Intervertebral disc degeneration (IDD) is characterized by excessive apoptosis of nucleus pulposus (NP) cells and hyperactive extracellular matrix (ECM) catabolism. Our previous studies revealed the relationship between human islet amyloid polypeptide (hIAPP) and NP cell apoptosis. However, the role of hIAPP aggregates in IDD has not yet been investigated. This study aimed to determine whether the accumulation of hIAPP aggregates promotes IDD progression. The aggregation of hIAPP increased in human NP tissues during IDD. The deposition of hIAPP aggravated the compression-induced IDD that promoted NP cell apoptosis and ECM degradation via IL-1β/IL-1Ra signaling in an ex vivo rat disc model. Moreover, neutralizing IL-1β augmented the protective effects of hIAPP overexpression by decreasing hIAPP aggregation in human NP cells. These results suggest that the aggregation of hIAPP promotes NP cell apoptosis and ECM degradation ex vivo and in vitro by disrupting the balance of IL-1β/IL-1Ra signaling. Intervertebral disc degeneration (IDD), is caused in part by a protein, hIAPP, that is secreted by the cells that make insulin, a blood sugar regulator. In IDD, the nucleus pulposus in disc degenerate, causing low back pain. Recent research linked formation of hIAPP aggregates with death of intervertebral disc cells. Given inflammation’s known role in IDD, Cao Yang at the Huazhong University of Science and Technology, Wuhan, China, and colleagues investigated how hIAPP and inflammation interact in IDD. Using a method they developed to model IDD by applying compression to cultured rat intervertebral disc cells, they showed that accumulation of hIAPP aggregates triggered inflammation and accelerated cell death. Blocking the inflammation decreased hIAPP aggregation and slowed IDD progression. Methods to disrupt hIAPP aggregation hold promise for treatment of IDD. Many factors, such as obesity, smoking and diabetes mellitus (DM), are thought to be involved in IDD progression3. However, the etiology and pathogenesis of IDD remain unknown. The intervertebral disc (IVD) functions as a buffering and conjunction organ for bearing mechanical loads and maintaining spinal stability; it is composed of the inner nucleus pulposus (NP) and the outer annulus fibrous (AF)4. IDD is characterized by the depletion of resident cells and the degradation of the extracellular matrix (ECM)5. An increase in the apoptosis level of NP cells contributes to excessive cell death, which accelerates the progression of IDD. Recent studies have reported that the amelioration of NP cell apoptosis exerts a therapeutic effect on the progression of IDD8. However, further research on the prevention and reversal of IDD progression is necessary.Low back pain (LBP) associated with intervertebral disc degeneration (IDD) is a common cause of disability worldwide; ~80% of people will suffer from LBP at some point in their lives10. Recent studies have revealed that the aggregation of hIAPP contributes to cell death by inhibiting autophagy, inducing oxidative stress, and promoting inflammatory cytokine release and cell membrane disruption15. IAPP is a normally soluble protein that is released by cells and can form insoluble toxic aggregates11. Previously, we showed that hIAPP expression is related to the development of IDD and that IAPP may regulate ECM metabolism and control the crosstalk between apoptosis and autophagy in NP cells17. Many studies have reported that hIAPP aggregates are toxic and result in islet cell apoptosis in vitro and in vivo. The aggregation of hIAPP has been identified in the pancreas and other extra-pancreatic tissues18. Previous studies have revealed that extracellular components, such as glycosaminoglycan, can accelerate the deposition of hIAPP, especially in an acidic environment20. NP tissues are abundant in the ECM, and a relatively low pH has been confirmed in NP tissues during IDD21. This suggests that hIAPP aggregates may play an important role in IDD progression. However, the aggregation and deposition of hIAPP in NP tissues during IDD has not yet been investigated.Human islet amyloid polypeptide (hIAPP), a 37–amino acid polypeptide, has been confirmed to damage cells through the formation of aggregates24. The proinflammatory cytokine interleukin-1β (IL-1β) is highly expressed in degenerative IVD tissues and has been proposed to be a major mediator of inflammation in the progression of IDD25. The stimulation of IL-1β leads to a significant increase in the apoptotic rate in NP cells and AF cells26. Moreover, as a proinflammatory mediator, IL-1β regulates the production of various cytokines, chemokines, and matrix metalloproteinases (MMPs), as well as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), resulting in inflammation and ECM degradation during IDD26. In degenerative disc tissues, IL-1β increases rapidly, while IL-1 receptor antagonist (IL-1Ra) decreases consistently28. An imbalance between IL-1β and IL-1Ra accelerates the progression of IDD to some degree. A recent study showed that hIAPP aggregates induce FAS upregulation, caspase activation, and cell apoptosis by stimulating the expression of IL-1β and reducing the release of IL-1Ra29. Moreover, IL-1β impaired IAPP processing, thereby potentiating the formation of aggregates and facilitating the activation of cell death signaling29. We surmise that IL-1β/IL-1Ra signaling may be involved in IDD by interacting with the deposition and aggregation of hIAPP.Accumulating evidence suggests that inflammation plays a critical role in the pathogenesis of IDDAs of now, the role of the deleterious hIAPP aggregates in IDD progression has not yet been investigated. We hypothesized that hIAPP aggregates may accelerate NP cell apoptosis and ECM degradation and may also be related to IL-1β/IL-1Ra signaling. To test this hypothesis, we conducted our experiments using tissue samples and an ex vivo rat disc model Fig. . The depThe experimental protocols were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from all participants involved in our study. Degenerative human NP tissues were collected from patients who underwent disc excision and spinal fusion surgery for IDD, while normal human NP tissues were obtained from patients who underwent deformity correction surgery for scoliosis. Detailed information for each patient is listed in Supplemental Table 2 to simulate the physiologically hypoxic disc environment. The culture medium was replaced twice each week, and the cells from the second or third passage were prepared for the following experiments.Isolated NP tissues were cut into pieces and