The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs . The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s The majority of recombinant glycoprotein therapeutics are manufactured in CHO (Chinese Hamster Ovary) cells due to their high productivity (1–10 grams per liter), genetic stability, and ability to be grown in large-scale suspension culture . HoweverBaL, prevented proteolysis at the GPGPR/AF sequence in the V3 domain. As this cell line is uniquely developed to express BaL-rgp120, it cannot be used for the expression of other recombinant proteins. Therefore, a C1s knockout CHO cell line, that can be used as the cell substrate for the expression of any other recombinant protein, is needed.Recently, we reported that the major CHO cell protease responsible for cleavage of clade B gp120s was the complement component 1 protease, C1s . C1s is -/- CHOK1 2.E7 is a CHOK1 cell line where the C1s gene has been inactivated by gene editing. The second cell line, C1s-/- MGAT1- CHOK1 1.A1 is also a CHO K1 cell line where both the C1s and MGAT1 genes have been inactivated. Inactivation of the MGAT1 gene limits N-linked glycosylation to mannose-5 and earlier intermediates in the N-linked glycosylation pathway. Limiting N-linked glycosylation via the MGAT1 cell line has been shown to improve the antigenic structure of gp120 as measured by the ability to bind multiple bN-mAbs . Th. Th61]. A model of CHO C1s was made using SWISS-model using the crystal structure of human C1s as a template (PDB 1ELV) . This stA closer look into the interactions between the P2 residue of the substrate and surrounding residues of the protease explains the ability of thrombin, but not C1s to cleave residues with a proline in the P2 position. Mutating C64, the P2 residue of gigastasin, to proline shows potential clashing . One of -/- CHOK1 2.E7 cell line and the C1s-/- MGAT1- CHOK1 1.A1 cell line, and the analysis of C1s substrate specificity has created a practical solution to solve the proteolysis problem in the large-scale production of clade B, HIV vaccine immunogens and other recombinant proteins.Although CHO cells are widely used for the production of most protein therapeutics, limited proteolysis, or clipping, is a common problem in large scale production of recombinant proteins. Clipping can be cell line-specific or protein-specific, and there are multiple CHO cell lines with varying chromosomal structures and genome sequences that are still in the process of being characterized for protease activity . The assThese cell lines, except the C1s-/- MGAT1- CHO-S cell line, are available for transient or stable transfection of any recombinant protein. All cell lines were produced in our lab.One of the existing solutions to prevent proteolysis of gp120s is mutating Arg315 of the GPGR motif to a Glutamine (Q). This mutation has been done for both gp120 monomers and SOSIP trimers found to undergo proteolysis, and highlights the applicability of the C1s-knockout cell lines ,71. WhilA number of groups are creating stable CHO cell lines expressing HIV envelope proteins for clinical trials. One such study hopes to evaluate different adjuvants with a multivalent vaccine using the RV144 subtype AE immunogen A244 and a subtype B immunogen, designated 6240 . Other gIn these studies, we explored the differences between the substrate specificity of C1s and thrombin. Although thrombin has long been known to cleave rgp120 at the GPGR/AF sequence in the V3 region, it was interesting to discover that C1s also cleaved this sequence ,23. Alth-/- CHOK1 2.E7 and C1s-/- MGAT1- CHOK1 1.A1 cell lines described in this report can be used for the expression of all recombinant proteins susceptible to proteolysis by C1s. The finding that CHO cell-derived C1s only cleaves substrates with a small amino acid prior to the arginine in the P1 position will help predict C1s cleavage sites in other proteins. Data mining of the MEROPS database for protein substrates with the sequences Gly-Arg or Ala-Arg in exposed loop regions may reveal a number of proteins that cannot be expressed in the normal CHO cell line. This information will guide how these proteins are expressed whether it be through the use of the C1s-/- CHOK1 cell line, mutating away protease cleavage sites as was done for the CHO production of MN-rgp120 or use of an alternate cell line [With the knowledge of the substrate specificity of C1s, future issues of proteolysis with recombinant proteins can be avoided. The prevalence of proteases in the regulation of physiological processes suggests there will be continued recombinant expression of proteases and protease substrates. Of the approximately 20,000 proteins in the human proteome, 570 are proteases, or about 2.6% of the proteome ,81. Protell line .In summary, we have shown that gene editing can solve the problems of proteolysis and glycosylation heterogeneity encountered in the expression of HIV vaccine immunogens. In principle, this same approach can be employed to eliminate proteases and other glycan modifying enzymes that interfere with the manufacturing and production of other recombinant proteins of pharmaceutical interest.S1 FigThe length in amino acids (AA) and number of potential N-linked glycosylation sites (PNGS) are listed for each of the HIV Env domains for the expressed Envs: A244, EN3_071.(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


It is known that single or isolated tumor cells enter cancer patients’ circulatory systems. These circulating tumor cells (CTCs) are thought to be an effective tool for diagnosing cancer malignancy. However, handling CTC samples and evaluating CTC sequence analysis results are challenging. Recently, the convolutional neural network (CNN) model, a type of deep learning model, has been increasingly adopted for medical image analyses. However, it is controversial whether cell characteristics can be identified at the single-cell level by using machine learning methods. This study intends to verify whether an AI system could classify the sensitivity of anticancer drugs, based on cell morphology during culture. We constructed a CNN based on the VGG16 model that could predict the efficiency of antitumor drugs at the single-cell level. The machine learning revealed that our model could identify the effects of antitumor drugs with ~0.80 accuracies. Our results show that, in the future, realizing precision medicine to identify effective antitumor drugs for individual patients may be possible by extracting CTCs from blood and performing classification by using an AI system. EGFR and RAS expression from colorectal cancer resected specimens and biopsy specimens selects anticancer drugs and molecular targeted drugs that can be expected to have therapeutic effects [The anticancer chemotherapy is an important first-line treatment in unresectable advanced tumors, such as colorectal cancer . In the  effects .The ability to predict the effect of an anticancer drug by liquid biopsy would be useful, because it is a minimally invasive procedure. Circulating tumor cells (CTCs) can be used as a type of liquid biopsy . RecentlConversely, computer-based analyses of large volumes of data recently have become widely used, as computer performance has improved ,8. Many Techniques using AI trained on convolutional neural networks (CNNs) that mimic optical neural networks have recently been developed. To use it in medical applications, there have been various attempts to train AI on medical imaging. Esteva et al. made the first report on the analysis of clinical information using AI . They trIn the field of cancer research, CNN, one of the deep learning algorithms, is rapidly being adopted for analyzing medical images . TherefoUsing two types of colorectal cancer cell lines, DLD-1 and HCT-116, we constructed the discriminant model using the VGG16 deep learning process . We usedTo use CTC for diagnosis in the future, we attempted discrimination at the single-cell level. In this step, we attempted to construct a discrimination model with machine learning using HCT-116 cell images that are either resistant or non-resistant to FTD anticancer drugs. As in the case of the confluence level training described above, 50 value between the control and resistant cells, HCT-116 in FTD resistance, was 31.1 μM, whereas DLD-1 in 5-FU resistance exceeded an estimated 80.0 μM (data not shown). This is the reason why we selected the HCT-116 cell line for machine learning at the single-cell level. Conventionally, in many reports on cancer cell imaging, much effort has been put into classifying cell populations. However, in this study, for the first time, machine learning could discriminate the characteristics of cancer cells, even at the single-cell level. In this study, it was possible to classify cell populations according to their characteristics with more than 0.98 accuracies, using the modified VGG16 neural network model, as shown in Considering that our model maintained ~0.80 accuracies, even with a cell line having such a low level of resistance, it is no exaggeration to say that we have established a foundation that is a great step forward in devising a single-cell-level character recognition system that is adaptable for examining circulating tumor cells. 2 in a humidified incubator. In our previous study [In this study, we used human colorectal carcinoma cell lines, HCT-116 and DLD-1, as controls. These were purchased from the American Type Culture Collection  and maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS at 37 °C and 5% COus study  on resis3 cells per well in 96-well plates and then pre-cultured for 24 h. They were exposed to various concentrations of FTD and 5-FU antitumor drugs, for 72 h. The in vitro cytotoxic effects were assayed using the Cell Counting Kit-8 .The cell lines were seeded at a density of 4 × 10The phase-contrast images of the colorectal cell lines, HCT-116 and DLD-1, were obtained with a microscope . The machine learning datasets were comprised of two categories with 9000 images of cell confluence and 1100 images of single cells. The images in each dataset were 240 × 240 pixels in size. The confluence category training dataset had 7500 images and its test set had 1500 images. For the single-cell dataset, 1000 images were used as the training set, and 100 images were used as the test set. During preparation, the samples were all converted to gray-scale images.A convolutional neural network (CNN) is a machine learning model, which is a system of convolutional, pooling layers and fully connected layers ,15. The


