PSI+] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI+] by the same mechanism– inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [+PSI] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [+PSI] and inactivation of Hsp104 cures [+PSI] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.[ Saccharomyces cerevisiae and in the fungus Podospora anserina. Yeast prions have served as convenient models to study the mechanism of prion propagation. The misfolded yeast prion is transmitted as seeds from mother to daughter during cell division, and these seeds then cause conversion of the properly folded protein to the misfolded prion form Prions are infectious proteins that self-propagate by changing from their normal folded form to a misfolded amyloid form. In mammals, prions are implicated in various fatal neurodegenerative diseases, such as scrapie in sheep, bovine spongiform encephalopathy in cattle, and Crueutzfeld-Jacob disease in humans. Prions have also been found in the yeast PSI+], occurs when the translation termination factor Sup35 is transformed into its aggregated amyloid form. Sup35 has three domains, N, M, and C, which are respectively an N-terminal Asn/Gln rich aggregation-prone domain, a middle domain, and a C-terminal domain that mediates translation termination. The [PSI+] phenotype has impaired termination of protein translation as opposed to the [psi-] phenotype in which Sup35 is present in its properly folded form. Maintenance of [PSI+] and other misfolded yeast prions requires molecular chaperones, in particular Hsp104 One of the best characterized yeast prions, [PSI+] phenotype -psi] yeast, whereas it forms fluorescent foci in [PSI+] yeast. However, NMG does not retain the functional properties of the parent molecule. More recently, Sup35 has been engineered with a GFP inserted between its N- and M- domains to produce NGMC -psi] yeast and forms foci in [PSI+] yeast. Consistent with this difference in appearance, the diffusional mobility of NGMC is much faster in [-psi] than in [PSI+] yeast One of the techniques that have been employed to understand the role of Hsp104 in prion propagation is confocal microscopy. Initially, the truncated Sup35 fluorescent fragment, NMG, was used as a reporter of the [PSI+] yeast. The Chernoff laboratory observed that when NMG was expressed from the SUP35 promoter on a centromeric plasmid, large fluorescent NMG foci were observed when [PSI+] was cured by overexpressing Hsp104-2KT PSI+] by guanidine treatment, they observed that the NMG fluorescence became diffusive PSI+] by overexpression of Hsp104-2KT was much faster than curing by guanidine treatment PSI+] by a different mechanism than overexpression of Hsp104-2KT. In contrast, Tuite and coworkers found that inactivation of Hsp104 by guanidine treatment or overexpression of Hsp104-2KT cured [PSI+] with similar kinetics To understand the role of Hsp104 in prion propagation, changes in both NMG and NGMC fluorescence have been examined following inactivation of Hsp104 in [PSI+] by guanidine treatment. This defect in transmission was observed either when NGMC was expressed from the MFA1 promoter GAL1 promoter SUP35 promoter showed that during curing by guanidine treatment, the NGMC foci became diffusive, acquiring mobility similar to their mobility in [-psi] yeast even though the yeast were still [PSI+] Two recent microscopy studies have shown a defect in the transmission of prion seeds from mother to daughter during the curing of [+PSI] yeast were cured either by overexpression of Hsp104-2KT or by guanidine treatment to better understand the role of Hsp104 in prion propagation. Our data showed that even though the seeds appeared to become much smaller during curing by guanidine treatment than by overexpression of Hsp104-2KT, the number of cells with foci correlated exactly with the number of [PSI+] cells regardless of the method of curing. These results showed that the NGMC fluorescent foci are indeed the prion seeds that maintain [+PSI] and curing occurs because of inhibition of the severing activity of Hsp104. In addition, when [+PSI] was cured by depleting the yeast of Hsp104, the NGMC foci remained prominent, which establishes that the loss of detectable foci during guanidine treatment is due to an Hsp104-dependent activity. Measurements of the seed size showed that the loss of detectable seeds occurred because the seeds became much smaller during the curing of [PSI+] by guanidine treatment than by overexpression of Hsp104-2KT. Therefore, our data are consistent with the thesis that during curing by guanidine treatment, Hsp104 retains an activity that reduces the size of the seed without changing their number of seeds.In the present study, we reinvestigated the changes in NGMC fluorescence that occur when [+PSI] yeast strain, used routinely throughout this study, has SUP35-GFP (NGMC) integrated into the genomic locus of SUP35. This strain was derived from wild-type strain 779-6A  MATα, kar1-1, SUQ5, ade2-1, his3Δ202. trp1Δ63, ura3-52, leu2-3, 112, sup35::SUP35-GFP) is a weak [PSI+] [-pin] strain derived from 74-D694 strain  TRP1-based centromeric plasmid and has HSP104 containing two amino acid mutations (K218T and K620T) under the control of GAL1 promoter, was transformed into these strains as described GAL1 promoter in yeast transformed with either pRS314-GAL1-HSP104(K218T) or pRS314-GAL1-HSP104(K620T). Plasmid pRS314-GAL1-SSA1 contains the SSA1 coding region with its terminator region (287 bp) fused to GAL1 promoter. GFP on a centromeric plasmid pJ543 was expressed by the SUP35 promoter in the 779-6A strain.The 1074  as well as the Δhsp104 strain with the wild type copy of HSP104 on centromeric plasmid pJ312 The 600≤0.6) by periodic dilution with fresh medium.Yeasts were grown at 30°C on synthetic defined medium  with complete supplement mixture (CSM) or the appropriate amino acid dropout complete supplement mixture for selection and maintenance of the particular plasmid. Synthetic galactose  medium contains both 2% galactose and 2% raffinose in place of glucose. ½YPD solid medium used in the plating assays contains 0.5% yeast extract, 2% peptone, and 2% glucose. Yeast were grown in synthetic medium rather than yeast peptone medium since the latter medium had background fluorescence that interfered with imaging NGMC. Cultures were always maintained in active growing conditions  to cure [PSI+] either in SD or SGal, as indicated. Yeast were grown in SGal to induce expression of the different Hsp104 mutants and Flp recombinase. To determine the extent of curing, yeast were plated on ½YPD plates as described previously PSI+].To express the different mutants of Hsp104, cells from growing culture in SD medium were shifted to SGal medium and continued to grow until curing of  yeast by overexpression of the Hsp104 dominant negative mutant, Hsp104-2KT. The 1074 yeast strain with NGMC integrated into the genomic SUP35 locus was used to express NGMC at the endogenous Sup35 level. Starting with [+PSI] yeast, we clearly detected NGMC foci when [+PSI] yeast were scanned using the Zeiss live confocal microscope. When Hsp104-2KT was induced by galactose, the foci became much more prominent after growing 1.5 h in SGal  yielded red colonies indicating [psi-] prion phenotype, while cells with three or more foci (n = 33) yielded white or white-red sectored colonies, indicating [+PSI] prion phenotype. However, 74% of the cells with only one focus (n = 27) and 27% of the cells with two foci (n = 26) gave rise to red colonies, indicating that the progeny of these [+PSI] cells were [psi-]. This in turn suggested that curing might be occurring on the plate after Hsp104-2KT expression was turned off, but before the remaining Hsp104-2KT was diluted out by cell division, as was proposed by Ferreira et al +PSI]  always produced one or more colonies that were either totally white or white-red sectored, which showed that the single focus was indeed a prion seed. In addition to the white or white-red colonies, many of the daughter cells formed red colonies, indicating that inhibition of severing by Hsp104-2KT persisted for several generations until the Hsp104-2KT was finally diluted out. It is this persistence of Hsp104-2KT that causes yeast with a single focus to produce many daughter cells that are [+PSI] was cured by overexpression of Hsp104-2KT as predicted by the cell division model of Tuite and coworkers Next, we examined whether the number of fluorescent foci is halved each generation when [+PSI] cells, the number of foci was counted in [+PSI] cells after 2 h of Hsp104-2KT induction. At this time the foci had become pronounced, but the yeast had not yet divided in the SGal medium. After fixing the cells, a value of 220±16 (n = 18) foci per cell was counted from the Z-stack confocal images, which is in good agreement with the number of foci that were derived from the curing curve The imaging of NGMC foci also provides a method to determine the number of NGMC molecules per focus provided that we can count the number of foci in each yeast cell. Since we could not clearly resolve the individual foci in [+PSI] cells were fixed and bud scars were stained. Daughter cells in the population were identified by the faint staining birth scar. The NGMC foci per cell were counted in daughter cells and in mother cells with either 2 or 3 bud scars. +PSI] yeast with active Hsp104 The transmission of foci from mother to daughter was also examined to determine whether the daughter cells inherited 40% of the total foci with the mother cells retaining 60%. This ratio is based on the seeds segregating with equal probability between the mother and daughter cells when corrected for the volume difference between mother and daughter cells +PSI] was cured by guanidine, the NGMC fluorescence became diffusive even though the cells were not yet cured +PSI] by guanidine. In agreement with our previous study -psi] yeast even though the cells were all [+PSI] based on the colorimetric colony assay. Interestingly, if curing of [+PSI] were done in SGal medium to induce expression of Hsp104-2KT, as well as 5 mM guanidine, the NGMC foci remained bright, but this did not occur in control cells with an empty vector when these cells were cured in SGal medium with 5 mM guanidine  formed red colonies, while cells with one (n = 15) or more foci (n = 17) formed white or occasionally white-red sectored colonies. Therefore, the foci are indeed the prion seeds, although the seeds do not always have to be visible as foci.In contrast to curing by overexpression of Hsp104-2KT, there was excellent agreement between the percent  by guanidine. After growing the yeast for 5 or more generations in the presence of guanidine, the yeast were starved for 1 h in water prior to fixation to enable detection of the foci. By imaging confocal Z-stacks of the fixed cells, we determined the average number of foci as a function of generation time. In agreement with Cox et al. +PSI] by guanidine lags by 2–3 generations compared to the curing by overexpression of Hsp104-2KT. Therefore, Hsp104 appears to retain some residual severing activity in 5 mM guanidine, in contrast to the complete inhibition of severing activity observed upon overexpression of Hsp104-2KT.We next examined whether the number of seeds halved each generation during the curing of [+PSI] yeast with guanidine, at which time the cells all showed diffuse fluorescence but still were [+PSI], the yeast were incubated in water for 1 h to make the foci prominent. The yeast were returned to SD medium with guanidine, which again caused diffuse fluorescence and further curing. At