enzymatically digested in 0.2% type II collagenase and 0.25% trypsin  for 3 h. After being filtered and washed in PBS, the suspension was centrifuged, and the isolated cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 15% fetal bovine serum  and 1% penicillin/streptomycin . The cells were incubated at 37 °C under 5% O2) with saturated humidity. The culture medium was replaced twice each week.IVDs were collected from Sprague-Dawley rats  with the ethical approval of the Animal Experimentation Committee of Huazhong University of Science and Technology. Caudal discs with complete endplates were isolated and cultured in DMEM containing 15% fetal bovine serum and 1% penicillin/streptomycin . The osmolarity of the culture medium was adjusted to 400 mOsm, and physiological conditions were approximated by the addition of 1.5% of a 5 M NaCl and 0.4 M KCl solution. Samples were incubated under a hypoxic atmosphere  of double-distilled water to maintain a humidified atmosphere. After mixed compressed air was pumped into the compression apparatus, it was applied to deliver 1.0 MPa static compression for 2 weeks.IVD organs or NP cells were cultured in a compression apparatus with a pressure of 1.0 MPa, as described previously32.The NP tissues or rat discs were washed in PBS, fixed with buffered formaldehyde  for 12 h, decalcified in a 10% formic acid solution, dehydrated by ethanol, and embedded in paraffin. The specimens were sectioned at 4 µm, and the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE), safranin-O/fast green, Masson’s trichrome or alcian blue. The histological grades of the specimens were assessed according to standards established previously17. Briefly, NP cells or tissues attached to slides were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100  in PBS. The slides were washed in PBS, blocked with 2% bovine serum albumin in PBS for 2 h at 37 °C and then incubated with primary antibodies against caspase-3 , caspase-9 , IL-1β , IL-1Ra , COL2A1 , and FAS . After being washed twice, the slides were subsequently treated with a secondary goat anti-rabbit antibody  at 37 °C for 2 h. Nuclei were costained for 5 min with 0.1 g/mL 4-6-diamidino-2-phenylindole . Images were captured under a microscope .Immunofluorescence analysis was performed, as previously describedTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to assess cell apoptosis. Cells were fixed in 4% paraformaldehyde for 1 h and then treated with 0.5% Triton X-100 for 10 min. After being washed with PBS, the cells were incubated with reagents from a TUNEL reaction kit  according to the manufacturer’s instructions. Thioflavin S (Th-S) staining was used to evaluate the deposition of hIAPP aggregates. After being washed with PBS, the fixed cells were incubated with Th-S solution . All of the images were captured under a fluorescence microscope .Total RNA was extracted from NP cells with Trizol reagent  and then reverse-transcribed and amplified by RT-qPCR according to standard protocols. The sequences of the primers used for RT-qPCR are listed in Supplemental Table 16. The following antibodies were used: hIAPP , FAS , FASL , Bax , Bcl-2 , VDAC1 , cyto-c , cleaved caspase-3 , cleaved caspase-9 , MMP3 , MMP9 , MMP13 , ADAMTS5 , SOX9 , aggrecan , COL2A1  and GAPDH . Horseradish peroxidase-conjugated secondary antibodies  were used, and the protein bands were visualized and detected using an enhanced chemiluminescence system. GAPDH was used as a loading control. The experiment was performed in triplicate.Proteins were isolated from cells or tissues using a Western cell lysis kit (Beyotime) according to the manufacturer’s instructions. The procedures and analysis were performed using standard protocolsg for 20 min to remove dead cells and debris. The IL-1β or IL-1Ra content in the supernatant was analyzed using an ELISA kit for IL-1β or IL-1Ra  according to the standard protocol. The experiment was performed in triplicate.The cell culture supernatant was collected and centrifuged at 1000 × 33. The shRNA sequences are listed in Supplemental Table NP cells were seeded and cultured in six-well plates and reached 70–80% confluence before transfection. For hIAPP overexpression, NP cells were infected with pLVX-mCMV-ZsGreen-PGK-Puro or pLVX-mCMV-ZsGreen-PGK-Puro-hIAPP according to the manufacturer’s instructions. For shRNA knockdown, three shRNA plasmids were designed, and their efficiencies were tested Fig. . To exclt-test and one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test were used to assess the changes in the effects for each experimental group. Statistical significance was set to P < 0.05 and determined using GraphPad Prism 7 software .All experiments were performed independently at least in triplicate, and the data are presented as the mean ± standard deviation (SD). Student’s Histologic staining was used to evaluate the specimens from patients with scoliosis (IS) or IDD diseases. The results indicated that, compared to IS tissues, IDD tissues showed degenerative changes by HE, Masson’s, and alcian blue staining Fig. . To deteTo evaluate the effects of hIAPP aggregates on IDD, we used an ex vivo model of IVD organ culture. rat discs were treated with exogenous hIAPP  and cultured in a compression apparatus to induce IDD progression Fig. . Th-S stTo further investigate the effects of hIAPP aggregates on IDD, the levels of proteins involved in ECM metabolism and cell apoptosis were measured by Western blot analysis Fig. . hIAPP d17. In addition, the effect of neutralizing IL-1β on ECM remodeling and cell apoptosis was evaluated by Western blot analysis  and the downregulation of the expression of anabolism genes  were observed in IDD in our compression-induced degeneration model. Meanwhile, pro-apoptotic proteins  were increased and the anti-apoptotic protein Bcl-2 decreased in compression-induced degeneration. These results indicated that mechanical loading effectively induced IDD in this ex vivo model. Studies have shown that the structure of mouse IAPP is different from that of human IAPP and that mouse IAPP does not form toxic aggregates44. In an ex vivo rat model, exogenous hIAPP was applied to rat discs during compression-induced IDD. The disc samples from the exogenous hIAPP group showed a higher histological grade and obvious characteristics of degeneration.The ex vivo culture system in our study simulated the in vivo IVD environment. The IVD organ culture, including the intervertebral disc and adjacent endplates, allowed nutrients and outer substances to penetrate into the inner disc while leaving the IVD structure and native