A Trop‐2‐positive EC xenograft model was used to determine the in vivo activity of SG. Moderate‐to‐strong staining was detected in 84% (120/143) of EC samples, whereas 43% (3/7) of the primary EC cell lines tested overexpressed Trop‐2. EC cell lines overexpressing Trop‐2 were significantly more sensitive to SG compared to control ADC (P = 0.014 and P = 0.005). Both SG and the unconjugated parental antibody hRS7 mediated high ADCC against Trop‐2‐positive cell lines. Moreover, SG induced significant bystander killing of Trop‐2‐negative tumors cocultured with Trop‐2‐positive tumors. In the xenograft model, intravenous administration of SG twice weekly for three weeks was well tolerated and demonstrated impressive tumor growth inhibition against poorly differentiated, chemotherapy‐resistant EC xenografts (P = 0.011). In summary, SG is a novel ADC with remarkable preclinical activity against poorly differentiated EC cell lines overexpressing Trop‐2. These findings warrant future clinical trials.Endometrial cancer is the most common gynecologic malignancy in developed countries. The antibody–drug conjugate (ADC) sacituzumab govitecan (SG) targets trophoblast cell‐surface antigen‐2 (Trop‐2) – a cell‐surface glycoprotein highly expressed in many epithelial tumors – and delivers the active metabolite of irinotecan SN‐38 to Trop‐2‐positive tumor cells. We evaluated Trop‐2 expression in endometrial endometrioid carcinoma (EC) tissues and the activity of SG against primary poorly differentiated EC cell lines and xenografts. Trop‐2 expression was assessed in 143 formalin‐fixed–paraffin‐embedded tumors and seven primary tumor cell lines by immunohistochemistry and flow cytometry, respectively. Cell viability of primary tumor cell lines was assessed following exposure to SG, or control antibodies. Antibody‐dependent cell cytotoxicity (ADCC) against Trop‐2‐positive and Trop‐2‐negative EC cell lines was measured  TROP‐1 is overexpressed in the majority of endometrial cancers. Sacituzumab Gavitecan (SG) is highly effective, both in vitro as well as in vivo, against primary endometrial cancer cell lines and xenografts overexpressing TROP‐2. Clinical studies with SG in patients with recurrent, chemotherapy‐resistant endometrial cancer overexpressing TROP‐2 are warranted. ADCantibody–drug conjugateADCCantibody‐dependent cellular cytotoxicitycontrol ADCnontargeting control antibody–drug conjugateDARdrug‐to‐antibody ratioECendometrial endometrioid carcinomahRS7unconjugated monoclonal antibodyPBLsperipheral blood lymphocytesSGsacituzumab govitecanSCIDsevere combined immunodeficiencyTMAtissue microarrayTrop‐2trophoblast cell‐surface antigen‐21et al., Endometrial endometrioid carcinoma (EC) is the most prevalent gynecologic malignancy in the United States, with approximately 61 880 newly diagnosed cases and 12 160 deaths in 2019. Unlike ovarian and cervical cancer, the incidence and mortality rates from EC are globally increasing. If current trends continue, the incidence of EC in the United States will double by 2030  precisely target tumor cells as they are composed of a monoclonal antibody specific to surface antigens present on particular tumor cells and then kill these tumor cells with a highly potent chemical linker. ADCs may therefore optimize tumor targeting TACSTD2 gene on chromosome 1p32, which is differentially expressed in a variety of epithelial tumors  is a 45 kDa transmembrane glycoprotein encoded by the et al., et al., et al., et al., et al., et al., et al., et al., et al., Sacituzumab govitecan or IMMU‐132 (SG) is a novel ADC combining the humanized RS7 antibody targeting Trop‐2 coupled to a hydrolyzable linker that allows for a time‐dependent release of the payload, SN‐38, the active metabolite of irinotecan (7‐ethyl‐10‐hydroxycamptothecin) to the tumor tissue  approved this study, and patient consent was obtained per institutional guidelines prior to tissue collection. The EC cell lines were established from fresh tumor biopsy samples and de‐identified as described previously from our group (El‐Sahwi 2.2n = 143). Representative areas from primary EC were selected in hematoxylin/eosin‐stained preparations by a trained gynecologic surgical pathologist, and 0.6 mm cores were obtained and arrayed in a recipient block. In order to increase representation and capture possible marker heterogeneity, two cores were obtained from different areas of each tumor were included in the TMA samples. Sections of the resultant TMA samples were cut at 5 μm and then transferred to glass slides for further histologic staining and processing. The tissue samples were collected with either specific consent or a waiver of consent under an approved Yale Human Investigation Committee protocol. Next, purified goat polyclonal antibody against the recombinant human Trop‐2 extracellular domain  was applied for 1 h. After that, a secondary biotinylated anti‐goat antibody  and the streptavidin–biotin complex  were applied. Subsequently, 303‐diaminobenzidine (Dako) was used as a chromogen and the sections were counterstained by hematoxylin (Dako). For all of the above, appropriate positive and negative controls were used. The percentage of tumor cells with membranous Trop‐2 immunoreactivity was estimated, and the staining intensity was measured semiquantitatively using the following scale: 0 for no staining; 1+ for weak; 2+ for moderate; and 3+ for strong staining. In order to obtain the final immunoreactivity score, staining intensity  was multiplied by the percentage of positive tumor cells. The final immunoreactivity score was then classified into four ordinal categories: 0–9 negative (score 0), 10–99 weak (score 1), 100–199 moderate (score 2), and 200–300 strong (score 3).A retrospective cohort of stage I‐IV endometrioid EC tissue microarray (TMA) format samples were used in this study 2 immunoglobulin (FITC) that was used as a secondary reagent . The data were acquired using cell quest software . Mean fluorescence intensity (MFI) was evaluated using cell quest and prism 8. Cell lines with MFI greater than 100 were determined to have 3+ expression of Trop‐2 and with MFI of 51–100 2+, 21–50 1+, and 20 or less were considered negative for Trop‐2 expression.Flow cytometry to determine Trop‐2 expression was performed on primary poorly differentiated EC cell lines that were cultured 2.4m stock solution for the in vitro experiments. Drug‐to‐antibody ratio (DAR) of SG and control ADC was 6.78 and 6.84, respectively. For the in vitro experiments, the dosage of the drug was adjusted according to the DAR, in order to expose cells treated with SG and control ADC to equivalent quantities of SN‐38. For in vivo experiments, SG and the control ADC were reconstituted in sterile 0.9% sodium chloride as a 5 mg·mL−1 solution. hRS7 IgG (molecular weight: 150 kDa) was obtained in liquid form from Immunomedics, Inc., as a 10 mg·mL−1 solution.Sacituzumab govitecan (hRS7‐CL2A‐SN‐38), a nontargeting control ADC (h679‐CL2A‐SN‐38), and unconjugated monoclonal antibody hRS7 IgG were obtained from Immunomedics, Inc. . Lyophilized SG and control ADC were dissolved in sterile 0.9% sodium chloride as a 2 μ2.551Cr) release assays were performed in order to measure the cytotoxic reactivity of Ficoll–Hypaque‐separated peripheral blood lymphocytes (PBLs), in combination with the drug SG, the control ADC, and the hRS7 IgG against the EC cell lines at an effector to target ratio (E : T) of 5 : 1 and 10 : 1. The release of 51Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to a concentration of 2.5 µg·mL−1 of SG, the control ADC, or hRS7 IgG. Negative controls for this experiment included tumor cells incubated with PBLs and no ADCs. The positive control condition included 1% SDS to achieve complete lysis of target cells. Chimeric anti‐CD20 mAb rituxan (2.5 μg·mL−1) was used in all bioassays as a negative control for hRS7 IgG. The release of 51Cr by cytolysis from the target cells was counted in a gamma radiation counter . The percentage cytotoxicity of SG, the control ADC, or hRS7 IgG was calculated by the following formula: % cytotoxicity = 100 × (E − S)/(T − S), where E is the experimental release, S is the spontaneous release by target cells, and T is the maximum release by target cells lysed with 0.1% SDS. The results were reported as mean ± SEM.Standard 4‐h chromium . The viable cells were then quantified using a flow cytometry‐based assay that has been previously characterized  to identify the percentage of live versus dead cells that were present in each well. The bystander effect was assessed by comparing the percentage of live Trop‐2‐negative cells (GFP‐ARK4) when they were cocultured with Trop‐2‐positive cells (ARK2) and treated with either SG versus the control ADC.ARK4, a Trop‐2‐negative uterine serous cell line stably transfected with a green fluorescence protein (GFP) plasmid  was plated (80 000 cells/well) alone in a 6‐well plate (2 mL per well). A 1 : 1 ratio of this ARK4 cell line stably transfected with GFP was admixed with END(K)265, a 3+ Trop‐2 expressing EC cell line, and then plated in a 6‐well plate (2 mL per well). After overnight incubation, cells were treated with either SG or the control ADC at a concentration of 1 n2.8in vivo antitumor activity of SG, the control ADC, and hRS7 IgG was tested in xenograft models using the Trop‐2 + END(K)265 cell line, a grade 3 EC with mixed endometrioid and clear cell histology. Each mouse ; ENVIGO, Indianapolis, IN, USA) received a subcutaneous injection of 14 million END(K)265 cells suspended in 300 µL of a 1 : 1 solution of sterile PBS containing cells and Matrigel® (BD Biosciences). When a tumor volume of 0.3 cm3 was obtained, the mice were then randomized into four groups (six mice/group): saline control, SG, control ADC, and hRS7 IgG. SG, the control ADC, and hRS7 IgG were given at a dose of 500 μg in 100 μL IV twice per week for three weeks by retro‐orbital and/or tail vein injection. Tumor volume was measured twice weekly using Vernier calipers. Determination of tumor volume was obtained using the