During recent years, small molecules containing five-member heterocyclic moieties have become the subject of considerable growing interest for designing new antitumor agents. One of them is 1,3,4-thiadiazole. This study is an attempt to collect the 1,3,4-thiadiazole and its derivatives, which can be considered as potential anticancer agents, reported in the literature in the last ten years. Cancer is one of the major causes of death globally. Based on type and stage of cancer, treatments include radiation therapy, chemotherapy, surgery and targeted therapies. Despite significant advances in medicine, the treatment of cancer remains a serious challenge. Therefore, new, effective drugs and anticancer strategies are still being sought.One of the methods of searching for new compounds is to modify the structure of known derivatives with documented activity. During recent years, small molecules containing five-membered heterocyclic moieties have become the subject of considerable growing interest for designing antitumor agents. One of them is 1,3,4-thiadiazole. Bioactive properties of thiadiazole are connected generally with the fact, that this heterocyclic ring is a bioisostere of pyrimidine, which in turn is the skeleton of three nucleic bases. Therefore, 1,3,4-thiadiazole derivatives have the ability to disrupt processes related to DNA replication. This permits them to inhibit replication of both bacterial and cancer cells.Thiadiazole derivatives have four isomeric forms: 1,3,4-thiadiazole; 1,2,3-thiadiazole; 1,2,4-thiadiazole; and 1,2,5-thiadiazole. In published studies, 1,3,4-thiadiazole derivatives tend to show the most significant therapeutic potential , antidepThis study is an attempt to collect the 1,3,4-thiadiazole and its derivatives, which can be considered as potential anticancer agents, reported in the literature between the years 2009 and 2020.50 (the concentration causing 50% cell growth inhibition) values in the range of 0.74–10.0 μg/mL against the human colon cancer cell line HCT116, the human lung cancer cell line H460 and the human breast cancer cell line MCF-7. The most active compound (1) against the HCT116 cell line and H460 cell lines, with GI50 values of 3.29 and 10 μg/mL, respectively, is depicted in In 2009, Morsy and co-workers reported anticancer activity in a series of bis-sulfonamides with 1,3,4-thiadiazole heterocyclic rings. The compounds have been tested as inhibitors of CAI, CAII, CAXII and CAIX (carbonic anhydrases). All compounds were rather modest inhibitors of isozymes CA I and XII; but much more efficient as inhibitors of the cytosolic CA II and transmembrane CA IX, with inhibition constants in the range of 21–129 nM against hCA II, and 23–79 nM against hCA IX, respectively. The compounds showed inhibition of growth of several tumor cell lines (ex vivo), with GI2–6 have shown inhibition of growth of the three tumor cell lines: CHO (Chinese hamster ovary), HL60 (human leukemia) and L1210 (mouse leukemia) pyrimidine  designed and synthesized a series of compounds as antitumor agents. The new compounds were prepared by condensation of 1,3,4-thiadiazole-2(3rimidine . The com14) and (oxo)thiadiazoloquinazoline-2-sulfonamide (15) derivatives have shown inhibition of growth activities against the human breast carcinoma (MCF-7) cell line. Anticancer activity of active compounds was higher than bleomycin and ethidium bromide used as reference drugs pyrimidine with a 1,3,4-thiadiazole heterocyclic ring . The in ficantly .18)   showed t18 also showed good effects  ..19) and N-(4-acetyl-5-(4-hydroxyphenyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)-acetamide (21)  of 4.27 µg/mL. A structure–activity relationship study revealed that the nature of substituent on the C-5 phenyl ring of 1,3,4-thiadiazoles is important for their cytotoxic activity  sulfonamide derivatives. New compounds were obtained from carbonic anhydrase inhibitor drug acetazolamide. All the synthesized compounds were investigated for anticancer activity towards cell lines HEK 293 , BT474(breast cancer cell line) and NCI-H226 (lung cancer). The results were compared with standard drug indisulam (ISL). The results demonstrated a strong dose-dependent growth inhibition intreated cell lines. The most active compounds are: N-({5-[(2-methoxybenzylidene)amino]-1,3,4-thiadiazol-2-yl}sulfonyl)benzamide 28  and N-({5-[(furan-2-ylmethylidene)amino]-1,3,4-thiadiazol-2-yl}sulfonyl)benzamide 29 (CTC50 = 0.913 µM against lung cancer cell line)  . None of50 values from 0.04 to 23.6 µM. 1,2,4-triazole derivatives exhibited much better potency than 1,3,4-tiadiazole analogs. The most active thiadiazole derivative was with tert-butyl substituent  30  reported synthesis and anticancer activity of a new 1,2,4-triazole and 1,3,4-thiadiazole. The cytotoxicity of the synthesized compounds were tested on five human tumor cell lines, including human pancreatic cancer cell line (BxPC-3), human lung adenocarcinoma cell line (H1975), human ovarian carcinoma cell line (SKOV-3), human melanoma cell line (A875) and human colorectal adenocarcinoma cell line (HCT116) by the MTT assay with adriamycin as a positive control. Some compounds have shown good activity in the test, with ICline) 30  25]..50 valueN-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide were reported by Aliabadi in 2013. The compounds were evaluated for their anticancer activity against three human cancers: prostate (PC3), breast (MDA) and glioblastoma (U87). The best activity was observed against MDA breast cancer cell line. Compound 31 (50 = 9 μM compared to Imatinib (IC50 = 20 μM) in MDA breast cancer cell line ..50 value40), 2-(4-bromophenylamino)-5--1,3,4-thiadiazole (41), 2-(4-fluorophenylamino)-5--1,3,4-thiadiazole (42)  and from 70 to 170 μM (MDA-MB-231) thiadiazole (100) . Triazol 0.15 μM .101–103) (1/R2 = cyclopropyl/H (101); cyclopropyl/CH3 (102); cyclopropyl/C2H5 (103) have shown the best activity with IC50 values of 0.20, 1.2 and 2.5 μmol/L ..50 value105  [Sunil and co-workers (2011) reported synthesis and anticancer evaluation of new compound: 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-3-[2-naphthyloxy)methyl]triazolothia-diazole 105 . It demo cancer) .H-pyrazol-4-yl]-3-[(2-naphthyloxy)methyl]triazolothiadiazole (106) (The same authors published results of anticancer investigations of 6-triazolothiadiazoles was obtained by Chowrasia et al. in 2017. The compounds were evaluated for their anticancer activity against cancer cell lines MCF-7, SaOS-2 and K562. Some new derivatives have shown good cytotoxicity against investigated cells, with the best ICle (108) . The intty (110) .50 value was 12 μM for 6-((4-fluorobutyl)sulfanyl)-3--3-yl)-1,2,4-triazolothiadiazole (111)  cell lines by MTT assay. The best ICle (111)  66]..50 valueb]thiadiazol-6-yl)flurobenzonitrile (DTTF) 112  by abrogating NF-κB activation cascade by a series of condensed-bicyclic 4,6-substituted 1,2,4-triazolo-1,3,4-thiadiazoles were described by Ningegowda and co-workers in 2017. The compounds were evaluated for their anticancer activity against SiHa , Caski , and HeLa  cell lines, by MTT assay. 5--triazolo-1,3,4-thiadiazole. The compounds were evaluated for their cytotoxicity against HeLa , SMMC-7721 (hepatocarcinoma) and A549 (human lung carcinoma) cell lines. The compounds with substituents RC4H9113  have sho114)   were repell line .115 , MKN45 (human gastric cancer) and EBC-1 (human lung cancer) cell lines. Compound 115  exhibiteound 116  has the igations .117–119  presented synthesis of twenty three 3,6-disubstituted 1,2,4-triazolo-1,3,4-thiadiazole derivatives and their antiproliferative activities in vitro against human hepatocarcinoma (SMMC-7721), HeLa, human lung carcinoma (A549), and mouse fibroblasts (L929) cell lines by the CCK-8 assay. The bioassay results demonstrated that all tested compounds exhibited antiproliferation with different degrees, and some compounds 117–119  showed btert-butoxycarbonyl)piperidine-4-carboxylic acid with 1-methylpiperazine. The most active compound 120  thiadiazoles were synthesized by Karki et al. (2011). The compounds demonstrated inhibition of the growth leukemia cell lines. Compound activity  with thee arrest .-5 M) in full NCI 60 cell panel studies. 5-Bromo-6-(4-chlorophenyl)-2-cyclopropylimidazothiadiazole (122)   indicate123) (H-chromen-3-yl)imidazothiadiazole-5-carboaldehyde and 2-(4-chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazothiadiazol-5-yl were most potent against all cell lines thiadiazoles (123) . The comll lines .50 value was 2.58 μM for compound 124   cell line using sulforhodamine B assay. The majority of the compounds have shown good cytotoxicity but lower than the reference drug doxorubicin. The best ICound 124 , but oneμM (102) . The aut50 values: 0.17–0.67, 0.25–0.87, 0.042–0.61 μM, respectively (126–128) , FM3A (murine mammary carcinoma), human T-lymphoblastoid (CEM) and human cervix carcinoma (HeLa) cell lines. Most of the tested compounds have shown good antiproliferative activity, with IC126–128) . The aut50 values from 0.13 to 3.8 μΜ. Next, the conjugates were evaluated for cell cycle analysis, tubulin polymerization assay and apoptosis. The three most active conjugates (129–131)   were indIn conclusion, this paper gives an overview of cytotoxic properties of various 1,3,4-thiadiazole derivatives. There are definitely more reports on simple 2,5-disubstituted derivatives of 1,3,4-thiadiazole than condensed systems of thiadiazole with other rings. It seems to be a very promising group of chemical compounds. Some of them have promising anticancer activity, higher than reference drugs . Both cy


Targeted therapies have improved the outcome of cancer, but their efficacy is intrinsically limited by the emergence of subclones with a mutation in the gene encoding the target protein. A few examples of collateral sensitivity have demonstrated that the conformational changes induced by these mutations can create unexpected sensitivity to other kinase inhibitors, but whether this concept can be generalized is unknown. Here is described the development of a model to screen a library of kinase inhibitors for collateral sensitivity drugs active on the Bruton Tyrosine Kinase (BTK) protein with the ibrutinib resistance mutation C481S. First, we demonstrate that overexpression of the constitutively active mutant of BTK harboring the E41K mutation in Ba/F3 cells creates an oncogenic addiction to BTK. Then, we have exploited this phenotype to perform a screen of a kinase inhibitor library on cells with or without the ibrutinib resistance mutation. The BTK inhibitors showed the expected sensitivity profile, but none of the drugs tested had a specific activity against the C481S mutant of BTK, suggesting that extending the collateral sensitivity paradigm to all kinases targeted by cancer therapy might not be trivial. With the development of targeted therapies, the emergence of subclones with a resistance mutation has become a major concern of cancer treatment. The collateral sensitivity concept is a promising strategy to overcome this problem: the conformational changes induced by a mutation conferring resistance to a given compound can create a specific sensitivity to another compound . FollowiB cell receptor (BCR) signaling has been recognized as critical in B cell lymphomagenesis, leading to the activation of oncogenic pathways such as NF-kB, PI3K/AKT and MAP-Kinases . Bruton In CLL, about a quarter of the patients treated with ibrutinib show disease progression during the first four years . ProgresHere, we describe the development of a cellular model to screen large chemical libraries for the identification of compounds with collateral sensitivity against BTK-C481S cells.WT  or human BTKE41K with the E41K mutation in the pleckstrin-homology domain, conferring constitutive activation of the kinase  [E41K. But not in Ba/F3 cells overexpressing BTKWT  versus BTKE41K (GFP positive) expressing Ba/F3. Whereas we did not observe any modification of the clonal composition when the cells were cultured with IL3 (49% GFP positive cells), we observed a positive selection of BTKE41K cells after IL3 deprivation (93% GFP positive cells) . The Ba/F3 cell line is a murine pro-B cell line, which requires murine IL3 in the culture medium to survive. This cell line has been largely used as a model to demonstrate the transforming potential of oncogenes, which are able to confer IL3 independence to the Ba/F3 cell line . We overackbone) . After Ing BTKWT . Accordi did not B. To cone cells) C. Of note of IL3 D.E41K-C481S). As expected, the C481S mutation conferred resistance to ibrutinib (IC50 10µM vs. 100 nM for BTKE41K)  D and aboBTKE41K) E.In order to maximize our chances to identify compounds able to exploit the collateral sensitivity created by the C481S mutation, we have chosen to test a library of 590 kinase inhibitors . The output of the screen was the cell viability (as compared to vehicle (DMSO)), estimated by automated image analysis of GFP positive cells after a 48 h culture using the Operetta device.E41K or BTKE41K-C481S in IL3 depleted medium, in the presence of drugs at 100 nM (screen 1) or 1 μM (screen 2) or equivalent quantities of DMSO. We calculated a differential score as follow:The screen compared the viability of Ba/F3 overexpressing either BTKE41K should fall in the upper right quadrant, whereas the drugs with preferential activity against BTKE41K-C481S  should fall in the lower left quadrant.For each drug, we plotted the value of d at 100 nM and 1 μM for graphical representation of the screens . AccordiE41K expressing cells (WT or BTKC481S in a human diffuse large B-cell lymphoma cell line sensitive to ibrutinib (TMD8). We confirmed that BTKC481S confers ibrutinib resistance in this cell line, but we did not detect any differential effect with idelalisib treatment  which showed more pronounced activity on BTKE41K-C481S at least at one concentration (100 nM or 1 μM), and tested them on a large dose range in independent experiments. The insulin receptor IGF-1R inhibitor BMS536924 was the only drug with a confirmed specific activity against Ba/F3 overexpressing BTKE41K-C481S over a large range of doses, but this was not confirmed in the lymphoma cell line TMD8 .We have developed a cellular model allowing the screening of a large number of compounds for their capacity to inhibit BTK with or without the C481S mutation. The use of the Ba/F3 cell line is a strategy which could be applied with other mutated oncogenes able to transform these cells, such as FLT3-ITD  BRAF V60E41K as compared to BTKE41K-C481S in the Ba/F3 cell line is unclear. Of note, we should keep in mind that we introduced the E41K activating mutation of BTK to confer IL3 independence to the Ba/F3, and we cannot exclude that this additional mutation might explain the preferential effect of PI3K inhibitors on BTKE41K expressing Ba/F3. Accordingly, the differential sensitivity to PI3K inhibitors was not confirmed in lymphoma cell lines overexpressing BTKWT or BTKC481S without the E41K mutation.Our data show that this screening strategy has a strong discriminating power, because BTK inhibitors have the expected profile of efficacy. However, some hits should be interpreted with caution (such as the PI3K inhibitors), because the basis of their preferential effects against BTKThe cell lines were cultivated in RPMI-1640 media  supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal calf serum. Ba/F3 were seeded at 5.105/mL in RPMI 1640 supplemented or not with murine IL3 . The BTK inhibitor ibrutinib was purchased from Selleck Chemicals  and the kinase inhibitor library was purchased from MedChemExpress .WT, BTKE41K, BTKC481S, or BTKE41K-C481S in Ba/F3 and TMD8 cells. A murine retroviral vector, pMSCV-IRES, allowing co-expression of the human BTK gene and mCherry (Addgene ref #52114) or GFP (Addgene ref #20672) via an IRES element was used for transduction of Ba/F3 cells . A lentiviral vector, pHIV-SFFV-MSC [We used murine retroviral vectors to express human BTKSFFV-MSC  allowingSFFV-MSC . BrieflyThe murine retroviral vectors were produced following the same protocol using 3 μg VSV-G envelope encoding plasmid, 8.6 μg of MLV-gagpol and 8.6 μg of vector or MSCV vector.E41K) and Ba/F3 cells expressing BTK with both the activating mutation and the ibrutinib resistance mutation C481S on the same allele (BTKE41K-C481S) were seeded in 384-well plates at a density of 10,000 cells in 20 µL/well. The MedChemExpress library of 590 kinase inhibitors  was then added to a final dose of 1µM or 100 nM (duplicates were performed, For compound screening, Ba/F3 stably expressing BTK with the activating mutation E41K (BTKThe absence of collateral sensitivity hits in this screen is disappointing from a clinical point of view. However, this result is important, because it suggests that the concept of collateral sensitivity is probably not so easily extendable to all kinases targeted by cancer therapy. Of course, other screens with different targets and different libraries will be necessary to comprehensively assess to what extent the concept of collateral sensitivity is useful to repurposing drugs for patients with drug resistance mutations. Regarding BTK C481S, the platform presented is a valuable tool for exploratory drug screening.


Canine prostate cancer represents a unique model for human prostate cancer. In vitro systems offer various possibilities but Xenograft in vivo imaging allows studying complex tasks as tumor progression and drug intervention longitudinal. Herein, we established three canine prostate carcinoma cell lines stably expressing fluorescent proteins allowing deep tissue in vivo imaging.Three canine prostate carcinoma (cPC) cell lines were stably transfected with fluorescent proteins in red, far-red and near infra-red spectrum, followed by G418 selection. Fluorescent protein expression was demonstrated by microscopy, flow cytometry and a NightOWL LB 983 in vivo imaging system. Cellular and molecular characteristics of the generated cell lines were compared to the parental cell line CT1258. Cell proliferation, metabolic activity and sphere formation capacity were analyzed. Stem cell marker expression was examined by qPCR and genomic copy number variation by genomic DNA whole genome sequencing.6. Furthermore, we demonstrated a similar sphere formation capacity in the fluorescent cell lines. Interestingly, the clone selected CT1258-mKate2C, showed increased sphere formation ability.Three stably fluorescent protein transfected cPC cell lines were established and characterized. Compared to the parental cell line, no significant difference in cell proliferation and metabolic activity were detected. Genomic copy number variation analyses and stem cell marker gene expression revealed in general no significant changes. However, the generated cell line CT1258-mKate2C showed uniquely no distal CFA16 deletion and an elevated metabolic activity. The introduced fluorescencent proteins allowed highly sensitive detection in an in vivo imaging system starting at cell numbers of 0.156 × 10Starting from a well characterized cPC cell line three novel fluorescent cell lines were established showing high cellular and molecular similarity to the parental cell line. The introduction of the fluorescent proteins did not alter the established cell lines significantly. The red fluorescence allows deep tissue imaging, which conventional GFP labeling is not able to realize.As no significant differences were detected between the established cell lines and the very well characterized parental CT1258 the new fluorescent cell lines allow deep tissue in vivo imaging for perspective in vivo evaluation of novel therapeutic regimens. Canine prostate cancer (cPC) is a very aggressive disease, which is usually diagnosed at very late stages in veterinary patients . In contIn general, cell lines represent key tools in cancer research allowing the generation of neoplasias in animal models mimicking closely the initial tumors in vivo. Thereby, the combination of early stage in vitro settings and advanced stage in vivo models provides several possibilities to study therapeutic approaches and thus is prerequisite for rapid bench-to-bedside translation of anticancer therapies.Advanced experimental approaches targeting complex tasks require the establishment of tumor specific in vivo animal models. Thereby, the characterization of early tumor development and the possibility to monitor tumor cell migration is of major interest for the evaluation of therapeutic agents. In previous studies we characterized the in vivo behavior of cPC derived cell line CT1258 in NOD/SCID mice and monitored tumor development in early stages by contrast enhanced 7T MRI , 16. WhiThe stable introduction of DNA coding for fluorescent proteins as eGFP and YFP offers an alternative allowing long term in vivo imaging without signal loss. While these early developed fluorescent marker proteins proved to be sufficient for most in vitro applications , in vivoFluorescence/luminescence-based monitoring of cancer development in vivo requires ideally a stable and long lasting expression of the fluorescent marker. Thus, a stable insertion of the acting recombinant proteins is a key for studies spanning observations in individual animals for several weeks. Commonly, lentiviral shuttle systems are used to stably transduce primary cells as well as cell lines for in vivo imaging applications . While tHerein, we describe the establishment, characterization and validation of three canine prostate cell lines: CT1258-FusionRed, CT1258-mKate2C and CT1258-TurboFP650, that stably express different far-red and near infra-red fluorescent proteins. These cell lines provide valuable tools for study canine prostate cancer in vivo.CT258 cells transfected with pFusionRed, pmKate2-C and pTurboFP650 expression vectors showed distinct red fluorescence 24 - 48 h post transfection, and achieved stable expression after approximate 30 days of cultivation and selection Fig. a.Fig. 1aFlow cytometry revealed specific fluorescence in the fluorescent target wavelength compared to non-transfected CT1258 control cells. All transfected cell lines were measured three times after 2 months G418 selection using passage 1, 11 and 12. A mean percentage of 68.4% CT1258-FusionRed, 93.9% CT1258-mKate2C, and 47.56% CT1258-TurboFP650 positive cells were achieved under constant selection pressure , FusionRed was 66.2% (CT1258-FusionRed) and TurboFP650 was 48.9% (CT1258-TurboFP650). No differences were determined compared with the cells maintained under selective pressure  of 29.2 h, CT1258-FusionRed of 28.6 h, CT1258-mKate2C of 29.4 and CT1258-TurboFP650 of 27.2 h. No significant difference was observed among the four cell lines.The cell growth curves of native CT1258 and the three fluorescent cell lines were generated by four consecutive days of cell counting. CT1258 cells were used of passages 287, 290 and 294. Fluorescent cell lines were used from passage 4, 7 and 11. All fluorescent cell lines showed comparable growth characteristics to the untransfected CT1258 cell line  and were identified by whole genome sequencing. The variations are presented in a Circos plot  of 226.13. Fluorescence of 0.156 × 10eak Fig. .Fig. 4InIn order to characterize if the clone selected cell line CT1258-mKate2C shows cancer stem-like cell behavior, sphere formation and CD49f expression were investigated. Under serum-free condition spheres with various sizes have formed from the cell