These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC Colorectal cancer (CRC) is the third most commonly diagnosed cancer, and the third leading cause of cancer-related deaths in the US and worldwide . In the Cell proliferation is sustained by signal transduction pathways that relay external growth signals to regulate cellular metabolism and gene expression. Cancer cells often rely on constitutively activated signaling pathways that short-circuit normal signaling and regulatory processes. The most prominent oncogenic signaling proteins are soluble and receptor protein tyrosine kinases (PTKs) , moleculTargeted therapy for CRC has largely relied on monoclonal antibodies against EGFR and VEGFR ,6. The aIn this study, we evaluate the responses of 10 CRC cell lines to the treatment of a panel of kinase inhibitors. Six of the 10 cell lines are inhibited by multiple inhibitors blocking independent signaling pathways, including the Mek inhibitor AZD-6244, the insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) inhibitor BMS-754807, and the Src kinase inhibitor dasatinib. Mechanistic analysis and drug combination studies indicate that the proliferation of these cells are supported by multiple pathways, and can only be effectively blocked by combination of drugs that respectively block these pathways. This study provides a mechanistic explanation for why targeted therapy has not worked well for CRC, and a rational strategy for combination targeted therapy. d values below 20 nM are shown in http://lincs.hms.harvard.edu/kinomescan/). Two of the PTK inhibitors, BMS-754807, specific for the kinases in the IR family and Met . Be. Be28]. The responses by most of these CRC cells to multiple kinase inhibitors are intriguing. For effective targeted therapy by a single agent, the cancer cell needs to be addicted to a single oncogene or a network ,31. Yet Even though AZD-6244 is a specific Mek inhibitor, it strongly activated Mek phosphorylation (Ser217/Ser221) in all three cell lines, but inhibited the phosphorylation of Erk (Thr202/Tyr204), a direct substrate for Mek. The fact the AZD-6244 blocked Erk activation and inhibited the proliferation of all three cells indicated that the function of Mek plays an important role for the proliferation of these cells. This is consistent with the fact all these cells contain KRAS mutations. AZD-6244 activation of Mek phosphorylation was somewhat surprising, but it could be a feedback mechanism. Mek phosphorylation on Ser217/Ser221 is catalyzed by Raf, blocking Mek activity by AZD-6244 appears to result in hyper-phosphorylation of Mek, perhaps as an attempt to compensate for the loss of Mek function. This is most likely a feedback mechanism in these cells compensating for the blockade of function by AZD-6244. Such a feedback mechanism has been observed for Akt inhibitor, GSK-690693 in other systems . NeverthWe attempted to determine the protein levels and the phosphorylation status of IR and IGF-1R in all three cells under treatments. While the IR and IGF-1R proteins could be readily determined B, WesterDasatinib inhibited Src autophosphorylation on Tyr416 and Src phosphorylation on Tyr527, catalyzed by another PTK, Csk, in all three cell lines. This result indicated that dasatinib did block Src kinase activity as well as Csk kinase activity, consistent with its reported specificity. Although normally down-regulation of Tyr527 phosphorylation correlates to activation of Src kinase activity, dasatinib also directly inhibited Src activity, thus the net effect of dasatinib on Src was likely inhibitory. A comparison of the activation levels of Src among the three cell lines suggests that LS-174T contains the highest level of phosphor-Tyr416 in control and under AZD-6244 and BMS-754807 treatments, suggesting that Src activation level was higher in LS-174T than in the other two cell lines. This is consistent with the fact that LS-174T is the only cell line among the three that is sensitive to inhibition by dasatinib. Although phosphor-Tyr416 in LS-1034 and LS-513 was clearly detectable and it was clearly inhibited by desatinib, Src apparently did not play a major role in the proliferation of these two cell lines, as dasatinib did not significantly inhibit the proliferation of these two cells. The above results together demonstrate that each of these drugs independently blocks a specific pathway. Thus, they support that hypothesis that these CRC cancer cells are indeed multi-driver/multi-pathway dependent cancer cells. The fact that multiple independent pathways are contributing to the proliferation of these cells explains the observations that these cells are sensitive to multiple inhibitors, but the inhibition by any one of them is only mild.The results from the previous sections suggested that CRC cell proliferation was driven simultaneously by IR/IGF-1R/AKT pathway, the MAP kinase pathway and/or the Src kinases. One logical prediction of the multi-driver proliferation model is that complete inhibition would require a combination of drugs that collectively block all the activated pathways in a given cell line. We tested this prediction by determining the responses of selected cells to combinations of drugs that were individually only mildly effective. 50’s were mostly in the high nM to low μM range. We determined if these cells would be more effectively inhibited by the combination (1:1 mixture) of the two drugs. As shown in 50’s were summarized visually in 50 for the two-drug combination ranged from 46 nM for NCI-H747 to 153 nM for LS-1034. These nM level IC50 values indicate that these CRC cell lines are extremely sensitive to the combination of AZD-6244 and BMS-754807. As shown in 60 and IC70 values  as a function of percentage of inhibition . Synergy0 values B for the50’s of 1.41 μM and 0.61 μM, respectively, but the two drugs combined had an IC50 of 102 nM. The DRI value also increased dramatically with the increase in the percentage of inhibition, reaching 17-fold at 70% inhibition. The benefit was even higher at 80% inhibition, however, the DRI could not be calculated because AZD-6244 did not reach 80% inhibition up to 20 μM. These results are consistent with the mechanistic understanding that both Src kinase activity and the MAP kinase pathway independently contribute to the proliferation of LS-174T, and further suggest that a cancer based on this mechanism would be subject to combination targeted therapy with dasatinib and AZD-6244.While inhibition by AZD-6244 and BMS-754807 seems to be a common feature of CRC cells, LS-174T, NCI-H747 and SK-CO-1 displayed sensitivity to dasatinib . Interes50 of 40 nM, marginally better than that of the AZD-6244 and BMS-754807 combination, at 52 nM. This could be due to the fact the dasatinib was a much weaker inhibitor than AZD-6244 and BMS-754807 for this cell line.Based on the results in the previous sections, most of the CRC cell lines were inhibited by AZD-6244, BMS-754807 and/or dasatinib, and two cell lines, SK-CO-1 and NCI-H747 were inhibited by all three. The synergistic effects in pair-wise combinations led us to determine if the three-drug combination would be even more potent than the two-drug combinations. To test this proposition, we tested SK-CO-1 against the 1:1:1 mixture of the three drugs at a series of concentrations A. The tr50s ranging from 10 nM for NCI-H747 to 1061 nM for LS-123, with seven of the 10 cell lines inhibited by the triple combination with IC50’s below 84 nM. The extremely low IC50 of 10 nM for NCI-H747 is consistent with the observation that it is more sensitive to each drug alone than the other cell lines  of imatinib inhibiting BCR-Abl dependent CML cell lines [50’s of the double and triple combinations are also shown to Although different cell lines were more potently inhibited by one combination or another, we thought that the triple combination may be a one-size-fits-all formulation for most CRC cells . We tested all 10 cell lines against the triple combination (1:1:1 ratio) B. The cell lines . Interesll lines . FurtherThe molecular mechanisms driving the proliferation of CRC cells have been extensively studied ,39. The 50, suggesting that receptor PTKs in the PDGFR, EGFR, FGFR, MET, and soluble PTKs in the Abl families do not play major roles in driving the proliferation of CRC cells. This is largely consistent with the conclusion from genetic analysis that receptor PTKs are rarely mutated and are unlikely to play any major roles in driving CRC proliferation [It is also noteworthy what kinase inhibitors did not inhibit the proliferation of the CRC cells. PTK inhibitors sunitinib, BGJ398, crizotinib, and imatinib did not inhibit any of the cell lines significantly, while gefitinib only inhibited one cell line, LS-513, with a modest ICferation . It is sferation . The preFour of the cell lines did not respond to the individual kinase inhibitors, and interestingly three of them contained hypermutated genomes. At least two scenarios are plausible. Because these cells’ genomes contain 10–20 times as many mutations as other cancer cell lines, it is possible that many signaling pathways are affected. About 8% all cancers contain hypermutated genomes. A recent study determined that normal cancers contain an average of four driver mutations/tumor, and hypermutated genomes likely contain many times more . It is pDespite the success of targeted therapy in treating some cancers, it has not reached most cancers. One of the most basic challenges targeted therapy faces is that it has worked effectively only for cancers that are addicted to a driver oncogene or pathway. Successful applications of Abl inhibitor for BCR-Abl-dependent chronic myeloid leukemia, EGFR inhibitors for EGFR mutation dependent non-small cell lung cancer, and ErbB2 inhibitors for ErbB2-dependnet breast cancer are all examples of both the power of targeted therapy and the challenge it faces. However, most cancers contain multiple driver genes that may independently contribute to the proliferative advantage of a cancer cell. A good strategy for dealing with this has not been worked out. Most of the CRC cells we studied here appear to be multi-driver cancer cells. None of the 10 CRC cell lines displayed an exclusive reliance on any one gene or pathway, as most of them contain activated MAP kinase, IR/IGF-1R/AKT, and/or the Src kinase pathways.Even though there is a certain level of heterogeneity among the tested cells, the level of similarity is also surprising. While it is not surprising that all six sensitive cells responded to AZD-6244, due to the mutation of BRAF and KRAS in these cells, it was surprising that five of the six sensitive cells responded to BMS-754807, because none of these cells contains a mutation in IR or IGF-1R. However, the relationship between IR/IGF-1R signaling is complicated by cros-talks and feedback loops ,41,42,43The inhibition of several cell lines by dasatinib suggests that Src is playing a role in driving the proliferation of these cells. Western blot analyses confirmed that dasatinib indeed blocked Src activation by autophosphorylation, and thus Src kinase activity. Indeed Src has been reported to be associated with some CRC cell lines ,45,46. TThe multi-driver nature of CRC cell proliferation could explain why CRC cannot be effectively treated by individual kinase inhibitors, even though these inhibitors clearly block their intended targets, and their intended targets clearly play important roles in CRC cell proliferation. The multi-driver nature of CRC cells also dictates that effective targeted therapy against CRC would require the combination of several inhibitors, each blocking one activated pathway. Our results confirmed this hypothesis. If the multi-driver mechanism applies to other solid tumors, then the combination of kinase inhibitors may be an effective and necessary approach of targeted therapy. With the realization of the limits of mono-agent targeted therapy ,48, combA key feature of the AZD-6244/BMS-754807 