ECM intact45. Accumulating evidence has suggested that the oligomers rather than the monomers of hIAPP are responsible for cell apoptosis11. The mature monomer form of hIAPP has been found to be nontoxic to cells while prone to aggregation in T2D individuals; however, the underlying mechanism is currently unknown48. Our previous studies showed that the overexpression of hIAPP in human AF or NP cells inhibits apoptosis and that the downregulation of hIAPP expression induces the death of disc cells17. In the present study, the analysis of IVD tissue samples suggested that hIAPP deposited and aggregated in degenerative IVD tissues. In an ex vivo model, the catabolism- and apoptosis-related genes were upregulated, while ECM degradation and excessive NP cell apoptosis were observed to increase along with the number of hIAPP aggregates. These results demonstrated that the formation of toxic hIAPP aggregates might occur as a consequence or as a cause of IDD. Several studies have reported that hIAPP aggregates faster in acidic environments and maintains a soluble nontoxic form at a higher pH44. IDD has been confirmed to be related to an increase in lactic acid levels and the intradiscal pH49. This partly explains the significant deposition of hIAPP aggregates in IDD tissues. However, the underlying mechanisms of hIAPP aggregation in IVD tissues remain unclear and require further study.Human IAPP is always deposited in matrix-abundant tissues and has been confirmed as the primary culprit in pancreatic cell loss12. A recent study showed that IL-1β plays a dual role in hIAPP-induced cell damage29. IL-1β impairs the processing of hIAPP, resulting in the aggregation of its immature form, which is easily deposited in human organs or tissues. Moreover, hIAPP aggregates also stimulate the expression of IL-1β, causing a vicious cycle. During the progression of IDD, the increased level of IL-1β expression has also been detected, and IL-1β has been confirmed as a critical mediator of IDD51. It has been suggested that excessive IL-1β promotes the aggregation of hIAPP during IDD progression and impairs the formation of nontoxic mature hIAPP monomers. Inflammation and ECM degradation play critical roles in the IDD process. Hyperactive ECM catabolism and inflammation have been characterized as the major phenotypes in degenerative IVD tissues24. Accumulating evidence has shown that mechanical compression affects IVD biology and that static compression promotes the progression of IDD52. Moreover, an overload of mechanical compression has been confirmed to induce the expression of proinflammatory cytokines and promote ECM catabolism in NP cells53. During the process of disc degeneration, IL-1β is upregulated, while IL-1Ra levels decrease, leading to hyperactive IL-1β signaling and accelerating the progression of IDD27. Park et al. reported that increased IL-1β levels impair the processing of pro-hIAPP and promote the deposition of immature hIAPP aggregates, which ultimately damages islet cell function29. In our ex vivo disc model, cotreatment with neutralizing IL-1β antibodies (nIL-1β) and hIAPP ameliorated IDD progression under static compression, while cotreatment with IL-1Ra (nIL-1Ra) antibodies and hIAPP accelerated the progression of IDD. Our results suggested that mechanical loading may promote the aggregation of hIAPP in NP tissues by regulating IL-1β/IL-1Ra signaling.The pathogenic mechanism of hIAPP aggregates has been confirmed to be closely related to the production of IL-1β16. This finding may be explained by the observation that hIAPP overexpression increases the formation of nontoxic hIAPP monomers and decreases the ratio of monomeric hIAPP aggregates. The number of hIAPP aggregates and the ratio of IL-1β/IL-1Ra both increased during compression-induced NP cell degeneration. However, the levels of IL-1β and IL-1Ra both decreased when NP cells directly overexpressed with hIAPP, indicating a balance in IL-1β signaling. Moreover, the blockade of IL-1β signaling with nIL-1β reduced the deposition and aggregation of hIAPP, which ameliorated the toxicity of hIAPP aggregates in the nIL-1β group compared to the control group. In contrast, the toxicity of hIAPP aggregates in NP cells was enhanced upon treatment with nIL-1Ra. These results suggested that hIAPP aggregates exacerbated the IDD process by accelerating the imbalance in IL-1β/IL-1Ra signaling.Interestingly, we observed that the overexpression of hIAPP decreased the level of IL-1β, reduced ECM degradation and attenuated apoptosis in NP cells. Consistent with our previous study, the overexpression of hIAPP exerted a mostly protective effect in NP cells in vitro11. A previous study showed that treatment with hIAPP induces FAS expression as well as the transcription of downstream genes54. The classical pathway is mediated by FAS receptor binding, resulting in the activation of the FAS-associated death receptor pathway. Caspase-8 is then activated and released into the cytoplasm, leading to the activation of caspase-9 and caspase-3 or the release of other pro-apoptotic factors, such as Bax, VDAC1, and cyto-C55. Moreover, the intrinsic apoptotic pathway can be elicited by various kinds of cellular stresses. The abnormal aggregation of hIAPP has been demonstrated to induce severe endoplasmic reticulum (ER) stress and oxidative stress as well as the unfolded protein response (UPR)48. Prolonged or severe UPR promotes the degradation of abnormal and normal cellular proteins and the expression of pro-apoptotic genes, ultimately causing cell apoptosis57. Inhibiting ER stress reduces the formation of hIAPP aggregates and ameliorates cellular dysfunction9. Therefore, severe cellular stresses, such as the mechanical loading-induced cellular stress used in our study, are potential mediators of hIAPP-induced NP cell apoptosis. In addition, several studies have reported that hIAPP aggregates enhance cell death by impairing autophagic flux; the activation of autophagy can attenuate the detrimental effects of hIAPP aggregates59. Autophagy is considered to be a cell self-protective mechanism, and impaired autophagic flux has been confirmed to promote the deposition of hIAPP aggregates13. Consequently, we hypothesize that hIAPP damages cells by impairing the balance of cellular protein processing, such as through autophagic degradation and ER stress, which accelerates the formation of hIAPP aggregates. In fact, the pathogenic mechanisms of hIAPP may be involved in the activation of the apoptotic signaling pathway, excessive cellular stresses, autophagic dysfunction, and more. A great deal of research