different time points during the curing cycle, the cells were incubated in water for 1 h, followed by Z-stack confocal microscopy. The maximum Z-stack projections of the yeast cells imaged at indicated generations in To further check that this residual severing activity does not account for the loss of detectable foci during curing by guanidine, we repeatedly imaged the foci during the final stages of curing. After 5 generations of growing [+PSI] was cured by overexpression of Hsp104(K218T), just as occurred during curing by guanidine, the NGMC foci became undetectable in the majority of the cells even though the cells were still [+PSI] (The Hsp104-2KT mutant has point mutations at the active sites of both NBD1 (K218T) and NBD2 (K620T), whereas it has been suggested that guanidine inhibits the activity of just the NBD1 domain l [PSI+] . Along w+PSI] by overexpression of Hsp104(K218T). However, addition of guanidine caused faster curing of [+PSI] by Hsp104(K218T) (+PSI] curing by the combination of overexpression of Hsp104(K218T) and 5 mM guanidine was essentially the same as that observed when [+PSI] was cured by overexpression of Hsp104-2KT. Therefore, this combination essentially eliminated the severing activity of Hsp104. At the same time, NGMC foci were not visible in cells that were still [+PSI]  . Specifil [PSI+] , which r+PSI] yeast, the cells were imaged and plated on ½ YPD plates. In agreement with the prediction, prominent foci were still present both at 3 and 8 generations after induction of Hsp104(K620T) . Therefore, the NBD2 domain, as well as the NBD1 domain of Hsp104, functions in the severing activity of Hsp104, but the NBD1 domain appears to have a greater severing activity than the NBD1 domain. This difference in severing activity between the NBD domains of Hsp104 has the caveat that there may be less hexameric Hsp104(K620T) compared to Hsp104(K218T) in the yeast cytosol based on in vitro studies showing that the Hsp104(K620T) mutant has impaired oligomerization The above results predict that the NBD2 domain of Hsp104 is acting to make the NGMC foci undetectable during curing. To test this prediction, we expressed Hsp104(K620T), which contains a point mutation only at the active site of the NBD2 domain of Hsp104. After inducing Hsp104(K620T) expression by galactose in [4(K620T) . Fig. 4B+PSI] by guanidine is dependent on the yeast strain by using the L2885 yeast strain derived from the wild-type strain 74-D694 SUP35 genomic locus using the NGMC construct engineered in the Serio laboratory +PSI] either by overexpression of Hsp104-2KT or by guanidine treatment. With this strain, there were also prominent foci in cells grown for 6 generations in SGal to overexpress Hsp104-2KT as compared to the absence of detectable foci in yeast grown in 5 mM guanidine . We next examined whether native Sup35 protein shows a similar difference in the amount of soluble Sup35 depending on the method of Hsp104 inactivation. This was done by growing [+PSI] yeast strain 779-6A, the parental strain of 1074, for 6 generations in either SD medium with guanidine or in SGal medium to induce overexpression of Hsp104-2KT, at which point the plating assay showed the yeast were all still [+PSI]. Western blot analysis of yeast lysates prepared from these cells was used to determine the total amount of Sup35 in the lysates and the amount in the supernatants after high speed centrifugation. As expected based on previous observations +PSI] yeast strain showed less soluble Sup35, than the 1074 [+PSI] yeast strain that expresses NGMC . Therefore, whether or not Sup35 is tagged with GFP, significantly more soluble Sup35 was present in [+PSI] yeast partially cured by guanidine than by overexpression of Hsp104-2KT. Since both methods show a similar time course of curing, as was shown previously by Ness et al +PSI] by guanidine is not dependent on the use of GFP-tagged Sup35.All of the above studies on curing [ses NGMC . Neverth+PSI] prion. Specifically, regardless of whether Hsp104 was inactivated by guanidine or by overexpression of Hsp104-2KT, the fraction of [+PSI] yeast with NGMC foci directly correlated with the extent of prion curing as determined by the red/white colony assay. None of the yeast in growing culture showed large agglomerates of immobilized NGMC nor was there evidence for dead-end aggregates of NGMC; whenever cells contained visible NGMC foci, the cells and their progeny were [+PSI]. The one exception was yeast that were left with only one focus when [+PSI] was cured by overexpression Hsp104-2KT. This was not a dead-end focus because when a cell with a single focus was grown overnight in SD medium a small fraction of its daughter cells were [+PSI]. Evidently, when these cells were plated, high levels of Hsp104-2KT were still present for a time in the daughter cells resulting in the curing of most of these cells, and these cured daughter cells obscured the limited number of [+PSI] daughter cells present thus giving the colony a red appearance.In this study, we first established that the NGMC foci were the seeds responsible for propagating the [+PSI] by Hsp104 inactivation is due to inhibition of severing of the prion seeds by Hsp104, in agreement with the model of Tuite et al. +PSI] by guanidine treatment compared to overexpression of Hsp104-2KT, as well as the difference between our data and the theoretical line obtained by halving the number of foci each generation. It is perhaps not surprising that there is residual severing activity in the presence of guanidine since in vitro experiments showed that the ATPase activity of Hsp104 was not completely inhibited by guanidine Second, we established that curing of [SUP35 promoter formed large fluorescent agglomerates during curing of [+PSI] by overexpression of Hsp104-2KT, as well as in a [+PSI] yeast strain expressing low levels of Hsp104 PSI+] in yeast expressing NMG from the GAL1 promoter SUP35 promoter, which probably induced lower levels of NMG expression than the GAL1 promoter, the Chernoff laboratory observed diffuse NMG fluorescence during curing of [+PSI] by guanidine +PSI] yeast during curing by Hsp104 inactivation was also examined by the Serio laboratory. By expressing NGMC under the MFA1 promoter, they observed that the foci were largely immobilized when Hsp104 was inactivated either by guanidine treatment or by overexpression of Hsp104-2KT Previously, the Chernoff laboratory observed that NMG expressed from the +PSI] by overexpression of Hsp104-2KT. Our data clearly show that the foci are transmitted based on their abundance in the daughter cells relative to the volume of the mother and daughter cells, as proposed by the Byrne et. al. model +PSI] prion, Serio’s laboratory has detected a defect in the transmission of seeds to daughter cells +PSI] yeast, Hsp104 is actively remodeling the seeds, which could make the seed size heterogeneous, whereas when Hsp104-2KT is overexpressed, there is no remodeling of the seeds and all the seeds tend to be large Studies overexpressing NMG suggest it forms large agglomerates that cannot be significantly reduced in size or transmitted to daughter cells. With regards to this study, we examined whether there was a defect in the transmission of the NGMC foci to daughter cells during curing of [+PSI], led us to incorrectly propose that cell division was not essential for the curing of [+PSI] +PSI] yeast were either starved or Hsp104-2KT was overexpressed. Since severing activity is 80% inhibited in 5 mM guanidine, severing activity cannot account for all of the seeds becoming undetectable. Furthermore, when [+PSI] was cured by overexpression of Hsp104(K218T) in the presence of guanidine, the curing kinetics showed severing was completely inhibited, but still the seeds became undetectable. This again shows that severing is not causing the absence of detectable foci.Although the mechanism of curing by inactivation of Hsp104 by guanidine treatment and by overexpression of Hsp104-2KT was similar, our data show that the NGMC foci became undetectable during curing by guanidine treatment. The fact that the NGMC appeared diffusive during curing by guanidine even though the cells were still [+PSI] yeast during curing by guanidine treatment is due to a reduction in the size of the foci. This reduction in size is dependent on Hsp104 since yeast in which the HSP104 gene was conditionally deleted showed prominent NGMC foci during curing of [+PSI] both in the absence and presence of guanidine. Therefore, our data are consistent with Hsp104 having an activity in the presence of guanidine that reduces or “trims” the size of the prion seeds without severing them into new seeds. This trimming activity was also observed when [+PSI] was cured by overexpression of Hsp104(K218T), which contains only a point mutation at the ATPase site of the NBD1 domain of Hsp104. Apparently, trimming activity removes Sup35 molecules from the prion seeds, but these released Sup35 molecules do not form a new seed. In fact, a size threshold for misfolded Sup35 oligomers to propagate new seeds rather than to dissolve into monomer has been incorporated into previous models of prion propagation FCS microscopy observations suggested that the loss of detectable foci in [+PSI] by guanidine, but there is also evidence that trimming occurs in propagating yeast. When Ssa1 was overexpressed, the time course of [+PSI] curing by guanidine treatment was not affected, but the NGMC foci remained prominent until the yeast were cured. This shows that SSa1 does not affect severing in the presence of guanidine even though it markedly reduces trimming. If trimming also occurs in propagating [+PSI] yeast, Ssa1 should have the same effect here, that is, without affecting severing it should increase the size of the foci. Indeed this is what occurred in the 1074 yeast strain of propagating [+PSI] yeast. Ssa1 markedly increased the size of the foci without curing [+PSI] even after 20 generations of Ssa1 overexpression. These results are consistent with inhibition of trimming of prion seeds in [+PSI] yeast in the presence of excess Ssa1. As for why the foci are larger in [+PSI] yeast with active Hsp104 than in [+PSI] yeast partially cured by guanidine, this may occur because as Hsp104 severs the prion seeds, there may be less Hsp104 available to trim the seeds.Not only does trimming by Hsp104 occur during curing of [+PSI] cells in the presence of guanidine would be expected to cure yeast even if cell division were prevented. Of course, as we pointed out above, this did not occur in our previous experiments, suggesting that perhaps the prion core has a special structure that makes it resistant to trimming. Finally, not only inactivation of Hsp104 cures [+PSI] prion, but overexpression of Hsp104 also cures +PSI] by overexpression of Hsp104, Hsp104 partially solubilizes Sup35 aggregates Our proposal that Hsp104 both severs and trims seeds is in agreement with observations that have been made on other triple-A ATPases. In mammalian cells, two different triple-A ATPase proteins, spastin and katinin, are able to both trim and sever microtubules Movie S1Transmission of the prion aggregates NGMC foci from mother to daughter cell during curing by Hsp104-2KT overexpression. Cells of 1074 strain having pFL39-GAL-HSP104KT were grown for 6–7 generations in SGal and imaged using Zeiss LSM780 confocal microscope. After first imaging the daughter cell, it was photobleached for 5 s, and then imaged every second for the next 2 min. More than 20 budding cells were imaged and a representative one is shown in this movie.(AVI)Click here for additional data file.