or AZD-6244/dasatinib drug combination is that the synergy increases dramatically with the level of inhibition. While the synergy at 10–20% inhibition is often minimal, it increases dramatically as the level of inhibition increases. At 60–70% inhibition, the dose reduction index often reached higher than 10, and as high as more than 30. This feature is fully consistent with the multi-driver proliferation mechanism, and would be particularly beneficial for combination targeted therapy. To inhibit the proliferation of these cells to a significant level, for example 70–80%, with an individual drug would require concentrations of 20 μM or higher even for the most potent inhibitors. Signaling inhibitors at such high concentrations would likely cause off-target effects and significant toxicity ,51. This50 values below 100 nM. As a benchmark for targeted therapy, CML cells dependent on BCR-Abl for proliferation are inhibited by Abl inhibitor imatinib with IC50 values in the range of 80–600 nM (38). Projecting these results from this study into patient treatment provides at least two treatment approaches that are worthy of further investigation. First, if a patient can be pre-tested for the drug sensitivity to decide which drug combination the patient would respond to, then an effective drug combination can be selected for the patient. This is the ideal scenario of targeted therapy or precision therapy. Second, if a drug sensitivity pre-test is not feasible, the triple combination of AZD-6244, BMS-754807 and dasatinib may be an attractive treatment option because majority of CRC cell lines can be strongly inhibited by the combination (IC50 ≤ 84 nM). The treatment with a triple combination may have another benefit. Increasing evidence demonstrates significant intra-tumor genetic heterogeneity, where a solid tumor may contain heterogeneous sub-population of cancer cells driven by different genetic mutations [The study demonstrated that double or triple combinations of AZD-6244, BMS-754807 and dasatinib are particularly effective in blocking the proliferation of most CRC cell lines, with many ICutations ,53. If t2. Kinase inhibitors were purchased from Selleckchem , LC Laboratories  or AdooQ Bioscience . All CRC cell lines were purchased from ATCC , and were used within 6 months of their purchase. ATCC authenticates all its cell lines by short tandem repeat profiling, cell morphology monitoring, karyotyping, and cytochrome C oxidase I testing. The cells were grown in ATCC-formulated McCoy’s 5a Medium Modified , RPMI-1640 Medium , or Eagle’s Minimum Essential Medium , containing 10% FBS and 1% penicillin-streptomycin . All cells were cultured at 37 °C in humid atmosphere containing 5% COCells were plated into 96-well plates at 25,000 cells per well, and were incubated with drugs at indicated concentrations in 100 μL medium containing 1% DMSO for 72 h. For adherent cells , at the end of the incubation, 10 μL of staining solution containing 5 mg/mL MTT dye -2,5-diphenyltetrazolium bromide) (Thermo Fisher Scientific) was added into each well. After incubation at 37 °C for another 4 h, the medium in the wells was removed by aspiration. DMSO (100 μL) was added into each well, and when purple formazon was dissolved, absorbance at 490 and 750 nm for each well was determined using a VersaMax Tunable Microplate Reader with SOFTmax Pro v5 . The A490-A750 values were taken as indicator of cell viability. For semi-suspension cells (NCI-H747), cell viability was determined by staining the cells with Biolog Redox Dye Mix MB . At the end of 72 h drug treatment, 10 μL Biolog Redox Dye Mix MB was added into each well, and the plates were incubated at 37 °C for 2 h before the absorbance of the water soluble formazan product at 590 and 750 nm was determined using the microplate reader. The A590-A750 values were taken as indicator of cell viability. All cell growth and drug inhibition experiments were performed in triplicates at least twice. Statistical analysis was performed on six repeat data sets for each assay.50 of the cell line for the drug was also determined using a 16-concentration 2x dilution series of the drug, ranging from 0.6 nM to 20 μM. The IC50 values were estimated from the dose response curves as the concentrations that resulted in 50% inhibition of the cell’s viability. To determine if the proliferation of a cell line is sensitive to inhibition by the kinase inhibitors, each cell was screened against a panel of ten kinase inhibitors  at four To determine the effects of the kinase inhibitors on the signaling network in the CRC cell lines, the cell lines were cultured to 70% confluency, and then treated by the indicated drugs. The culture medium was then replaced by fresh medium containing kinase inhibitors at designated concentrations. The cells were incubated for 2 h under treatment. After the treatment, the culture medium was aspirated, and the cells were then resuspended in the SDS-PAGE loading buffer containing a protease inhibitor cocktail for mammalian cell and tissue extracts  and protein phosphatase inhibitors  and lysed in a 95 °C heat bath immediately. The lysates were then cleared by centrifugation and the supernatants were fractionated by SDS-PAGE, and immunoblotted by various antibodies recognizing selected signaling proteins and their specific phosphorylated forms. The loading was normalized based on the β-actin expression levels in these lysates determined by an antibody specific for this protein. All antibodies were purchased from Cell Signaling Technology .x) was determined manually from the graphs of drug dose response curves. The combination index (CI) was calculated for a given percentage of viability inhibition according to Chou [40, IC50, IC60, etc., were determined from the dose response curves, and the CI at a given level of inhibition (X%) was calculated by the following equation: CI = ICxab/ICxa + ICxab/ICxb, where ICxab is the concentration of the drug combination that resulted in X% inhibition, and ICxa and ICxb are the concentrations of drug a and drug b, respectively, that caused X% inhibition to cell proliferation. The dose reduction index (DRI), a measure of how many folds the combination concentration may be reduced, for a given percentage of inhibition, compared with the doses of each drug alone, is calculated as: DRI = 1/CI [To determine if two drugs were synergistic in their inhibition of a cell line’s proliferation, the cell line viability was determined by incubating the cells in the presence of each drug alone or both drugs (1:1 ratio) at 16 concentrations ranging from 0.6 nM to 20 μM. The concentration of a drug or drug combination that inhibited cell proliferation at X%  using Excel with the assumption of two tailed distribution and equal variance. The P values are reported in the appropriate figure legends.We performed three types of statistical analysis to evaluate the data. First, the IChttps://cancer.sanger.ac.uk/cosmic) [Genomic data for the CRC cell lines were collected from the COSMIC database, the Catalogue of Somatic Mutations in Cancer (/cosmic) .


Bacopa monnieri. Previously, we showed that bac II reduced endothelial cell migration and tube formation and induced apoptosis in colorectal cancer cell lines. The aim of the current study was to examine the effects of treatment with combined doses of bac I and bac II using four cell lines representative of the breast cancer subtypes: triple negative (MDA-MB-231), estrogen receptor positive (T47D and MCF7) and human epidermal growth factor receptor 2 (HER2) positive (BT-474). Drug treatment outcome measures included cell viability, proliferation, cell cycle, apoptosis, migration, and invasion assays. Relationships were analysed by one- and two-way analysis of variance with Bonferroni post-hoc analysis. Combined doses of bac I and bac II, each below their half maximal inhibitory concentration (IC50), were synergistic and reduced the viability and proliferation of the four breast cancer cell lines. Cell loss occurred at the highest dose combinations and was associated with G2/M arrest and apoptosis. Migration in the scratch wound assay was significantly reduced at apoptosis-inducing combinations, but also at non-cytotoxic combinations, for MDA-MB-231 and T47D (p < 0.0001) and BT-474 (p = 0.0003). Non-cytotoxic combinations also significantly reduced spheroid invasion of MDA-MB-231 cells by up to 97% (p < 0.0001). Combining bac I and II below their IC50 reduced the viability, proliferation, and migration and invasiveness of breast cancer cell lines, suggesting synergy between bac I and II.Bacopaside (bac) I and II are triterpene saponins purified from the medicinal herb  Breast cancer remains the second most common cancer in women worldwide and the fourth leading cause of cancer death . Breast Bacopa monnieri [Terpenoids are a diverse family of natural products found in a variety of fruits, vegetables and medicinal plants . Classifmonnieri ,12,13,14monnieri ,15, withmonnieri ). We theTo further support the use of bac I and bac II in cancer, we previously showed that these drugs block the functional activity of the transmembrane protein aquaporin (AQP) 1 . AQP1 isTogether, these studies provide a strong rationale to investigate the effects of bac I and II in breast cancer. We therefore tested the anti-cancer activity of the drugs individually and combined on breast cancer cell lines from the major molecular subtypes, MDA-MB-231 (TNBC), T47D (ER+/PR+), MCF7 (ER+/PR−), and BT-474 (HER2+), and looked for potential synergy between the two drugs.50s) shown in 50 values for bac I for MDA-MB-231, T47D and MCF7 were 99 μM  91–109), 89 μM (95% CI 73–109), and 83 μM (95% CI 79–87), respectively, whilst BT-474 was more sensitive at 59 μM (95% CI 56–63) , 19 μM (95% CI 17–20) and 16 μM (95% CI 15–17), respectively, whilst T47D was less sensitive at 29 μM (95% CI 9–96) (A viability assay (MTS) was performed on the four breast cancer cell lines treated with varying concentrations of either bac I or bac II. The cell lines showed a dose response effect to both drugs with half maximal inhibitory concentrations (ICI 56–63) a. The ICCI 9–96) b.50 5.5-fold lower than for bac I for MDA-MB-231, we used a constant dose of bac II well below the IC50—specifically, 2.5 μM—whilst the concentration of bac I was varied; this was done to determine the effect of bac II, at a dose that did not reduce viability, on the IC50 of bac I. For MDA-MB-231 cells, the IC50 of the combinations was determined as 13 μM bac I (95% CI 10–16 μM) + 2.5 μM bac II. Bac II shifted the IC50 for Bac I from 99 μM (95% CI 91–109) to 13 μM (95% CI 10–16)  of 0.270 (95% CI 0.240–0.301)  c, indica0–0.301) d. Isobolp < 0.0001 relative to vehicle at day 3). Relative to day 0, a decrease in absorbance occurred at day 1 with this dose for all cell lines (p ˂ 0.0001), as well as with 10 μM bac I + 2.5 μM bac II (p < 0.0001) for MDA-MB-231 and BT-474 (p = 0.0002) for BT-474 , and by 13% and 20% with 10 μM bac I + 5 μM bac II for T47D (p < 0.0001). Sub-G1 events, suggestive of dead cells and/or debris, increased with increasing combinations of bac I and bac II, most notably for MDA-MB-231 and BT-474.Based on the cell loss induced with combination doses, cell cycle analysis was performed, comparing vehicle and drug-treated cells. For each breast cancer cell line, there was an increase in the percentage of cells in G2/M with increasing doses of bac I and bac II. The dose combinations at which this occurred were cell-line dependent. Compared to the vehicle, with a dose of 5 μM bac I + 5 μM bac II, the proportion of cells in G2/M increased by 17% and 9% for MDA-MB-231 a,b, and for T47D c,d, and for T47D e,f, respThe morphology of breast cancer cell lines treated with each drug alone and in combination was observed microscopically after three days in culture. Bac I and bac II, each at 5 μM, and bac I, at 10 μM, had no observable effects on morphology compared to the vehicle control, while 10 μM bac II