is necessary to explain the underlying mechanisms.Our results showed that hIAPP aggregates accelerated IDD progression and caused excessive cell apoptosis. During the IDD process, hIAPP aggregates may induce NP cell death by activating apoptotic signaling. The immature form of hIAPP, which presents as a toxic molecule that induces death signaling, including the FAS and caspase-3-dependent signaling pathways, is more likely to aggregate and be depositedThe present study has certain limitations. First, the ex vivo model used in our experiments to mimic the IDD process has more advantages than monolayer cell culture, but it still does not fully represent in vivo conditions, especially during long-term IDD progression. Second, exogenous hIAPP treatment was used in our ex vivo experiments because mouse hIAPP cannot form aggregates spontaneously. However, the results would be more convincing if hIAPP transgenic mice has been used in this study. Third, the pathogenic mechanisms of hIAPP aggregates discussed in our study may represent only a small fraction of the mechanisms at play. More specific mechanisms of hIAPP aggregates in the progression of IDD still require further investigation.In summary, we demonstrated that hIAPP aggregation in NP cells is associated with IDD progression. The deposition of the toxic hIAPP aggregates promoted NP cell apoptosis and ECM degradation by disturbing the balance of IL-1β/IL-1Ra signaling in ex vivo and in vitro systems. Therefore, methods aimed at inhibiting the formation of hIAPP aggregates in IVD tissues may prove beneficial for the treatment of IDD.Supplementary Information


This study investigated the impact of immune-related pathway inhibition, among them indolamine 2,3-dioxygenase (IDO), alone and together with immune cells on growth and viability of colorectal cancer (CRC) cells. A panel of patient-derived CRC cell lines with different molecular characteristics  was included. Initial phenotyping of CRC cell lines (n=17) revealed high abundance of immunosuppressive checkpoint-molecules in general, but an individual profile for IDO. Presence of immune-related molecules was independent of the molecular subtype. Selective treatment of CRC cell lines showing high or low IDO expression (n=2 cell lines each) was performed with single agents and combinations of Indoximod, Curcumin, and Gemcitabine with and without the addition of peripheral blood lymphocytes (PBL) in an allogeneic setting. All substances affected CRC cell growth in a cell line specific manner. The combination of Curcumin and Gemcitabine proved to be most effective in tumor cell elimination. Functional read-out analyses identified cellular senescence, after both single and combined treatment. Curcumin alone exerted strong cytotoxic effects by inducing early and late apoptosis. Necrosis was not detectable at all. Addition of lymphocytes generally boosted antitumoral effects of all IDO-inhibitors, with up to 80 % cytotoxicity for the Curcumin treatment. Here, no obvious differences became apparent between individual cell lines. Combined application of Curcumin and low-dose chemotherapy is a promising strategy to kill tumor target cells and to stimulate antitumoral immune responses. Vice versa; their high immunogenicity forces MSI+ tumor cells to escape the otherwise effective immune attack by creating an immunosuppressive microenvironment. Additionally to downregulate major histocompatibility complex (MHC) class I on the tumor cells' surface and inactivation of the antigen-processing machinery, upregulation of immune-checkpoint-molecules (such as programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1(PD-L1)) represents another escape mechanism . Authenticity for these lines was verified by means of short tandem repeat fingerprinting  from genomic DNA according to  and cho+ cell lines. CIMP-associated cell lines' response was weaker.Substance combinations enhanced antitumoral effects in almost all cases . Best kiγ pretreatment, described to induce IDO expression and rendering cells more vulnerable to cytolysis [data not shown).Sequential treatment (preincubation with Indoximod for 72 h) did not boost effects of the monotherapies. Thus, the direct cytotoxic potential of Indoximod on CRC cells was rather low. Even interferon-ytolysis , did notμM led to a heterogeneous expression profile, with an increased expression of DNA-damage response gene ATM in MSI+ cell lines HROC257 T0 M1 and HROC50 T1 M5. Expression of CDKN2A and CCNE1, both being involved in cell cycle regulation, was largely unchanged ( ATM and CDKN2A (p < 0.05 versus control). CCNE1 and MDM2 were also upregulated in this combination  and HROC257 T0 M1 (IDOhigh) cells.We next aimed to unravel the underlying mechanisms of biomass reduction under treatment. Autophagy induction was studied by immunofluorescence based on staining of acidic vesicles with acridine orange. Frequency of autophagy was quite low . By contreatment . Most prhigh) cells; sub-G1 defines dead cells). The best antiproliferative effect was inducible in HROC183 T0 M2 (IDOlow) cells after Indoximod/Curcumin treatment  cells, apoptosis was two to three times higher than in controls (early apoptosis: 36 ± 20 % versus 11 % ± 2 % control and late apoptosis: 26 % ± 10 % versus 15 % ± 8 % control). Adding Gemcitabine to Curcumin further enhanced cytotoxic effects  cell line.All substances reduced tumor cell numbers in this test system. The best cytotoxic effect could be induced by Curcumin, resulting in a massive tumor cell reduction in all four cell lines (> 80 % versus control). Of note, combining Curcumin either with Indoximod or Gemcitabine even enhanced this toxic effect with nearly complete elimination of tumor cells . Best tuHowever, even with 72-hour incubation time, a specific antigenic activation is unlikely to occur and thus the observed effects are most likely due to a more unspecific stimulation of lymphocytes by the tested drugs.In this study, we describe (I) the expression profile of immune-modulating molecules on a panel of molecularly well-characterized patient-derived CRC cell lines and (II) the effects of IDO-inhibition directly on CRC cells as well as in coculture experiments with immune cells. in vivo [+ lymphocytes and clinical patients' outcome [ In vitro, IDO decreases tumor cell proliferation and mediates mediating drug resistance [data not shown), we indeed found some overlap between IDO level and sensitivity towards clinically used drugs, such as 5-fluorouracil .Specific IDO blockade with Indoximod identified an inverse correlation between biomass reduction and IDO1 