lines CT1258 and CT1258-mKate2C. CT1258 derived spheres could be generated when more than 16 cells per well were initially seeded resulting in up to two spheres. At the density of 128 cells per well, the number of spheres ranged from 0 to 15. CT1258-mKate2C derived spheres were achievable already by single cell seeding in a well. Wells containing 128 cells resulted in 14 to 61 spheres  and TurboFP650 protein (near infra-red), respectively. Under G418 selective condition, resistant cells outgrow non-resistant cells, resulting in a polyclonal population of stably expressing CT1258 cells. These populations maintained their heterogeneous character of the initial cell line CT1258.CT1258-mKate2C represents a selected subline generated by limiting dilution assay. In CT1258-mKate2C, more than 90% cells are mKate2 positive and able to maintain a considerable fluorescent expression level after long-term cultivation even without selective antibiotic pressure. All cell lines have shown stable long time expression of their respective fluorescent protein. Further, fluorescent detection potential was verified by NightOWL LB 983 in vivo imaging system which is frequently used to monitor labelled cells in single animals over considerable time spans. The combination of the high NightOwl imaging sensitivity and the red/fR/NIR properties of the herein generated cell lines represent a valuable tool to study canine prostate cancer development and drug efficacy in vivo.Stable undirected DNA integration can easily result in alteration of basic cell line characteristics. Comparative analyses of basic cell biologic characteristics as metabolic activity and doubling time revealed that the recombinant cell lines do not differ significantly from their parental cell line CT1258. While the major characteristics remained stable, an interesting observation could be made with clone selected cell line CT1258-mKate2C. During culture of the three fluorescent cell lines, cell cluster formation in CT1258-mKate2C was observed similar to a previous study in which sphere formation was achieved by serum-free CT1258 culture . MatchinFurther genomic profiling of the generated fluorescent cell lines showed in general no significant changes in the composition of copy numbers. However, again CT1258-mKate2C differed as this is the only compared cell line which does not show a distal CFA16 deletion. DAVID pathway analyses revealed three pathways which are modulated by twelve of the genes located in the preserved CFA16 part.Matching the general genomic CNV data the analyzed stem cell marker expression also showed no significant variation. Consequently key aspects of the generated cell lines remained comparable to the parental line allowing transferability of earlier generated functional results achieved with CT1258 , 17.In general, in this study we established and characterized stably transfected canine prostate carcinoma cancer cell lines in vitro for deep tissue in vivo imaging. The generated CT1258-FusionRed, clone selected CT1258-mKate2C and CT1258-TurboFP650 cell lines kept their parental cell line characteristics and showed stably strong fluorescent protein expression. As the cell lines express their respective marker in red, far-red and near infra-red spectrum the generated cell lines provided a valuable option for deep tissue in vivo imaging and with that the possibility for later in vivo evaluation of novel therapeutic regimens.The canine prostate adenocarcinoma cell line CT1258 established and characterized previously by us was used as parental cell line , 22. Celr) allowing selection of stably transfected cells using Geneticin® Selective Antibiotic (G418) . The vectors were transformed into thermocompetent E. coli DH5α cells according standard procedures. Plasmid DNA was extracted from isolated and expanded culture bacteria using NucleoBond® PC 500 plasmid DNA purification Kit .Three mammalian vectors pFusionRed-C, pmKate2-C and pTurboFP650-C  were used for transfection. The vectors respectively encode red fluorescent protein FusionRed, far-red fluorescent protein mKate2 and near infra-red fluorescent protein TurboFP650. All three vector backbones contain a neomycin resistance gene  according to the manufacturer’s protocol. Briefly, X-tremeGENE HP DNA Transfection reagent, plasmid DNA and transfection diluents Opti-MEM Reduced Serum Media (Life Technologies) were allowed to equilibrate to room temperature (RT). For each transfection, 2 µg plasmid DNA was diluted in Opti-MEM media to a final volume 200 µl. Following, in each sample 6 µl transfection reagent were added into the medium containing the plasmid DNA and mixed gently. The transfection complex was incubated 15 min at RT. After adding the transfection complex to the respective cells, the respective plates were incubated in a humidified 5% CO2 incubator for 48 h at 37 °C. The same number of cells was respectively seeded in three additional wells  serving as untransfected control. The expression of the fluorescent protein was verified using a Leica DMI 4000B fluorescence microscope .The CT1258 cells were plated 5 × 10The G418 kill curve generation of CT1258 was performed previously by us . Post tr6 cells in 500 µl phosphate buffered saline (PBS), and examined using the FL-3 channel in a FACS Calibur . Data analyses were performed with Cell Quest soft-ware .During G418 selection, the three transfected cell lines were controlled by fluorescence microscopy weekly. The respective amount of fluorescent transfected cells were analyzed by flow cytometry. Therefore, the cells were trypsinized and dissociated into single cell suspension, adjusted 1 × 10To further characterize the stability of the fluorescent cell lines, the respective fluorescent cell rates were measured by flow cytometry after 96 h cultivation without G418.In order to preserve the heterogeneous character of the cell line CT1258, the transfected cell lines are preferable to keep the original CT1258 polyclonal character. However, despite the G418 selection a population of untransfected cells remained in the cell lines. We identified and selected single clone from CT1258-mKate2 transfection by limited dilution and cultivated them separately further. This cell line was named CT1258-mKate2C.The selection was done as follows: CT1258-mKate2 cells were diluted to a density of 10 cells/ml by selection medium and seeded 100 µl per well in 96-well plate. After four days, the number of colonies was assessed in the wells. Only wells with one colony per well were marked for further analyses. Fluorescence expression was identified by fluorescence microscopy of the remaining colonies, two positive colonies were selected for further expansion. Fluorescence was further verified by flow cytometry after three passages, the clones showing highest expression were kept in culture and frozen for long-term storage.5 cells per well in four 12-well plates adding 2 ml of medium without G418. The number of viable cells was determined at 24, 48, 72 and 96 h by trypan blue staining using a conventional cell count chamber. Population doubling time (PDT) was calculated by the formula PDT = 1/[3.32(logNH-logNI)/(t2 − t1)] . Herein, cells were harvested after 72 h to calculate the PDT.The CT1258, CT1258-FusionRed, CT1258-mKate2C and CT1258-TurboFP650 cells were seeded 2.5 × 104 cells per well were plated in triplicate in 150 μl medium without G418. Metabolic activity was analyzed after 48, 72 and 96 h using tetrazolium compound WST-1 reagent . Absorbance at 450 nm and the reference wavelength at 750 nm were determined by GloMax®-Multi Detection System . All experiments were repeated three times independently.Metabolic activity measurements were performed by WST-1 assay. In a 96-well plate, 1.5 × 10As stem cell marker expression is crucial in cancer cell lines a distinct marker panel covering the canine genes CD44, CD133, c-KIT, CD34, ITGA6, MYC, NANOG, DDX5, KLF4, SOX2, MELK and OCT4  wasTotal RNA was extracted from CT1258 and fluorescent cells using RNeasy mini Kit . cDNA synthesis was carried out using 500 ng of total RNA in 20 µl according to the manufacturer’s protocol for the QuantiTect Reverse Transcription Kit . The qPCR reactions were performed using the ViiA™ 7 Real-Time PCR System (Life Technologies) and QuantiTect SYBR green qPCR Kit (Qiagen). ß-actin (ACTB) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) were used as endogenous control. The qPCR results were analyzed using the delta delta CT (ΔΔCT) method relative to CT1258 cells. For each cell line, three samples of different passages were used. All samples were analyzed in triplicates including non-template and non-reverse transcriptase controls for each reaction.® Tissue Kit  following the manufacturer’s instructions.As Genomic DNA was extracted from cultured CT1258 and CT1258 fluorescent cell lines using the NucleoSpinhttp://picard.sourceforge.net) the sequence reads in 500 kbp bins were counted, the numbers were corrected for mappability and GC content and log2-transformed copy-number ratios were called using the QDNAseq R-package [200 ng genomic DNA was ultrasonically sheared and sequencing libraries were prepared using the NEBNext Ultra DNA Library Prep Kit  according to manufacturer’s instructions. Shallow shotgun sequencing  was conducted on a NextSeq 500  yielding an average of 14 M reads (SD: 6 M). Reads were aligned to the canine reference genome (version: Broad canFam3.1). After duplicate removal using Picard Tools (-package . Obtaine-package .https://david.ncifcrf.gov/tools.jsp) for the analysis of associated pathways. Furthermore, the gene list was converted by DAVID Gene ID Conversion tool and partial genes were submitted to DAVID Functional Annotation tool for pathway analysis.The genes in the chromosomal deletion region of CFA16 (chr16:18500001-59500001) were identified based on Ensembl database. The complete gene list was uploaded to the DAVID Functional Annotation tool . Photo was taken using a filter with excitation of 590 nm and emission of 655 nm.Viable cells were counted and plated at a density of 2.5 x 10The capacity of sphere formation of CT1258 and CT1258-mKate2C cell lines were examined additionally. The cells were prepared as a single-cell suspension in serum-free medium. The medium consisted of DMEM/F12 , supplemented with 5 µg/ml Insulin , 20 ng/ml human epidermal growth factor (EGF) (Biochrom), 20 ng/ml human basic fibroblast growth factor (bFGF) (Life Technologies), and 2% B27 (Life Technologies). Different cell numbers varying from 128 to 1 cell/per well were seeded in 96-well plates with duplicates. The number of generated spheres was counted after 10 days cultivation. The medium was changed every 3 days. The assay was performed three times independently.6 cells were placed in 100 µl PBS with 1% BSA in a flow tube. Cells were incubated with 1 μg rat anti-human CD49f  at 4 °C for 30 min in the 100 µl PBS [CD49f (a.k.a. ITGA6) expression on the cell surface was detected by flow cytometry. CT1258 and CT1258-mKate2C cells were trypsinized, washed with PBS twice and then resuspended in PBS. For each measurement, 1 × 100 µl PBS . After wt test, where a p-value of less than 0.05 was considered to be statistically significant.The Significant differences were calculated using Student’s Additional file 1. Genes located in the chromosomal area chr16:18500001-59500001.