induced small vacuoles in MDA-MB-231 only; therefore, the highest doses of each drug selected for use in combination were doses of ≤10 μM bac I and ≤5 μM bac II . Combinep < 0.001, p < 0.0001 respectively) (Cells were treated with a vehicle at a dose that slowed proliferation but did not cause cell loss and one or more doses that induced cell loss. ctively) a,b,g,h. p < 0.0001). MCF7 cells were more resistant (p < 0.0001). For BT-474 (p < 0.0001). Representative images of wound closure assays are shown in We assessed the effects of bac I and bac II on breast cancer cell migration, since AQP1 is known to be important in this process and these drugs target AQP1 function. esistant c: an inhr BT-474 d, at dosp < 0.0001). Furthermore, the doses of 2.5 μM bac I + 2.5 μM bac II and 5 μM bac I + 2.5 μM bac II significantly reduced invasion, by 68% and 97%, respectively (p < 0.0001)—doses that did not induce apoptosis , being very low in T47D and BT-474, and undetectable in MCF7 (data not shown). Cell lines expressing AQP1 transcript were treated for 24 h with a non-cytotoxic combination of bac I and bac II that reduced migration: specifically, 5 μM bac I + 2.5 μM bac II for MDA-MB-231 and BT-474, and 10 μM bac I + 2.5 μM bac II for T47D. Cells were also treated with the 5 μM bac I + 5 μM bac II combination that induced apoptosis. Compared to vehicle, AQP1 transcript expression in MDA-MB-231 cells was reduced with the non-cytotoxic dose (p = 0.004) and by 2.5-fold with the cytotoxic dose (p < 0.0001) (AQP1 transcript expression was significantly higher in MDA-MB-231 relative to the other cell lines ( 0.0001) a. For T4 0.0001) b,c. ThesThe cell membrane permeabilisation effects of high concentrations of saponins lead to hemolysis and cytotoxicity. At lower doses, however, anti-cancer properties such as the inhibition of proliferation, induction of apoptosis and attenuation of invasiveness have been demonstrated (reviewed in ). Here, B. monnieri containing approximately 20% bac II w/w [B. monnieri extract was cytotoxic to human cancer cell lines of the colon, lung, cervix, and breast [B. monnieri extract in skin carcinogenesis [There are no studies investigating the anti-cancer effects of combining bac I and II. Previous studies have focused on the anti-cancer effects of either the whole plant extract, bacoside A (a mixture of saponins from c II w/w ), or bacd breast ,30,31; bd breast  and gliod breast  cell lind breast . Similard breast  and in ed breast . We haved breast . In vivod breast  and of Bogenesis ,36, as wogenesis , and Ehrogenesis , respectThese studies provide strong evidence to investigate the anti-cancer efficacy of bac I and bac II combined in breast cancer cell lines from each of three main molecular subtypes, as performed here for the first time. We found that all cell lines were sensitive to the synergistic effects of bac I and bac II combined, but particularly MDA-MB-231 and BT474, representative of the more aggressive subtypes of breast cancer of TNBC and HER2+, respectively. We also found that certain dose levels and combinations were cytotoxic, consistent with previous studies using the whole extract and its derivatives.B. monnieri extract [A novel aspect of our study, however, was the finding that lower doses of bac I and II combined, at doses that did not induce apoptosis, also had potent anti-cancer activity in vitro. Previously, we showed that non-cytotoxic concentrations of bac II alone inhibited endothelial cell tube formation—an in vitro measure of angiogenesis —but othe extract . This pr extract .B. monnieri extract containing bac I and bac II at levels higher than these. A large single oral dose of 5000 mg/kg of whole extract, containing 51.5 mg/kg bac I and 91 mg/kg bac II, caused no acute toxicity in Sprague-Dawley rats; in addition, rats receiving up to 1500 mg/kg of whole extract daily for 270 days, containing 15.5 mg/kg bac I and 27.3 mg/kg bac II, showed no evidence of chronic toxicity [B. monnieri whole extract at too low a dose to be comparable to the amounts of bac I and bac II used here [In terms of safety, the highest doses used in our in vitro study to induce apoptosis were 10 μM bac I and 5 μM bac II, equivalent in mice to approximately 7 mg/kg and 4 mg/kg, respectively, while the human equivalent dose  would betoxicity . Others toxicity  and clintoxicity  and micetoxicity —5 to 8 tsed here ,45,46,47sed here , the estThe rationale for combining the two drugs was based on their ability to block AQP1 ion and water channel function. Both bac I and bac II block the water channel function of AQP1, but only bac I blocks the ion channel function . We therB. monnieri and bacoside A, the latter of which contains bac II but not bac I. Both induced the death of human glioblastoma cell lines in vitro through macropinocytosis, a process whereby cell membrane buckling leads to the accumulation of extracellular fluid inside large vacuoles within the cell [There have been reports of other suggested mechanisms of anti-cancer activity for extracts of the cell . This buthe cell . They althe cell . The potbacopa monnieri, were obtained from ChromaDex , solubilised in methanol at 10 mM and 1.5 mM stock solutions, respectively, and stored at −20 °C. Cell lines MDA-MB-231, T47D, MCF7 and BT-474 were purchased from the American Type Culture Collection . Cells were maintained in complete medium, either in Dulbecco’s modified Eagle’s medium  for MDA-MB-231, MCF7 and BT-474 or Roswell Park Memorial Institute (RPMI) 1640 medium  for T47D, containing 10% foetal bovine serum (FBS) , 200 U/mL of penicillin, 200 μg/mL of streptomycin  and 2 mM L-alanyl-L-glutamine dipeptide . Cells were grown under standard culture conditions at 37° C with 5% CO2 in air and used within 4 passages.The analytical standards bacopaside I  and bacopaside II , derived from the medicinal herb 4 cells per well of a 96 well plate and incubated under standard culture conditions overnight. Next, cells were treated with either vehicle (1% methanol), bac I, bac II or combinations of bac I and bac II, for 24 h. MTS assay was performed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay  according to the manufacturer’s instructions. Absorbance was read at 492 nm and results were calculated as the mean absorbance normalized to the vehicle control. The half maximal inhibitory concentration (IC50) for each drug alone and in combination was determined by non-linear regression analysis.The effect of bac I and bac II, alone and in combination, on cell viability was determined by MTS assay as described previously ,21. Cell50 of bac I and bac II combined, bac II was used at a constant concentration of 2.5 μM whilst the concentration of bac I was varied. The isobologram method [50s for bac I and bac II alone on the y- and x-axis respectively. The IC50 combination of bac I and bac II was also plotted on the same graph. Its position would indicate whether the IC50 combination was antagonistic, additive, or synergistic, depending on whether the point was above, on or below the theoretical line respectively. This was also reflected by the combination index, calculated by the formula, CI = d1/Dx1 + d2/Dx2, where Dx1 was the IC50 of bac I, Dx2 was the IC50 of bac II, and d1 and d2 were the doses of bac I and bac II respectively that combined gave an IC50. The combination was antagonistic, additive or synergistic if the combination index was greater than, equal to or less than one [To determine the ICm method  was used3 (MDA-MB-231 and MCF7), 3 × 103 (T47D) or 4 × 103 (BT-474) cells per well of a 96 well plate followed by overnight incubation. Cells were then treated with the vehicle or bac I and/or bac II, and crystal violet absorbance at 595 nm was measured on days 0, 1 and 3 of treatment.The crystal violet assay was used to measure cell proliferation and was performed as described previously . Briefly5 cells were seeded per well of a six-well plate, incubated overnight, then treated with either the vehicle or combinations of bac I and bac II for 24 h. Cells were resuspended in ice-cold Dulbecco’s phosphate buffered saline (DPBS) with drop-wise addition of 100% ice cold ethanol to a final concentration of 70% ethanol. Cells were centrifuged at 300× g for 5 min and cell pellets resuspended in 0.25% Triton X-100 in DPBS. Cells were stained for 2 h in darkness with 25 μg/mL propidium iodide  and 40 μg/mL bovine pancreas ribonuclease A  in DPBS and analysed on the BD FACSCanto II cell analyser . Doublet populations were excluded and 50,000 single cell events were captured per sample. Data was analysed using FlowJo software v10.4.0 , specifically by applying the Watson model to determine the percentage of cells in each stage of the cell cycle.Cell cycle analysis was performed as previously described ,52. Brie5 per well of a six well plate, cultured overnight then treated with either vehicle or bac I and bac II for 24 h. Cells were harvested and stained with annexin-V and propidium iodide for 15 min in darkness at room temperature then run on a BD FACSCanto II cell analyser . Compensation was performed to account for the spectral overlap between fluorochromes. Cells were gated to exclude debris and doublets and at least 10,000 single cell events were acquired per sample. Data was analysed using FlowJo software v10.4.0 .Apoptosis was determined by annexin-V and propidium iodide (PI) staining, using the Annexin-V-FLUOS staining kit , as previously described . Briefly4 (T47D and MCF7), 4 × 104 (MDA-MB-231), or 5 × 104 (BT-474) cells per well of a 96 well plate, incubated under standard conditions until 80–90% confluent, then serum starved overnight. A circular wound was made with a p10 pipette tip. Cells were resuspended in complete medium supplemented with 1 μg/mL mitomycin C  to inhibit cell proliferation, and either vehicle or bac I and/or bac II. The area of wound closure was measured using NIS Elements software .The scratch wound assay was performed as described previously ,53. Cell® 3D culture spheroid cell invasion assay  was set up as per the manufacturer’s instructions using the kit contents, including spheroid formation extracellular matrix (ECM) and invasion matrix. MDA-MB-231 cells were seeded in the spheroid formation ECM at 3000 cells per well of a Costar® 96-well ultra-low attachment plate  followed by incubation for 72 h. Spheroids were subsequently embedded in invasion matrix and vehicle or drug treatments were added. The area of invasion on day 3 was measured using NIS elements software  and was expressed normalised to the mean invasion of the vehicle control.The Cultrex5 cells per well in six-well plates and incubated until 70 to 80% confluent. RNA was isolated with the PureLink RNA mini kit  at time 0 and following 24 h treatment with vehicle or combinations of bac I and bac II. The iScript cDNA Synthesis Kit  was used to reverse transcribe 200 ng of RNA. Transcript expression was determined as previously described [−ΔCt relative quantification method, normalizing to CCSER2 reference gene.Breast cancer cell lines were seeded at 3 × 10escribed , using mData were analysed using GraphPad Prism version 7.0c for Mac OS X  and is presented as means ± standard deviation (SD). Statistical analysis was done by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test unless otherwise stated.This study is the first of its kind to test the anti-cancer potential of bac I and bac II combined on breast cancer cell lines from each of the three molecular subtypes. Bac I and bac II combined behaved synergistically, the combination was more potent than either drug alone, and all subtypes were susceptible to their effects; however, the degree of susceptibility was cell line-specific. In particular, the more aggressive TNBC (MDA-MB-231) and HER2+ (BT-474) cell lines were the most sensitive. Further studies will explore the toxicity and pharmacokinetics of the two drugs in mouse models of breast cancer and elucidate the exact mechanisms underlying the observed effects.