abundance in our patient-derived cell lines. Of note, IDOmembrane . Additiomembrane . However in vitro encouraged us to use combinations with other IDO-blocking substances. Curcumin and Gemcitabine were selected based on the following criteria: (I) interference with IDO; (II) direct cytotoxic activity against tumor cells (via induction of immunogenic cell death) in (pre-)clinical studies, (III) safe application with minimal toxic side effects (at least in low-doses) in patients, and (IV) enhanced antitumoral effects when given in conjunction with other drugs. These experiments identified Curcumin as very promising candidate for IDO-blocking cancer therapy. Biomass of tumor cells significantly reduced and oncolytic effects even increased when Curcumin was combined with Gemcitabine, a quite surprising finding. In functional analyses, senescence and apoptosis could be identified as underlying molecular mechanisms. Effects were cell line and treatment specific, but again largely independent from IDO-expression status. Cytotoxicity of Curcumin is mainly based on apoptosis induction, without significant impairment of the cell cycle. This was somewhat unexpected, since short-time treatment with Curcumin has been reported to decrease cyclin D1 expression and to induce G1-/S-phase arrest [The missing direct cytotoxic potential of Indoximod monotherapye arrest , 35. In vivo, IDO blockade by Indoximod breaks immune tolerance by increasing the level of inflammatory molecules, such as C-reactive protein and IL6 [Finally, a coculture system mimicking tumor-immune cell interactions was applied to test the impact of IDO-inhibition for reconstitution of immune function. and IL6 . We coul and IL6 , 36. Gem and IL6 . However colon ascendens. HROC60 cells show high abundance of immunosuppressive molecules, i.e., IDO, PD1/PD-L1. It is therefore of particular interest whether such Curcumin-based combinations provide a real opportunity for selected CRC patients in the future.Interestingly, there was a trend towards highest susceptibility of HROC60 cells, established from a 71-year-old male patient with a low-grade CIMP tumor in theHowever, it is worth mentioning that coculture experiments were performed in an allogeneic setting using naïve and thus “unprimed” immune cells from healthy volunteers, without tumor-induced T-cell dysfunction. Prospective studies will have to test if comparable strong oncolytic effects can be achieved in an autologous setting with partially exhausted lymphocytes from cancer patients.Although we were unsuccessful to directly link IDO-level with treatment response, we would like to bring forward the argument that this molecule plays a central role in tumor-induced immunosuppression. To this end, not only pharmacological agents, but also other means of IDO1 targeting may provide clinical benefits to patients. A recently performed phase I study even described disease stabilization in patients after application of an IDO1-peptide vaccine. In most patients with metastatic non-small cell lung carcinoma, detectable IDO-1-specific T cell responses were accompanied by significantly improved overall survival , 39. ComWe report effects of IDO-inhibition on a panel of patient-derived colorectal cancer (CRC) cell lines. IDO blockade was induced by different substances, either directly (Indoximod) or indirectly  interacting with IDO. We have shown that Curcumin-based treatment is promising by inducing apoptosis in CRC cell lines, with even higher cytotoxic activity when Gemcitabine is added in low and thus nontoxic doses. Coculture of tumor and immune cells revealed a boosted antitumoral effect of Curcumin-based therapies by massive activation of unspecific immune responses, counteracting IDO-induced immune tolerance.


We found that the expression of EGFR/ERBB2 and activation of ERBB pathway was significantly related to Lapatinib sensitivity. GO (Gene Oncology) analysis of top 10 pathways showed that the sensitivity of Lapatinib was positively correlated with cell keratin, epithelial differentiation, and cell-cell junction, while negatively correlated with signatures of extracellular matrix. Forty-four differentially expressed lncRNAs were found between the Lapatinib sensitive and resistant groups . Gene set variation analysis (GSVA) was performed based on 44 lncRNAs and genes in the top 10 pathways. Five lncRNAs were identified as hub molecules. Co-expression network was constructed by more than five lncRNAs and 199 genes in the top 10 pathways, and three lncRNAs  and 47 genes were identified as close-related molecules. The three lncRNAs in epithelium-derived cancers were differentially expressed between sensitive and resistant groups, but no significance was found in non-epithelium-derived cancer cells. Correlation analysis showed that SPINT1-AS1  and GIHCG  were correlated with the IC50 of epithelium-derived cancer cells. In further experiments, GIHCG knockdown enhanced cancer cell susceptibility to Lapatinib, while high level of SPINT1-AS1 was a sensitive biomarker of NCI-N87 and MCF7 cancer cells to Lapatinib. In conclusions, lncRNAs GIHCG and SPINT1-AS1 were involved in regulating Lapatinib sensitivity. Up-regulation of GIHCG was a drug-resistant biomarker, while up-regulation of SPINT1-AS1 was a sensitive indicator.Lapatinib is a small molecule inhibitor of EGFR (HER1) and ERBB2 (HER2) receptors, which is used for treatment of advanced or metastatic breast cancer. To find the drug resistance mechanisms of treatment for EGFR/ERBB2 positive tumors, we analyzed the possible effects of lncRNAs. In this study, using CCLE (Cancer Cell Line Encyclopedia) database, we explored the relationship between the lncRNAs and Lapatinib sensitivity/resistance, and then validated those findings through  Lapatinib is a small molecular drug that has been shown to be a dual tyrosine kinase inhibitor, which is involved in the EGFR/HER1 and ERBB2/HER2 pathways and suppresses the autophosphorylation of these receptors. Clinically, it has been used in combination therapy with capecitabine in patients with advanced or metastatic breast cancer that overexpressed ERBB2/HER2 in the cases of previous treatment with anthracyclines, taxanes, or trastuzumab (Herceptin)  are a large class of transcribed RNA molecules that are longer than 200 nucleotides but do not encode proteins. In addition to the regulation of diverse cellular processes, such as epigenetics, cell cycle, and cell differentiation, they have been found to play important roles in carcinogenesis, tumor development, and treatment resistance  is an open access resource with the most completely