formula (A2 × B)/2, where B represented the largest tumor diameter size and A was the smallest perpendicular tumor diameter. The mice were subsequently euthanized when the group's average tumor volume reached 1.5 cm3. All animal care and euthanasia were carried out according to the regulations and rules established by the Institutional Animal Care and Use Committee (IACUC), which are in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.The 2.950 extrapolations were performed using graphpad prism 8 . Five drug concentration points were used for the calculation of IC50 in agreement with the previous literature  was performed using a TMA containing 143 EC patient samples. Moderate‐to‐strong (score 2–3) Trop‐2 expression was found in 84% (120/143) of these tumors  (2 μg·mL−1) at E:T ratios of 5 : 1 and 10 : 1, they were resistant to PBL‐mediated cytotoxicity  of hRS7 IgG, SG, and the control ADC revealed that hRS7 IgG and SG were highly effective in inducing ADCC against primary EC cell lines expressing Trop‐2 at high levels 265 cell line) but the control ADC did not. As shown in Fig. E:T ratios of 5 : 1 and 10 : 1, a mean cytotoxicity ± SEM = 12.3 ± 3.1% for SG and 19.7 ± 5.4% for hRS7‐IgG was determined, respectively, while low/negligible killing  was induced by control ADC 265) and Trop‐2‐negative cell line 361) were both tested for their potential sensitivity to PBL‐mediated cytotoxicity using standard 4‐h 3.4P = 0.014 and P = 0.005, respectively)  .Subsequently, three primary EC cell lines (two were Trop‐2‐positive and one was Trop‐2‐negative) were treated with scalar concentrations of SG, the control ADC, and hRS7 IgG. There was a significant increase in cell cytotoxicity induced against Trop‐2‐positive EC cell lines treated with SG 265 and END(K)254, respectively) than that of those treated by the control ADC (3.5P = 0.001).In order to evaluate the potential of SG to mediate a bystander killing effect toward EC cell lines with heterogeneous Trop‐2 expression, we admixed cells with high Trop‐2 expression 265 cells) and those with low or negligible Trop‐2 expression  for 72 h. As shown in Fig. 3.6in vivo experiments comparing the potential antitumor activity of SG, the control ADC, hRS7 IgG, and saline. Within 4 days of therapy initiation , SG demonstrated a significant antitumor effect in these chemotherapy‐resistant tumors compared to all controls, including nonspecific ADC . This SG‐mediated inhibition of tumor growth was evident at all subsequent time points up to day 15 when the control ADC animals were euthanized secondary to progression of disease, which was defined as a tumor volume greater than 1.5 cm3 (P < 0.01). This antitumor effect translated into a significant survival benefit for SG‐treated animals in comparison with all the controls . Median survival for the SG group was 40 days versus 15 days for the control ADC, and 11 days for hRS7 IgG and saline 265 tumor cells in SCID mice, these xenograft models were chosen for the 4et al., et al., et al., et al., et al., High expression of Trop‐2 has been discovered in a variety of human epithelial tumors including cervical, uterine, and ovarian cancers and confers tumor cells with an increased ability to proliferate and migrate  of the EC samples evaluated by IHC and 43% of the primary EC cell lines assessed by flow cytometry. Importantly, we have shown Trop‐2‐positive EC cell lines to be highly sensitive to treatment with SG when compared to treatment with the control ADC and hRS7 IgG. In contrast, no differences were found in Trop‐2‐negative cell lines. These results demonstrate the need for Trop‐2 receptor expression on tumor cells for the induction of SG cytotoxic activity against EC.et al., et al., et al., Furthermore, we demonstrated that the mechanism by which SG induces tumor cell killing is not only due to the internalization of the ADC and the consequent intracellular release of the toxic payload SN‐38 but it may also be potentially mediated by immune system cells . Indeed, both SG and hRS7 IgG demonstrated significant induction of ADCC toward Trop‐2‐positive EC cell lines in the presence of PBL, while the control ADC generated only a low level of cytotoxicity. The killing activity was Trop‐2‐specific as demonstrated by the negligible ADCC induced by SG against Trop‐2‐negative tumors. These findings support previous results from our group evaluating the activity of the unconjugated humanized anti‐Trop‐2 monoclonal antibody hRS7 in ovarian cancer  demonstrated that a few injections of SG were highly effective in inducing regression of EC xenografts. Indeed, twice‐weekly administration of SG demonstrated a statistically significant difference in tumor growth inhibition of END(K)265 when compared to administration of the control ADC. It was considered that a possible limitation in the use of SG in the clinical setting is its potential toxicity. Thus, it is important to note that there was no evidence of acute or chronic toxicity detected in the animals treated with SG for the entire duration of the study. These results are the first to demonstrate the efficacy and safety profile of the in vivo activity of SG against biologically aggressive and poorly differentiated EC. These findings warrant the support of the use of SG in clinic trials.Nonetheless, the in vitro and in vivo results in biologically aggressive endometrial cancer, results from a phase I/II clinical study reported acceptable toxicity and encouraging therapeutic activity of SG against multiple recurrent human epithelial cancers including urothelial cancers and lung cancers  but more uniquely SG demonstrates a significant bystander killing effect, which may aid in the treatment of tumors with heterogeneous antigen expression. Nonetheless, EC xenografts overexpressing Trop‐2 were found to be highly sensitive to treatment with SG. Our results combined with recent phase II data demonstrating significant clinical responses in tumors resistant to standard chemotherapy regimens support the implementation of clinical trials for patients with recurrent and poorly differentiated EC that harbor Trop‐2‐positive tumor expression.Trop‐2 overexpression is found in over 80% of EC, and our All authors fulfill the conditions required for authorship. ADS declares research funding support to Institution from ImmunoMedics. AM declares that she receives research funding support from Spanish Medical Oncology Society.ADS designed research; EP, SL, BZ, SB, AB, LZ, AM, EB, PM, NB, GA, CH, GM, ER, D‐AS, MA, JTR performed research; PES, GS contributed new reagents/analytic tools, ADS and EP wrote the paper.


N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG.Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2–7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate- Differentiated NMTC can be grossly subdivided in follicular and papillary variants. About 90–95% of differentiated NMTC can be cured by a combination of surgery and radioactive iodide treatment. Recurrence of differentiated NMTC is mainly seen in a subset of BRAF-mutated papillary thyroid cancers (PTC) and in Hürthle cell carcinomas (HCC). Although HCC explains only 3–10% of consecutive cases of differentiated NMTC, HCC accounts for up to 40% of recurrent cancers due to abrogation of sodium iodide (NaI) transport over the cellular membrane that impairs radioactive iodide therapy. HCC is now considered a distinct subtype of thyroid cancer2.Non-medullary thyroid cancer (NMTC) is the most common endocrine malignancy3. Analysis of the mitochondrial DNA (mtDNA) in oncocytic lesions from different origins revealed frequent inactivating/disrupting variants, concentrating in the genes encoding subunits of complex I (CI) of the electron transport chain (ETC), thus suggesting an impact on metabolism4. However, other studies also identified numerous mtDNA variations in other types of NMTC6. Furthermore, a substantial fraction of HCCs entirely lack mtDNA variants9. Thus, mtDNA mutations cannot solely explain the oncocytic phenotype and might be a secondary effect due to mitochondrial hyperplasia caused by a coupling defect11. It has even been suggested that the oncocytic phenotype may be caused by mutations in oncogenes and/or tumour suppressor genes, with mtDNA variations accumulating as a late effect12.The characteristic oncocytic metaplasia found in HCC is caused by abnormal proliferation of inadequately functioning mitochondria14. Near-haploidy can be easily detected by multiparameter DNA content analysis15 and is frequently followed by endoreduplication or genome doubling of the entire near-haploid genome13, a common trait of solid tumours18. Endoreduplication of an entire near-haploid genome is sometimes referred to as ‘uniparental disomy’20. Thus, many HCCs show a ‘near-homozygous genome’ (NHG), a term that covers both a near-haploid genome status and the status after endoreduplication. We recently suggested that NHGs in HCC are the net result of a continuous process of whole-chromosome instability (w-CIN). w-CIN may be driven by factors such as increased intracellular production of reactive oxygen species (ROS) as a by-product of metabolic changes21 following the loss of CI functionality22. Interestingly, a recent study of renal oncocytomas found that recurrent CI loss was the earliest detected genetic alteration accompanied by altered metabolism and affected the production of the ROS scavenger glutathione23.In addition to an oncocytic phenotype, HCCs frequently show near-haploidy due to loss of complete chromosomes, but with retention of chromosomes 5, 7 and 12 in a heterozygous state24 in two TCCLs with NHG  and two TCCLs without a NHG (SW579 and BHP 2–7). Furthermore, we performed spectrophotometric activity measurements of OXPHOS complexes in TCCLs. In addition we quantified superoxide and hydroxyl radical generation under normal culture conditions and after complex III (CIII) inhibition.The aim of this study was to elucidate the metabolic features of thyroid cancer cell lines (TCCLs), focusing on ROS-driven w-CIN. To do