Steroid-resistant graft-versus-host disease (GvHD) is a major challenge after allogeneic stem cell transplantation and associated with significant morbidity and mortality. There is no therapeutic standard defined beyond calcineurin inhibitors (CNI) and steroids. Furthermore, some patients may have contraindications against CNI or high-dose steroids. Efficacy of ruxolitinib against GvHD has been described recently. Ruxolitinib was used for treatment of acute or chronic GvHD in eight patients. The patients either needed intensification of therapy or had contraindications against use of CNI or high-dose steroids. Supplementation of therapy in acute GvHD with severe diarrhea with ruxolitinib was unsuccessful. All these patients died from acute GvHD. Introduction of ruxolitinib into therapy and relapse prophylaxis in other patients was successful in 4/4 cases . Indications for ruxolitinib were contraindications against CNI due to aHUS in two cases and the need for steroid sparing in two other cases. None of these patients suffered from diarrhea at the initiation of ruxolitinib. Ruxolitinib was effective for therapy of acute and chronic GvHD in higher lines in patients without severe diarrhea. Ruxolitinib could replace successfully CNI and high-dose steroids. Further investigations are necessary to define the position of ruxolitinib in GvHD-therapy. Still five decades after introduction of allogeneic transplantation of haemopoietic stem cells graft-versus-host disease (GvHD) is a leading cause for morbidity and mortality after transplantation. The incidence of GvHD may be as high as >50% . The staRuxolitinib is an inhibitor of Janus kinases 1/2 developed for therapy of myeloproliferative diseases. Spoerl et al. described in 2014 the reduced proliferation of t-effector cells and a suppression of proinflammatory cytokine production and positive effects of ruxolitinib in experimental murine GvHD .Here, we describe our experience with ruxolitinib in therapy of acute and chronic graft-versus-host disease. The reasons for the choice of the JAK-2 inhibitor were an unsatisfying response of GvHD to preceding therapy lines, the necessity to spare steroids, or a contraindication against CNI.Eight patients received ruxolitinib for therapy of acute or chronic GvHD. One patient  was female and the other patients  were male. The indications for allogeneic SCT  were acute myeloid leukaemia in three cases  and small cell lymphocytic lymphoma, chronic lymphocytic leukaemia, multiple myeloma, follicular lymphoma, and osteomyelofibrosis in one case  each. The median age was 57 years (range 36-68 years) at alloSCT. Seven  patients were grafted from unrelated donors (10/10 match) and the woman suffering from multiple myeloma received a graft from her HLA-identical sibling . In all Diagnosis and grading of acute and chronic GvHD followed published criteria , 7. HistTwo patients were admitted for primary chronic GvHD #3) and for chronic GvHD as part of an overlap-syndrome (#6). Both had severe pulmonary involvement. Therapy of acute and chronic GvHD followed international standards with steroids as backbone  and for . In addiRuxolitinib was administered in higher therapy lines in doses between 5mg and 15mg according the publication from Zeiser et al. [th to 5th) therapy lines in conjunction with other immunosuppressive agents, e.g., cyclosporine-A, steroids, and mycophenolate mofetil, to achieve control about uncontrolled acute GvHD with severe diarrhea. In these patients the supplementation of GvHD-therapy with ruxolitinib did not led to improvement and all of these patients  have died from uncontrolled GvHD between day +133 and +209 after allogeneic SCT  or with primary chronic GvHD (#3) responded within 14 days to treatment with ruxolitinib. However, no patient suffered from active GvHD of the gut with diarrhea when the JAK-2 inhibitor was started. GvHD of skin, liver, mucosa, and even severe pulmonary symptoms in patients #2 and #6 responded favorable to supplementation of GvHD-therapy with ruxolitinib. In patient #1 ruxolitinib was given for replacement of CNI due to atypical HUS and for steroid sparing. Ruxolitinib has been initially combined with steroids and MMF. Signs of GvHD regressed completely under continued monotherapy with ruxolitinib and even this therapy could be discontinued on day +804 at a patient's Karnofski score of 90%. Patient #3: chronic GvHD, pulmonary manifestation included, improved strikingly under combined treatment including ruxolitinib. Unfortunately, the patient died from pulmonary infection under double immunosuppression with MMF and ruxolitinib. Patient #6 developed an Aspergillosis under conventional treatment of cGvHD. Extracorporal photopheresis and steroids had to be discontinued and pulmonary GvHD exacerbated. In addition, the patient developed severe side effects of steroids. The combination of ruxolitinib with MMF and steroids in decreasing doses led even here to a clear improvement of pulmonary GvHD and nightly artificial respiration could be terminated. The patient is alive on day +1392 with a Karnofski score of 60%. Patient #7 developed stage III acute GvHD from day +30 after alloSCT. Steroids in conjunction with cyclosporine-A and MMF improved the GvHD and terminated diarrhea. However, GvHD of skin, liver, and mucosa exacerbated under steroid tapering and extracorporal photopheresis was initiated. Control of GvHD was unsatisfying, even under this combination, and ruxolitinib was added. Signs of GvHD regressed completely under this combination, steroids were terminated, and intervals of photopheresis could be extended. The patient is well and alive without active GvHD at day +565 after alloSCT (Karnofski score 90%) .In summary, the response of GvHD to supplementation of GvHD-therapy with ruxolitinib was excellent in patients without active gut involvement . There were no long-term hematological as well as nonhematological toxicity; especially there were no opportunistic infections. In four patients with uncontrolled acute grade IV GvHD and severe diarrhea , no response was seen (NC=4) .Therapy of Graft-versus-Host disease with ruxolitinib in conjunction with other drugs led to a response rate of 50% (4/8) in this investigation. Three remissions were complete and one partial, respectively. Generally, these results are in accordance with those published by other investigators. Zeiser et al. reported in their landmark paper data from 95 patients with steroid-refractory acute (n=54) and chronic (n=41) GvHD and described for both groups a response rate above 80% . AnotherThe published data suggest that ruxolitinib is active against acute and chronic GvHD, independently, from the affected organ. Even for pulmonary and intestinal GvHD positive effects have been described , 12. HowFurthermore, our report shows that ruxolitinib is not only an option in steroid-refractory GvHD; it can be used as a substitute for other drugs like CNI in aHUS after alloSCT or to spare steroids . Since sThe optimal dose of ruxolitinib and the length of therapy need further research. The definite position of ruxolitinib in prophylaxis and treatment of GvHD has to be clarified by prospective trials , 19.