Universal phenotyping techniques that can discriminate among various states of biological systems have great potential. We applied 557 fluorescent library compounds to NCI's 60 human cancer cell-lines (NCI-60) to generate a systematic fluorescence phenotypic profiling data. By the kinetic fluorescence intensity analysis, we successfully discriminated the organ origin of all the 60 cell-lines. A principal challenge for functional genomics is to identify genotypes that are associated with a specific phenotype. Recent advances in gene expression profiling and next-generation sequencing (NGS) technology have driven significant improvements in high-throughput genotyping. We envisioned that a fluorescent probe library would have great potential to identify a set of universal phenomic parameters due to their uniform phenotypic readout , easy dose controls, high sensitivity to micro-environments, and structural diversity at the molecular level. Our group has recently developed fluorescent libraries and reported a series of imaging probes in glucagon secreting cells, To demonstrate the phenomic scale of fluorescence image-based screening methodology, 60 human cancer cell lines (NCI-60) of the National Cancer Institute's Developmental Therapeutics Program were chosen. These cells were selected based on the following the two reasons. (i) it is the largest standard collection of broad cancer cell types. The NCI-60 set contains cancers of 9 different origins, including leukemia, melanomas, renal, breast, colon, lung, CNS, prostate, and ovarian carcinomas. (ii) rich biochemical background information about the collection is available to public. The characteristics of the NCI-60 set have been profiled at the genomic, RS) and BODIPY (BD) fluorophore libraries (240 rosamine λabs = 480∼616 nm) and fluorescence emission (λem = 530∼656 nm) ranges with a broad structural diversity. To maximize the information from the cell based assays, we utilized a whole cell fluorescence image-based screening platform, which would provide integrated cellular characteristics, so called high-contents screening. The large-scale profiling was performed using an automated fluorescence microscope system . NCI-60 cells were plated in thin-bottom 384-well plates at 70% confluency, and 557 fluorescent compounds were added to different wells at two final concentrations of 250 nM and 500 nM. The fluorescence images were obtained both in the FITC and the TRITC channels to cover the broad range of spectra of the probes. Fluorescent images were taken from two independent sites of each well for 3 time points , and all the experiments were duplicated. All together, 1,604,160 images (557 probes×60 cancer cells×2 concentrations×2 channels×2 sites×3 time points×2 duplicates) were collected. A representative fluorescence image data of BD-46 is presented in BD-46.Combination of rosamine  exhibited similar response pattern in the cell lines for same cancer origin , we decided to use fluorescence intensity fold change over the time (intensity kinetic profile), rather than the absolute value of the intensity. Firstly, we measured the mean values of the fluorescence intensity of the cells, and the intensity pattern for different cell-lines were analyzed by their intensity kinetic profile . Then, we analyzed the overall fluorescence intensity pattern change of the NCI-60 set of cell-lines by heatmap analysis and hierarchical clustering . In gener origin .RS-K series of compounds. These compounds initially showed low fluorescence intensity, but after 24 h, their fluorescence was dramatically increased only in specific set of cell-lines . Moreover, fluorescence response profiles were highly correlated with the probe structure and cancer cell type  exhibited fluorescence turn-on effects in lung and colon cancer cells  showed turn-on responses in lung cancer cells  that up-regulated in KM12 cells (Log2(Fold) >1.5) were selected, and their gene annotation information were analyzed. KEGG pathway and gene ontology analyses identified five pathways that exhibit significant difference  between KM12 cells and all the other NCI60 cells, such as cell communication, hematopoietic cell lineage, urea cycle and metabolism of amino groups and p53 signaling pathway  among 60 cancer cell lines after 24 h pre-incubation.Another cell line specific profile was observed with the ll lines . Compare pathway , S4. Witjackknifed cross-validation  to classify the cancer cell types. LDA is a commonly used classification method especially for high dimensional data for reduction of dimensionality. By applying LDA to kinetic profiles, it was possible to evaluate fold change responses of each chemical probe for optimum cancer cell identification, and generate score function to predict the identity of cells even each probe does not show specific response to single cell line. The minimum fluorescent probe set that could discriminate 9 cancer cell types was chosen by a forward step-wise variable selection algorithm, and 37 differentially responding fluorescent probes were selected . Surprislidation . LDA scolidation . It is wes alone . This rein vitro cancer cell lines, our results provided a clue that combinations of fluorescent probes could distinguish complex and subtle differences of cancer cells. Based on the response pattern, the better identification result was achieved when more fluorophore scaffolds were used, and the fluorescence intensity profiles were highly correlated with the chemical structure of the fluorescent probes and cancer types. These findings demonstrate the practical utility of the diversity-oriented fluorescence library approach in universal cell phenotyping.In short, we report the first fluorescence intensity-based phenotype profiling of 60 human cancer cell-lines (NCI-60) using synthetic fluorescent probes. Structural diversity of the fluorescent probes seems to be a critical factor for the generation of distinct phenotypes, and our results showed that 60 cancer cell-lines are all successfully discriminated in terms of 9 cancer cell origins. The most emphasis in cancer research has been focused on pathogenesis and metastasis, and metastasis research has been stagnated because of a lack of reliable tracking technique that could visualize a specific origin of cancer cells. Fluorescence compounds that probe specific origin of cancer cell could not only provide a new window for metastasis research, but also be utilized for cancer diagnosis and progression monitoring. Although this study has limitations in that we performed identification of cancer cells origin using only The rosamine and BODIPY library compounds were prepared according to previously reported protocols, and the compound structures and characterization data has been reported. The NCI-60 cell-lines were obtained from the National Cancer Institute. All NCI-60 cell-lines were cultured as described in the distributor's manual. Briefly, cells were cultured in a high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% antibiotics.cat function in Matlab to optimize IO processing. To minimize the background effect, only the fluorescence intensity values for area that were positive for Otsu's threshold were used to calculate the average value. The graythresh and im2bw functions in the Matlab image processing module were used to calculate Otsu's threshold and the cell area selection. All quantitative image processing batch jobs were computed using a 16-node PC cluster for 3 days.Mean values of the fluorescence intensities were calculated using Matlab 7.9.0 (R2009b). In our dataset, 4 images represented each experimental condition (2 duplicate×2 images/well). We combined the 4 images using the Linear discriminant analysis (LDA) was performed using fold change values of the fluorescence intensities between 48 hour and 1 hour incubation points. Probe selection was processed by forward-stepwise variable selection algorithum in SYSTAT (version 13).The mRNA expression patterns for the NCI-60 set of cell lines were downloaded from the NCBI gene expression omnibus (GEO) database. The GSE5846 data were analyzed using GenePlex v3.0 DEG finding and Pathway analysis modules .Figure S1Schematics of NCI-60 assay format. We used 384 well plate for high throughput fluorescent imaging. 60 cells were divided into 13 subsets (each set contains less than 5 cell lines), and 2 fluorescent probes are tested with each of the subsets in an individual well plate. Since the culture media evaporate fast in edge wells, first and last two columns were not used for the assay.(TIF)Click here for additional data file.Figure S2SAR of fluorescent probes in phenotype profile. Hierarchical cluster of fluorescent response phenotype reveals structural relationship of fluorescent probe. Fluorescent intensity changes pattern of 557 fluorescent probes (x-axis) against 60 cancer cells (y-axis) were clustered. Fold = (fluorescent intensity after 48 h incubation)/(fluorescent intensity after 1 h incubation).(TIF)Click here for additional data file.Figure S3Fluorescence intensity profile of RS-K series. (a) Fluorescence response profile of RS-K15 in NCI60 cells and fluorescence images of lung cancer cell lines. First and second row images were taken after 1 h and 24 h after probe treatment respectively. All 4 images for each experimental condition are shown. (b) Fluorescence intensity bar graph pattern of RS-K series towards 60 cancer cell line.(TIF)Click here for additional data file.Figure S4Fluorescence intensity profile of RS-E series.(TIF)Click here for additional data file.Figure S5Cell line specific response of RS-E series compounds among renal and colon cancer origin. Fluorescence intensity bar graph of RS-E1, RS-E3, and RS-E7 compounds for cancer cells from (a) colon and (b) renal origin.(TIF)Click here for additional data file.Figure S6Structures of 37 selected fluorescence probes from LDA. Probes were choosed based on step-wise forward automatic variable selection algorithm with alpha-to-enter: 0.150 and alpha-to-remove: 0.150 criteria using SYSTAT v13.(TIF)Click here for additional data file.Table S1Jackknifed cross-validation matrix for three set of fluorescent probes.(XLS)Click here for additional data file.Table S2LDA score function and coefficient of selected fluorescent probes.(XLS)Click here for additional data file.Table S3Selected KEGG pathways based on DEGs of KM12.P-values were calculated by Fisher's exact test.(XLS)Click here for additional data file.Table S4Top 20 biological processes from gene ontology annotation of DEGs in KM12 cell.(XLS)Click here for additional data file.