Hematopoietic cell transplantation (HCT) provides potentially curative therapy for high-risk hematological malignancies, predominantly through alloreactivity mediated by donor immune effectors directed at a recipient's malignant cells; this is termed graft vs. leukemia (GVL) effect . This bepotential for developing either GVL, using tumor specific antigen (TSA) burden, or likelihood of developing GVHD, using minor histocompatibility antigens (mHA). Historically haematopoietically restricted mHA   of transplant donors and recipients was performed in a group of HLA matched donors and recipients, and demonstrated an average of >6,000 non-synonymous single nucleotide polymorphisms (SNP) per HLA matched donor-recipient pair (DRP) . These p ver2.0)  and repr ver2.0)  and in H ver2.0) –53, as wWhile the sheer number of mHA alone vastly outnumbers the potential TSA in hematologic malignancy, these numbers do not tell the whole story. Whether the potential mHA result in a T cell proliferation depends on several factors, such as peptide cleavage potential, antigen binding affinity, and critically, T cell clones bearing receptors that might recognize the mHA-HLA complexes. Crucially, the T cell receptor affinity for HLA-mHA or HLA-TSA complexes also needs to be adequate to ensure T cell engagement and activation. Mathematical modeling of T cell expansion in response to these HLA-antigen complexes has given important insights into the quantitative principles at hand in these processes. First, the expansion of donor T cells recognizing specific antigens will be proportional to the amount of antigen available, i.e., the expression level of the antigen bearing protein will determine the extent of T cell expansion. Secondly, this T cell expansion is likely governed by the affinity of the antigen to the HLA molecule, and the affinity of the T cell receptors for antigen-HLA complex. This is an exponential relationship, with T cell growth increasing non-linearly in response to changing affinity. An important clue to this is provided by the T cell clonal frequency distribution which follows Power Law when these are plotted out for T cell clones present in normal individuals .r values in the logistic equation, these simulations, showed that the simulated organ-specific alloreactive T cell clonal growth had marked variability, with orders of magnitude of difference between different HLA matched DRPs (N = 78). This was because of the differences in the unique polymorphic peptide sequences and their binding to the many different HLA types. In this study higher total and organ-specific T cell counts were associated with the incidence of moderate to severe GVHD  SCT DRP , each responding T cell clone's proliferation was determined by the logistic equation of growth . Assuminere GVHD . T cell ere GVHD . This mo SCT DRP . Such esWith these data in mind, when the relative number of tumor specific antigens and minor histocompatibility antigens are examined it becomes obvious that the relatively small number of TSA compared with mHA, may in most individuals result in outcompeting of tumor specific targets, by normal tissue targets setting up the field for GVL occurring in the company of GVHD . Thus, pAll is not lost in the mathematical medley of chaos and combinatorics. It is clear that the quantitatively driven T cell responses depend on relative antigen abundance and HLA affinity. Traditionally HSCT is done with patients in remission, and as immunosuppression is withdrawn, they may develop chronic GVHD, which confers protection from relapse, and in a few patients GRFS might be observed. This likely depends on both the extent of T cell clonal diversity emerging after transplantation, as well as the balance of antigen expression. It is therefore critical to understand the notion of relative antigen abundance , such thIn conclusion, mathematical modeling of immune reconstitution, guided by NGS, along with an in-depth analysis of the relative expansion of donor T cell clones in response to the differentially expressed TSA and normal recipient antigens in individual patients, may allow a deeper understanding of the apparently stochastic nature of clinical outcomes observed at a population level. Mathematical modeling of T cell responses has revealed the chaotic dynamics of post-transplant immune responses, when multiple antigens with different HLA binding affinities and tissue expression levels are studied , 61, 62.EK and AT developed the idea and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epithelial-to-mesenchymal transition (EMT) relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation generating mesenchymal-like cells with newly acquired migratory and invasive properties. In cancer cells, EMT leads to drug resistance and metastasis. Moreover, differences in genetic backgrounds, even between patients with the same type of cancer, also determine resistance to some treatments. Metabolic rewiring is essential to induce EMT, hence it is important to identify key metabolic elements for this process, which can be later used to treat cancer cells with different genetic backgrounds. Here we used a mathematical modeling approach to determine which are the metabolic reactions altered after induction of EMT, based on metabolomic and transcriptional data of three non-small cell lung cancer (NSCLC) cell lines. The model suggested that the most affected pathways were the Krebs cycle, amino acid metabolism, and glutathione metabolism. However, glutathione metabolism had many alterations either on the metabolic reactions or at the transcriptional level in the three cell lines. We identified Glutamate-cysteine ligase (GCL), a key enzyme of glutathione synthesis, as an important common feature that is dysregulated after EMT. Analyzing survival data of men with lung cancer, we observed that patients with mutations in GCL catalytic subunit (GCLC) or Glutathione peroxidase 1 (GPX1) genes survived less time than people without mutations on these genes. Besides, patients with low expression of ANPEP, GPX3 and GLS genes also survived less time than those with high expression. Hence, we propose that glutathione metabolism and glutathione itself could be good targets to delay or potentially prevent EMT induction in NSCLC cell lines. Epithelial-mesenchymal transition (EMT) is a natural cellular phenomenon that converts epithelial cells to mesenchymal-like, giving them motile, and invasive properties . This prConstraint-based modeling integrates a set of algorithms that combine computational modeling of genome-scale metabolic reconstruction with omic data to differentiate and predict metabolic alterations in biological systems . Among tIn this paper, we analyzed the metabolic alterations in metabolism that follow EMT induction. Our study used Dycone to predict alterations in enzyme activity during EMT in three non-small cell lung cancer (NSCLC) cell lines , using available metabolome data . These cp-value was lower than 0.05 and log fold change >1, unless otherwise indicated.Microarray and metabolome data was obtained from Sun et al. . All codt-test.Metabolome and microarray data was analyzed with R 3.5.3. Metabolites concentrations were converted to log scale and compared using a Microarray data was normalized using the R package fRMA from Bioconductor and analyzed with the R limma package, unless otherwise indicated data was considered significant with a p-value lower than 0.05 and absolute fold change higher than 1. Code is available upon request.Enrichment analysis for metabolome data was done in MSEA web platform with the concentration table of each of the cell lines  , 13. TheTo investigate the metabolic reprogramming before and after EMT in a system-level approach, we have constructed a medium-size but essential metabolic network , which iReactions had to be included in enriched metabolic pathways.Reactions which had concentrations measured for substrates, products, or both.Reactions that connected the network and are not isolated from others.Reactions important to complete gaps even though substrates or products are not in data.Finally, adjacent reactions where intermediates did not have measured concentrations were joined in a single reaction.https://github.com/cdiener/dycone)  , 10. Brihttps://portal.gdc.cancer.gov/). We used the portal to search survival data from male patients with bronchus and lung cancer as a primary site. We compared two cohorts of patients, the first patients with mutations in genes of interest and the second patients without mutations on these genes. Cumulative survival time was calculated by the Kaplan–Meier method and analyzed by log-rank testing. Survival analysis of patients with different expression levels was obtained from Kaplan Meier plotter (p < 0.05).Genomic Data Commons Data Portal was used for the survival analysis of patients with mutations  and Glutathione reductase (GSR), plus an upregulation of Alanyl Aminopeptidase (ANPEP) after EMT in the three cell lines . GPX andp-value 0.0183)  . Besides 0.0183) . This sup = 6.2e−07), while patients with low expression of ANPEP, GPX3 and GLS survive less time than those with high expression   . Overallin silico approach predicted that some set of reactions changed in all three cell lines. One of these reactions was glutaminase (GLS), which we expected as the original paper showed impairment in the conversion of glutamine to glutamate  in cancer is one of the first steps for metastasis and drug resistance. EMT induction rewires metabolism, and some metabolites and enzymes have been identified as essential features for this process, such as the accumulation of fumarate, changes of succinate dehydrogenase and high levels of ROS in the cell, among others , 11, 16.lutamate .On the other hand, the model predicted that reactions involved in glutathione metabolism changed in the three cell lines, paired with an alteration of the expression of genes encoding enzymes involved in this pathway. Glutathione oxidation and reduction are usually carried out by glutathione peroxidases (GPXs) and glutathione reductase (GSR). At the transcriptional level, EMT downregulated the expression of GPX2 and GSR in A549, HCC827, and NCI-H358 cell lines. Furthermore, GSR and GPX genes have already been reported as essential features to block EMT in melanoma cells, cervical cancer, hepatocellular carcinoma, gastric, pancreatic, breast, and colon cancers , 42, 43.in silico metabolic analysis of A549 and NCI-H358. ANPEP has been correlated with EMT markers at the proteome level, finding higher amounts of this protein when EMT markers are highly produced, however, this study is comparing two different cell lines with different genetic backgrounds, and they do not induce EMT in any form . GCL catalyzes the first and rate-limiting step in the production of the cellular antioxidant glutathione. Our computational modeling predicted that GCL had changes in HCC827. Furthermore, GCLC, the gene that encodes the catalytic subunit of this enzyme, was downregulated in A549 and NCI-H358. From a clinical point of view, there is evidence that the high expression of GCLC predicts a better prognosis for melanoma patients . We alsoIn conclusion, we presented a systems biology approach that integrates computational modeling, metabolome, and microarrays and can serve as a quantitative framework to predict and identify important reactions in EMT of NSCLC cell lines. These predictions are potentially useful in the design of therapeutic targets, as we compared NSCLC cell lines with different genetic backgrounds, hence having differences in their metabolism. Thus, finding similarities and differences between these cell lines help us understand the underlying mechanisms of the metabolic rewiring during EMT. Notably, the proposed genes depicted in this work have already been reported in other cancer as essential features for EMT induction. Overall, we highlight that our model can serve as a quantitative framework to predict and identify important reactions in EMT of NSCLC cell lines. Specifically, we highlight the role that glutathione metabolism has into EMT, a pathway that can be used to improve the outcome of patients in the future.https://cancerandmetabolism.biomedcentral.com/articles/10.1186/2049-3002-2-20). All code used for analysis is available at https://github.com/Meztlimg/EMTanalysis.Publicly available datasets were analyzed in this study. This data can be found here: Sun et al.,  SupplemeMM-G and OR-A developed the methods and MM-G performed the analysis. MM-G and OR-A wrote the paper. CZ and ER helped with the manuscript revision and writing. All authors contributed to the article and approved the submitted version.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.