One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective  MDR is considered as the major impediment to the success of chemotherapy3, and leads to an unprecedented decrease in the survival rate4 of cancer patients. The development of MDR occurs at an alarmingly high rate during the treatment phase of various cancers5 and the underlying mechanisms of MDR in cancer and subsequent relapse have puzzled researchers worldwide6. Only a small subset of tumor cells has been reported to be sufficient for progressive resistance to chemotherapy, leading to the development of MDR in at least 50% of cancer patients7. The basic underlying MDR mechanism is associated with 5 events: (i) increased drug efflux, (ii) decreased drug influx, (iii) increased drug metabolism, (iv) increased DNA repair and (v) decreased apoptosis9. The major players involved in drug efflux related MDR mechanisms are the ATP-binding cassette (ABC) transporter proteins, such as P-glycoprotein (P-gp/MDR1), multidrug resistance-associated protein 1 (MRP-1), and breast cancer resistance protein (BCRP)10. Overexpression of P-gp/MDR-1, a membrane-bound active drug efflux pump, appears to be the most prominent contributor to MDR development in cancer cell lines12.Cancer drug resistance is an unmanageable outcome of a series of cascade events that are altered in cancer cells during disease progression. Multidrug resistance (MDR) is one of the mechanisms of cancer drug resistance, which is described as the resistance to multiple chemotherapeutic drugs with different structures and functional activity14. The significance of drug transporters has been extensively studied by Dirk et al.15, the study showed the role of drug transporters for MDR in HNSCC. Subsequently, other researchers have also established a correlation between HNSCC and MDR17. Presently, there are limited well-defined in vitro models and assay systems available to classify resistance into MDR and non-MDR categories. First of the two presently employed strategies uses treatment-sensitive in vitro cell lines that are exposed to a specific therapeutic anticancer drug until the designated cell line attains a resistance genotype18. The second strategy uses a genotype-based assay to focus on the identification of genetic anomalies arising in the treatment-resistant cell lines19. These two tactics have been exploited to integrate numerous MDR pump inhibitors into cancer treatment modalities; however, the outcomes were not sufficiently effective for clinical translation20. These strategies have been associated with various discrepancies concerning the differentiation between treatment-sensitive and treatment-resistant cancer cells in vitro10.A directly proportional relationship exists between elevated ABC transporters levels and the progress of MDR, as has been previously reportedin vitro model21. MCSs are self-assembled aggregates of cancer cells, which can mimic the complex micro-environmental milieu of the tumor tissue observed in vivo22. Sutherland’s integration of in vitro three-dimensional (3D) culture methods into cancer research nearly four decades ago, triggered increased interest in the application of MCSs in drug discovery and understanding of the basic biological mechanisms underlying tumor progression and response to treatment23. MCSs show an intermediate but clinically relevant complexity between in vitro 2D cell cultures and in vivo solid tumors and they have been assigned a relevant platform for in vitro drug screening24. They mimic the complex cell-cell adhesion and cell-matrix interactions in solid tumors, which results in the metabolite gradient generation for nutrients and growth factor signals as observed in vivo22. As result of the metabolite gradient and a complex microenvironment, MCSs contain proliferating, quiescent, and necrotic zones, much like the internal milieu of human tumors25. In addition, owing to their multicellular nature, MCSs spontaneously develop MDR against many chemotherapeutic drugs27, thus making them the appropriate model system for the purpose of the present study. Recently, members of our group reported a marked treatment response difference between 2D cell cultures and MCSs of HNSCC pertaining to epithelial-mesenchymal transition and stem cell characteristics, suggesting that 3D cell cultures are a clinically relevant superior model to 2D monolayers for the investigation of new therapeutic targets28. However, there is no well-defined in vitro method or criteria for the identification of the resistance status of cancer cells. Furthermore, the integration of these two approaches into translational research is challenging and unlikely to be implemented in the near future.Multicellular tumor spheroids (MCSs) are considered to be the most relevant pre-clinical, high throughput in vitro model and assay system for the profiling of drug resistance status in cancer cells using MCSs obtained from PTPD HNSCC. This report constitutes a comparative investigation between 2D and MCSs for the assessment of drug resistance profile of the same cell. Our strategy combines drug screening, real-time fluorescence microscopy, and flow cytometry for rapid identification of drug resistance status using MCSs, so that a beneficial personalized treatment regimen can be offered to patients. The cell lines used were previously established by the members of our group29. Briefly, we have investigated the drug response profiles of LK0917 (gingiva), LK0902 (tongue), and LK1108 (hypopharynx) cells to doxorubicin, cisplatin, and methotrexate in 2D and MCSs. In order to establish the drug response profiles for these cell lines, we first investigated their efflux pump activities by assessing the differential uptake of calcein acetoxymethyl ester , a substrate for the P-gp and MRP1 efflux pumps30, using real-time live cell fluorescence imaging31. We further studied the reactive oxygen species (ROS) generation in both in vitro models using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay, in order to have a better understanding of the MCS microenvironment of the PTPD HNSCC cell lines, which we then used for further assessment of MDR status. Finally, we validated our findings with a flow cytometry-based (FACs) assay for functional detection and profiling of MDR phenotypes in 2D cell cultures and MCSs by assessing calcein-AM uptake in the presence of specific efflux pump inhibitors.In the present study, we describe a fast and robust in vitro experimental workflow used for determining the MDR profile is provided below . The research adhered to the tenets of the Declaration of Helsinki.The schematic representation of the low Fig. . The cel29, biopsies were excised from the tumors of gingiva, tongue, and hypopharynx and harvested immediately for establishing 2D cell lines in vitro. MCSs and 2D monolayer cells were developed using the same cells. Categorical segregation of MDR and non-MDR cancer cells was performed using a combination of anticancer drug screening on 2D and MCSs, differential uptake of calcein-AM in time-lapse fluorescence microscopy, monitoring of real-time ROS generation in the MCSs and 2D cultures, and a flow cytometry-based MDR assay. Finally, MCSs obtained from LK0917 gingiva tumor (referred to as MCS17 hereafter), LK0902 tongue tumor (referred to as MCS02 hereafter), and LK1108 hypopharynx tumor (referred to as MCS08 hereafter) were randomly selected for the development of a multidrug cancer resistance model system.As reported earlier2 atmosphere at a temperature of 37 °C. Once cells reached 80% confluence, single cell suspensions were prepared by detaching the cells via mild enzymatic dissociation using 0.25% trypsin and 0.02% EDTA solution (Thermo Fisher Scientific). Trypsin was inactivated by adding complete KSFM medium. The number of live cells/mL were determined by adding 10 µL of 0.4% trypan blue (Thermo Fisher Scientific) to 10 µL of single cell suspension, mounting the mixture on Luna cell counter slides, and counting the cells on the automatic Luna cell counter . For the generation of MCSs sized 300–500 µm, 200 µL of LK0917 (MCS17), LK0902 (MCS02), and LK1108 (MCS08) single cell suspensions were seeded in ultra-low attachment (ULA) plates  at varying cell densities in the range of 0.25–0.75 × 105 cells/mL. The plates were incubated at a humidified 5%CO2 atmosphere at 37 °C (48–72 hrs) for maturation and assessment of MCSs diameter variation with respect to cell density. Progression of spheroid formation was imaged on a daily basis using a bright field microscope  with 5× or 10× objectives and further image analysis was performed. Formation of MCSs was also monitored every 3 hours by live-cell imaging using Incucyte Zoom™  throughout the entire spheroid formation process with a phase-contrast set up using the 10× objective and the images were analyzed.The PTPD HNSCC cell lines LK0917, LK0902, and LK1108 were revived from frozen stocks in 10 mL complete keratinocyte serum-free growth medium , supplemented with 10% fetal bovine serum , and penicillin 50 IU/mL and streptomycin 50 µg/mL (Thermo Fisher Scientific) and incubated in a humidified 5% CO2 atmosphere for 24 hours before drug treatment. After 24 hours, the culture medium was carefully aspirated and 2D cultures of three cell lines were treated with cisplatin (3.33–333 µM), doxorubicin (170–17000 µM), and methotrexate (2.2–220 µM) prepared from their stock solutions  in complete KSFM medium. Cells were treated with drugs for 72 hours. Generation of MCSs was performed as described in the previous section. The cell density for the cytotoxicity assays was 0.7 × 105 cells/mL for both MCS17 and MCS02 and 0.5 × 105 cells/mL for MCS08. Tumor spheroids were incubated at 37 °C and 5% CO2 atmosphere for 48 hours. After 48 hours of spheroid formation, MCS17, MCS02, and MCS08 were treated with different doses of cisplatin, doxorubicin, and methotrexate at the same concentrations used for the 2D cell cultures, by replacing 50% of the culture medium with freshly prepared drug-supplemented32 medium, followed by incubation at 37 °C and 5% CO2 atmosphere for 72 hours. For each drug concentration, 8 MCSs were used in triplicates, with effective drug concentrations equivalent to those used for the 2D cell cultures. Cell cytotoxicity in the drug-treated 2D cell cultures was assessed using the CellTiter96® AQueous One Solution Cell Proliferation Assay . Briefly, at the end of 72 hours, the drug supplemented medium was replaced with 317 µg/mL MTS reagent-supplemented medium. For a total volume of 200 µL, 40 µL of the MTS reagent was added into each well and the plates were incubated at 37 °C and 5% CO2 atmosphere for 3 hours. At the end of the incubation period, absorbances at 490 nm and 650 nm were recorded using a microplate reader . Three independent set of experiments were performed with triplicates for each concentration of drugs per study.Single cell suspensions (2D) of LK0917, LK0902, and LK1108 cell lines were seeded in 96-well flat bottom plates at a cell density of 8000 cells/well in 200 µL complete medium at 37 °C and 5% CO31. In the present context, we have utilized the enhanced efflux properties of MDR tumor cells to generate separate calcein-AM uptake kinetic profiles for 2D and MCSs. We monitored the real-time calcein uptake and intracellular calcein accumulation in 2D cell cultures and MCSs of LK0917, LK0902, and LK1108 using live-cell fluorescent imaging over a period of 11 hours (images represent up to 10 hrs) with image acquisition at 20-minute intervals.Acetoxymethyl ester (AM) derivatives of fluorescent probes such as calcein are actively pumped out of cancer cells with higher MDR1 and MRP1 expression5 cells/mL (2D) and cultured for 24 hours before the start of the experiment. MCS17, MCS02, and MCS08 were generated as described in the section above. After spheroid formation, the KSFM growth medium was carefully decanted without disturbing the spheroids. 