integrated datasets of cancer cells genomes and drug effectiveness. It includes the experimental datasets of drug treatment of 24 kinds of chemical compounds in almost 1,000 cancer cell lines of various human cancers  database , three were melanoma cell lines , three were hepatocarcinoma cell lines , three were thyroid cancer cell lines , two were breast cancer cell lines (MCF7 and SK-BR-3), two were pancreatic cancer cell lines (TCC-PAN2 and BxPC3), and two were colorectal cancer lines (DLD-1 and NCIH-747). Cell lines NCI-N87, MuM-2C, LM3, MV3, Huh7, SGC-7901, CAL-62, AGS, MCF7, C643, 97L, SK-BR-3, KHM-5M, A-375, TCC-PAN2, MKN-45, and BxPC3 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . Cell lines DLD-1 and NCIH-747 were purchased from The Global Bioresource Center ATCC . The cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum in a humidified incubator at 37°C with 95% air and 5% CO2.Nineteen cancer cell lines were used for validating experiments SPINT1-AS1 and GIHCG siRNAs were transfected into cancer cells by Lipofectamine 2000  according to the manufacturer's instructions. The siRNA sequences are shown in Table S1.Total RNA was isolated using the TRIzol solution . The cDNA was synthesized using Reverse Transcription kit . Real-time PCR was performed in 10 μl reaction mixtures with the HT 7900  using SYBR™ Select Master Mix . The sequences of primers were designed and synthesized by Sunny Biotech : The primer sequences are shown in Table S1.Five thousand cells of different cancers were placed in each well of 96-well plates (100 μl/well). Different concentrations of Lapatinib  were incubated for 48 h. After adding 10 μl CCK-8 for 2 h, OD value was measured at 450 nm by spectrophotometry .Gene Set Variation Analysis,) in R language, which utilizes non-parametric unsupervised method for evaluating gene set enrichment (GSE) in transcriptomic data . Student's t-tests were performed by GraphPad Prism 6.0. P < 0.05 was considered statistically significant.The “corrplot” and “pheatmap” package in R software were utilized for visualizing pearson correlation analysis and cluster analysis by “euclidean” method. The Benjamini and Hochberg method was used to calculate P. adjust value. By means of “clusterProfiler” package in R, GSEA  and GO (Gene Ontology) analyses were carried out to explore involved gene clusters. GSEA is a computational method based on previous publication by Subramanian et al. . GO analn = 302) and low_IC50 (n = 118). Since EGFR and ERBB2 are the targets of the Lapatinib drug, the expression levels of EGFR, and ERBB2 in high_IC50 and low_IC50 groups were analyzed. The expression levels of EGFR and ERBB2 were significantly higher in low-IC50 group than in high_IC50 . The distribution tendency of 22 types of solid cancer cell lines in high-IC50 (up to 8 μM) and low_IC50 (lower than 8 μM) groups is presented in P < 0.002, p. adjust = 0.064; regulation of ERBB signaling pathway NES = −1.69, P < 0.002, p. adjust = 0.064).The CCLE data of Lapatinib IC50 of the selected 420 cell lines was shown in P < 0.001, P. adjust < 0.001).To illustrate the mechanism of Lapatinib resistance, we selected genes with fold-change >1.5 times to perform GO analysis (Table S2). In the top 10 involved pathways, Lapatinib sensitivity was positively associated with cell keratin, epithelial differentiation, and cell-cell junction, while negatively related to signatures of extracellular matrix (P < 0.01). Then, we selected genes in the top 10 pathways and 44 differential lncRNAs for the construction of the co-expression network. The enrichment scores of the top 10 pathway genes in every cancer cell lines were calculated and determined by GSVA analysis. Five lncRNAs were highlighted as the hub factors in the top 10 regulating pathways  and non-epithelium derived group . The differential expression levels of the three lncRNAs between the two groups are presented in P < 0.05). In the non-epithelium groups, there was no significant difference of the three lncRNAs between Lapatinib high_IC50 and low_IC50 groups. Higher expressing level of SPINT1-AS1 was found in epithelium-derived cancer cells, and higher expressing levels of MAGI2-AS3 and GIHCG were observed in the non-epithelium group.We divided the 420 cancer cell lines into epithelium derived group .Differentially expressed genes (1.5-fold change) between the Lapatinib high_IC50 and low_IC50 groups in epithelial group (Table S3) were utilized to perform GO analysis. Enhanced signatures of cell keratin, epithelial differentiation, and cell-cell junction were observed in Lapatinib low_IC50 group, and decreased signature of extracellular matrix were observed in Lapatinib low_IC50 group , while higher expression levels of GIHCG were significantly related to higher Lapatinib IC50 .In validating experiments, we examined expression levels of GIHCG and SPINT1-AS1 in seven types of cancer cell lines  and Lapatinib IC50 of the same cancer cell lines. Correlation analysis showed that higher expression levels of SPINT1-AS1 were significantly associated with lower Lapatinib IC50 (The sensitive cancer cell lines of NCI-N87 (gastric cancer) and MCF7 (breast cancer), as well as the resistant cancer cell lines of NCIH-747(colon cancer) and BxPC3 (pancreatic cancer) were selected for a subsequent validating study. After knocking-down expression levels of GIHCG and SPINT1-AS1 by small interfering RNAs, Lapatinib IC50, and inhibitory rate of cancer cells were detected. Among three small interference sequences of GIHCG and SPINT1-AS1 mRNAs, siRNA sequence 3 of GIHCG Si3, , and siRG, suppression of GIHCG in Lapatinib resistant cancer cell lines NCIH-747 and BxPC3 could induce up-regulation of SPINT1-AS1 (P < 0.05), while knockdown of SPINT1-AS1 did not change the expression level of GIHCG (P > 0.05).Knocking-down of GIHCG could significantly enhance the sensitivity to Lapatinib in MCF7 and BxPC3 cancer cell lines , while dLncRNA is an important regulatory molecule in drug resistance during chemotherapy or gene targeted therapy (Li et al., PI3K/Akt, Ras/Raf/MEK/ERK1/2, and PLCγ pathways are downstream pathways of EGFR and ERBB2 and play important roles in cell proliferation and survival of multiple cancers (Roskoski, Although the role of lncRNAs in cancer progression and Lapatinib resistance have been reported in other studies (Russell et al., In conclusion, the current study proposed a group of lncRNAs related to Lapatinib sensitivity based on pan-cancer cell lines analysis. By subsequent experimental study, lncRNAs GIHCG and SPINT1-AS1 were firstly identified as crucial lncRNAs in regulating