Mutations in BRAF V600E oncogene (BRAFMT) occurs in 8-15% of colorectal cancer (CRC) patients1. This mutation constitutively activates MAPK signalling, resulting in a proliferative and survival advantage for the tumour cells and oncogenic BRAF status has been linked with poor prognosis2. Despite introduction of the BRAFMT specific inhibitor Vemurafenib in metastatic melanoma3, there is no effective treatment strategy for BRAFMT CRC patients. This study aimed to assess the effectiveness of Ganetespib (HSP90 inhibitor), the multi-kinase inhibitor (CRAF/VEGFR/PDGFR) Sorafenib and the BRAFMT inhibitor Vemurafenib in BRAFMT CRC cell line models.BRAF MT RKO F6-8 (MT/WT) and isogenic wild-type T29 (null/WT) cell lines were used. MTT assays were used to determine IC50 values. Protein expression was determined by Western Blotting. Levels of apoptotic cells were assessed by flow cytometry.The RKO BRAFMT cell line was equally sensitive to Sorafenib and Ganetespib compared to the isogenic BRAFWT clone (IC50 1.4µM vs. 0.9 µM and 0.86 nM vs. 0.79nM respectively). Vemurafenib treatment resulted in a strong decrease in MAPK signalling and showed greater specificity towards BRAFMT cells (IC50 of 2.4µM vs. 3.3 µM) than BRAFWT cells in cell viability assay. Western blotting showed that neither Sorafenib nor Ganetespib had an observable effect in targeting mutant BRAF. Sorafenib did have some effect at targeting downstream MAPK signalling however Ganetespib had no observable effect on targeting the MAPK pathway in BRAF mutant cell lines.. Vemurafenib targeted BRAF and downstream MEK activation in both mutant-type cell lines but not in the wild-type cell lines. We also found that treatment with Vemurafenib induced activation of the STAT3 survival pathway, highlighting a resistance pathway which may be activated in response to treatment in BRAFMT cell lines.Despite the number of compounds which can target BRAFMT cell lines through inhibition of mutant BRAF directly or indirectly through inhibition of other pathways, our studies have shown that Vemurafenib is the most effective treatment strategy in the cell line model tested. Effects of Ganetespib or Sorafenib treatment did not display specificity towards BRAFMT cells alone nor an effect on mutant BRAF. Vemurafenib has shown selectivity towards BRAFMT cells in which a reduction in MAPK signaling is achieved along with induction of apoptosis. Evidence of the emergence of a potential resistance mechanism via STAT3 following Vemurafenib treatment was also found giving insight into the kinome reprogramming event which takes place following treatment.


Adenovirus (Ad) vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue). Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA) contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed. Despite this issue HEK293 was and still remains a major host cell line for Ad vector development including development of master cell banks and development and optimization of Ad-manufacturing processes [The disadvantage with the HEK293 cell line is the potential for the generation of replication-competent adenovirus (RCA) contamination within the viral preparation. This can occur because many first-generation Ad vectors are deleted from nt 400 to 3500 and thus still retain significant homology with the cellular DNA allowing for a double crossover recombination event to take place  Figure . While trocesses .In the 90s, alternate E1 producer cell lines were being evolved . One of Like the 911 cell line, PER.C6 is also based on HER cells. The Ad5 sequence incorporated into the cell genome was the E1A- and E1B encoding sequences, nt 459–3510, under control of the human PGK promoter to compensate for the removal of the 5′ endogenous E1A promoter. Furthermore, the coding region for the minor capsid pIX protein which is in the 3′ region following the E1B 55-kDA coding region was also deleted in this construct. The smaller fragment of Ad5 sequence thus incorporated into the cell line has limited homology to many E1-deficient adenoviral vectors and double homologous recombination is not possible when using an appropriately designed Ad vector, thereby eliminating RCA. However, further studies indicated that rare unconventional recombination can still occur if the Ad vector genome is not perfectly matched. There only needs to be a short stretch of homology between cellular DNA and the vector genome to allow recombination  Figure  resultinDue to the RCA issue and potential licensing issue several other laboratories have developed E1 complementary cells that contain smaller adenoviral E1 DNA segments than HEK293 cells and can propagate vectors to high titers similar to those seen on HEK293 –11 Tabl. All of The requirement of the pIX sequence in first generation Ad vector producer cells has been questioned ,8. Of thApart from the HEK293 cell line which was generated with sheared Ad5 DNA, the main strategy for generating producer cell lines for E1 trans-complementation has involved insertion of the contiguous sequence of E1A and E1B into the cell line genome utilizing DNA plasmid based systems. However, new and novel approaches are being taken to reduce and eliminate RCA, while maintaining trans-complementation. One approach utilized adenoviral E1A and E1B gene integration into the A549 genome at separate locations . This waA unique approach is being explored in our own laboratory as an alternate means to overcome RCA. In this instance, the Ad5 sequence in HEK293 cells would be modified to contain a large ∼8 kb spacer sequence of DNA, inserted at nt3510 . To achiFirst generation Ad vectors are the workhorse vector for many vaccine and gene therapy strategies, although they do have limitations as discussed in the following sections. The E1 complementing packaging cell lines developed to produce these vectors form the basis for all cell lines utilized with other types of genome deleted Ad vectors. The most important aspect to arise from this work is overcoming RCA, and this relies not only on how much of the Ad5 E1 sequences are incorporated into the cell line genome, but also careful design of the vector genomes themselves. Therefore the future for new cell line development is the incorporation of advantageous genes into the genome to improve productivity, adaptability and robustness as described for the 293-VLI model.2.Second generation Ad vectors contain further genome deletions, in the E2 and/or E4 regions see . The E2 In the early-80s and early-90s it was demonstrated that E2 defective Ad vectors ,47 and Etet operator sequences in the CMV promoter region of the E2A cassette. In the presence of tetracycline, E2A is not expressed, but in the absence E2A is expressed. Viral production in 293-C2 is about 10- to 30- fold below those of E2 wild type vectors [E2 complementing cell lines either express E2A (DBP) or E2B  viral products. DBP is required to be expressed at high levels for successful viral replication and therefore most E2A complementing cell lines are prepared with controlling elements. The 293-C2 line  was cons vectors , while E vectors . Other E vectors  and AE1- vectors  rely on  vectors . In the  vectors .It is known that E2B mRNA is expressed at low levels in Ad-infected cells  and therInitial studies with E1/E4 deleted vectors utilized the incorporation of full length E4 into cell lines ,49. VL2-Despite extensive development of second-generation Ad vectors and cell lines throughout 1995 to 2000 many of these vectors have faded into the background. Yields of these vectors can be reduced when compared to respective first generation vectors, depending on the cell line and vector system used. Furthermore comparative studies between E2A or E4 and their respective isogenic E1 deleted vectors indicate that E2A deletions confer limited improvement ,64,65 wh3.in vivo result in long-term, high-level transgene expression combined with negligible toxicity . The ma (review . The req (review ) but theloxP sites flanking the packaging site [To reduce contamination of HAd a helper-dependent system was developed that involved generating a cell line expressing Cre recombinase, 293Cre4, in combination with modifying the HAd to contain ing site . Due to ing site –75 and/oing site ,77 baseding site ,77–79 aning site  and C7 [ing site  , but physical separation of the HC-Ad and HAd through CsCl ultracentrifugation can address this issue. For small preparations this is generally acceptable, however for industrial-scale clinical grade vector production further measures have to be taken , especia4.Within the human population there are high titers of pre-existing neutralizing antibodies against Ad5 and Ad2 serotypes –85 due tHuman subgroup B adenoviruses, in particular, Ad11 and Ad35 are emerging as strong contenders for replicating defective vectors ,98,100. In the second approach new packaging cell lines CRE35G3  and PER.Recently a new strategy for Ad35 has been developed relying on further modification of the Ad vector genome . It has Several non-human serotypes derived from bovine , canine One of the interesting observations to arise from the use of producer cell lines for alternate Ad serotypes is that in many cases there was a shift from developing new producer lines towards further vector genome modifications. While it seemed necessary to generate specialized producer lines for the human subgroup B viruses, once their structure and biology was further delineated, it was realized that it is simpler to incorporate further vector modifications in the E4 region that allow for their propagation on a well-characterized E1 complementing cell line. This paradigm was also utilized with respect to simian based Ad vectors and is worth considering if there is rapid need to move a novel Ad vector from bench to bedside.5.Throughout the last 3 decades a wide array of producer cell lines have been developed for the production of Ad vectors. There are still only a few cell lines approved for cGMP production of replication-defective Ad5 vectors, as the regulatory requirements include a very extensive process of cell banking and testing. As far as we know at the present time only HEK293, PER.C6, N52.E6, 293-ORF6 and 293FLP cell lines have approval for cGMP production of non-replicating Ad vectors, although unmodified A549 cells have been approved for cGMP production of replicating Ad vectors. While other laboratories develop additional cell lines, either as research tools to generate novel Ad vectors, to overcome RCA or licensing issues, regulatory considerations for cell lines should always factor into the terms of moving Ad vectors from bench to bedside in a time appropriate manner.