O-methylcimigenol-3-O-α-L-arabinopyranoside as the most potent candidate. Interestingly, not only the methyl group at position C-25 increased the cytotoxic effect but also the arabinose moiety at position C-3 had an impact on the activity. The variety of cimigenol derivatives, moreover, allowed a detailed discussion of their structure–activity relationships, not only for their effect on multiple myeloma cells but also with regard to previous studies on the cytotoxicity of black cohosh triterpenoids.Black cohosh is a well-established medicinal plant and preparations of its rootstock are used for the treatment of mild climacteric complaints. The compounds considered responsible for the therapeutic effect are triterpene glycosides, characterized by a cycloartane scaffold and a pentose moiety. Because some of these triterpenoids were found to exhibit relevant cytotoxic effects against human breast cancer cells, we decided to investigate their activity on multiple myeloma cell lines NCI-H929, OPM-2, and U266. In a systematic approach, we initially tested three known cytotoxic compounds of three different triterpenoid types, revealing the cimigenol-type triterpenoid as the most active constituent. In a second round, seven naturally occurring cimigenol derivatives were compared with respect to their sugar moiety and their substitution pattern at position C-25, leading to 25- Actaea racemosa (syn. Cimicifuga racemosa), commonly known as black cohosh, is an important medicinal plant, which was already used by the Native Americans for the treatment of rheumatism and gynecological disorders [1) and 23-epi-26-deoxyactein were found to exhibit beneficial effects against postmenopausal osteoporosis promoting cell growth and differentiation of osteoblast as well as osteocalcin production and mineralization [isorders ,2. Preseisorders ,4,5,6. Cisorders . These tisorders . Becauseisorders ,10. In tlization ,12,13, tlization .1), 23-epi-26-deoxyactein and cimiracemoside A on the growth inhibition of MCF7 breast cancer cells with IC50 values of 14, 21 and 41 µg/mL, respectively [1) found that the inhibition of cell growth is associated with activation of specific stress response pathways and apoptosis [O-acetylshengmanol-3-O-β-d-xylopyranoside (2), 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (3), 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (4) and cimigenol-3-O-β-d-xylopyranoside (6) on MCF7 and doxorubicin-resistant-MCF7 (R-MCF7) cells, with IC50 values of 39 and 60 µg/mL for compound 2 and IC50 values between 4.0 and 5.3 µg/mL for compounds 3 and 4 [1) exhibits anti-angiogenic and immunomodulatory effects and decreased tumor size and metastasis in lung and liver [Apart from the activities related to the traditional usage, several studies are dealing with the cytotoxic activity of black cohosh triterpenoids. Einbond et al., for example, investigated the impact of actein , 23-O-acetylshengmanol-3-O-β-d-xylopyranoside  and 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside .The metabolite pattern of black cohosh rhizome is unusually complex with, so far, more than 40 known different triterpenoids . Because1) showed only weak to moderate cytotoxic effects, which increased after 48 h of incubation. However, at 25 µM almost no effects were observed, independently from the time of incubation. 23-O-acetylshengmanol-3-O-β-D-xylopyranoside (2), at a concentration of 50 µM, exhibited slightly stronger effects than actein (1) after 24 h of incubation and similar effects after 48 h, with the only difference that OPM-2 cells were more affected than U266 cell lines. At a concentration of 25 µM the picture was more or less the same than that of actein (1), with only little effects against all cell lines tested (and at both incubation times). 25-O-acetylcimigenol-3-O-α-L-arabinopyranoside (3) had a significantly higher impact on all three myeloma cell lines, reducing cell viability to 10% (NCI-H929), 13% (U266) and 20% (OPM-2), respectively, at a concentration of 50 µM after 24 h of incubation and below 10% for all cell lines after 48 h. In contrast to compounds 1 and 2, the cimigenol derivative (3) also exhibited moderate effects at a concentration of 25 µM.At a concentration of 50 µM and after 24 h of incubation, actein  in the same setting. Promising, the average rate of cell death induction was slightly but constantly higher in the malignant myeloma cell lines compared to the healthy cells, and the extent was depending on the cell line tested. The induction of cell death is likely due to activation of apoptotic pathways since Annexin V/propidium iodide double-staining can be observed and necrosis (propidium iodide only staining) is missing . InductiActaea racemosa were investigated in order to eventually identify more active constituents and to gain further insight into the structure–activity relationships of this particular compound class , 25-O-acetylcimigenol-3-O-β-d-xylopyranoside (4), 25-O-methylcimigenol-3-O-α-l-arabinopyranoside (7) and 25-O-methylcimigenol-3-O-β-d-xylopyranoside (8). Compounds 5 and 6, which both consist of an underivatized cimigenol scaffold, showed no activity on any of the cell lines tested. Neither did 25-anhydrocimigenol-3-O-α-l-arabinopyranoside (9), which is characterized by the absence of a hydroxy-group in position C-25.Of the seven cimigenol derivatives tested, four compounds showed considerable effects against myeloma cell lines NCI-H929, OPM-2, and U266, namely 25-50 values of the four active constituents are given in 50 values after 48 h of incubation [ICcubation  and Einbcubation ,16,17. A4 showed similar effects as its arabinose counterpart (3) on NCI-H929 cells, a slightly higher impact on OPM-2 cells and lower activity against the U266 cell line. Comparison of the methylated cimigenol derivatives 7 and 8 with the two abovementioned acetylated forms 3 and 4, showed that methoxylation at position C-25 increased the activity against all cancer cell lines more than acetoxylation. For the methylated derivatives, an exchange of an arabinose with a xylose moiety did neither affect the activity against NCI-H929 nor OPM-2 cells. However, against myeloma cell line U266 the IC50 value of the xyloside was about twice as high (31.1 vs. 16.2 µg/mL), thus indicating that the sugar moiety contributes to the cytotoxic effect in this setting.Compound Actaea racemosa is a well-established medicinal plant, which was investigated for its effectiveness and safety in around 20 clinical studies involving over 6000 patients [1) or 23-epi-26-deoxyactein. However, black cohosh not only contains a variety of different triterpenoids but also structurally very divers triterpene types (1 and 3), for example, consist of a six-ring scaffold (without counting the cyclopropane-ring), whereas shengmanol-type triterpenoids (2) show a four-ring scaffold and thus a more steroid-like structure. Regarding their polarity, acteol- and shengmanol-type triterpenoids show a higher grade of oxidation and therefore are usually more polar than cimigenol-type triterpenoids. This is even more pronounced, if the cimigenol scaffold underwent derivatization in position C-25 .patients , and itspatients . Most ofne types . Acteol-O-acetylshengmanol-3-O-β-d-xylopyranoside (2) was exceeding that of actein (1) and 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (3) by more than the factor 5 [3 was significantly active on all three myeloma cells lines, compounds 1 and 2 only affected NCI cells remarkably -receptors, where the effect of 23-factor 5 . In the markably . For theActaea species [Actaea racemosa, cimigenol-3-O-α-l-arabinopyranoside (5) and cimigenol-3-O-β-d-xylopyranoside (6) display two of the most abundant triterpenoids [3, 4, and 7–9, but also hydroxylation and subsequent acetylation at other positions, e.g., 1α, 7β or 12β, can be found [Actaea triterpenoids most interesting results, apart from actein (1), were obtained for cimigenol-3-O-β-d-xylopyranoside (6) and some of its acetylated derivatives [6 to be almost as effective as actein (1) on cell proliferation in MDA-MB-453 (Her2 overexpressing) human breast cancer cells. In the same assay, 25-acetyl-7,8-didehydrocimigenol-3-O-β-d-xylopyranoside displayed the most active compound, whereupon the acetyl group at position C-25 was considered important for the growth inhibitory activity.Cimigenol-type triterpenoids represent one of the major triterpenoid classes in black cohosh and several other  species ,14. In Arpenoids , but alsrpenoids . These dbe found . Regardiivatives ,18. Einb3 and 4 were the two most active compounds against MCF7 and R-MCF7 cell lines, with IC50 values of 4.0 and 4.3 µg/mL (MCF7) as well as 5.3 and 4.8 µg/mL (R-MCF7), respectively. Similar activity was achieved when the acetoxy-group was replaced by chlorine; however, the fact that cimigenol-3-O-β-d-xylopyranoside (6) showed no activity in their study indicated the importance of lipophilic substitution at position C-25. 23-O-acetylshengmanol-3-O-β-d-xylopyranoside (2) was investigated in the same study but showed only moderate effects with about ten times higher IC50 values.The importance of hydrophobic groups in position C-25 was also mentioned in a study by Fang et al. , in whic3 and 4 to be significantly more active than its hydrophilic congeners 5 and 6 and as the structurally diverse triterpenoids actein (1) and 23-O-acetylshengmanol-3-O-β-d-xylopyranoside (2). Moreover, the fact that compound 9, which can be considered the most lipophilic compound in our study, had no effect on the cell lines tested indicates that the overall lipophilicity of the molecules is not the key criterion. It rather seems that a lipophilic group at position C-25 increases the effect in contrast to a hydrophilic group such as a hydroxyl-group, but that the absence of a functional group at this position causes a loss of activity. This can be explained with the need for electron donor groups, which are provided by methoxy- and acetoxy-groups as well as by the chlorine atom in the study of Fang et al. [7 and 8 exhibited stronger effects than acetylated compounds 3 and 4, furthermore, suggests that the size of the functional group might as well have an impact on the activity. Having the same hydrogen accepting capacity and nearly the same polarity, methoxy and acetoxy-groups show significantly different van der Waals volumes and surface areas, which consequently affect receptor binding and specificity.The results obtained in our study corroborate these findings. Similar to Einbond et al.  and Fangg et al. , though 3, 4, 7 and 8) the arabinose-bearing derivatives showed higher activity than their xylose-bearing counterparts on U266 myeloma cells, which was most evident for compounds 7 and 8. For this cell line, the effect of the sugar moiety was even surpassing an exchange of acetyl and methyl groups in position C-25, demonstrated by the higher growth inhibitory activity of 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (3) compared to 25-O-methylcimigenol-3-O-β-d-xylopyranoside (8). L-arabinose is an epimer of d-xylose at position C-4, showing an axial hydroxy-group at this position and thus only differing by the orientation and not by the exchange of a functional group. Still, the axial orientation of the hydroxy-group leads to a slightly higher polarity and to a higher ability for eventual intermolecular hydrogen bonding.Apart from the impact of different functional groups in position C-25, our study revealed that slight differences in the sugar moiety – and thus differences at the other end of the molecule – also contribute to the antimyeloma effect of cimigenol-type triterpenoids. Of the four active components  as the most potent triterpenoid. Reports on cytotoxic activities of compound 7 are, so far, limited to a single study, in which moderate effects against human squamous cell carcinoma (HSC-2) cells and normal human gingival fibroblasts (HGF) were reported [O-methylcimigenol-3-O-α-l-arabinopyranoside (7) as anti-cancer drug might be of interest. Because of the observed antimyeloma activity in our study, investigations on other hematopoietic cancer cell types, e.g., leukemia, will also be reasonable.The structure-guided approach, moreover, led to the identification of 25-reported . With thApart from the identification of cytotoxic constituents in black cohosh, the present study demonstrates the need for systematic investigations in the field of phytopharmacology. In medicinal plants, different compound classes are present and even if one compound class is dominating, as e.g., in black