Lapatinib resistance or sensitivity in epithelium-derived cancer cell lines. SPINT1-AS1 is a Lapatinib sensitivity predictor, while GIHCG is a predictive molecule for Lapatinib resistance.The protocols used in this study were approved by Rui Jin Hospital Ethics Review Boards. Written informed consents were obtained from all human material donors in accordance with the Declaration of Helsinki. Animals were used according to the protocols approved by Rui Jin Hospital Animal Care and Use Committee.KL and YY conceived and designed the experiments. ZX, ShS, ZZ, JG, and QL performed the experiments. ZX, ZZ, SaS, WS, YY, and KL analyzed the data. ZX, ShS, ZZ, SaS, WS, YY, and KL contributed reagents, materials, and analysis tools. ZX, YY, and KL wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current review focuses on the role of PTPs in controlling CD4+ T cell activity in the intestinal mucosa and how disruption of PTP activity in CD4+ T cells can contribute to intestinal inflammation.Protein tyrosine phosphatases (PTPs) play a critical role in co-ordinating the signaling networks that maintain lymphocyte homeostasis and direct lymphocyte activation. By dephosphorylating tyrosine residues, PTPs have been shown to modulate enzyme activity and both mediate and disrupt protein-protein interactions. Through these molecular mechanisms, PTPs ultimately impact lymphocyte responses to environmental cues such as inflammatory cytokines and chemokines, as well as antigenic stimulation. Mouse models of acute and chronic intestinal inflammation have been shown to be exacerbated in the absence of PTPs such as PTPN2 and PTPN22. This increase in disease severity is due in part to hyper-activation of lymphocytes in the absence of PTP activity. In accordance, human PTPs have been linked to intestinal inflammation. Genome wide association studies (GWAS) identified several PTPs within risk loci for inflammatory bowel disease (IBD). Therapeutically targeting PTP substrates and their associated signaling pathways, such as those implicated in CD4 The gastrointestinal track is a large mucosal surface at which the host's immune system is juxtaposed with a dense microbial population and a diverse array of dietary antigens. Immune recognition of enteric antigens however, is minimized by physical compartmentalization. Bacteria and dietary products are retained within the gut lumen, while the host immune system is localized in the mucosal tissue. This physical separation is preserved by the intestinal mucosal barrier, which includes a mucin layer, a single epithelial cell lining sealed by tight and adherens junctions and a continuous secretion of anti-inflammatory soluble mediators Figure  1–5).–5.1–5).The mucosal barrier is not absolute however, and a small number of bacteria do translocate from the lumen to the underlying lamina propria. In such instances, a complex network of innate and adaptive immune cells impedes the spread of intestinal bacteria in a manner that limits tissue damage , 7. ThisPhysical compartmentalization and mucosal immunity therefore establish and maintain microbiome-host mutualism . The los+ T cell development and function. Examples include IL23R, JAK2, and STAT3  have been shown to induce remission in both CD and UC patients , Ustekinumab  and Infliximab   –33. It s+ T cells are classified into distinct subsets based on their inducing cytokines, transcription factor expression, and effector cytokine secretion. The initial classification of CD4+ T cells as TH1 IFNγ producers vs. TH2 IL-4 producers, has been broadened to include multiple additional subsets (H9 (IL-9), TH17 , TH22 (IL-22), T follicular helper TFH (IL-21) cells, as well as thymic-derived and peripherally-induced T regulatory cells   –40 , co-stimulatory molecules, and the T cell receptor (TCR), converge to induce gene expression profiles that drive CD4+ T cell activation and differentiation into distinct subsets  expressed on antigen presenting cells. Upon TCR engagement, Src-family kinases  are activated and phosphorylate tyrosine residues within the immune-receptor tyrosine-based activation motifs (ITAMs) in the TCR-associated CD3 and zeta chains –46. Phos+ T cell differentiation  cells are generated primarily from CD4+ Foxp3− T cells exposed to antigen under tolerogenic conditions or during homeostatic proliferation .–62.+ T c+ T cell activation and differentiation. The current review will examine the role of protein tyrosine phosphatases (PTP) in safeguarding this network, and how PTP deletion can perturb CD4+ T cell function and consequently contribute to intestinal inflammation.Thus a network of signaling pathways, heavily dependent on tyrosine phosphorylation, directs CD4The PTP family comprises a heterogeneous set of enzymes that were first defined by Tonks and colleagues by their capacity to dephosphorylate phospho-tyrosine residues and by their structurally related phosphatase catalytic domain , 64. PTPThe human PTP family members are divided into distinct classes based on their structural and biochemical properties Figure . The majMore recent studies characterized enzymes using alternate nucleophilic catalytic residues which were then added to the list of PTP family members. These two additional classes are the aspartic-based and histidine-based acid phosphatase enzymes. Thus, within this classification frame, Alonso and Pulido expanded the PTP gene superfamily to 125 members .Tyrosine phosphorylation is a reversible mechanism that promotes protein–protein interactions and propagates signaling cascades in all cell types. It therefore stands to reason that the PTPs targeting these phosphorylated amino acids play important roles in modulating the strength and duration with which a signaling pathway is activated. Moreover, given that PTPs are a heterogeneous gene family  it is not surprising that PTPs have been associated with many distinct pathologies in both animal models and in human disease . Of impoThe immune system relies heavily on the use of protein phosphorylation to recognize and transmit intra- and extracellular signals. Signaling cascades initiated at the cell surface by receptor kinases, or from the recruitment of cytoplasmic kinases such as the JAK and Src-like kinase families, translate into multiple immune cell responses like cellular adhesion, division, migration and others. Hence, from the list of over 100 classical Cys-based PTPs, Arimura and Yagi demonstrated that between 58 and 76 of them are expressed in various cell lineages of the immune system  and thatMultiple genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) linked