Neuroendocrine (NE) phenotypes characterize a spectrum of lung tumors, including low-grade typical and intermediate-grade atypical carcinoid, high-grade large-cell NE carcinoma and small cell lung carcinoma. Currently, no effective treatments are available to cure NE lung tumors, demanding identification of biological features specific to these tumors. Here, we report that autophagy has an important role for NE lung tumor cell proliferation and survival. We found that the expression levels of the autophagy marker LC3 are relatively high in a panel of lung tumor cell lines expressing high levels of neuron-specific enolase (NSE), a key NE marker in lung tumors. In response to bafilomycin A1 and chloroquine, NE lung tumor cells exhibited cytotoxicity whereas non-NE lung tumor cells exhibited cytostasis, indicating a distinct role of autophagy for NE lung tumor cell survival. Intriguingly, in certain NE lung tumor cell lines, the levels of processed LC3 (LC3-II) were inversely correlated with AKT activity. When AKT activity was inhibited using AKTi or MK2206, the levels of LC3-II and SQSTM1/p62 were increased. In contrast, torin 1, rapamycin or mTOR knockdown increased p62 levels, suggesting that these two pathways have opposing effects on autophagy in certain NE lung tumors. Moreover, inhibition of one pathway resulted in reduced activity of the other, suggesting that these two pathways crosstalk in the tumors. These results suggest that NE lung tumor cells share a common feature of autophagy and are more sensitive to autophagy inhibition than non-NE lung tumor cells. Lung cancer is currently the leading cause of cancer death in men and women, causing more deaths than colon, breast and prostate cancer combined . Lung caAutophagy is an important cellular recycling process to overcome limited availability of nutrients, which is mediated by a constitutive lysosomal degradation pathway . Under sRecent studies have demonstrated the significance of autophagy in pulmonary epithelial cell proliferation and survival. For example, it has been demonstrated that increased autophagy contributes to the pathogenesis of chronic obstructive pulmonary disease by promoting epithelial cell death . It has Here, we demonstrate that, in a panel of human lung cancer lines, steady-state levels of the autophagy marker, LC3, are relatively high in the cell lines expressing neuron-specific enolase (NSE), a key NE marker in lung tumor ,15. We tμg of streptomycin per ml. Bafilomycin A1 and chloroquine were purchased from Sigma . Torin 1, rapamycin, AKTi and MK-2206 were purchased from TOCRIS , Cell Signaling Technology , EMD Chemicals Inc.  and Active Biochem , respectively.The human lung cancer lines, DMS53, NCI-H209, NCI-H82, NCI-H69, NCI-H889, NCI-H345, SHP-77, A549, NCI-H23, NCI-H460, NCI-H1155, NCI-H358, NCI-H727, NCI-H125 and NCI-H1770 (ATCC), were maintained in phenol red-deficient RPMI-1640  supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin and 100 μg/ml before use. Viral titer was determined by scoring cells resistant to puromycin.The lentiviral pLKO.1-shRNA vector targeting mTOR (Addgene plasmid 8454) was previously described . For lenTo determine cell survival rates, cells were seeded in 24-well plates  at a density of 5,000 cells per well. Cell proliferation and death was determined by counting trypan blue-stained cells using hemocytometer. For cell cycle analysis, cells were washed with ice-cold 0.2% BSA in PBS, resuspended in 250 mM sucrose/40 mM citrate buffer (pH 7.6) containing 0.5% DMSO. Nuclei were prepared, stained with propidium iodide  and analCells harvested at various times were lysed and analyzed for protein concentration using the BCA reagent , as previously described . ProteinTo profile steady-state levels of autophagy in different lung tumor types, we analyzed LC3 levels in 15 human lung tumor cell lines, including 7 SCLC and 8 NSCLC cell lines. In this panel, cell lines were also examined for NSE expression as a marker of NE phenotype. All SCLC cell lines except for DMS53 expressed high levels of NSE whereas among the NSCLC cell lines, only the known NE lines, NCI-H1155 and NCI-H1770 , expressIn this cell line panel, we detected relatively high LC3-I levels in strong correlation with high NSE levels . BecauseIn this cell line panel, we also examined the mTOR and ERK1/2 pathways, which are often deregulated in cancer. However, neither ERK1/2 activity, as indicated by phosphorylation of their activation loop (Thr202/Tyr204 of ERK1 and Thr183/Tyr185 of ERK2), nor activity of the mTOR pathway, as indicated by phosphorylation of 4E binding protein 1 (4E-BP) and the ribosomal protein S6, were significantly correlated with the low LC3-II levels . 4E-BP iTo determine the significance of autophagy in NE lung tumor cells, we examined the effects of inhibition of steady-state autophagy in NCI-H69, NCI-H209 and NCI-H1155 cells using bafilomycin A1 and chloroquine, the lysosomotropic agents that inhibit lysosomal degradation of autophagosome. As expected, these cells exhibited highly increased LC3-II and p62 levels within 48 h in response to the inhibitor treatment , which iSubsequently, we analyzed NCI-H69, NCI-H209 and NCI-H1155 cells in comparison with the non-NE lung tumor cell lines, NCI-H23, NCI-H460 and NCI-H727 for their sensitivity to different doses of bafilomycin A1. We found that bafilomycin A1 could effectively suppress proliferation of these tumor lines regardless of NE phenotypes . HoweverThe correlation between low LC3-II levels and high AKT phosphorylation in NCI-H69, NCI-H209 and NCI-H1155 cells  led us tSince mTOR complex (mTORC) 1 is well known for its antagonizing effect on autophagy ,17 and bAKT can regulate mTORC1 activity via phosphorylation of tuberous sclerosis complex 2 or PRAS40 –29. ConvAKTi and MK-2206 treatments mildly but consistently decreased phosphorylation of 4E-BP1 and S6K1 with the decreases being more significant under the low serum culture condition , suggestThe effect of torin 1, which inhibits both mTORC1 and mTORC2 activity, was confirmed by a lentiviral shRNA construct that was previously used to specifically knockdown mTOR activity in cells . ConsistThis study demonstrates that human NE lung tumor cell lines maintain relatively high LC3 levels and sensitivity to autophagy inhibition when compared with non-NE lung cancer types, suggesting that autophagy may have important roles in NE lung tumors.in vitro, suggesting that autophagy has a role in regulating lung epithelial cell survival  . However0–80% . HIntriguingly, it has been suggested that NSE expression is associated with the degree of tumor malignancy and, thus, NSE has been proposed as a marker for staging and monitoring of NE lung tumor ,35,36. T


We assessed expression of p85 and p110α PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines.Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor, NVP-BEZ235 alone and with an EGFR inhibitor.50s in the nanomolar range and resultant down-regulation of pAKT and pP70S6K. Adding Erlotinib to NVP-BEZ235 resulted in synergistic growth inhibition.p85 and p110α tend to be co-expressed (p<0.001); p85 expression was higher in adenocarcinomas than squamous cell carcinomas. High p85 expression was associated with advanced stage and poor survival. p110α expression correlated with mTOR (ρ = 0.276). In six NSCLC cell lines, addition of rapamycin to LY294002 or NVP-BKM120 was synergistic. Even very low rapamycin concentrations (1 nM) resulted in sensitization to PI3K inhibitors. NVP-BEZ235 was highly active in NSCLC cell lines with ICin vitro, and a dual PI3K/mTOR inhibitor was highly active. Adding EGFR inhibition resulted in further growth inhibition. Targeting the PI3K/AKT/mTOR pathway at multiple levels should be tested in clinical trials for NSCLC.The association between PI3K expression, advanced stage and survival in NSCLC suggests that it might be a valuable drug target. Concurrent inhibition of PI3K and mTOR is synergistic  Lung cancer is the leading cause of cancer-related death worldwide. In the United States alone, about 222,520 new cases were diagnosed and about 157,300 deaths occurred due to lung cancer in 2010 The phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway impacts many aspects of cell growth and survival A PI3K is the most widely implicated type in cancer and will be referred to as “PI3K” in the remainder of this manuscript. PI3K is a heterodimer consisting of a p85 regulatory and a p110 catalytic subunit.The PI3Ks are a family of enzymes that phosphorylate phosphatidylinositol biphosphate to phosphatidylinositol triphosphate. PI3Ks are most often activated by receptor tyrosine kinase (RTK) signaling such as through EGFR, IGF1-R and HER2/neu S6K) and 4E-binding protein (4E-BP). Phosphorylation of 4E-BP leads to the release of the translation initiation factor eIF4E which has been demonstrated to exhibit transforming and anti-apoptotic activites in vitroPhosphatidylinositol triphosphate mediates the activation of AKT PIK3CA, the gene encoding p110α, and changes in phosphorylated AKT (pAKT) expression have been described in premalignant bronchial epithelial cells and NSCLC PIK3CA are relatively infrequent in lung cancer, PIK3CA copy number gain has been reported in 33.1% of squamous cell lung cancer and in 6.2% adeno lung cancer in one large series K-RASin vivo in a mouse model of NSCLC In NSCLC, PI3K/AKT/mTOR signaling is frequently deregulated due to mutations affecting one of its upstream regulators, the EGFR receptor, and other components within the pathway Inhibition of PI3K/AKT/mTOR signaling through pharmacologic and genetic approaches induces antiproliferative effects on certain NSCLC cell lines A PI3K in two large independents cohorts of NSCLC specimens and to assess the association with clinical and pathological variables including previously published mTOR expression. To obtain more precise, objective expression measures, we used a newly developed method of automated, quantitative analysis (AQUA) of tissue microarrays The purpose of our study was to characterize the expression of p85 and p110 α subunits of Class IA NSCLC cohort was obtained from the H. Lee Moffitt Cancer Center . The Moffitt Cancer Center cohort (MTMA) contains cores from primary NSCLC tumors of patients diagnosed between 1991 and 2001. Follow-up time ranged between 0.8 months and 146.4 months, mean follow-up time of 52.3 months. Age at diagnosis ranged from 40.8 to 84.4 (mean age 69 years). The cohort included 54.5% males and 45.5% females.The Yale University cohort (YTMA) was constructed from paraffin-embedded, formalin-fixed tissue blocks obtained from the Yale University Department of Pathology Archives. The specimens were resected between 1995 and 2003, with a follow-up range between 0.1 months and 182.25 months, and a mean follow-up time of 41 months. Age at diagnosis ranged from 21 to 90 (mean age 65 years). The cohort included 51% males and 49% females.TMAs were constructed as previously described TMA slides were stained for each of the two target markers, PI3K p85 and p110α subunits. Staining was performed for AQUA as described previously Images were analyzed using algorithms that have