this, we used metabolomics analysis25, FTC-236, (derived from an FTC metastasis)26, SW579 (derived from a poorly differentiated thyroid carcinoma and deposited by A. Leibovitz) and XTC.UC1, the only model for HCC27. Cell lines were authenticated by a multiplex PCR , cultured for 20 passages and tested for mycoplasma at regular intervals (in-house PCR method). For detailed information, see Supplementary Table 2. Standard RPMI-1640 media contains 3.3 µM reduced glutathione (GSH).The following cell lines were used: BHP 2–7 (derived from a primary papillary thyroid cancer)For metabolite analysis, cells were cultured for two weeks in DMEM/F-12 or RPMI-1640. Subsequently, ten replicates of each cell line were grown for 72 hours. Six and 24 hours before cell extraction, culture media were refreshed (controls) or replaced by glucose-free DMEM/F-12 or RPMI-1640 supplemented with 5 mM galactose (as a glucose substitute). In parallel, cell-free DMEM/F-12 or RPMI-1640 were also incubated for the same time periods and used as controls to calculate the consumption and production of metabolites.The levels of ATP, ADP and AMP, as quantified from NMR spectra (see section “NMR spectroscopy and quantification of metabolites” below), were used to assess the adenylate energy charge (AEC) for each cell line. AEC values were 0.89 ± 0.02 for XTC.UC1, 0.90 ± 0.02 for FTC-236, 0.90 ± 0.01 for SW579 and 0.89 ± 0.01 for BHP 2–7.3:H2O, 8.1:0.9:1 (v/v/v)). Mixtures were stored at −80 °C for at least 30 min, allowing protein precipitation. The mixtures were then centrifuged at 16000 × g for 10 min at 4 °C. Supernatants were dried under nitrogen flow and samples were stored at -80 °C until further processing.Extracellular metabolites: Culture medium (100 μL) was collected prior to harvesting and immediately mixed with two volumes of a pre-chilled (−28 °C) solution of methanol/chloroform/water (MeOH:CHCl2 was discarded and intracellular metabolites were extracted at 4 °C with 1.5 mL pre-chilled (−28 °C) methanol/chloroform/water  and detached and lysed with the aid of a cell scraper. The solutions were transferred to 2 mL Eppendorf tubes and centrifuged at 16000 × g for 20 min at 4 °C. Supernatants were dried under nitrogen flow.Intracellular metabolites were extracted as follows. Cells were quickly washed with 5 mL MilliQ water at 37°C. Metabolic activity was immediately quenched by adding 15 mL liquid nitrogen to the petri dish and samples were stored on dry ice until further processing. The liquid N2HPO4/KH2PO4 buffer (pH = 7.4) prepared with 99.9% deuterated water (D2O) containing 0.2 mM NaN3 and 0.4 mM trimethylsilylpropionic-d4 acid sodium salt (TSP-d4). From each sample, 190 µL was transferred to 3 mm NMR tubes using a Gilson 215 robot. Remaining volumes from each sample were pooled according to each condition and used for 2D NMR experiments.Extracts were reconstituted in 250 μL of 0.15 M KCells were harvested using trypsin/EDTA and washed with HBSS. Cell counting was performed with a Muse cell analyser (Merck-Millipore) using the count and viability kit (product # MCH100102) according to the manufacturer’s instructions, after first calibrating the system with the Muse system check kit (product # MCH10010).24. Briefly, NMR data were recorded using a 14.1 T NMR spectrometer  under standardized instrumental settings for all samples. Per sample, a 1D 1H spectrum was collected using the noesygppr1d pulse sequence, which is implemented in the standard library of the NMR manufacturer. The spectral data were phase and baseline corrected and referenced to TSP-d4 methyl protons at δ 0.00 ppm and subsequently imported into Chenomx NMR suit 8 , allowing metabolite quantification. Metabolite assignment was based on the Bruker Bbiorefcode and Chenomx databases. The structures of all annotated metabolites were then confirmed in 2D NMR experiments of the pooled samples. The quantification of each metabolite was performed by integration of its proton peaks in the NMR spectrum using the deconvolution fitting algorithm of Chenomx NMR Suit. The calculated integrals were subsequently transformed to concentrations (mM), based on the known concentration of the internal standard TSP-d4 in each sample (0.05 mM). Finally, all concentrations were normalised to the cell population of each cell line. The quantities of ATP, ADP and AMP calculated from the NMR data were used to calculate the adenylate energy charge (AEC) of each sample. Uptake and release rates of metabolites by the cells, from and to the medium, respectively, were calculated using the following formula: r = (Cspent − Cfresh) × V × 1000/A, where Cspent (mM) is the concentration of each metabolite as quantified in the culture media after 72 h of incubation and Cfresh (mM) is the concentration of the same metabolite in a parallel incubation of cell-free medium; V in litres is the volume of the culture medium  and A is the area under the growth curve. The latter was calculated using the formula A = [(cells × d)/ln2] × (1-2−t/d), where cells represent the population of cells at 72 h, d is the doubling time and t is the time of incubation (i.e. 72 h). A positive value of r indicates release of a metabolite from the cell and a negative value indicates uptake in units of μmol/h/106 cells. To represent uptake and release in the figures, the r values of each metabolite were divided by the mean r of that metabolite and reported as a relative change in the figure legend.A detailed protocol for NMR spectroscopy and quantification of metabolites has been described previouslySuperoxide and hydroxyl radicals were analysed semi-quantitatively by flow cytometry. Cells  were treated and labelled according to the following scheme  after daily calibration. Fluorescence was collected using a 450/50 nm bandpass filter for detector B , and a 610/20 bandpass filter (MitoSOX RED fluorescence) and a 530/30 nm bandpass filter (CellROX Green) for detectors D and F (blue 488 nm laser line), respectively. Cyto-Cal beads FC3MV  were analysed allowing comparison of different measurements over time and expressing data as median equivalent fluorochrome (MEF). The experiment was repeated three times.Data were analysed using WinList 8.0 . Ten thousand single live cell events were selected after doublet and dead cell discrimination using SSC (side light scatter)-W (pulse width) vs. SSC-H (pulse height), FSC (forward light scatter)-W vs. FSC-H, and FSC-A (pulse area) vs. DAPI-A dot plots, respectively. Median relative fluorescence intensities (MFI) were converted to MEF values. An ANOVA test was used to study statistical significance.29.Isolated mitochondria were used for enzymatic measurements. Enzyme activities of the OXPHOS complexes were measured as previously describedPrimary antibodies: anti-α-Tubulin Mouse, 1:50,000 ; anti-SOD2 Rabbit mAb, 1:1000 . Secondary antibodies: Goat anti-Rabbit IRDye 800CW, 1:10000 ; Goat anti-mouse IRDye 680LT, 1:10000 .30. Cells were washed with ice cold PBS and lysed with Hot-SDS buffer containing PhosSTOP  and cOmplete .Western blotting was performed as described previouslyProtein concentrations were determined with the Bio-Rad DC Protein Assay . Ten µg lysates were mixed with 4x Laemmli Sample Buffer  containing βME, heated for 5 minutes at 100 °C and loaded onto a 1.5 mm 10% acrylamide gel . Electrophoresis was performed at 50 V. For blotting, the Bio-Rad semi-dry Trans-Blot Turbo Transfer System was used . Blots were then washed in Tris-buffered saline (TBS), blocked in Odyssey Blocking Buffer  at a 1:1 ratio with TBS for 1 hour, washed in TBS and incubated overnight at 4 °C with primary antibodies in TBS, 0.1% Tween-20, 5% BSA .The next day, blots were washed in TBS and incubated for one hour with the secondary antibodies in TBS/0.1% Tween-20/5% BSA protected from light. After washing, blots were air dried and imaged at high-resolution with the Odyssey Infrared Imaging System  using intensities 1.5 and 3.0 for the fluorescent colour channels red (700) and green (800), respectively. Images were analysed using Image Studio Lite Ver 5.2 software package .http://www.R-project.org/) using the packages pheatmap, dplyr, ggplot, ggsci and RColorbrewer, as well as GraphPad Prism version 8.0 . Pairwise comparisons of metabolite levels between the cell lines were performed using the Mann Whitney U test and p-values were corrected using the False Discovery Rate method of Benjamini-Hochberg (BH). Cluster analysis of cell lines and metabolites was performed in R using the factoextra package, with Spearman correlation analysis.Analysis and visualization of metabolomics data was performed in R statistical software  leads to increases in oxidized glutathione (GSSG), so higher levels of intracellular GSSG generally indicate increased ROS production. We used quantitative NMR to measure the intracellular levels of GSH and GSSG. Our analysis showed only minimal differences in the ratio of GSH/GSSG in the four cell lines, with XTC.UC1 and BHP2–7 showing a positive ratio (more GSH) while FTC-236 and SW579 had more GSSG than GSH Fig. . It shouWe also measured GSH/GSSG after challenging the cells by culturing in glucose-depleted medium for 6 and 24 h, as lack of glucose shifts dependency to OXPHOS in terms of energy needs. We found that both GSH and GSSG were only slightly decreased Fig. , and GSH2O2 scavenger, differ between the cell lines we then asked whether ROS production under normal culture conditions correlates with glutathione levels. We used quantitative flow cytometry to measure superoxide anion (O2−) under basal conditions and during inhibition of oxidation of the ETC component ubiquinol (CIII) using 100 µM antimycin A (AMA) for 2 hours . AMA treatment also induced a strong increase of CellROX Green fluorescence at this concentration, a ROS indicator mainly responsive to OH−, an increase that could be significantly inhibited in all cell lines when simultaneously treated with N-acetyl cysteine (NAC), an H2O2 and OH. scavenger. The signal amplitude of MitoSOX Red, which mainly reacts with the