cohosh, several structure-types may co-occur and may possess different biological activities or potencies, respectively. A successful systematic approach is shown in a previous study on copaiba, in which both the cytotoxic as well as the antimicrobial activity were attributed to certain compound classes and the necessary feature for the antifungal activity was identified . This waThis leads to another interesting aspect/challenge in phytopharmacology, where studies often focus on the investigation of extracts and/or the most abundant constituents. However, if the major components only exhibit moderate effects, it is very likely that no further experiments with minor constituents are considered necessary, especially if these constituents demand high efforts for their isolation. Thus, possible drug candidates might be overseen. Of course, when it comes to in vivo experiments, certain amounts of drug substance are required, which can hardly be obtained when the respective compound is only present in small quantities. Still, once a potential candidate is identified other ways of yielding the compound may be feasible, such as semi-synthetic approaches or isolation of the compound from closely related plant species.Dried and cut plant material was obtained from Caesar and Loretz GmbH . LC-MS grade acetonitrile, methanol and water and other  solvents and reagents were purchased from VWR International GmbH . LC-MS grade formic acid was obtained from Sigma Aldrich Co. . Ultrasonication was accomplished with a Bandolin Sonorex ultrasonic bath (Type RK 1028 CH) and evaporation of solvents was done on a Heidolph Laborota 4000 efficient vacuum evaporator connected to a Vacuubrand PC 2001 vario pump. TLC was performed on silica gel 60 F254 plates  using dichloromethane-methanol (85:15) as mobile phase and vanillin-sulfuric acid for detection. Semi-preparative HPLC was carried out on a Waters Alliance e2695 Separations Module coupled to a 2998 PDA, a 2410 RI and a WFC III fraction collector and a Phenomenex Aqua column . Extracts, fractions, and pure compounds were analyzed by a VWR-Hitachi Chromaster Ultra RS equipped with a 6170 binary pump, 6270 autosampler, 6310 column oven, 6430 DAD and a VWR 100 evaporative light scattering detector using a Phenomenex Synergi Max-RP column . LC-MS analysis was carried out with a Shimadzu LC-MS 8030 triple quadrupole mass spectrometer using a Phenomenex Kinetex RP-18 column  and atmospheric pressure chemical ionization.n-hexane-ethyl acetate-methanol (10:10:0.5 to 10:10:3) in a gradient manner, yielding 11 subfractions (C1–C11). Fraction C9 gave 16.38 mg of actein (1) and fraction C7 yielded 5.65 mg of 23-O-acetylshengmanol-3-O-β-d-xylopyranoside (2). Fraction B (1.17 g) was subjected to Sephadex LH-20 column chromatography (100 × 2 cm) using dichloromethane-aceton (85:15) as solvent mixture yielding 8 subfractions (B1–B8). Subfraction B4 (180 mg) was separated with semi-preparative HPLC using isocratic elution with water-acetonitrile (50:50) yielding 6.16 mg of 25-O-acetylcimigenol-3-O-α-l-arabinopyranoside (3). Compounds 4 to 9 were isolated as described in one of our previous studies [Dried and ground rhizomes (700 g) were extracted three times with 5 L of acetone using ultrasonication, and the solvent was evaporated under reduced pressure to afford 42 g of a crude extract. The extract was re-dissolved in a mixture of dichloromethane-methanol (9:1) and applied to 300 g of silica gel (dry load). The sample was then subjected to silica gel column chromatography (50 × 7.5 cm) and eluted with dichloromethane-methanol  yielding six fractions (A–F). Fraction C (1.98 g) was chromatographed over a silica gel column (100 × 3.5 cm) using  studies . Furtherl-glutamine 100 µg/mL, and penicillin-streptomycin 100 U/mL. PBMCs from healthy donors were used after obtaining written informed consent at the University Hospital Salzburg . Cells were subjected to Ficoll separation (Ficoll PaqueTM) and incubated in RPMI-1640 Media with supplements as above. In brief, 0.5 × 106 myeloma cells/mL or similar numbers of PMBCs were incubated for 24 h and 48 h with or without the tested compounds dissolved in dimethyl sulfoxide at different concentrations. At least three analyses in triplicates were performed for each cell line and a solvent control was always included. As a positive control, the proteasome inhibitor bortezomib (BTZ) was used at concentrations of 10 and 5 nM. The extent of non-apoptotic cells (AnnexinV/propidium iodide negativity) was calculated as percentage of viable cells in respect to the untreated control. Data are shown as mean percentage of viable cells and standard error of the mean (SEM) (error bars).Induction of apoptosis was measured in myeloma cell lines NCI-H929, OPM-2, and U266 as well as in peripheral blood mononuclear cells (PBMCs) of healthy donors by flow cytometry using established protocols [O-methylcimigenol-3-O-α-l-arabinopyranoside as the most potent constituent. The availability of a variety of cimigenol derivatives thereby allowed a detailed discussion of their structure-relationships and revealed an impact of the sugar moiety for one of the tested cell lines. Apart from the identification of black cohosh triterpenoids with in vitro antimyeloma activity and the structural features responsible for these effects, the present study, moreover, demonstrates the need for systematic approaches in phytopharmacology, in order to fully exploit the potential of this interesting research field.In the present study, the effect of black cohosh triterpenoids on multiple myeloma cell lines NCI-H929, OPM-2, and U266 was investigated. Following a structure-guided approach, we found apolar cimigenol-type triterpenoids to exhibit higher activity than other (more polar) triterpenoid types. Subsequent studies on cimigenol derivatives, focusing on the sugar moiety at position C-3 and the substitution pattern at position C-25, led to the discovery of 25-


Calea pinnatifida and the structural characterization and determination of the absolute configuration of the isolated compounds as well as their antitumor potential. The HPLC fractionation of the CH2Cl2 phase of the MeOH extract from the leaves of C. pinnatifida led to the isolation of two related sesquiterpene lactones (STLs): calein C (1) and calealactone B (2). Additionally, during the purification process, a derivative of calein C (3) was formed as a product of the Michael addition of MeOH. The structures of Compounds 1–3 were established based on spectroscopic and spectrometric data, while the absolute stereochemistry was established by vibrational circular dichroism. In order to evaluate the effect of the conjugated double bonds on the cytotoxic activity of STLs, Compounds 1–3 were tested against anaplastic (KTC-2) and papillary (TPC-1) thyroid carcinoma cells. Calein C was the most active of the STLs, and displayed activity against both KTC-2 and TPC-1. On the other hand, the calein C derivative (3) was the least cytotoxic of all the compounds tested. These results are promising and suggest the importance of studying sesquiterpene lactones isolated from C. pinnatifida in terms of antitumor activity, especially considering the effects of α,β-unsaturated carbonyl systems.This work describes the chromatographic fractionation of the aerial parts of  Calea pinnatifida (R. Br) Less. (Asteraceae), popularly known as aruca, cipó cruz or quebra-tudo, occurs in Brazil, mainly in the “cerrado” biome + corresponding to the molecular formula C21H26O8Na . All signals in the 1H- and 13C-NMR spectra were attributed by the analysis of the 1H–1H COSY, HMQC, HMBC and NOESY  experiments and calculations were used to determine its absolute stereochemistry for the first time in the literature. VCD associated with density functional theory (DFT) calculations has been demonstrated as a powerful probe for chirality in natural products + peak at m/z 439.1980, corresponding to the molecular formula C22H30O9 . According to 1H-NMR data  that suggested the presence of a methoxy group replacing the exocyclic double bond in the lactone ring. In the 13C-NMR, the presence of the signal δC 66.4 attributed to C-13, which appears shifted upfield compared to that observed for calein C (δC 126.3), indicated the absence of the exocyclic double bond at C-11/C-13 as present in the lactone ring of Compounds 1 and 2. Additionally, the 13C data revealed the presence of an extra carbon δC 59.2, attributed to the methoxyl carbon. According to HMBC spectrum, it was possible to observe a correlation of H-13 (δH 3.70/3.35) with C-22 (δC 59.2) and C-12 (δC 174.1), as well as the correlations between H-7 (δH 3.08) and C-13 (δC 66.4), confirming the position of the methoxyl group at C-13  was also investigated in terms of cytotoxicity against anaplastic and papillary thyroid cancer cell lines.In order to define the importance of the conjugated system in the lactone ring to the biological activity, Compound (1) than to the other substances tested (1) (NIH-3T3). Calealactone B (2) resulted in similar cytotoxic values against the cell lines tested when compared to calein C (1), probably due to their analogous chemical structures. The calein C derivative (3) was shown to be the least cytotoxic of the STLs tested in all cell lines, including fibroblasts.Cytotoxic assays revealed that KTC-2 and TPC-1 tend to be more sensitive to calein C . In addition to that, anaplastic cells KTC-2 were also affected by the treatment with calein C (IC50 1.67 μM). The literature also reports the cytotoxicity of calein C against the breast cancer cell lines MCF-7, MDA-MB-231 and Hs578T. The data suggest that calein C is able to reduce cell viability as well as inhibit cell cycle progression, consequently resulting in cell death [In general, it is possible to observe that all substances were cytotoxic against all of the cell lines. However, the ICll death .50 values were slightly higher than those obtained for calein C. This difference might be due to the presence of an epoxide in positions C-2 and C-3 instead of the double bond present in calein C.Similarly to what is observed with calein C, calealactone B was also shown to be cytotoxic against both of the cell lines, whereas the IC3) was the least effective against both cell lines tested, for anaplastic (IC50 25.3 μM) and papillary (IC50 23.6 μM). It was possible to see that the addition of the methoxy group at C-13, replacing the double bond, drastically changes the biological response.On the other hand, the cytotoxic potential of the calein C derivative ; genistein, an isoflavone naturally found in numerous plants; and epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, induced a strong and significant reduction of the viability of ATC cell lines [Other phytochemicals, such as resveratrol, a phytoalexin present in several plant species; curcumin, a phenolic compound obtained from the rhizomes of turmeric  and benzene-d6 (Aldrich) were used as solvents and as the internal standards. Silica gel flash  and Sephadex LH-20  were used for column chromatographic separations, while silica gel 60 PF254 (Merck) was used for analytical thin-layer chromatography (TLC). HPLC analysis was performed in a Dionex Ultimate 3000 chromatography system , using a Luna Phenomenex RP-18 column  and UV-DAD detector. IR and VCD spectra of Compounds 1 and 2 were recorded with a Single-PEM ChiralIR-2X FT-VCD spectrometer  using a resolution of 4 cm−1 and a collection time of 6 h. The optimum retardation of the ZnSe photoelastic modulator (PEM) was set at 1400 cm−1. Minor instrumental baseline offsets were eliminated from the final VCD spectra of 1 and 2 by subtracting their VCD data from those obtained for the solvent under identical conditions. The IR and VCD spectra were recorded in a BaF2 cell with a 100 µm path length, using CDCl3 as the solvent. The samples were prepared as follows: 5 mg of 1 were added to 120 µL of CDCl3 and 3 mg of 2 were added to 110 µL of CDCl3.Calea pinnatifida (R. Br) Less. (Asteraceae) were collected from the Atlantic Forest area of São Paulo City, SP, Brazil , in October 2012. A voucher specimen  has been deposited in the SPF Herbarium of Botany Department from the Biosciences Institute of University of São Paulo.Leaves of C. pinnatifida (300 g) were dried, ground and then exhaustively extracted using MeOH at room temperature. After evaporation of the solvent under reduced pressure, the obtained crude extract (10 g) was resuspended in MeOH/H2O (2:1) and then partitioned with dichloromethane. The CH2Cl2 phase (650 mg) was subjected to column