2D and MCSs were incubated in serum-free KSFM medium containing non-fluorescent calcein-AM (1 mM in Dimethyl sulfoxide (DMSO), Sigma AB, Malmo, Sweden) at a final concentration of 1 µM, for 12 hours at 37 °C and 5% CO2 atmosphere. During the 12-hour incubation period, phase contrast and green fluorescence  images of the monolayer/spheroids were acquired every 15 minutes using time-lapse fluorescent microscopy. A 10× objective was used for image acquisition . Three independent sets of experiments were performed with triplicates for each individual study.In brief, cells were seeded in 96-flat bottomed plate (2D) and ULA (for MCSs) at a density of 0.7 × 1017, MCS02, and MCS08 were prepared using various cell densities. Here, we seeded 1 × 104, 1.5 × 104, and 5 × 104 cells/well for MCSs formation and assessed the calcein-AM uptake of the generated MCSs using the same procedure described in the section above.For this experiment, MCS5 cells/mL and cultured in complete KSFM medium for 24 hours before the experiment. MCS17, MCS02, and MCS08 were generated as previously described, and the complete growth medium was replaced with serum-free KSFM containing DCFDA (20 µM). DCFDA is a hydrophobic fluorogenic dye used in the measurement of intracellular ROS activity. After cellular uptake, DCFDA is deacetylated by cellular esterases into a non-fluorescent hydrophilic compound that cannot exit cells and which is further oxidized into a highly fluorogenic product in the presence of ROS, which can be detected by fluorescence microscopy. The cell monolayers and the ULA plates containing the spheroids were immediately incubated in the Incucyte ZoomTM (Sartorius AG) live-cell imaging microscope at 37 °C and 5% CO2 atmosphere. Green fluorescence images were automatically obtained every 20 minutes for a total duration of 60 minutes. Three independent experiments were conducted for the study with triplicates for each set of experiment. 2D cells were seeded at a density of 0.7 × 106 cells/mL were prepared in complete KSFM medium. For each sample to be assayed, 4 sets of tubes were prepared and each set-in triplicates.For the experiments on 2D cell cultures, single-cell suspensions of LK0917, LK0902, and LK1108 cell lines were prepared by trypsinization and counted using an automated cell counter as described in the previous section. For each cell line, 1 × 1017, MCS02, and MCS08 was initiated 48 hours before performing the assay. After spheroid formation, single cell suspensions were prepared from MCSs using 0.25% trypsin and 0.02% EDTA solution. The trypsinization time for MCS17 and MCS02 were 10 minutes and for MCS08 was 20 minutes. Immediately following trypsinization, complete KSFM medium was added in a 1:1 ratio. The MCSs were gently pipetted several times for complete dissociation. In the following step, different MDR pathway inhibitors such as novobiocin , verapamil , and MK-571  provided with the MDR assay kit (Abcam 204534), were added to the reaction tubes. Complete KSFM medium containing 1% DMSO was used as control. The reaction tubes were then incubated at 37 °C for 5 minutes after gentle mixing, followed by the addition of efflux green detection reagent, gentle mixing, and incubation at 37 °C for 30 minutes. Following 30 minutes of incubation, 5 µL of propidium iodide (PI) provided with the kit was added to the reaction mixture before performing flow cytometry. The cellular green fluorescence signal of efflux green detection reagent was measured using BD FACSARIA III  in the PI-negative cell population using identical PMT voltage settings. Median fluorescence intensity (MFI) values were calculated for each triplicate set of reaction tubes using the DIVA software (BD Biosciences) and FlowJo 2.0 (BD Biosciences).For the experiments on MCSs, the spheroid formation for MCSTo obtain the automated real time drug uptake information, accurate segmentation of MCSs is important for quantitative analysis of red and green channel fluorescence of the spheroids. Intensity inhomogeneity over the spheroids and poor contrast in the boundary of spheroids are the major bottlenecks for acceptable segmentation. Traditional image segmentation techniques such as thresholding, region growing, and level set methods are unable to segment the spheroid with sufficient accuracy.33, which takes an entire image as input and give a dense segmentation. The detailed architecture of P-Net is shown in Fig. 34 was used in P-Net to preserve the resolution of feature maps and enlarge the receptive field to incorporate larger contextual information. The images are resized to 512 × 512 pixels to reduce the time of segmentation. Several augmentation techniques such as flip and rotation were performed to increase the number of training images. A stochastic gradient-based optimization ADAM35 was applied to minimize the cross-entropy based cost function.Therefore, we have used the P-Net Fig.  based fu36. The background and foreground weights were maintained at 1:10 ratio and training was performed up to 20 epochs.The learning rate for the ADAM optimizer was set to 0.0001 and over-fitting was reduced by using dropoutThe hyper-parameters were determined based on the validation dataset. The qualitative segmentation results are shown in Fig. 2D cell culture and MCS image analyses were performed using MATLAB 2016b . ANOVA and Tukey’s multiple comparison test was performed in GraphPad Prism 8  for comparing different data sets. Values are presented as mean ± S.D. A p value < 0.05 was considered as statistically significant. All experiments were performed in triplicates.We studied the drug response profiles of 2D and MCSs obtained from LK0917, LK0902, and LK1108, to doxorubicin Fig. , cisplat38. In 2D and MCSs of all cell lines, we observed lowest sensitivity to doxorubicin (LK0917 displayed lowest IC50 in comparison to the other two cell lines i.e. LK0902 and LK1108) for both the in vitro model, followed by cisplatin and methotrexate. In terms of drug resistance, LK1108 appeared to be the least sensitive cell line to treatment having the highest IC50 values for all the three drugs tested, followed by LK0902, and LK0917. However, the IC50 in case of methotrexate (2D) remains the same for all the tested cell lines. Interestingly, although large differences could not be observed between the drug responses of 2D cell cultures and MCSs obtained from LK0902 and LK0917 cell lines to cisplatin and methotrexate, significant differences were observed between the drug responses of LK1108 2D cell cultures and MCSs to doxorubicin. In addition, the difference in response of LK1108 2D cell cultures to cisplatin and methotrexate could be observed in the LK1108 MCSs , MCS02 (middle panel), and MCS08 (lower panel) as exemplified in Fig. 17 followed by MCS02 and MCS08. This suggests that the efflux pump activity was lowest in MCS17 followed by MCS02 and MCS08, which is consistent with the drug response profiles provided in Fig. 08 was the most resistant to drug treatment, indicated by significantly higher IC50 values  and indeed, the MCSs from these cells showed the highest efflux of calcein-AM over time, indicated by lowest green fluorescence over time, without penetration to the spheroid core. Likewise, MCS17 was the least resistant to drug treatment, indicated by significantly lower IC50 , which showed the lowest efflux of calcein-AM over time, indicated by the highest green fluorescence over time, with penetration into the spheroid core and without complete expulsion over a period of 12 hours. These findings suggest that MCS17, MCS02, and MCS08 can be characterized as treatment sensitive, moderately resistant, and highly resistant, respectively, based on their efflux pump activity  values for the 2D cell cultures and MCSs obtained from LK0917, LK0902, and LK1108 cell lines in the presence and absence of the specific efflux pump inhibitors verapamil, novobiocin, and MK-571 against MDR1, BCRP, and MRP1, respectively, using flow cytometry analysis44, out of which 90% are HNSCCs. Early-stage disease progression is curable by surgical removal of tumor tissue and radiotherapy, but the prognosis for recurrent disease onset is still challenging and puzzling45. Presently, chemotherapeutic drugs such as cisplatin, 5-fluorouracil (5-FU), and taxanes such as paclitaxel and docetaxel, are the standard treatment options for recurrent or advanced HNSCC. However, the variability and robustness of these treatment modalities are not very well understood47. MDR against cytotoxic drugs is regarded as the main clinical impediment in using chemotherapy in HNSCC. MDR is a result of the interplay between a diversity of factors, which include overexpression of the transporter molecules MDR1, MRP1, and BCRP54. In spite of this well-known phenomenon, effective detection methods are still lacking for correct characterization of MDR status in cancer cells. Development of strategies that enable this characterization can prove to be highly effective in devising targeted treatment regimens against sensitive and resistant cancer cells. Presently, commonly used detection methods include polymerase chain reaction (PCR), in-situ hybridization (ISH), and RNase protection assays (RPAs) for the quantification of MDR1 mRNA levels. Western blotting and immunohistochemistry have also been used for the detection of MDR proteins59. Previously, researchers have shown that the expression of MDR1, MRP1 and MRP7 are refractory factors in head and neck cancer chemotherapy58. In another study, it has been shown that the expression of hypoxia-inducible factor 1α (HIF-1α) is significantly correlated with MDR1 expression in human laryngeal cell carcinoma (LSCC) and that these two proteins might serve as potent biomarkers for predicting the progression of malignant and metastasis of human LSCC59. In the present study, we have attempted to simplify the identification of MDR status in PTPD HNSCCs by combining drug screening, measurement of the difference in calcein-AM uptake studied using live-cell fluorescence imaging, fluorescence-based assessment of ROS activity, and flow cytometry-based prediction of ABC transporter involvement. We performed a comparative assessment of 2D cell cultures and MCSs and observed noticeable differences between the two in vitro systems. Tumor spheroids were introduced as model systems by Sutherland et al., owing to their resemblance to solid tumors in many structural and microenvironmental aspects, and they serve as the most reliable in vitro model for investigating therapeutic and mechanistic approaches60.The global annual occurrence of head and neck cancers exceeds 0.5 million50 values compared to the 2D cultures. Interestingly, LK1108 cells required the highest dose for all three tested drugs in order to achieve 50% inhibition, irrespective of the culturing method, which indicated that among the three cell lines, LK1108 showed the highest resistance to treatment. On the other hand, the lowest IC50 values were observed for LK0917, indicating this cell line as the most drug sensitive among the three cell lines. These findings suggested the MDR status of LK0917 < LK0902 < LK1108.In the present study, we have used 2D cell cultures and MCSs obtained from the PTPD LK0917, LK0902, and LK1108 HNSCC cell lines. Firstly, we performed a treatment response cell viability assay in the presence of cisplatin, doxorubicin, and methotrexate to assess drug cytotoxicity in both 2D cell cultures and MCSs (Fig. 64. We chose live-cell fluorescence imaging because dynamic cellular changes can be observed using this method, unlike fixed-cell imaging. In addition, real-time fluorescence microscopy is less prone to experimental artifacts, rendering the outcomes more reliable. We did not observe any difference between the calcein-AM uptake profiles of 2D cell cultures obtained from LK0917, LK0902, and LK1108, with total accumulated calcein trapped inside the cells remaining comparable for the three cell lines, hence making it extremely problematic to detect differences in drug resistance activity of these cell lines. However, using MCSs as the in vitro model system we were able to detect differences in drug resistance, indicated by calcein accumulation over time. Over a 12 h time window, we observed the highest calcein accumulation in MCS17, followed by