O2− radical, was only slightly reduced by NAC.Basal levels of Ones Fig. . However2− to H2O2 and O2. BHP2-7 and XTC.UC1 showed relatively higher expression of SOD2 compared to SW579 and FTC-236, the latter showing the lowest level , creatine, phosphocreatine (PCr) and acetylcarnitine were similar in XTC.UC1 and FTC-236 and significantly (p < 0.05) higher compared to the non-NHG SW579 and BHP 2-7 cell lines   Fig. . XTC.UC1ine Fig. , while cine Fig. . In additle Fig. . Finallytle Fig. .Figure 56 cells and 25.61 +/− 5.59 µM/106 cells, respectively), and these ratios were significantly higher than in XTC.UC1 (9.28 +/− 0.76 µM/106 cells) and FTC-236 (9.25 +/− 0.59 µM/106 cells). Collectively, these data indicate that XTC.UC1 is able to utilize multiple sources to produce high amounts of aspartate 21. The loss of entire chromosomes has been attributed to aberrant interactions with reactive oxygen species (ROS)21. Interestingly, recent findings showed that ROS vary with the cell cycle phase and reach their highest levels during mitosis33. Thus, in cells with malfunctioning mitochondria, e.g. oncocytic cells such as XTC.UC1, mitosis might lead to greater cellular stress, increased levels of ROS and subsequent ROS-induced aneuploidy by chromosomal missegregation35. This mechanism may be regulated via enhanced CHK2 phosphorylation, as seen in mitotic cells after AMA treatment21, which could in turn contribute to lagging chromosomes and missegregation36.Metabolic adaptation plays an important role in the development of cancers characterized by abundant malfunctioning mitochondria (oncocytic phenotype), including thyroid Hürthle cell carcinoma (HCC)2−) and hydroxyl radical (OH−) production in thyroid cancer cell lines with (XTC.UC1 and FTC-236) and without an NHG (SW579 and BHP 2–7), and studied the effects of inhibiting CIII using antimycin A. We also studied OXPHOS complexes in TCCLs by spectrophotometric activity measurements and used standard and glucose-depleted cultures to evaluate glycolytic activity, GSH synthesis and additional metabolic alterations.In this study we investigated the basal levels of superoxide  in the four TCCLs tested, despite differences in mtDNA variants21. This suggests that under standard culture conditions mitochondrial H2O2 is rapidly catabolized by enzymes such as catalase, glutathione peroxidase and peroxiredoxin 338, and only a fraction is converted to OH−, as shown by the limited effect of NAC. Interestingly, efficient detoxification of ROS is a suggested mechanism in HCC39. When we stressed the cell lines with 100 µm AMA, the NHG cell lines XTC.UC1 and FTC-236 generated significantly more O2− than BHP 2–7 and SW579. Simultaneously a strong increase of CellROX fluorescence was observed, which could be reduced significantly by NAC. This shows that AMA treatment drives redox homeostasis towards H2O2, which can be neutralized by NAC. By contrast, MitoSOX fluorescence (which reacts mainly with O2−) was left relatively unaffected by NAC, indicating that SOD2-mediated dismutation to H2O2 is likely favoured above scavenging by NAC in the presence of active SOD240.Similar to earlier studies of cybrid models carrying homoplasmy and various degrees of heteroplasmy41 and levels increase significantly during AMA treatment42. Our data indicate that the ETC in TCCLs is  functional even in the presence of a disruptive and/or damaging mtDNA variants21 and CI impairment in XTC.UC1 and FTC-236. Furthermore, overexpression of CIII and CIV, as described by Baris et al., suggests a compensatory mechanism for the relative loss of electron flow39. A functional ETC in the presence of CI mtDNA disruptive/damaging alterations might also be explained by the formation of respiratory supercomplexes that partially compensate for an impaired CI activity, as found in XTC.UC1 and in FTC-236 and as demonstrated in Ndufs4−/− mice43. This process might also play a role in HCC44 and in renal oncocytomas showing loss of CI activity45. The latter studies showed that, like XTC.UC1, renal oncocytomas produce high levels of glutathione as a defence against partial impairment of CI of the ETC. Furthermore, Gopal et al.23 found that cysteine and glycine, precursors of glutathione, were reduced in renal oncocytomas, suggesting that their utilization may be increased due to flux towards glutathione synthesis. We observed no difference in the uptake of cysteine between XTC.UC1 and FTC-236 46 and de novo synthesis of aspartate47, together with its precursors pyruvate, fumarate, malate and citrate, and comparable to what was found in XTC.UC1 cells. This might reflect cytosolic GOT1 activity in the context of an impaired CI48, which partly counterbalances loss of mitochondrial aspartate synthesis49. The use of α-KG might indicate direct scavenging and the utilization of reductive carboxylation of α-KG by XTC.UC1 and FTC-236 to support growth compromised by defective mitochondria51. In addition, all TCCLs exhibited increased aspartate synthesis  and the NRF1 and TFAM transcription factors are upregulated in HCC56, highly comparable to the observations in acetylcarnitine-supplemented aging rats. The downstream effects of PRC were confirmed in XTC.UC111. Thus, the high levels of acetylcarnitine in XTC.UC1 and FTC-236 might be a response to oxidative stress57, and the poor condition of the mitochondria in XTC.UC1 and FTC-236 might reflect accelerated aging55. Further support is found in the significantly lower levels of GSH, glutamate and glycine in FTC-236 (derived from a follicular thyroid cancer) compared to XTC.UC1 (HCC). This could indicate that the ROS scavenging capacity of FTC-236 is greatly reduced in comparison to XTC.UC1, BHP 2–7 and SW579, or that the GSH to GSSG turnover in FTC-236 is higher than in the other three cell lines. Yet another factor is the level of SOD2 expression, which is about two-fold lower in FTC-236 compared to XTC.UC1. Thus, in FTC-236 the conversion of O2− to H2O2 might be rate-limiting due to lower SOD2 expression compared to XTC.UC1. Interestingly α-KG, likewise identified as an H2O2 scavenger59, was significantly lower in XTC.UC1 and FTC-236 compared to SW579 and BHP 2–7, which might indicate an active role in H2O2 scavenging in the first two cell lines. Despite the lack of excess O2− and OH− under standard culture conditions, in XTC.UC1 we observed not only higher GSH but also more GSH compared to its oxidized form GSSG, suggesting lower consumption due to a low proliferative activity and a balanced flux of GSH synthesis, as long as there is a constant supply of nutrients.Both XTC.UC1 and FTC-236 showed high levels of acetylcarnitine, which plays a key role during β-oxidation of fatty acids to generate ATP61. We showed that XTC.UC1 and FTC-236 are less glycolytic and less OXPHOS competent compared to the BHP2-7 and SW579 cell lines. At first glance, FTC-236 appears to show classic Warburg-like characteristics due to low levels of TCA intermediates and low oxidative respiration. However, and similarly to XTC.UC1, FTC-236 showed lower levels of intracellular (Fig. 62. This is in line with the increased levels of pyruvate in XTC.UC1 and FTC-236 (see Fig. 63. Furthermore, lactate has also been identified as a TCA cycle carbon source in non-small-cell lung cancers. This was most apparent in patients with an aggressive tumour showing a high 18fluorodeoxyglucose uptake64, as can be observed in HCC65. In this context it is noteworthy that both XTC.UC1 and FTC-236 has been derived from metastasis. Low levels of lactate might also contribute to the relative low proliferative activity of XTC.UC1 compared to the other TCCLs (Supplementary Table Many earlier studies reported an increased dependency of tumours on glycolysis. More recently, the importance of other metabolic pathways in tumorigenesis, tumour progression and survival has become clearlar Fig.  lactate lar Fig.  and 4d. see Fig.  compared69, in line with a relatively quiescent glycolytic state70. Apparently, glucose is used to fuel other glycolytic pathways, such as the hexosamine pathway which supports UDP-GlcNAc synthesis (Supplementary Fig. The high levels of creatine and phosphocreatine might point to an alternative route to ATP generation in XTC.UC1 and FTC-236 and direct ROS scavenging via creatine71 and associates with formate and alanine excretion, found at high levels in all four TCCLs. This underscores this pathway as an interesting target for inhibition when combined with decreased glycolytic ATP production.The important role of glutamine in the metabolism of XTC.UC1 is reflected in an approximately four-fold increase after 6 h in a glucose-depleted medium see Fig. . This ma22, and which might correlate with a NHG in these tumours. Furthermore, our data points to a subtle balance between ROS generation and scavenging, directly via creatine and/or α-KG and/or conversion of ROS intermediates72. This might explain ROS-induced w-CIN in HCC and perhaps other rare cases of follicular thyroid cancer showing w-CIN73. Moreover, our data suggests phosphocreatine cycling and β-oxidation as alternative routes for generating ATP in XTC.UC1 and FTC-236, otherwise relatively quiescent glycolytic cell lines. Besides the pivotal role of acetylcarnitine in β-oxidation, the high levels found in the NHG cell lines XTC.UC1 and FTC-236 might provide protection against aged mitochondria. Importantly, we also found a profound metabolic switch of the HCC cell line XTC.UC1 towards the synthesis of aspartate. Bearing in mind the recent findings regarding the role of aspartate in cancer metabolism47, to the best of our knowledge our data shows, for the first time, that novo aspartate synthesis also plays a key role in HCC cells and might be considered a treatment target in this type of thyroid cancer.To define common metabolic characteristics associated with NHGs in TC, we studied four distinct TCCLs with and without a NHG. We demonstrated that XTC.UC1 and FTC-236 are CI impaired, a phenomenon found earlier in HCCSupplementary information.Supplementary Tables.


