed as mean ± SD. The Statistical evaluations were measured through a statistical package program .M. nudicaulis in the different concentrations ranging from 3.12 to 200 µg/mL against A2780 cell lines was analyzed by cell growth inhibitory assay. Plant extract showed 86% ofinhibition  at the highest 200µg/mL concentration. It possesses similar significant cell growth restriction activity in the ovarian cancer cell lines of A2780 with a low concentration as 32.46±0.92 µg/mL, when compared with cyclophosphamide 10.17±0.53 µg/mL. The induction of cell death occurs in a shallow concentration range, like other potential anticancer drugs [M. nudicaulis holds significant anticancer activity, and it may be used to prepare good drug candidates for an anticancer agent.The in vitro anticancer activity of n-hexane extract from er drugs . Thus, iet al., (2004) found that HER2 increases the expression of CXCR4, which is also necessary for in vitroand in vivo mediated invasion by HER2 lung metastasis. HER2 also prevents ligand-induced degradation of CXCR4 [M. nudicaulis treated A2780 cells have the lowest mRNA expression and protein level provided the significant anticancer function.  The most potent antioxidant, anti-inflammatory, andanticancer activity were demonstrated by methanolic extract of M. nudicaulis Lam., which may cause the CXCR4 and HER2 expression controlled by M. nudicaulis.. It is well known that phytochemicals such as alkaloids, steroids,flavonoids, saponins, and terpenoids have the strongest anticancer effect, resulting in anticancer activity owing to M. nudicaulis. Also, another Mollugo sp. (pentaphylla L) showed important anti-inflammatory, antitumor and antioxidant effectsin animals [M. nudicaulis to produce some bioactive compounds that can regulate the expression and control inflammation of CXCR4 and HER2 expression.Chemokine receptor 4 (CXCR4) is also recognized as a receptor for chemokine (C-X-C motif) ligand 12, a seven-transmembrane G protein-coupled receptor. A piece of accumulating evidence has shown that various types of cells, like WBCs that are produced in thelymph nodes, stem cells, the layer of cells that line heart, blood and lymphatic vessels, and membranous, flattened cells that cover the internal organs, and tumor cells, express CXCR4 . In specet al., 04 found  animals , it alsoM. nudicaulis have anticancer activity with IC50 values of 32.46±0.92 µg/mL on A2780 cell lines. Moreover, CXCR4 and HER2 mRNA and protein expression were significantlyreduced in M. nudicaulis treated A2780 cell lines. Together this study can conclude that n-hexane extract of M. nudicaulis holds strong antiovarian cancer activity. It might be used to develop novel drug candidates for ovariancancer. Nevertheless, further studies are needed to authenticate the current finding.This study reports that, the n-hexane extract of