MCS02 and MCS08, which indicated that transporter activity was lowest in the MCS17, followed by MCS02 and MCS08. A comparison of the mean green fluorescence intensities between the 2D cell cultures and MCSs confirmed these findings. In order to assess whether spheroid diameter affected calcein-AM diffusion, we performed the same experiments with varying cell seeding densities and observed the same calcein accumulation pattern, indicating that spheroid diameter did not have any effect on the calcein-AM uptake profiles of MCSs, thus eliminating the possibility of false positives. After establishing the transporter activity based MDR status in these cell lines, we further validated our findings using the DCFDA assay for the detection of ROS activity.Next, we performed live-cell fluorescence imaging of calcein-AM uptake in the 2D cell cultures and MCSs, in order to assess transporter activity, as calcein-AM is a substrate of MDR1 and MRP117, indicated by green fluorescence and no ROS activity indicated by a lack of fluorescence in the MCS02 and MCS08 spheroids that we identified to have higher drug resistance compared to the MCS17. ROS activity is indicative of oxidative stress. It is expected that drug resistant cell lines that actively pump drugs out of the cell have acquired higher survival capacity compared to those that cannot. In this context, lack of ROS activity in the untreated MCS02 and MCS08 spheroids, indicates lack of ROS activity in these cells, which could be attributed to another mechanism of survival, as oxidative stress is detrimental to cell survival, thus indirectly supporting our finding that these cell lines are highly drug resistant, one of the mechanisms being high transporter activity and the other being acquired lack of ROS activity. On the other hand, the presence of ROS activity in the non-resistant MCS17 spheroids shows that these cells are more prone to oxidative stress and therefore they are more responsive to drug treatment. These findings indicate that by assessing drug transporter activity and ROS activity, the MDR status of patient cancer cells can be further characterized based on their drug transporter and ROS activities, which could potentially help determine patients that can benefit from a particular treatment regimen.We observed highest ROS activity in MCSWe have investigated drug resistance in 2D and MCSs to establish an assay system for the determination of cancer cell MDR status in cancers with unknown drug resistance profiles. Using our assay system, we were able to predict the efflux pump activities of three different PTPD-HNSCCs, which is important for determining cytotoxic drug vulnerability and the potential of developing MDR as a result of repeated drug exposure. The methods we described here could potentially be integrated into translational research for obtaining the MDR status of cancers and aiding in the determination of the optimal treatment strategy.Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids


A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2 Cholangiocarcinoma (CCA) is a rare subtype of liver cancer for which the highest incidence and mortality have been reported in northeastern Thailand ,2. The pOpisthorchis viverrini (Ov), through ingestion [Ov-associated CCA is increased xenobiotic metabolizing gene expression [Ov-associated CCA identified a distinct signature with higher rates of TP53, SMAD4, and ARID1A mutations but lower frequencies of IDH1/2 and BAP1 mutations when compared to other Asian and Western populations [Ov-associated CCAs were gathered in cluster 1 and cluster 2 in Jusakul’s study [CCA has high heterogeneity in nature . Severalngestion ,10. A unpression . Mutatioulations ,13,14. Tulations ,16,17. Hulations ,19. The ’s study . The mol’s study  and cyclThere are several models for CCA, including a previously established cell line, a cell line-transplanted xenograft, and a genetically engineered mouse model. The limitation of these models is disease homogeneity ,21. The The patient-derived xenograft (PDX) model is a promising tool for the propagation of a patient’s tumor in an immunodeficient mouse. This PDX is an invaluable asset for the advancement of cancer precision medicine, particularly for rare and aggressive cancers . A highe2 atmosphere.Four CCA cell lines—KKU-055, KKU-100 , KKU-213Sixteen CCA tissue samples were obtained from the Department of Pathology, Faculty of Medicine, Khon Kaen University, Thailand, after the clinical specimens were obtained from the operating room and the pathological specimens were taken as the standard protocol for pathological staging. All tissue samples were histologically diagnosed; 10 were of intrahepatic CCA (ICC), and six were of the extrahepatic subtype (ECC). The study protocol was reviewed and approved by the Ethical Committee for the Human Research of Khon Kaen University (HE571283), based on the Declaration of Helsinki of 1975. Written informed consent was obtained from each subject. Demographical and pathological characteristics of patients and CCA tissues are listed in 3 pieces (2–3 mm each dimension). Non-viable cells were removed. Each tissue was divided into two parts: (1) for transplantation and (2) for molecular characterization and paraffin-embedded tissue preparation. Tissues were transplanted into flank areas of Balb/c Rag-2/Jak3 double-deficient  [For transplantation, frozen CCA tissues were thawed and vigorously washed with PBS three times. Tissues were cut into 8–27 mmlb/c RJ)  or Balb/lb/c RJ)  subcutan2 atmosphere. Cells were maintained in vitro culture system at least 6 months to ensure the immortalization properties.For cell line development, fresh xenograft tissues were prepared as previously described . Cells wAll four newly established cell lines were deposited into the Japanese Cancer Research Resources Bank (JCRB), National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan.To demonstrate the liver cell origin of the cell lines, the expression profile of bile duct or hepatocyte-related genes including cytokeratin 7 (CK7), CK19, γ-glutamyl transferase (GGT), α-fetoprotein (AFP), and albumin (ALB) were determined as previously described . RNA wasPCR products were separated in 1.5% agarose gel in Tris-Borate-EDTA (TBE) buffer. A gel was stained with ethidium bromide solution  and the images were captured by Bio-Rad Gel Doc 2000 .® Identifiler® Plus PCR Amplification Kit . DNA was extracted by the QIAamp® DNA Micro Kit . PCR products were analyzed using ABI Prism 3130 Genetic Analyzer and GeneMapper® ID Software v3.2 (Applied Biosystems).To determine the genetic stabilities of the cell lines, 16 short tandem repeats (STR) of cell lines, original tumor tissues, and patient’s white blood cells (WBC) were compared using AmpFℓSTRTP53 gene mutation was analyzed as previously described in [ribed in . Briefly4 cells were injected intrasplenically as previously described [The xenograft transplantation ability of the cell lines was determined. One to two million cells of each cell line were transplanted subcutaneously into both flanks of Balb/c RJ mice. For intrasplenic transplantation, 5 × 10escribed . Tissues® DAB substrate kit .Hematoxylin and eosin staining of the original CCA tissues and transplanted tumors was performed regularly. For immunohistochemistry staining, a standard protocol using citrate buffer retrieval buffer was used. Signals were enhanced by EnVision-system-HRP  or the Vectastain Elite ABC standard kit . Detection was performed using the HistofineThe sources of antibodies were as follows: anti-CK19  was from Sigma-Aldrich, anti-Ki-67 (MIB-1) was from Dako, anti-epithelial cell adhesion molecule  and anti-Oct-3/4 (C-10) were from Santa Cruz Biotechnology , anti-Sox2 (D6D9) was from Cell Signaling Technology , and biotinylated goat anti-mouse IgG and biotinylated goat anti-rabbit IgG were from Vector Laboratories.The comparison of tissue architecture between the original CCA tissue and transplanted tissue was made by the pathologists. The images were taken by the BZ-8100 Biozero fluorescent microscope. For the quantitation, the immunoreactivity signals were quantified by BZ-II Analyzer  as previously described .r) was calculated using GraphPad Prism version 6.07 .For the correlation study, Pearson’s correlation coefficient  while six were of the extrahepatic subtype (ECC) based on the 7th edition of the AJCC cancer staging classification. The most extended storage duration with successful transplantation was 134 days (19–134 days). After defrosting and cleaning, tissues were transplanted into both flanks of Balb/c RJ mice. Thirteen tissue samples (eight ICC and five ECC) successfully grew in the subcutaneous areas of the mice . No massA similar experiment was carried out in nude RJ mice. Three samples were transplanted into both flanks of mice. The transplantation success rate was comparable to those in Balb/c RJ, but F0 growth in nude RJ took approximately 2 weeks longer. Thus, we selected the Balb/c RJ mice for our PDX generation and further testing.From our protocol, we successfully established the method for generation of the CCA-PDX model, which is highly efficient (67–80% success rate) . The durAmong 12 serially transplantable tissues, four tissues were successfully developed into cell lines. All cell lines were of intrahepatic origin; three were histologically characterized as well-differentiated subtypes  and one was characterized as a mixed adenosquamous subtype . KKK-D068 and KKK-D131 were established from the F0-transplanted tumor while KKK-D138 and KKK-D049 were established from the F1 and F2 tumors, respectively. The morphologies of the cell lines are presented in The authentications of cell lines were performed by STR analysis . DNA froFurther characterization of cell lineage marker was performed using RT-PCR b. ComparTP53 mutation analysis revealed CCC to CGC at codon 72, which will cause missense P72R mutations in KKK-D138 but is not detected in KKK-D068 and KKK-D131. The TP53 mutation of KKK-D049 has not yet been analyzed.n = 4). Tumors were grown in the mice for a month, and tumor masses were observed twice a week. We observed tumor masses from all injected sites . Spleen, liver, and lungs were removed at 1 month after injection. Tumors were observed in 88% of transplanted livers and spleens (7/8 mice)  b. No tum/8 mice) . No tumoThe subcutaneous tumor masses were prepared for the histological comparison with the tumor tissues from the patients . These tOpisthorchis viverrini (Ov) infection has been demonstrated by distinctive molecular signatures [Ov-related CCA might be vulnerable to distinct treatment. Several attempts were made to provide opportunities for Ov-related CCA treatment [Cholangiocarcinoma (CCA) is a rare, aggressive tumor of liver found worldwide ,2. The wgnatures ,13,18,19Patient-derived xenograft (PDX) models are preclinical models that are ideally developed for the implementation of personalized medicine. PDX models are a powerful tool for cancer propagation that retain disease complexity and heterogeneity . MoreoveThe newly developed CCA-PDX model is highly efficient, with 75% transplantable efficiency compared to 6–35% engraftment rates in the previously described models ,28. ThisPDX-derived cell lines developed in this study show some degree of heterogeneity in vitro and in vivo, yet keep the characteristics of tissue organization, which are common in PDX models ,26,27. TWe propose the platform for anti-cancer drug screening using a PDX-derived cell line, and a PDX mouse model as demonstrated in In conclusion, in this study we established a highly effective CCA PDX model and highly transplantable PDX-derived cell lines. Cell authentications and characterization have been demonstrated. The cellular heterogeneity and preserved tissue architecture have been confirmed in PDX-derived cell lines and cell line xenografts. These PDX-derived cell lines and PDX models might be a promising platform for anti-CCA development. The advantage of our PDX model for personalized medicine seems to be limited by the long transplantation duration in some cases.