In a grouped analysis based on AR status, there were 2,127 down-regulated circRNAs in androgen independent cell lines compared to 2,236 in androgen dependent cell lines, thus identifying a potential circRNA signature reflective of androgen dependency. Through a bioinformatics approach, the parental genes associated with the top 10 differentially expressed circRNAs were identified such as hsa_circ_0064644, whose predicted parental gene target is RBMS3, and hsa_circ_0060539, whose predicted gene target is SDC4. Furthermore, we identified three circRNAs associated with the parental gene Caprin1 . Other studies have shown the importance of Caprin1 in PCa cell survival and drug resistance. Given the modified circRNA expression signatures identified here, these hypothesis generating results suggest that circRNAs may serve as potential putative diagnostic and predictive markers in PCa. However, further validation studies are required to assess the true potential of these markers in the clinical setting.Circular RNAs (circRNAs), a recently discovered non-coding RNA, have a number of functions including the regulation of miRNA expression. They have been detected in a number of malignancies including prostate cancer (PCa). The differential expression pattern of circRNAs associated with PCa and androgen receptor (AR) status was investigated in this study. circRNA profiling was performed using a high throughout microarray assay on a panel of prostate cell lines, which consisted of normal, benign, and malignant cells ( Non-coding RNAs (ncRNAs), which include microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) , play anThe aim of this study was to profile circRNAs in a panel of prostate cell lines and identify potential signatures associated with malignancy and androgen dependency. We propose that circRNAs have the potential to serve as useful biomarkers to improve diagnostic screening for PCa and/or identify men who are at risk of developing castration-resistant disease.A panel of malignant and benign prostate cell lines were used in this project, all of which were purchased from the ATCC . The benign prostate cell lines  were cultured in Keratinocyte Serum Free Media , containing 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL Epidermal growth factor (EGF) (Thermo Fisher Scientific) and 1% Penicillin Streptomycin (P/S) . The benign prostatic hyperplasia (BPH-1) and PCa cell lines  were cultured in RPMI-1640 (Merck KGaA) supplemented with 10% Fetal Bovine Serum (FBS) (Merck KGaA) and 1% P/S. PC-3 was cultured in ATCC-formulated F-12K Medium (Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S.® Reagent (Thermo Fisher Scientific). For phase separation, 200 μL chloroform (Merck KGaA) was added to the cell lysate. The sample were vortexed vigorously for 15 s, incubated at RT for 5 min and centrifuged at 12,000 × g for 15 min at 4°C. The upper aqueous phase was transferred into a fresh tube and the RNA precipitated from the aqueous phase, by the addition of 400 μL isopropyl alcohol (Merck KGaA). The sample was mixed, incubated for 5 min at RT and centrifuged at 12,000 × g for 15 min at 4°C. One milliliter 75% EtOH (Merck KGaA) was added to pellet, and centrifuged at 12,000 × g for 10 min at 4°C. The EtOH was removed and a repeat pulse centrifuge was performed. Surplus EtOH was removed and the pellet air-dried. The sample was re-suspended in 30 μL molecular grade H2O before DNase treatment with Ambion® TURBOTM DNase (Thermo Fisher Scientific), and an additional RNA clean-up using standard EtOH precipitation.Total RNA was prepared from cell lines  according to manufacturer’s instructions. Briefly, cells were lysed directly with 2 mL TRIzolThe panel of cell lines  were profiled using the Arraystar Human circRNA Array version 2.0 . The sample preparation and microarray hybridization were performed according to manufacturer’s instructions. Briefly, total RNA was digested with RNAse R  to remove linear RNAs and enrich for circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method with Arraystar Super RNA Labeling Kit (Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8 × 15 K). The array slides were washed and scanned on the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.t-test was used to identify significantly altered circRNAs. The false discovery rate (FDR) was applied to determine the threshold of p-value. An FDR of < 0.05 was used. Changes in expression were identified using unsupervised clustering analysis (euclidean distance measure and the “average” agglomeration method) and associated heat-maps generated.Quantile normalization and subsequent data processing were executed using the R software package . circRNAThe human circular RNA microarray (Arraystar) version 2.0 covers 13,617 previously discovered human circRNAs. Our cell line panel consisted of malignant , benign (BPH-1), and normal  cell lines. AR expression in 22Rv1, LNCaP, and VCaP was confirmed using qPCR . In totap-value  according to FC and p-value . We firsp = 0.001), located on chromosome 3  and NFIX (hsa_circ_0049657), both known to be involved in the regulation of the AR  in PCa  has been shown to be involved in the inhibition of PCa stem cell invasion, migration and proliferation, as has KRT7 (hsa_circ_0026358), which has been identified along with FAM129A, MME and SOD2 as part of a four-gene androgen regulated panel  in PCa  via tran) in PCa . hsa_cire in PCa . Similared panel .p < 0.001) is an exonic circRNA located on chromosome 11 and is associated with the gene SIAE . Caprin1 plays an important role in PCa cell survival and its upregulation has been shown to be associated with drug resistance, and is increased in SPOP mutated cell lines  and androgen independent/castration resistant) cell lines DU145, PC-3). Response to androgen ablation was demonstrated by confirming the presence or absence of AR-FL across cell lines see . CircRNA5, PC-3. ene SIAE . mRNA exh cancer . We idenll lines .p < 0.0001) located on chromosome 18 and is associated with the gene MTCL1 (FLNA (hsa_circ_0091934 and hsa_circ_0091894) located on the X chromosome, which plays a significant role in PCa development and progression, and has been highlighted as a possible biomarker for disease screening and detection  and SDC4 (hsa_circ_0060539). Interestingly, we identified three circRNAs associated with the parental gene Caprin1  and two circRNAs associated with FLNA (hsa_circ_0091934 and hsa_circ_0091894). Many of these genes are currently under active investigation as biomarkers in PCa, and identifying their associated circRNAs may contribute to ongoing research in this field (Parental genes of interest were is field .There is evidence to suggest that circRNAs regulate miRNA function by competing for the pool of miRNA binding sites to influence the activities of miRNAs in regulating gene expression . Interesin vitro study which identified multiple potential genes and ongoing validation is required in cell lines and in vivo to confirm our findings here.This study had several limitations. Firstly, we used a circRNA microarray platform to detect circRNAs compared to RNA-seq methods which may have higher sensitivity and accuracy to detect circRNAs. Secondly, this was a preliminary In conclusion, circRNAs were modified between benign and malignant cell lines, as well as androgen dependent and independent cell lines, which suggests a role for circRNAs in PCa initiation and progression. This study highlights the importance of circRNAs in PCa and their potential role in identifying the disease and monitoring response to treatment. The dysregulated signature identified here may prove useful for the development of a blood-based assay with both diagnostic and predictive value in this disease. However, this would require validation in larger patient cohorts.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DKK3 is a secreted protein, which belongs to a family of Wnt antagonists and acts as a potential tumor suppressor in gallbladder cancer. To further understand its tumor suppressor functions, we overexpressed DKK3 in 3 GBC cell lines. We have employed high-resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides to check the downstream regulators. In this study, we reported for the first time the alteration in the phosphorylation of 14 kinases upon DKK3 overexpression. In addition, we observed DKK3 induced hyper phosphorylation of 2 phosphatases: PPP1R12A and PTPRA, which have not been reported previously. Canonical pathway analysis of altered molecules indicated differential enrichment of signaling cascades upon DKK3 overexpression in all the 3 cell lines. Protein kinase A signaling, Sirtuin signaling pathway, and Cell Cycle Control of Chromosomal Replication were observed to be differentially activated in the GBC cell lines. Our study revealed, DKK3 overexpression has differential effect based on the aggressive behavior of the cell lines. This study expands the understanding of DKK3-mediated signaling events and can be used as a primary factor for understanding the complex nature of this molecule. The family of dickoppfs are majorly involved in embryonic development through its interaction and regulation of the Wnt signaling pathway . Multiplmyopathy . In HEK2myopathy . DKK3 ovmyopathy ,8,9. AltProteins account for the larger part of the cell molecular machinery and as a part of organized functional modules and networks. It performs various cellular activities which determine the cell fate . In receGallbladder cancer (GBC) is highly invasive and molecularly heterogenous cancer. The molecular mechanisms underlying the progression of this disease are not well studied. Isocitrate dehydrogenase (IDH), fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (ERBB), KRAS, ROS1 remain to be the most promising therapeutic targets to date ,19. Iden2.3 gallbladder cancer cell lines TGBC24TKB, OCUG-1, G-415 were grown and maintained in DMEM (dulbecco’s modified eagle’s medium) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% COpCS2-hDKK3  and X-tremeGENE HP DNA transfection reagent . Briefly, DNA was diluted in opti-MEM to a final concentration of 1 µg plasmid DNA per 100 µL medium (0.01 µg/µL) and mixed gently. X-tremeGENE