been described JMP version 5.0 software was used . The prognostic significance of parameters was assessed using the Cox proportional hazards model with progression free survival and overall survival as an end point. Univariate survival analyses were depicted using the Kaplan-Meier method. The association between continuous AQUA scores and other clinical/pathological parameters was assessed by analysis of variance. All reported p-values are based on two-sided significance testing. ANOVA and t-test were used to compare continuous measurements.PIK3CA, K-RAS and EGFR are shown in Table S1. The adenocarcinoma cell lines H1650, HCC2935 and HCC827 have wild-type PIK3CA and K-RAS but mutant EGFR. All squamous carcinoma cell lines SK-MES-1, H2170 and SW900 have wild-type EGFR and wild-type PIK3CA. SK-MES-1 and H2170 have wild-type K-RAS. SW900 has mutant K-RAS. We were interested in comparing the effects of PI3K inhibition in adenocarcinoma to squamous carcinoma cell lines and comparing with EGFR mutant to EGFR wild-type cell lines. All human lung cancer cell lines were cultured under standard conditions (37°C in 5% CO2 atmosphere) and grown in RPMI  supplemented with 10% FBS. HBE135-E6E7 cell line, a non-transformed bronchoalveolar cell line, was purchased from the American Type Culture Collection  and grown in Keratinocyte-Serum Free medium supplemented with 5 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, 0.005 mg/ml insulin and 500 ng/ml hydrocortisone.Squamous carcinoma cell line H2170 and adenocarcinoma cell lines H1650, HCC2935 and HCC827 were kindly provided by Drs. John Minna and Michael Peyton (Southwestern University). Squamous carcinoma cell lines SK-MES-1 and SW900 were obtained from American Type Tissue Culture. The characteristics of each cell line including mutational status of 473), phosphorylated p70S6K (Thr389), PI3K p110α, phosphorylated S6 Ribosomal Protein (Ser235/236), PARP  at 1∶1000, and mouse monoclonal anti-human PI3K p85 or anti-caspase-2  at 1∶1000. A monoclonal mouse β-Actin  was utilized as a control to standardize sample loading. Detection of proteins was done with peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies  and ECL (PerkinElmer).Protein concentrations of cell lysates were calculated by the BCA (Bicinchoninic Acid) assay . Proteins (50 µg) were diluted in a sample buffer  and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed by standard methods using the following primary rabbit anti-human antibodies: phosporylated AKT (Ser3 cells were plated in triplicate in a 96 well microtiter plate (BD Bioscience) and allowed to grow for 24 hours to an approximate confluence of 30%. For drug inhibition studies, NVP-BEZ235 and NVP-BKM120  and LY294002  were used to treat lung cells at various concentrations. For combination experiments, the mTORC1 inhibitor, rapamycin and EGFR inhibitor erlotinib (LC Laboratories) were utilized.For cell viability assays, 1×1050 values were determined by the XLfit software .Cell viability was evaluated at 72 hours using the CellTiter-Glo™ Luminescent Cell Viability Assay, according to the manufacturer's instructions ; an equal volume of the Cell Titer-Glo™ reagent was added to the wells and after a 10 minute incubation, luminescence was recorded using a Victor™X multilabel plate reader (Perkin Elmer). The IC50 or IC25 concentrations. The IC50 and IC 25 values of each drug were first obtained from single-drug viability assays and then utilized to design drug combination experiments. The level of synergism was assessed over a wide range of drug concentrations, focusing on concentrations below or at the IC50 for either drug alone. Our results were analyzed for synergistic, additive, or antagonistic effects using CalcuSyn software . Synergistic effect is indicated by a Combination Index (CI) of less than 0.9, additive effect by a CI between 0.9 and 1.1, and antagonistic effect by CI greater than 1.1.The Chou and Talalay combination index analysis method was utilized to analyze results from combinatory drug experiments  and Table S2. Histospots were deemed un-interpretable if they had insufficient tumor cells, loss of tissue in the spot, or an abundance of necrotic tissue. Specimens with less than 5% tumor area per spot were not included in the AQUA analysis.For the MTMA, 166 and 190 specimens were fully assessable with regard to PI3K p85 and p110α subunit expression, respectively. PI3K did not show significant nuclear staining for either subunit, and we therefore analyzed only the cytoplasmic compartment. Staining patterns within the tumor mask within a histospot were highly homogenous for both subunits. AQUA scores ranged from 7.12 to 127.04  for the p85 subunit, and from 5.43 to 138.56  for the p110α subunit. An example of AQUA staining of histospots for p85 and p110α expression is shown in P<0.0001 for p85 and P = 0.0356 for p110α), as shown in , panel A & B.We assessed the associations between PI3K subunit expression and histologic subtype, by unpaired t-tests. Expression of both the p85 and the p110α subunits were significantly higher in adenocarcinomas than in squamous cell carcinomas (P<0.0002 for p85 and P = 0.0266 for p110α), as shown in , panel C & D. High p85 and p110α subunit expression correlated with advanced disease stage of disease , but not with age and gender (not shown).To validate the association between the PI3K subunits and adenocarcinoma, the YTMA was employed. As opposed to the MTMA which was primarily stage I (A and B) lung cancer (92%), the YTMA included 55% stage I, and 45% stage II–IV  lung cancer. For the YTMA PI3K p85 and p110α subunit expression was interpretable for 163 and 168 specimens, respectively. The AQUA scores for this cohort ranged from 4.18 to 120.73  for the p85 subunit, and from 9.17 to 86.91  for the p110α subunit. Unpaired t-test confirmed the findings from the MTMA; expression of the p85 subunit and the p110α subunit were significantly higher in adenocarcinomas than in squamous cell carcinomas (P = 0.0198). AQUA provides continuous output scores, rather than categories of “high” or “low”, as determined by pathologists interpreting standard immunohistochemistry. To visualize the association between continuous PI3K scores and survival, AQUA scores were dichotomized by the median, reflecting the use of routine statistical divisions in the absence of underlying justification for division of expression. Kaplan-Meier survival curves were generated for PI3K p85 subunit expression and survival and P-values were obtained by the Mantel-Cox log-rank method. As shown in , panel E & F, we found a significant association between high p85 expression and poor survival (P = 0.0075). No association was found between p110α expression and survival.By Cox univariate survival analyses of continuous AQUA scores, we found that high p85 expression correlated with decreased survival (P = 0.0000). No such association between p110α subunit expression and survival was noted.On multivariate analysis p85 expression did not retain its independent predictive value, presumably due to its association with histologic subtype and stage. The only variable associated with survival by multivariate analysis was Stage (p = 0.0006), while no association was found between levels of mTOR and p85 subunit.The association between the previously published mTOR expression in vitro activity of PI3K inhibitors was studied in six human lung cancer cell lines. Two PI3K inhibitors were utilized; we studied NVP-BKM-120, a clinical quality PI3K inhibitor being developed by Novartis, and LY294002, a commercially available compound that has been widely used to study PI3K inhibition in the preclinical setting. Cells were treated with either NVP-BKM120 at concentrations ranging from 0.01 nM to 8,200 nM or LY294002 at concentrations ranging from 6.4 nM to 20,000 nM. All experiments were done in triplicate. After 72 hours incubation, cell viability was evaluated, and the IC50 was calculated for each cell line, and averaged for repeat experiments. As shown in 50 for NVP-BKM120 and LY294002 inhibition ranged from 716 nM to 1265 nM and 4123 nM to 11925 nM, respectively.Due to the association between PI3K and advanced stage and poor survival, the 50 of these two drugs, respectively, in NSCLC cells. 13% growth inhibition was seen at 10 µM NVP-BKM120 but an IC50 could not be reached at higher concentrations, and a mere 25% growth inhibition was obtained with 50 µM LY294002 treatment.To test whether growth inhibition inflicted by NVP-BKM120 and LY294002 are specific or at least enhanced in malignant compared to normal cells, we used an immortalized, non-transformed bronchoaveolar cell line, HBE135-E6E7. These cells were derived from normal bronchial epithelium taken from a man undergoing lobectomy for squamous cell carcinoma Figure S1).Given that the expression levels of drug targets sometimes predict drug sensitivity, we studied the association between the degree of sensitivity/resistance to NVP-BKM120 and LY294002 and PI3K levels. We assessed the two PI3K subunits and total and phosphorylated AKT by immunoblotting. No clear association was found between pretreatment levels of PI3K (p85 or p110α) or total and phosphorylated AKT and sensitivity/resistance to NVP-BKM120 or LY294002 (S4). We then studied synergism between LY294002 and rapamycin. Concentrations of 5000 and 2500 nM of LY294002, were combined with a range of concentrations of rapamycin  in six lung cell lines. Synergism was seen in all six cell lines at all concentrations of LY294002 with all three concentrations of rapamycin as shown in Table S3. Resistance to PI3K inhibition has been noted in different diseases and attributed to numerous mechanisms. Constitutive PI3K pathway activation could result from AKT activation by mTOR-C2 or mTOR activation by MAP kinase pathway members despite specific PI3K drug inhibition. Due to the co-expression between p110α and mTOR seen in our clinical specimens, we studied synergism between the mTOR inbitor rapamycin and NVP-BKM120 and rapamycin and LY294002. Concentrations of 1000 and 500 nM of NVP-BKM120 were combined with a range of concentrations of rapamycin  in six NSCLC cell lines. Synergism was seen in five of the six cell lines at all concentrations of NVP-BKM120 with all three concentrations of rapamycin, and in the sixth cell line (H1650), synergy was seen at higher concentrations of NVP-BKM120 , and viability was measured at 72 hours with the Cell Titer Glo assay. As shown in Table S6, synergism was seen in all six cell lines studied, including SW900 and SK-MES-1 which are highly resistant to erlotinib alone. Notably, in all four erlotinib sensitive cell lines  synergy was relatively more enhanced at higher concentrations of erlotinib (Table S6). A graphic example of the combined effect of these two drugs in H2170 and HCC2935 is shown in and Table S7. Of note, the majority of erlotinib concentrations used in these experiments are well below the reported steady-state level of erlotinib achieved in actual patients which has been reported as 1200+/−620 ng/ml Our results so far show that mTOR blockade is necessary to enhance the inhibition of the PI3K pathway. Given that inhibiting