chromatography (CC) over Sephadex LH-20 using MeOH as a mobile phase to give seven fractions (A–G). Fraction B (420 mg) was subjected to CC over silica flash using increasing amounts of MeOH in CH2Cl2 as the solvent, to afford three fractions (B1–B3). Part of Fraction B1 (100 mg) was purified over semi-prep RP-18 HPLC, eluted with ACN/H2O (4:6) , to afford calein C  and calealactone B . In the course of extraction and fractionation, a calein C derivative  was produced and isolated.Fresh leaves of −1 of penicillin-streptomycin (ThermoFisher Scientific). NIH-3T3 was maintained in DMEM (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (ThermoFisher Scientific). All cell lines were cultured at 37 °C with 5% CO2.In order to assess the cytotoxic effects of the STLs, two thyroid cancer cell lines—KTC-2 (anaplastic thyroid cell line) and TPC-1 (papillary thyroid cancer cell line), donated by Professor Edna Kimura—and the murine fibroblast nontumor cell line NHT-3T3 were selected. KTC-2 and TPC-1 were maintained in RPMI-1640  supplemented with 5% and 10% fetal bovine serum (ThermoFisher Scientific), respectively, and 0.01 μg mL50) was estimated from the non-linear regression analysis of the dose response curve using Prism 5 . Cells were briefly treated with different concentrations of each compound for 72 h at concentrations ranging from 12.3 to 0.096 μM for calein C, 60.3 to 0.532 μM for calein C derivative and 17.7 to 0.137 μM for calealactone B. After 48 h treatment, 10 µL of PrestoBlue reagent was added to each well and incubated for 1.5 h. Fluorescence was also measured at M3. All experiments were performed in triplicate and data were expressed as the mean ± SEM.The cell viability after drug treatments was determined with PrestoBlue Cell Viability Reagent (ThermoFisher Scientific), following the manufacturer’s instructions. Briefly, the thyroid cancer cells (KTC2 and TPC1) and the nontumor cell line (NIH-3T3) were seeded into 96-well plates at an initial density of 1000 cells per well and incubated at 37 °C for 48 h. Cells were treated with three concentrations of calein C , calein C derivative  and calealactone B  for 72 h. A concentration of 10 μM of cisplatin was used as the positive control in the KTC2 and TPC1 cell lines for cytotoxicity. After 48 h of treatment, 10 μL of PrestoBlue reagent was added to each well and incubated for 1 h 30 min. Fluorescence (540 nm excitation/590 nm emission) was measured using a microplate reader, M3 . Vehicle-treated control cells (DMSO) were used to normalize the relative luminescence units from treated wells, and they were expressed as percentages of viable cells. The concentration to achieve 50% of cell death -1 and -2. Conformational searches were carried out at the molecular mechanics level of theory with the Monte Carlo algorithm, employing the MM+ force field incorporated in the HyperChem 8.0.10 software package. Initially, 34 conformers of -1 and 29 conformers of -2 with a relative energy (rel E.) within 10 kcal mol−1 of the lowest-energy conformer were selected and further geometry-optimised at the B3LYP/6-31G(d) and B3PW91/6-311G levels. The two conformers with rel E. < 4.0 kcal mol−1 for each compound, which corresponded to more than 99% of the total Boltzmann distributions, were selected for IR and VCD spectral calculations. DFT calculations were carried out at 298 K in the gas phase using Gaussian 09 software [−1 cm−1). Vibrational analysis at the B3LYP/6-31G(d) and B3PW91/6-311G levels resulted in no imaginary frequencies, confirming the considered conformers as real minima. Each spectrum was plotted as a sum of Lorentzian bands with half-widths at half-maximum of 6 cm−1. The calculated wavenumbers were multiplied by a scaling factor of 0.975. The final spectra were generated according to Boltzmann weighting of the lowest-energy conformers identified for 1 and 2 and plotted using the Origin 8 software.Calculations were performed for the arbitrarily chosen  were isolated: calein C (1) and calealactone B (2), while Compound 3 was obtained as a product of the Michael addition of MeOH to calein C. The absolute configurations of 1 and 2 were unambiguously confirmed as 4R,6R,7S,8S,9R,10R and 2S,3S,4R,6R,7S,8S,9R,10R, respectively, by means of VCD and DFT calculations. According to the biological data obtained, it was possible to observe that calein C, calealactone B and the calein C derivative were effectively cytotoxic against thyroid cancer cells, both papillary and anaplastic. However, differences in conjugated double bonds among the structures of the STLs were shown to influence their cytotoxic potential. KTC-2 and TPC-1 were shown to be more sensitive to calein C than to the other tested substances. Thus, the obtained results demonstrate how the presence of an α,β-unsaturated lactone ring might be important for the antitumor activity. To the best of our knowledge, this is the first report of the calein C derivative (3) and the structure–activity relationship among sesquiterpene lactone analogues from C. pinnatifida, suggesting the importance of conjugated systems in their biological potential.From the MeOH extract of the leaves of


Despite new therapies, the estimated 229 875 women living with ovarian cancer have a 5‐year survival rate of 47.6%. This cavity‐localized cancer lends itself to local administration of modalities, such as the oncolytic adenovirus (Ad) Ad5/3‐D24‐granulocyte‐macrophage colony‐stimulating factor virus (ONCOS‐102). Its repeated administration to a patient with chemotherapy‐refractory ovarian cancer induced CD8+ antitumor immune responses with the overall survival reaching 40 months. Here we probe the dominant receptor used by ONCOS‐102 in four established epithelial ovarian cancer cell lines. Ad3 can use the desmoglein‐2 (DSG2) and CD46 receptors on susceptible cells. DSG2 was nearly absent in A2780 cells but was expressed in more than 90% of OAW42, OVCAR3, and OV‐90 cells. After 96 hours, ONCOS‐102 treatment showed significant oncolytic activity (≧50%) in OAW42, OVCAR3, and OV‐90 cells, but minimal activity in A2780 cells, suggesting DSG2 as the dominant receptor for ONCOS‐102. Furthermore, retrospective analyses of phase I clinical trial of ONCOS‐102 treatment of 12 patients with varied tumors indicated a correlation between viral genomes in blood and DSG2 RNA expression. These data support the role of DSG2 expression on cancer cells in virus infectivity and the continued development of ONCOS‐102 for ovarian cancer treatment. These data support the role of DSG2 expression on cancer cells in virus infectivity and the continued development of ONCOS‐102 for ovarian cancer treatment. To assess the role of expression of the DSG2 and CD46 receptors on ONCOS‐102 treatment of patients with solid tumors from a previously reported phase I trial,22.1Two human ovarian carcinoma cell lines, A2780  and OAW42  were purchased from Sigma. Two human ovarian carcinoma cell lines OV‐90 (ATCC CRL‐117321/lot: 63990123) and OVCAR3 (ATCC HTB‐161) were purchased from ATCC. All cell lines were cultured in the indicated media with the indicated 10% to 20% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Table S1). Cells were passed at 80% of the confluence with trypsin‐EDTA and the doubling times ranged from 1 day to 7 days: A2780, 1 day; OAW42, 2 days; OVCAR3, 3 days; OV‐90, 7 days.2.25 cells/cell line) after a 30 minutes incubation at 4°C with PE‐conjugated anti‐DSG2 antibodies  or PE‐Vio770‐conjugated anti‐CD46 antibodies , respectively, and washed with PBS. CAR receptor cell surface expression were measured on the four human ovarian carcinoma cell lines (1 × 105 cells/cell line) after a 30 minutes incubation at 4°C with the primary anti‐CAR rabbit polyclonal antibody , washed and labeled with anti‐rabbit Alexa‐Fluor 488 secondary antibody  for 30 minutes at 4°C, and washed in PBS. Results were acquired on the Attune Nxt Flow cytometer on at least 104 events in duplicate in two independent experiments.Flow cytometry was used to quantify the cell surface expression of the three receptors for adenovirus: CAR, CD46, and DSG2. DSG2 and CD46 cell surface expression were measured on the four human ovarian carcinoma cell lines  or infected with ONCOS‐102 at 0.1, 1, 10, 100, or 1000 VP/cell in triplicate. Because the cultured cells were not confluent during infection, few tight junctions would have been present and thus both DSG2 and CD46 receptors would have been accessible during infection.Oncolytic efficacy was determined with the MTS cell viability assay  at 72 and 96 hours postinfection. Briefly, the human ovarian cell lines were plated at 2 × 102.510, 1 × 1011, and 3 × 1011 VP/injection, respectively, at each time point.Retrospective analyses were performed with previously obtained data from patients who had enrolled and participated in the escalating dose phase I clinical trial, NCT01598129.2.6All variables were analyzed by using GraphPad Prism (v8) software. The correlation was calculated using the nonparametric Spearman test . Statistical significance for in vitro studies was performed by two‐way analysis of variance.33.1Both A2780 and OAW42 ovarian carcinoma cell lines are categorized as nonserous adenocarcinoma cell lines and OVCAR3 as high‐grade serous by genomic profiles.3.2P < .01). ONCOS‐102 also modestly but not significantly reduced cell viability in OV‐90 (19%) . These data support that the ONCOS‐102 infection of ovarian epithelial carcinoma cells primarily relied on at least initial binding to DSG2 for its uptake.ONCOS‐102 treatment (1000 VP/cell) at 96 hours Figure , Table 13.3To investigate whether DSG2 expression level may affect ONCOS‐102 replication in patients with tumors, we retrospectively analyzed data from tumor biopsy and blood samples from the previously reported ONCOS‐102 phase I clinical trial NCT01598129.R = .4526; P = .1401; Figure R = .1287; P = .6865; Figure The DSG2 RNA expression levels in the tumor tissue from the 12 patients positively correlated, but not significantly with the number of viral genomes in the patients' blood on day 4 .As an update to the survival data from the phase I trial,4R = .4526) with the viral genomes released 3 days postinjection in ONCOS‐102‐treated patients with tumors of varied origin,P = .1401), possibly due to the cohort, was comprised mostly of other primary tumors (10 of 12). These data further support the role of DSG2 expression as a major receptor for ONCOS‐102 replication in primary tumor cells, including EOC cells.Ovarian cancer treatment, despite new modalities, has only modestly increased the 5‐year survival rate in the last decade.R = .1287; P = .6865). Whereas binding of Ad3 fiber knobs to DSG2 can open tight junctions and expose CD46 for interaction with Ad3,Expression of adenoviral receptors varies in primary isolates of EOC and established cell lines. CAR cell surface proteins were detected on average in 55% to 75% of the cells of the four established EOC cell lines , in agreement with its expression in 30% to 99% of cells from seven primary EOC, with a median of 92% positive CAR cells .4.1First, although the oncolytic activity of ONCOS‐102 was not detected in A2780 cells, A2780 is susceptible to the oncolytic activity of replication‐selective Ad5 agents.Furthermore, several primary ovarian cancer isolates were susceptible to a different conditional replicative Ad5/35 chimeric fiber knob vectorTherefore, oncolytic adenoviruses with a modified knob can successfully infect cancer cells expressing DSG2 and subsequently induce the development of antitumor immune responses in 11 of 12 patients5In conclusion, these data showed that the dominant receptor for ONCOS‐102 binding and replication was DSG2 in these ovarian cancer cell lines. Furthermore, these data support the continued development of ONCOS‐102 for the treatment of ovarian cancer. We speculate that patients with ovarian cancer expressing DSG2 may be more susceptible to the oncolytic activity of ONCOS‐102 and other oncolytic AD5/3 vectors than those with EOC without detectable DSG2 expression.LK and A‐SWM are employees and/or shareholders in Targovax Oy in Finland and Targovax ASA in Norway.LK and A‐SWM conceptualized the study. LK and A‐SWM gave the methodolgy. LK provided software, worked on validation, and conducted formal analysis. Data LK and A‐SWM did data curation. LK and A‐SWM wrote the orginal draft and reviewed and edited it. LK visualized. LK and AS supervised the study. LK and A‐SWM were responsible for project administration. LK acquired funding.Supporting informationClick here for additional data file.