HP DNA transfection reagent was added in 1:2 (DNA:reagent) ratio directly into the medium containing the diluted DNA without coming into contact with the walls of the tube. The complex was mixed gently and incubated for 15 min and added to the cell lines with swirling the wells to ensure even distribution over the entire plate surface [TGBC24TKB, OCUG-1, G-415 were used to determine the optimum concentration of  surface . Cell liPart of transiently overexpressed cell lines: TGBC24TKB, OCUG-1, G-415 were harvested using RIPA lysis buffer , 1% Triton-X-100, 0.1% SDS containing protease and phosphatase inhibitor cocktails) after serum starvation and sonicated to extract proteins. Western blot analysis was performed, as previously described, using 30 µg protein lysates . NitrocepCS2-hDKK3  was overexpressed transiently in TGBC24TKB, OCUG-1, G-415 cells for 72 h. The cells were serum starved in serum-free medium for 12 h, and then lysed in cell lysis buffer (2% SDS in 50 mM TEABC (triethyl ammonium bicarbonate). Protein concentration was estimated using the BCA method . Equal amount of protein from control and overexpressed TGBC24TKB, OCUG-1, G415 cells was precipitated using ice-cold acetone.Cell lysis and protein extraction was performed as previously described . pCS2-hDProteins were reduced and alkylated using 5mM DTT (dithiothreitol) and 20 mM IAA (iodoacetamide), respectively. Samples were then digested overnight at 37 °C using trypsin (1:20) . DigesteThe TMT labeled peptides were subjected to stage tip based SDB-RP (styrenedivinylbenzene-reverse phase sulphonate) fractionation to generate 3 fractions, as described previously . For pac3 . Dried peptide fractions were reconstituted in 80% acetonitrile/0.1% TFA. Peptide mixtures were enriched for phosphorylated peptides with 10 µL IMAC beads for 40 min. Enriched IMAC beads were loaded on Empore C18 silica packed stage tips. Stage tips were equilibrated with methanol followed by 50% acetonitrile in 0.1% formic acid and 1% formic acid. The beads with enriched peptide were loaded onto C18 stage tips and washed with 80% acetonitrile in 0.1% trifluoroacetic acid. Phosphorylated peptides were eluted from IMAC beads with 500 mM dibasic sodium phosphate, pH 7.0 .IMAC beads were prepared from Ni-NTA superflow agarose beads  that were stripped of Nickel with 100 mM EDTA and incubated in an aqueous solution of 10 mM FeCl5 and ion filling time set 50 milliseconds. The most intense ions with charge state ≥2 was isolated and fragmented using HCD (higher collision dissociation) fragmentation with 34% normalized collision energy and detected at a mass resolution of 50,000 at 200 m/z. The AGC target for MS/MS was set as 1 × 105 and ion filling time set 100 milliseconds dynamic exclusion was set for 30 s.Enriched phosphopeptides and peptides were analyzed on an Orbitrap Fusion Tribrid mass spectrometer  interfaced with Easy-nLC II nanoflow liquid chromatography system . Peptides were separated on an analytical column  C18) at a flow rate of 280 NL/min using a step gradient of 8–22% solvent B (0.1% formic acid in 90% acetonitrile) for first 70 min, followed by 22–35% up to 103 min. The total run time was set to 120 min. The mass spectrometer was operated in a data-dependent acquisition mode. A survey full scan MS (from m/z 350–1600) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z. The AGC (automatic gain control) target for MS1 was set as 4 × 10The mass spectrometry raw data were searched using Sequest HT search engines with Proteome Discoverer 2.1 (Thermo Fisher Scientific). Phosphopeptide-enriched fractions from each replicate were searched against the RefSeq protein database (version 89). The search parameter used as carbamidomethylation of cysteine residues as a fixed modification. Oxidation of methionine, the phosphorylations of serine, threonine, and tyrosine, were selected as dynamic modifications. Trypsin was set as the protease and a maximum of two missed cleavage were allowed. Precursor mass tolerance was set to 10 ppm, and a fragment mass tolerance of 0.05 Da was allowed. All peptide-spectrum matches (PSM) were identified at a 1% false-discovery rate (FDR). The probability of phosphorylation for each site was calculated by the phosphoRS node in Proteome Discoverer. Only phosphopeptides with >75% site localization probability were considered for further analysis. A 1.5-fold cut-off was used for dysregulated phosphopeptides compared to control cell line. High-confidence PTMs were profiled using PTM-Pro tool .https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis (accessed on 15 August 2020)). Significantly altered phosphopeptides in all the 3 cell lines were used to do the analysis. To generate the core analysis, Ingenuity Knowledge Base (genes only) repository was used. Core analysis was created using direct and indirect relationships and species human criteria. The cutoffs were set to 0.58 and −0.58 for hyperphosphorylated and hypophosphorylated molecules respectively. The rest of the filters were set to default to avoid loss of information and relationships. Core analyses identified the top canonical pathways, upstream regulators, etc., significantly enriched in our datasets. After performing core analysis for each cell line, comparison analysis was performed on the output of core analysis for all the 3 cell lines. The analysis results were schematically replicated using Adobe Illustrator .The core and comparison analysis were generated using IPA . Available online: http://www.kinhub.org/kinmap/index.html (accessed on 9 June 2020)) was used to build the kinome map. The list of identified kinases were plotted and highlighted on the kinome map as described previously [The KinMap tool (KinMap beta. Available online: eviously . Dysreguhttps://casecpb.shinyapps.io/ksea/ (accessed on 1 August 2020)) [z-scores are reported. NetworKIN predictions with scores 2 or greater. A negative score corresponds to a collective dephosphorylation of the kinase’s substrates; the inverse is true for a positive score. Bars that are colored blue or red are kinases whose score met the p < 0.05 score cutoff.The kinase-substrate enrichment analysis was done using the online KSEA tool ) . ProteinOur previous study , identifp-value significant in total dataset. Out of these proteins, 12, 41, 47 of molecules were found to be significantly up-regulated upon DKK3 overexpression in TGBC24TKB, OCUG-1, G-415 cells, respectively, compared to control cells. In addition, 6, 7, 27 of molecules were found to be significantly down-regulated upon DKK3 overexpression in TGBC24TKB, OCUG-1, G-415 cells, respectively, compared to control cells. The list of dysregulated proteins for each cell line are shown in Quantitative proteomic analysis of TGBC24TKB, OCUG-1, G-415 control, and DKK3+ cells resulted in identification of 4646, 4717, and 4419 proteins, respectively. Overall, 1904, 1800, and 2361 molecules were found to be The TMT labeled peptides from control and DKK3 overexpressed cells were enriched using IMAC beads as per manufacturers’ protocol and LC MS/MS analysis was carried out as described in methods section. To increase the reliability of our phosphoproteomic analyses, we included technical replicates in our study. The MS data were processed and searched against databases by SEQUEST-HT algorithms using the Proteome Discoverer 2.1 platform. The probability of Ser/Thr/Tyr phosphorylation sites on each peptide was calculated by the PTM-pro algorithm using a cut-off of >75%. We identified 2382 unique peptides with PTM modifications corresponding to 1110 proteins in TGBC24TKB, with a total of 2130 phosphosites identified: 1907 Ser residues, 211 Thr residues, and 12 Tyr residues . Overallp-value 2.5 × 10−4), Sertoli Cell-Sertoli Cell Junction Signaling , Role of p14/p19ARF in Tumor Suppression  , Sumoylation Pathway , BER pathway , DNA Double-Strand Break Repair by Non-Homologous End Joining , HOTAIR Regulatory Pathway  , Sirtuin Signaling Pathway , ATM Signaling , Cell Cycle: G2/M DNA Damage Checkpoint Regulation , Granzyme B Signaling   A. The to × 10−2) B. The to × 10−3) C.z score = 2) and inactivated in G-415 (z score = −1.667). Simultaneously, pathway leading to Cell Cycle Control of Chromosomal Replication was activated only in OCUG1 cell line with z score of 2.646. The Protein Kinase A Signaling pathway was activated in OCUG-1 (z score = 0.816) and inactivated in G-415 (z score = −1.633)  A. To fur −1.633) B. The meIn our phosphoproteomic dataset, we identified 79 non redundant kinases. In TGBC24TKB, 13 kinases were identified. In OCUG-1, 45 kinases were identified of which, 10 were hyperphosphorylated. In G-415 cell line, 49 kinases were identified, out of which 1 was hyperphosphorylated and 5 were hypophosphorylated. The list of dysregulated kinases along with their phosphorylation sites are provided in DKK3 also known as REIC plays an important role in several biological processes. Dysregulated DKK3 expression is linked to the development of a number of diseases like cardiac hypertrophy , systemiMultiple groups have applied conventional approaches to study the effects of DKK3 in different model organisms, and there are no studies till date which have analyzed downstream effects of DKK3 using high throughput platform. Our proteomics and phosphoproteomics data analysis revealed that alteration was majorly seen at phosphorylation level upon DKK3 overexpression in gallbladder cancer cells. To understand the downstream dysregulated pathways, we analyzed phosphoproteomic data for all the 3 cell lines distinctively. Ingenuity pathway analysis identified molecules based on their functional annotation were significantly associated with 9 molecular and cellular functional categories; RNA Post-Transcriptional Modification, Cellular Development, Cell Death and Survival, Protein Synthesis, Cell Morphology, Cellular Assembly and Organization, Cell Cycle, Gene expression, and Cellular Growth and Proliferation . DKK3 isThe IPA analysis compared the effect of DKK3 overexpression with respect to the invasiveness of the cell lines. Our comparison analysis identified the set of pathways predicted to be differentially activated in GBC cell lines. We