only one or two steps of the survival and proliferation pathways often proves to be therapeutically insufficient due to the various escape mechanisms, we studied the efficacy of multi-level targeting by adding EGFR tyrosine kinase inhibition to dual PI3K/mTOR inhibition in the same set of six NSCLC cell lines. Cells were treated with the PI3K inhibitor NVP-BEZ235 alone or combined with erlotinib, at their respective single drug ICPIK3CA, the gene encoding p110α, have been described in lung cancer, and a limitation of our study is that the PIK3CA genetic status is unknown in the tumor specimens of our cohorts. However, the expression of p85 might be a more valuable indicator to study, as several studies have demonstrated that p110α that is expressed in excess of p85 is unstable and rapidly degraded when not bound to p85 PIK3CA status. Furthermore, p85 has been proposed to have a regulatory function by associating with proteins other than p110 α such as IRS-1 and PTEN Deregulation of the PI3K/AKT/mTOR signaling pathway has been demonstrated in NSCLC. In this study, we assessed the expression of PI3K subunits p85 and p110α in NSCLC tumor specimens. In the two cohorts we studied there was higher expression of p85 in adenocarcinoma compared to squamous cell carcinoma across all stages. High p85 expression was prognostic of decreased survival on univariate analysis, but not on multivariate analysis, presumably due to the strong association with disease stage. Mutations or copy number gains of Our findings are consistent with previous observations in a smaller cohort that included 73 cases of primary NSCLC which indicated that high p85 expression was associated with higher tumor grade and metastatic disease 308 and Ser 473. Increasing evidence has emerged that a rapamycin-insensitive mTOR complex is the kinase responsible for AKT activation resulting from phosphorylation on Ser 473The association between high p85 expression and disease aggression demonstrated by our data and corroborated by results published by other investigators pointing to the role of PI3K in cancer, suggests that PI3K might be a valuable therapeutic target in NSCLC and warrants further investigation using novel and more effective PI3K inhibitors, such as those studied here. However, resistance to PI3K inhibition has been attributed to numerous mechanisms including negative feedback loops. One of the events downstream of PI3K activation is the activation of AKT through phosphorylation on ThrWhile our AQUA data show a higher expression of p85 and p110α in adenocarcinoma compared to squamous cell carcinoma, it does not appear in the limited number of cell lines that were studied that adenocarcinoma cell lines are more sensitive to PI3K inhibitors than squamous carcinoma cells.m-TOR inhibitors like rapamycin and its analogues  have cytostatic properties in preclinical models 50s in the nanomolar range and led to downregulation of pAKT and pP70S6K. This result is consistent with the effects of NVP-BEZ235 in NSCLC cell lines recently published by other investigators Dual inhibitors of PI3K and mTOR have demonstrated promising activity in a number of malignancies In the limited number of lung cancer cell lines that we studied the response to co-inhibition by PI3K inhibitors and rapamycin or to the dual inhibitor, NVP-BEZ235, was independent of wild-type or mutant EGFR status. This observation differs somewhat from a previous report by Faber et al. 50s for erlotinib which spanned four logs between the most sensitive and the most resistant cell lines, erlotinib potentiated the growth inhibition by NVP-BEZ235 in all cell lines studied. These results support a potential therapeutic role of co-targeting of EGFR and the PI3K pathway and suggest that this approach should be evaluated further for patients with NSCLC. Of note, the concentrations of erlotinib used in majority of these experiments were well below the reported steady state level of erlotinib achieved in actual patients We explored the possibility of targeting the PI3K/AKT pathway at multiple levels by adding the direct EGFR tyrosine kinase inhibitor erlotinib to the dual inhibition of PI3K and mTOR by NVP-BEZ235. Erlotinib is approved by the FDA as single agent therapy for NSCLC after first and second line chemotherapy In summary, here we showed that PI3K expression is associated with advanced stage and decreased survival in NSCLC, suggesting that it might be a good drug target for this disease. The p110α subunit was strongly co-expressed with mTOR. Concurrent inhibition of PI3K and mTOR was synergistic in all NSCLC cell lines studied and resulted in growth inhibition and apoptosis. It appeared that minimal amount of rapamycin in the nanomolar range was sufficient to potentiate the effect of the PI3K inhibitors, LY294002 and NVP-BKM-120. This could potentially translate into decreased toxicity and better clinical tolerability of the drug combination. Dual inhibition of PI3K and mTOR by NVP-BEZ-235 is promising and should be further evaluated in clinical trials for patients with NSCLC alone and in combination with EGFR inhibitors.Figure S1Expression of PI3K (p85 or p110α) and total and phosphorylated AKT in NSCLC lysates. Pretreatment PI3K, AKT and phosphorylated AKT levels were variable among cell lines and there is no apparent association between these levels and sensitivity/resistance to NVP-BKM120 or LY294002.(TIF)Click here for additional data file.Table S1Characteristics of cell lines. References for mutational status of cell lines: http://www.sanger.ac.uk/perl/genetics/CGP/cosmic/http://www.atcc.org.(XLS)Click here for additional data file.Table S2AQUA Score Distribution.(XLS)Click here for additional data file.Table S3Combination Indices (CI) for NVP-BKM120 and rapamycin or LY294002 and rapamycin in six lung cancer cell lines. (Synergistic combinations are highlighted in blue.)(XLS)Click here for additional data file.Table S4Viability data for NVP-BKM120 and rapamycin or LY294002 and rapamycin in H2170 and SW900 lung cancer cell lines.(XLS)Click here for additional data file.Table S550 of Erlotinib in a panel of six lung cancer cell lines.IC(XLS)Click here for additional data file.Table S6Combination Indices (CI) for NVP-BEZ235 and Erlotinib in six lung cancer cell lines. (Synergistic combinations are highlighted in blue.)(XLS)Click here for additional data file.Table S7Viability data for NVP-BEZ235 and Erlotinib in H2170 and HCC2935 cancer cell lines.(XLS)Click here for additional data file.


We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.The majority of melanoma cell lines were either sensitive  or hypersensitive  to E6201. This sensitivity correlated with wildtype in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.Our data demonstrate that E6201 elicits a predominantly cytocidal effect  Melanoma is the most lethal form of skin cancer. The prognosis for patients with metastatic disease is poor, with a median survival of 4–6 months and 5-year survival of 16% for patients with distant metastases. This, tBRAF-mutant metastatic melanoma, albeit tempered by the rapid emergence of resistance prior to being imported into CalcuSyn for calculation of the combination index. Any effect values that were less than 0  were set to 0.001 for analysis. As these values were artificially set at 0.001, any combination index values that were generated from drug combinations that were set to 0.001 were excluded from graphs of the combination index values. A combination index value of 1 indicates additivity, values < 1 indicate synergism, and values > 1 indicate antagonism.PTEN: Phosphatase and tensin homolog; Ras: Rat sarcoma; SRB: Sulforhodamine B.Akt: Protein kinase B; CI: Combination index; DMSO: Dimethyl sulfoxide; ELISA: Enzyme-linked immunosorbent assay; ERK: Extracellular signal-regulated kinase; IGF-1R: Insulin-like growth factor 1 receptor; MAPK: Mitogen-activated protein kinase; MEK: Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; mTOR: Mammalian target of rapamycin; PI3K: Phosphoinositide 3-kinase; This study was supported in part by a grant and drug from Eisai Inc., Andover, Massachusetts, USA. JWu and KN are both employees of Eisai Inc. JWa is an employee of H3 Biomedicine Inc. H3 Biomedicine Inc. is a subsidiary of Eisai Inc.in vitro experiments, including proliferation assays, cell cycle and cell death analyses, E6201/LY294002 combination studies and Western blots. SB also helped to draft the manuscript. DL performed some of the Western blots and helped to draft the manuscript. CW performed proliferation assays, cell cycle and cell death analyses, E6201/LY294002 and E6201/Rapamycin combination studies and Western blots. AW performed some of the Western blots and helped to draft the manuscript. JWu, JWa and KN carried out the xenograft experiments, participated in the study’s design and helped to draft the manuscript. PP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.SB carried out many of the  Figure S1. Efficacy of MEK1/2 inhibition with E6201. Western blots demonstrating phosphorylated ERK1/2 levels in our panel of melanoma cell lines following treatment with either vehicle (0.05% DMSO) or 500 nM E6201. Briefly, 500,000 cells from each cell line were plated in duplicate in a 6-well plate on day 0. The next day cells were washed twice with PBS and serum-starved in DMEM containing 0.2% FBS. Sixteen hours after serum starvation, cells were treated with either 0.05% DMSO or 500 nM E6201. After 6 hours of treatment protein lysates were collected. 30 μg of total protein were analysed on a 12% SDS-PAGE gel. Phosphorylated ERK1/2 protein was probed for with a phospho-specific antibody from Cell Signaling Technology . Immunoblots were then stripped and re-probed for total ERK1/2  and tubulin .Click here for file Figure S2. Efficacy of PI3K inhibition with LY294002. Western blots demonstrating phosphorylated AKT (serine 473) levels in UACC647, UACC558, UACC903, MM622, WM35 and MM540 cell lines following treatment with either vehicle (0.15% DMSO) or 20 μM LY294002. Briefly, 500,000 cells from each cell line were plated in duplicate in a 6-well plate on day 0. The next day cells were washed twice with PBS and serum-starved in DMEM containing 0.2% FBS. Sixteen hours after serum starvation, cells were treated with either 0.15% DMSO or 20 μM LY294002. After 6 hours of treatment, protein lysates were collected. 30 μg of total protein were analysed on a 12% SDS-PAGE gel. Phosphorylated AKT protein was probed for with a phospho-specific antibody from Cell Signaling Technology . Immunoblots were then stripped and re-probed for total AKT  and GAPDH .Click here for file Figure S3. LY294002 Single Agent Concentration Response Curves. Concentration response curves of UACC647, UACC558, UACC903, MM622, WM35 and MM540 melanoma cell lines to the PI3K inhibitor LY294002. The IC50 of LY294002 for each cell line is provided in the legend.Click here for file Figure S4. Concentration response curves for E6201 and LY294002 combinations normalized to DMSO. Concentration response curves of UACC647, UACC558, UACC903, MM622, WM35 and MM540 melanoma cell lines to increasing concentrations of E6201 (3 nM to 3 μM) in combination with LY294002  treatment.Click here for file


STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines  by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both  Liver cancer is one of the most common cancers worldwide. Based on a global cancer statistics study, liver cancer represents the fifth most frequently diagnosed cancer and the second most frequent cause of cancer death in menHCC had long been diagnosed at an advanced stage with progressive liver damage and there were no known effective therapeutic options. During the past twenty years, considerable progress has been achieved in diagnosis, therapies and life expectancy. Effective treatments include surgical resection, radio-frequency ablation, liver transplantation and percutaneous ablationPatient-derived xenograft (PDX) models have been regarded as valuable models for oncology drug development2 and all efforts were made to minimize suffering.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of AAALAC international. The protocol of human primary tumor establishment was reviewed and approved by Shanghai ChemPartner's IACUC (Permit Number: X998HL001). All HCC tumor tissues used for PDX model establishment were obtained from Nantong Tumor Hospital in accordance with protocols approved by the Institutional Ethics Committee of Nantong Tumor Hospital. All participants provided their written informed consent form. All tumor tissues were surgically resected and collected for the purposes of the study, and were appropriately de-identified except some information which was listed in 2.6–8 week-old female SCID mice  were used for human HCC fragments implantation. Mice were maintained under specific-pathogen-free (SPF) conditions. The fresh tumor tissue specimens were rinsed twice with Hank's balanced salt solution (HBSS) containing antibiotic and transported on ice. As described previously3. In vitro primary culture of HCC cells was initiated as described previously2. The culture medium was RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen), 10 µg/ml human recombinant insulin and 1% Antibiotic-Antimycotic  containing amphotericin B, streptomycin and penicillin. Cells were subcultured at 70–80% confluence and used for further experiments after the passage 20. These cell lines were deposited at China Center for Typical Culture Collection (CCTCC). The access numbers could be found in The tumor-bearing mice were sacrificed and the tumors were removed when the tumors grew to 500-700 mm2O2 solution to quench endogenous peroxidase activity, and blocked with 4% goat serum  in PBS. The cells were incubated with the following primary antibodies at room temperature for 60 min: anti-AFP , anti-CA19-9 , anti-CEA , anti-ki-67 , anti-Vimentin  and anti-cytokeratin . Anti-IgG1 was used as negative control. Then the slides were developed by DAB methods  and mounted. The slides were observed and visualized by Eclipse Motorized Advanced Research Microscope .For morphology studies, cells were plated in 10-mm plates and grew to approximately 90% confluence. Phase images were obtained via inverted phase-contrast microscope . For immunohistochemical staining, tumor cells were collected by centrifuging as cell pellet. Slowly added neutral formalin into the tube and fixed the cell pellet overnight. Then processed the cell pellet to paraffin block for embedding which could be used for making histological sections. After de-paraffinization and antigen retrieval procedure, cells were permeabilized with 0.1% Triton X-100, incubated with 0.3% HThe chromosome specimens of the established HCC cell lines were valued as previously describedGenomic DNA from tumor cells was isolated using AxyPrep Multisource Genomic DNA Miniprep Kit  and 9 STR loci were analyzed by AmpF/STR® Identifiler® PCR Amplification Kit .After establishment, growth kinetics of the tumor cells was obtained by seeding the cells at the density of 2000 cells/well into 96-well plates. The population doubling time was measured as previously described 6) were pre-seeded into T75 tissue culture flasks for 24 h. The harvested cells were washed with PBS and fixed with ice-cold 70% ethanol at −20°C overnight. The fixed cells were resuspended in PBS (containing 0.2 mg/mL RNAse and 10 µM PI) and incubated in the dark for 30 min at 37°C. A FACSCalibur flow cytometer  was used for flow cytometric analysis.The cell cycle distribution was analyzed by flow cytometry. Cells  according to the manufacturer's instructions. Reverse transcription was carried out with 0.5 µg of total RNA with PrimeScript™ RT Master Mix  and oligo (dT) 15 primer . Primer sequences used were obtained from the Primer-Bank database and sequences were provided in 50 was determined by XLFit software.The cell populations were further characterized by analysing their reeponse to chemotherapeutic agents. Sorafenib , docetaxel , doxorubicin (Sigma), vinblastine (Sigma), tarceva , gemcitabine , oxaliplatin (Sigma) and cisplatin (Sigma) were added into triplicate wells at the same time of cell inoculation. A solvent control was performed at same time as the final DMSO concentration in medium was 0.5%. Cell viability was measured by CellTiter-Glo method  after 96 h incubation and IC3, the mice were euthanized and the tumors were collected. The viable tumor tissues were fixed in 4% formaldehyde, paraffin embedded and diagnosed (H&E staining).6–8 week-old female SCID mice were used for the xenograft tumor generation. As described previously in vivo efficacy study. About 5.0×106cells was injected into the mice to generate xenograft tumor. When the tumor grew to 100–250 mm3, twenty tumor-bearing mice were selected and divided into two groups. One group was treated with sorafenib 30 mg/kg intragastrically daily for 3 weeks. The other group was vehicle control group. The tumor growth was measured twice a week. At the end of test, tumor tissues were fixed in 4% formaldehyde, paraffin embedded and diagnosed (H&E staining).LIXC006 and LIXC012 cell lines were used to perform t tests. Values of p<0.05 were considered to be statistically significant.We used SPSS 18.0 software (IBM) for statistical analyses. Data were expressed as means ± SEM or S.D and were compared using one-way analysis of variance (ANOVA) followed by Student's Human tumor tissues were obtained from surgical resection of Chinese patients. SCID mice were used to generate PDX models, and then primary culture of tumor cells was performed for cell lines establishment. We used 20 tumor tissues from different patients and got 7 PDX models, and then established 7 HCC cell lines which were designated as LIXC002, LIXC003, LIXC004, LIXC006, LIXC0011, LIXC012 and CPL0903. The information of patient related to cell line accordingly was listed in After establishment, cell lines were banked at low passages and cells passaged at least 20 times were used for characterization. All cell lines were free of contamination by bacteria or mycoplasma. Their growth kinetics was shown in Chromosome aberrations could be found in all cell lines. Most cell lines had more than 46 chromosomes except LIXC011 which had 35 chromosomes (Table.3). The result of immunohistochemical staining for some biomarkers was shown in βcatenin and cMet are two proto-oncogenes. Overexpression of β-catenin is associated with many cancers including HCCβcatenin than normal HL 7702 cell (P<0.05). cMet, which encodes hepatocyte growth factor (HGF) receptor, was up-regulated in LIXC006, but down-regulated in LIXC011. HGF- cMET axis activation was implicated in cellular invasion and metastases27kip1 was known as a protein of cyclin-dependent kinases inhibitor (CDKI)PTEN, which acts as a tumor suppressor gene through its phosphatase action, was often found lost its copies commonly in human cancersPTEN mRNA than HL 7702 (P<0.05)  .B. Bcl-2l of HCC .C. RetinOur results demonstrated the diversity of these cell lines in biomarkers and tumor related genes.50 results were demonstrated in 50 of sorafenib ranged from 0.93 to 13.57 µM in 6 cell lines. LIXC003 tolerated oxaliplatin but was sensitive to docetaxel. LIXC004 was resistant to docetaxel. LIXC12 was the most sensitive cell line among 7 cell lines to all drugs. We failed to detect IC50 in CPL0903 according to our dilute protocol. The cells might be clinical multiple drug resistant tumor cells. LIXC011 cell possessed moderately multiple drug resistance property with higher IC50 quantity for several drugs.New cell lines were used for validation of anti-tumor drugs. 8 chemotherapeutic drugs, including sorafenib, docetaxel, doxorubicin, vinblastine, tarceva, gemcitabine, oxaliplatin and cisplatin, were tested for their anti-proliferation effect. ICIn vivo tumorigenesis study was performed with all cell lines using SCID mice. Only LIXC006 and LIXC012 showed better tumorigenic ability. LIXC006 cell formed tumor with higher cell concentration and matrigel was necessary. LIXC012 cell formed xenograft tumor within 30 days, with lower cell concentration, no matter with or without matrigel. Tumor growth curve was plotted and presented in 3, the tumor tissue was collected and H&E staining was performed, as shown in in vivo drug reponse assay indicated that sorafenib showed anti-tumor efficacy in xenograft tumor of LIXC006  and its homologs, which function via nuclear RA receptors (RARs), are potent regulators of gene expression. Compounds that target these receptors are powerful anticancer drugsWe characterized the mRNA expression profile of some tumor related genes including proto-oncogenes and tumor suppressor genes. Unexpectedly, 3 cell lines overexpressed in vitro, so was in mice bearing xenograft tumor of LIXC006. LIXC012 cell, the most sensitive to sorafenib with 0.929 µM as IC50, lost its sensitivity to the drug in vivo. It indicated that results based on in vitro should be carefully validated. Plenty of work could be carried out in future to figure out the relevance between cell molecular profile and drug sensitivity.Cell systems as tools and models for basic clinical research have been progressively increased during last few decades. We performed drug validation using the new HCC cell lines and got very interesting results. 8 chemotherapeutic drugs, including doxorubicin, gemcitabine, sorafenib and so on, were validated in our study. The results were quite individual. LIXC006 and LIXC012 were more sensitive to all drugs among the 7 cell lines. Some cell lines, such as LIXC011, showed multiple drug resistance potential towards all tested drugs. Sorafenib, a multi-kinase inhibitor which has been approved worldwide as a new target agent for HCCThe interest of PDX models in preclinical research is more and more emphasized. Since it could reproduce the tumor microenvironment and tumor cell interactions with the innate immune system, PDX models offer a powerful tool for studying tumor biology and for evaluating anticancer drugsFile S1Contains the files: Table S1. The access number of novel cell lines in China Center for Typical Culture Collection (CCTCC). Table S2. The primer sequences. Method: Electrochemiluminescence immunoassay (ECLI). Table S3. Concentration of AFP, CEA and CA19-9 in culture medium.(DOCX)Click here for additional data file.