Brassica napus CMS  lines with their corresponding maintainer lines. By comparing anther developmental stages between CMS and maintainer lines, we identified that in the Nsa CMS line abnormality occurred at the tetrad stage of pollen development. Phytohormone assays demonstrated that IAA content decreased in sterile lines as compared to maintainer lines, while the total hormone content was increased two-fold in the S2 stage compared with the S1 stage. ABA content was higher in the S1 stage and exhibited a two-fold decreasing trend in S2 stage. Sterile lines however, had increased ABA content at both stages compared with the corresponding maintainer lines. Through transcriptome sequencing, we compared differentially expressed unigenes in sterile and maintainer lines at both (S1 and S2) developmental stages. We also explored the co-expressed genes of the three sterile lines in the two stages and classified these genes by gene function. By analyzing transcriptome data and validating by RT-PCR, it was shown that some transcription factors (TFs) and hormone-related genes were weakly or not expressed in the sterile lines. This research work provides preliminary identification of the pollen abortion stage in Nsa CMS line. Our focus on genes specifically expressed in sterile lines may be useful to understand the regulation of CMS.The interaction between plant mitochondria and the nucleus markedly influences stress responses and morphological features, including growth and development. An important example of this interaction is cytoplasmic male sterility (CMS), which results in plants producing non-functional pollen. In current research work, we compared the phenotypic differences in floral buds of different  A vacuum chamber was used to evacuate the air and volatiles from the sample bottles. Fixed floral buds were dehydrated by a graded series of ethanol  for one hour. Pre-infiltration and penetration by Technovit 7100 resin steps were undertaken to produce semi-thin sections ~3 μm thick. Samples were stained by 1% toluidine blue  for 3 min, and 5 specimens of each stage were observed to take images under an optical microscope . Pol CMS, Ogu CMS, Nsa CMS, and their corresponding maintainer lines were quantified for ABA and IAA phytohormones. Samples were collected and extracted using methanol compounds [About 60 floral buds  of ompounds . The extOgu CMS, Nsa CMS, and their corresponding maintainer lines) at the same time. Samples collected from each line were pooled, frozen in liquid nitrogen, and stored at −70 °C for RNA preparation. Total RNA from two stages of floral buds (<2.5 mm and >2.5 mm) of pol CMS, Ogu CMS, Nsa CMS, and their corresponding maintainer lines were extracted by using RNA kits  in accordance with the manufacturer’s protocol. The integrity of the total RNA was checked by 1% agarose gel electrophoresis. The concentration was detected by Nano-Drop  and purity of RNA was determined by Agilent 2100 Bio-analyzer . RNA (10 μL) was sequenced using the Illumina HiSeq 2000  and 150 bp of data collected per run. After removing adapters and low-quality data, the resulting clean data was aligned to the B. napus reference genome [About 60 floral buds were harvested from the plants of each line . Specific primers for differentially expressed genes were designed to amplify 600–750 bp sequences . RT-PCR Pol and Ogu CMS lines. Conversely, the pollen abortion stage in the Nsa CMS line had not been determined clearly. From this study, we identified the tetrad stage of Nsa CMS for pollen abortion by using the semi-thin sectioning of floral buds. This information will help support the application of Nsa CMS in plant breeding. Higher content of ABA and lower content of IAA was observed in sterile lines when compared to maintainer lines in all male sterile systems. This result may reveal that ABA and IAA play different roles in fertile pollen development. During the two stages, genes involved in energy production were enriched in maintainer lines in comparison to sterile lines for all CMS systems investigated.Considerable effort has been taken to identify the pollen abortion stage in


BIRC5) promoter, can be used to treat several cancers. As survivin is overexpressed in ATL, we examined the effects of Surv.m-CRAs on ATL-selective replication and survival.Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by long-term human T-cell leukemia virus type I (HTLV-1) infection. Survivin-responsive, conditionally replicating adenoviruses regulated by multiple tumor-specific factors (Surv.m-CRAs), in which the expression of the adenoviral early region 1A gene is regulated by the survivin , which is important for adenoviral infection, was high in the cell lines. In contrast, we observed the absence of survivin promoter activity and a low expression of CAR in activated peripheral blood lymphocytes (PBLs) from healthy subjects. Surv.m-CRAs actively replicated and induced cytocidal effects in five out of six cell lines; conversely, we observed minimal viral replication and no marked cytotoxicity in normal activated PBLs.This is the first report demonstrating that Surv.m-CRAs constitute attractive potential anti-ATL agents. Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of mature peripheral T lymphocytes that results from long-term infection with human T-cell leukemia virus type I (HTLV-1). Five to twenty million individuals are estimated to be currently infected with the virus, and only 3–5% of HTLV-1-infected patients develop ATL. HTLV-1 infection can lead to the insertion of oncogenic viral genes into the host genome, which activate NF-κB and Akt signaling, as well as cyclin-dependent kinases, and silence TP53 in host cells.The prognosis for patients with ATL after treatment with classical cytotoxic agents is still poor. New therapeutic approaches, distinct from standard cytotoxic chemotherapy treatments, are expected to lead to the eradication of ATL , 2. For E1A), the first transcribed gene after infection with wild-type adenoviruses, transactivates the viral and cellular genes essential for generating transmittable adenoviruses. m-CRAs replicate in a tumor-specific manner by replacing the innate E1A promoter with a tissue- and tumor-specific promoter [Conditionally replicating adenoviruses (CRAs), also termed oncolytic adenoviruses, which selectively replicate in and eradicate cancer cells, are smart anti-cancer agents . We devepromoter –15.Survivin (BIRC5), a member of the inhibitor of apoptosis gene family, is overexpressed in most cancers, but not in normal cells. Clinical trials have demonstrated a positive correlation between high survivin expression levels and poor prognosis, enhanced rate of recurrence, and increased resistance to chemotherapy in patients with malignancy .We developed survivin-responsive m-CRAs (Surv.m-CRAs), which express E1A under the transcriptional control of the survivin promoter. Surv.m-CRAs can kill a variety of cancers more efficiently and specifically than telomerase-responsive m-CRAs in vitro and in vivo . MoreoveAs survivin is overexpressed in ATL, especially in acute-type ATL , we exam2. Written informed consent was obtained from healthy subjects to participate of this research.The human ATL cell lines, S1T and Su9T01, and the HTLV-1-infected T-cell lines, Oh13T, K3T, F6T, and MT-2, were sustained in RPMI 1640  supplemented with 1% penicillin/streptomycin (Gibco/Invitrogen) and 10% fetal bovine serum (Gibco/Invitrogen). All the cell lines, except MT-2, were established from patients in our laboratory . MT-2 waE1A under the transcriptional control of the survivin promoter, E1B55KD under the transcriptional control of the CMV promoter, and EGFP under the transcriptional control of the CMV promoter; two types of E1-deleted, replication-defective adenoviruses expressing EGFP under the transcriptional control of the CMV promoter (Ad.CMV-EGFP) or LacZ under the transcriptional control of the survivin promoter (Ad.Surv-LacZ) were also proliferated and purified.The following viruses were proliferated and purified as described previously : Surv.m-5/plate) were infected with Ad.Surv-LacZ at a multiplicity of infection (MOI) of 30 for 24 h. We measured cellular β-gal activity using the β-Galactosidase Enzyme Assay System , after harvesting. The AGTE for each cell type in vitro was examined 48 h after infection with Ad.CMV-EGFP at an MOI of 30.Promoter activities and in vitro adenoviral gene transduction efficiency (AGTE) were examined as described previously with some modifications . Cells . Using flow cytometry, we assessed the expression of coxsackie and adenovirus receptor (CAR), which is recognized as the primary cropping receptor for coxsackie B viruses and members of the adenovirus family JM Bergelson, JA Cunningham, G Droguett, EA Kurt-Jones, A Krithivas, JS Hong, MS Horwitz, RL Crowell and RW Finberg . Cells wCells in 96-well plates were infected with Surv.m-CRA at MOIs of 0.3, 3, and 30 or Ad.CMV-EGFP at an MOI of 30. Cell viability was determined by MTT assay  after 3 and 5 days, according to the manufacturer’s protocol.t-test. P < 0.05 was concluded statistically significant.Data are shown as the mean ± standard errors (s.e.). Statistical significance was determined using Student’s We tested 2 ATL cell lines and 4 HTLV-1-infected T-cell lines. We infected the cells with Ad.CMV-EGFP or Ad.Surv-LacZ at an MOI of 30. All the cell lines had high AGTE and strong survivin promoter activation  gene in multiple cancers, especially in ATL. PD-L1 disruption could serve as a genetic marker to identify cancers that actively evade anti-tumor immunity through PD-L1 overexpression [Recently, immunotherapies using antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1) have proven to be powerful and effective treatments for patients with various advanced cancers in many clinical trials –30. The pression . CombinaThis is the first report demonstrating that Surv.m-CRAs represent a striking potential anti-leukemic agent that could efficaciously and specifically treat ATL. Because our experiments did not include any in vivo therapeutic data, we will focus on the methods of gene administration in future extensive studies.