identified several pathways which are not linked with the expression of DKK3 before. The Sirtuin signaling pathway and Protein kinase A signaling pathway were predicted to be activated in OCUG-1 cell line compared to G-415 cell line. However, the exact role of DKK3 in these pathways is not fully understood. Among the list of upstream regulators, we identified novel molecules that showed differential activation and are never linked to DKK3 expression before. From our analysis, we identified molecules such as P-TEFb, PPME, SET, CIP2A which showed differential activation pattern upon DKK3 overexpression with respect to the invasiveness of the cell lines. P-TEFb is a kinase that phosphorylates the C-terminal domain of the large subunit (RPB1) of RNA polymerase II and subsequently stimulates productive transcription elongation of multiple cellular genes. It is a master regulator involved in transcription elongation and its dysregulation is involved in multiple diseases including cancer. P-TEFb release and activation by CDK9 inhibitors or by other releasers and activators is known to promote cell growth arrest . ActivatWe further identified potential altered kinases upon DKK3 overexpression that could be validated to deduce the mechanism by which DKK3 acts. Among all the dysregulated kinases, CDKs (cyclin-dependent kinases) were majority. It is well established, a fundamental aspect of cancer is dysregulated cell cycle control and CDK being a major part regulating cell cycle control, our data identified 6 CDKs to be differentially phosphorylated. Among the differentially phosphorylated CDK’s CDK1 is only the kinase, which was hypophosphorylated in our data while CDK16, CDK11A/B, CDK2, and CDK18 were hyperphosphorylated. The exact mechanism for the differential phosphorylation for these kinases needs to be validated. Nevertheless, the result of these alterations is important in studying the tumor suppression function of DKK3. We also observed hyperphosphorylation of serine/threonine-protein kinase PRP4 homolog (PRPF4B) at 3 different positions S257, S292; S294, which is involved in pre-mRNA splicing. We also observed the hyperphosphorylation of kinase involved in myocyte cytoskeletal development: (SPEG), Oxidative Stress Responsive Kinase 1 (OXSR1), etc. A consolidated list for the available small molecule inhibitors that target against dysregulated kinases was adapted from Moret et al., and is depicted in Overall, the data generated from this study greatly expands the understanding of the downstream modulators upon DKK3 overexpression. Our study highlights the list of dysregulated molecules to be important in regulation of various process initiated by overexpressing DKK3. For the very first time the complexities and responses involved upon DKK3 overexpression and its effect on biological functions and cellular phenotype are identified using high-throughput method. We have characterized differential protein expression and phosphorylation events that occur upon DKK3 overexpression. We identified differential activation of regulators and pathways that hold potential significance with respect to DKK3 signaling and needs to be unfolded in time ahead. Our findings help in unraveling the complex biological nature of DKK3 molecule. However, as the next step of this study, a detailed analysis of the molecules identified in the study is needed, which is indeed a limitation for this study. Our study highlights downstream effect of DKK3 overexpression at global proteomic and phosphoproteomic levels in gallbladder cancer cell lines, however the deeper validation of these integrated signaling molecules would provide a more explicit visualization. Furthermore, assessing the involvement of regulatory networks described in our study needs to be widened across multiple cellular and biological models.


Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants  was selected as target and both nucleases were expressed by an egg‐cell specific promoter to assure efficient germline transmission  conferring Imazapyr resistance and one silent point mutation to avoid DSB induction within the donor. For donor excision, the donor molecule is flanked by the respective spacer sequence (Figure LbCas12a or ttLbCas12a, were transformed as T‐DNAs into Arabidopsis thaliana Col‐0 plants via the floral dip method. After transformation, the T0 plants were grown either at 22°C or at 28°C until maturity and the harvested seeds subsequently sown out on Kanamycin selection medium. The resistant T1 primary transformants were grown until maturity at either 22°C or 28°C, respectively. The harvested T2 seeds were sown out on Imazapyr medium to verify ipGT events. At least 90 lines were tested per approach (Figure LbCas12a outperforms the standard enzyme at 22°C by almost twofold. At 28°C, still one third more lines with GT events were detected for ttLbCas12a. The effect is even more pronounced if we take the number of GT events per line into account. For all lines, the individual GT frequencies were calculated by setting the total number of seeds into relation with the number of Imazapyr resistant seedlings. Mean GT frequencies were determined over all lines per approach. At 22°C, ttLbCas12a outperforms the native enzyme by 2.4‐fold and at 28°C still by 1.7‐fold. This is also obvious when we compare the targeting frequencies for the single lines individually for 28°C and for 22°C (Figure ALS gene as target (Wolter et al., LbCas12a are in about half of the cases true GT events indicating that the modified enzyme, ttLbCas12a, is at least as efficient as the native one, which showed perfect GT events in about one third of the cases. Furthermore, for ttLbCas12a at 28°C molecular analysis indicated that 3% of the analysed T2 lines already comprised a homozygous GT event, which did not occur in any other approach in this study.Therefore, we performed ipGT experiments in Arabidopsis testing e Figure . The GT h Figure . As expeC Figure . In prinLbCas12a‐mediated GT in plants (van Vu et al., LbCas12a (Schindele and Puchta, LbCas12a for GT might be even more promising as our data indicate. We were recently able to show that ttLbCas12a provides access to target sites that could hardly be edited at all before by LbCas12a even with a high‐temperature treatment (Schindele and Puchta, LbCas12a (Wolter and Puchta, LbCas12a might increase GT efficiencies at those loci that are more difficult to access even much stronger.Recent results already demonstrated the beneficial effect of increased temperature on The authors declare no conflict of interest.H.P. conceived the research. P.S., F.W., T‐K.H. and L.M. designed and L.M. executed the experiments. H.P., P.S. and L.M. wrote the article.


Dendrobium chryseum Rolfe, which is used in traditional and folk Chinese medicine, the protocorms were raised in Murashige and Skoog (MS) media in three strengths, full strength (FMS), half strength (1/2 MS), and quarter strength (1/4 MS), with or without the phytohormones 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) and coconut water (CW). The comparative cytotoxic activities of the wild and in vitro-raised protocorms were evaluated in human cervical carcinoma (HeLa) and human glioblastoma (U251) cell lines by MTT assay. In in vivo and in vitro, the methanol extracts of D. chryseum showed significant cytotoxic activities. Significant growth inhibition (%) and potent IC50 values were demonstrated in HeLa cell lines (49.79% (210.5 μg/mL) for in vitro-raised Dendrobium chryseum (DCT) versus 46.97% (226.5 μg/mL) for wild Dendrobium chryseum (DCW)). Similarly, activities against U251 cell lines exhibited also significant inhibition (28.76% (612.54 μg/mL) for DCW and 17.15% (1059.92 μg/mL) for DCT). The cytotoxic activities of both, wild and tissue-cultured samples, were superior in HeLa cells. In U251 cells, the wild sample was more active than the tissue-cultured one with a moderate cytotoxic effect. Hence, protocorm culture may therefore be a promising future tool for producing pharmacologically bioactive compounds in medicinal orchids. Such sustainable technology approach will minimize the pressure on the natural population of threatened but commercially important medicinal orchids.From the medicinal orchid  Orchidaceae is a highly developed and extensively distributed monocotyledonous family consisting of terrestrial, saprophytic, and epiphytic species , 2. OrchDendrobium is the second largest genus of the family Orchidaceae in Nepal. Thirty Dendrobium species are found in Nepal, distributed across the tropical, subtropical, and temperate climatic regions [Dendrobium chryseum is an epiphytic herb found throughout the western and eastern Himalayas at elevations from 1000 to 2150 m. It is popularly used in traditional and folk Chinese medicine for its antipyretic, eye benefiting, and immunomodulatory effects. Yang et al. reported the presence of particular compounds such as bibenzyl-phenanthrene and coumarin derivatives in D. chryseum. While the chemical compound isolated from the stem of D. chryseum exhibited antioxidant activity [D. aurantiacum var denneanum (D. chryseum non Rolfe.) possess tumor-inhibitory and blood glucose-reducing effects in vivo [D. chryseum Rolfe demonstrated antioxidant properties and an antidiabetic indication in various studies [in vitro techniques in plant conservation efforts designed to counter the rapid decline in the world's biodiversity [ex situ cultivation is one of the key elements in modern conservation strategies adopted to address the threats of increasing urbanization, population growth, and industrialization [in vitro techniques are highly useful for providing sustainable sources of optimal plant-derived natural products [ regions –17. Dendactivity , polysac in vivo . D. chry studies . Its wil studies –23. Receiversity , 24, 25.lization . Furtherproducts .in vitro [D. chryseum was cultured in vitro to produce protocorms for the assessment of potential cytotoxic activity and comparison of such activities with its wild counterparts. Protocorm formation is the peculiar stage in orchids in which an embryo transforms and develops into a specific spherical structure with unique characteristics. Protocorms undergo growth and differentiation stages during their development into mature plants [D. chryseum, especially for its potential as a source for antineoplastic agents. The objectives of the present study were to establish protocorm culture for the production of bioactive secondary metabolites. Subsequently, isolated extract material thereof was examined for its effect between substances originated from protocorms compared to wild plant material in terms of cell proliferation against two representative cancer cell lines: human cervical carcinoma (HeLa) and human glioblastoma (U251), respectively.In particular, orchid species have been cultured with various techniques to synthesize bioactive molecules in vitro , 35. In e plants , 36. Proe plants . This isD. chryseum, collected from Godavari, Lalitpur, Central Nepal, at 1579 m above sea level, were used as explants to produce protocorms in vitro. The plant was identified by the Nepalese taxonomist Dr. Keshav Rajbhandari as well as from the literature. A voucher specimen of this plant was deposited in the Tribhuvan University Central Herbarium (TUCH) (voucher number P07) in Kathmandu, Nepal. Material from the stems and leaves of the wild plants and the in vitro protocorms were subsequently analyzed for their cytotoxic activities.Immature capsules of D. chryseum for protocorm formation is already described and reported by us and is followed in the present experiment as well [D. chryseum were first washed under running water added by 2-3 drops of Tween-20 for half an hour to remove dust particles and then rinsed off with distilled water for 10 min. Before inoculation, the explants were surface-sterilized by immersing them first in a solution of 1% sodium hypochlorite and then 70% ethyl alcohol for 2 min each. Finally, they were rinsed off with sterile distilled water at least three times. The surface-sterilized capsules were transferred into Petri dishes and dried on a filter paper in a laminar airflow cabinet. Next, the sterile capsules were cut longitudinally into two equal halves using a sterile surgical blade exposing the seeds within  and placed in a sonicator for extraction. Then, the concentrated plant extract was placed in a rotary evaporator until the solvent was dry and the resultant crude extracts of plant materials were stored in a refrigerator at 4°C for later use.The stems of wild plants and protocorms of 2 incubator [4 U251 cells/well and 2 × 104 HeLa cells/well) in 100 μL of the medium and incubated in a 5% CO2 incubator at 37°C for 24 h. Following attachment and cell confluence, the cells were treated with different concentrations of the plant extracts  for 48 h. Crude extracts were diluted by dissolving them in 0.1% DMSO  and the EMEM culture medium. The assay was performed in triplicate. Following 48 h of incubation, the supernatant was removed and 150 μL of the medium was combined with 50 μL of MTT for each well. Following 4 h of incubation, a purple formazan crystal was produced. Then, 100 μL of 0.1% DMSO was added and the plates were incubated for a further 15 min at room temperature to dissolve the formazan crystals. The absorbance of samples was measured with a microplate reader  at a wavelength of 595 nm. A commercially available cis-platin drug was used as positive control. The percentage of cytotoxic activity was calculated by the following formula:The cytotoxic activity of extracts was evaluated in 96-well flat-bottomed microtiter plates (Corning) by using the standard MTT -2,5-diphenyl-tetrazolium bromide) colorimetric assay with a slight modification. For this purpose, HeLa human cervical carcinoma cells and U251 human glioblastoma cells were cultured in T25 flasks in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 1% of penicillin/streptomycin, and 1% L-glutamine and kept at 37°C in a 5% COncubator . The cel50 μg/mL of the extract). The cytotoxic capacities of extracts (IC50) were calculated using a regression equation.A dose-response curve was plotted for each extract to calculate the 50% inhibition of cell growth , FMS fortified with CW (5% and 10%) and MS medium supplemented with 0.5 mg/L BAP alone and in combination with 0.5 mg/L NAA, 1 mg/L BAP + 0.5 mg/L NAA, 1.5 mg/L BAP + 0.5 mg/L NAA, and 2 mg/L BAP + 0.5 mg/L NAA  for DCT and 46.97% (226.5 μg/mL) for DCW while the cytotoxic activities of wild and in vitro samples were nearly the same, being slightly better in the in vitro-grown samples  for DCW and 17.15% (1059.92 μg/mL) for DCT. The cytotoxic activities of wild and in vitro samples differed in such a way that the inhibitory action of samples derived from wild-grown plant material was better than that from in vitro-grown samples. However, in vitro-grown samples still provided substantial inhibition of the investigated human glioblastoma cell proliferation  and NAA (0.5 mg/L) took the most time to germinate. The size of protocorms varied depending upon the nature of the nutrient. Small protocorms were observed on 1/4 MS, 1/2 MS, and FMS media with both 5% and 10% CW. These small protocorms developed directly into shoots. In contrast, the protocorms which developed on FMS supplemented with NAA (0.5 mg/L) and BAP (0.5 mg/L) grew quite large by the 24th week but did not develop shoots. Maximum seed germination occurred on 1/2 MS medium which later developed into young protocorms. The result of mass propagation of D. chryseum by using protocorm culture is already reported by us [Dendrobium species with a high percentage of germination and protocorm formation included 67% MS [D. transparens seeds germinated in vitro on a 1/2 MS recipe with 2.0 mg/L of 6-benzyladenine (BA) [Vanda dearei (45%) and Taprobanea spathulata (92.73%). Based on the germination result, the present study revealed that all different strengths of MS medium favored seed germination up to the protocorm development. A similar result was also found for the in vitro germination of orchid seeds of Phaius tankervilleae and Cymbidium iridioides [In the present investigation, seeds of ed by us . A simild 67% MS . D. tranine (BA) . Half stidioides , 49.D. nobile showed cytotoxicity against human lung carcinoma, human ovary adenocarcinoma, and human promyelocytic leukemia cell lines [D. signatum exhibited cytotoxic activities against human MDA-23 1, HepG2, and HT-29 cancer cell lines [D. chrysanthum inhibited the proliferation of leukemia HL-60 cells [D. longicornu and D. moniliforme showed cytotoxic activities against HeLa and U251 cancer cell lines [Bulbophyllum kwangtungense showed antitumor activities against HeLa and K562 human tumor cell lines [Bulbophyllum odoratissimum was found to have cytotoxic activities against human cancer cell lines, such as human leukemia cell lines K562 and HL-60, human hepatoma BEL-7402, human lung adenocarcinoma A549, and human stomach cancer cell line SGC-7901 [in vitro versus wild plants has been reported.Previous studies revealed that orchids can be a potent source of anticancer agents. For instance, ll lines , 51. D. ll lines . D. chry60 cells . D. longll lines , 5, 35. ll lines  while Buin vitro grown plant samples of D. chryseum on HeLa cells revealed that tissue-cultured protocorms (in vitro samples) were slightly more active than wild plants. Both, however, showed a significant percentage of growth inhibition and highly potent IC50 values in a dose-dependent manner. Upon microscopic investigation, initially cultured HeLa cancer cells before the treatment regimen were elongated and spindle-shaped (D. chryseum extracts play a pivotal role in inhibiting the growth of HeLa cancer cells, whereas, against the U251 cell line, the activity of the wild sample was comparatively better than the in vitro-raised protocorms. Nevertheless, also for this cellular glioblastoma model system, significant percentage of cancer cell proliferation inhibition and corresponding IC50 values were demonstrated.A comparison of the cytotoxic activity of both wild and e-shaped . Soon afe-shaped . This cycis-platin drug was found active against HeLa cells and U251 cells with the lowest IC50 value and applied as the assay control.Similarly, a commercially available in vitro-grown protocorms may provide a good source for obtaining bioactive compounds efficiently.Protocorm suspension cultures have been established for the mass propagation of valuable orchid species , 29, 36 D. chryseum [D. chryseum. One of them, isolated from the stem, exhibited antioxidant activity [in vitro propagation of medicinal orchids and the application of other plant tissue culture techniques provide an avenue for the efficient production of selective metabolites, with medicinal use.A number of studies about the pharmacological properties of plant metabolites support the findings of this study. The majority of plant-based secondary metabolites are phenols, alkaloids, flavonoids, and tannins , 58. Allchryseum . Furtheractivity . The in D. chryseum, as a novel source of active agents with the potential for treating human malignancies. Both wild samples and in vitro-grown protocorm mass of D. chryseum exhibited cytotoxic activities in a dose-dependent manner against selected cancer cell lines of human cervical carcinoma and human glioblastoma, a type of brain tumor. In vitro-cultured protocorms were comparable to wild stems in inhibiting cancer cell proliferation in HeLa and U251 cell lines, respectively. Therefore, protocorm in vitro culture offers a highly suitable process for the production of the investigated pharmacologically useful compounds contained in protocorm samples. Further work on the isolation and pharmacological validation against a range of targets and medical indications of bioactive compounds derived from protocorms is needed. However, this may underscore the general attractive utility that protocorms are a potential source for the commercial production of desirable metabolites from in vitro culture. Such technology implementation would certainly augment to vast extent very much needed efforts to minimize the pressure on the natural population and their entire habitats of threatened but commercially important medicinal orchids in Nepal.The present findings, based on these first cellular studies, establish the orchid species,