with IBD, in or near PTP coding units , 71. ExaPTPN22 is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells . It has PTPN22 deficient mice exhibit a lymphoproliferative disease and accumulate memory-phenotype T cells with age, but do not display signs of spontaneous autoimmunity  tyrosine following TCR ligation and not due to changes to IL-2 or IL-15 receptor signaling  induced model, these studies attributed the increase in disease severity to changes in TLR4 signaling, type I interferon production and macrophage polarization , 95.PTPN2−/− CD4 and CD8 was identified as causing increased T cell activation due to hyper-phosphorylation of the activating tyrosine residue of Lck  presented higher expression of TH1 and TH17-related transcription factors and cytokines  in comparison to PTPN2 expressing CD patients. Such human studies solidify the important role of PTPN2 CD4+ T cell responses in the context of human IBD  is a cytoplasmic phosphatase expressed in all hematopoietic cell lineages throughout development and activation (me) and motheaten viable (mev) mice. These mice harbor mutations that result in undetectable and reduced PTPN6 expression respectively. Similar to PTPN2 deficient mice, me/me mice succumb to disease 2–3 weeks following birth . Similar to PTPN6, PTPN11 regulates signaling downstream of multiple surface receptors including growth factor receptors, cytokine receptors and integrins. Its broad tissue expression is in line with the embryonic lethality of PTPN11 knockout mouse at mid-gestation in –133.PTPN11 mutations in patients with multiple cancer types including leukemias, breast cancer and gastric cancer  differentiation is suppressed  mice were reconstituted with either WT or DUSP2−/− CD4+CD45RBhi T cells. In this model, an increase in body weight loss, severity of histopathology scoring, and colon thickening were observed following the transfer of DUSP2−/− T cells compared to controls. Such clinical signs were corroborated by a rise in the number of colon infiltrating CD4+ T cells expressing a TH17 gene signature. Moreover, such TH17 T cells in the inflamed tissue expressed elevated levels of pathogenic TH17-associated genes but not non-pathogenic genes  and is only increased minimally following PMA stimulation. The low levels of DUPS2 expression were found to be attributed to CpG methylation. Unfortunately, the levels of DUSP2 in colonic biopsies, or colonic T cells, were not quantified. As such, further evidence is required to definitely demonstrate that a reduction in T cell DUSP2 occurs in IBD patients and that this reduction impacts human T cell polarization in the disease state  signaling regulates multiple cellular responses to activation including T cell proliferation, cytokine production, survival and adhesion. CD4balance. –153.in vitro assays suggested a role for DUSP6 in the regulation of CD4+ T cell activation. Specifically, DUSP6 was shown to be upregulated following TLR4 stimulation and then restrained ERK activation and suppressed IFNγ production by TCR stimulation  that recognize and dephosphorylate the insulin receptor (IR). The regulation of IR activation by a PTP was hypothesized to decrease IR mediated signaling events, and subsequent entry of glucose into insulin receptive tissues. It was believed that a rise in the expression or activity of such a regulatory PTP would result in high levels of blood glucose in the circulation, which would then instigate type II diabetes. This hypothesis was confirmed by two seminal publications which described the PTP1B knockout mouse that presented with an increase in IR phosphorylation. These studies validated PTP1B as an outstanding candidate to be targeted for the treatment of type II diabetes , 159 andIn spite of all these reports, there are no small molecule inhibitors against PTP1B (or any PTP) that have been successfully developed beyond initial clinical trials. Multiple reviews have already examined the difficulties associated with developing PTP competitive inhibitors and this subject is beyond the scope of this review –163. In It is worthy to note that novel therapeutic strategies are also being developed to target PTPs. These include the forced dimerization and inhibition of receptors PTPs , 167, inex-vivo cell cultures. This allows for the modulation of PTP activity in a cell-type specific and temporary fashion. For example, we reported a protocol for dendritic cell (DC) vaccination that employs inhibitors of PTPN1-PTPN2 (Significant effort is also being made in the development of cell based therapies. Potential adverse effects that could be associated with systemically inhibiting PTPs are mitigated by using PTP inhibitors in N1-PTPN2 . This woFrom an IBD perspective, it is clear that inhibiting the PTPs presented above would most likely exacerbate disease by potentiating the effects of pro-inflammatory cytokines and promoting the differentiation of pathogenic T cells. Indeed this contention is supported by the fact that, biologics and small molecules that suppress cytokine receptor signaling have been clinically successful in IBD disease management. One example would be tofacitinib, a JAK inhibitor found to be effective in phase 2 and 3 trials in moderate to severe ulcerative colitis .Our understanding of PTP function in immune cells is expanding beyond cytokine receptor signaling, and our capacity to modulate cellular responses by titrating PTP expression is also evolving at a rapid pace. The cell-dependent context of positive or/and negative modulation bestowed by the nearly 80 PTPs expressed in immune cells, remains an exciting ground for study and clinical improvement. It remains to be seen if this renewed interest in PTP inhibitors would be applicable to inflammatory diseases such as UC and CD.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT.Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing.Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score <0.2 of proteins related to Wnt signaling. However, gene silencing did not change the Wnt pathway activation. Renewed analysis with respect to putative relations to Wnt signaling revealed that P-cadherin (CDH3) had three mutations in the coding region of the extracellular domain and was associated with high Wnt signaling. Silencing by siRNA not only in lowered Wnt activity, but also decreased levels of phosphorylated cSRC and sP-cad, and changed cell morphology towards spindle cell appearance.Conclusion: It is concluded that expression of mutated CDH3 is associated with activation of cSRC, stabilization of ß-catenin and a rounded morphology related to a stemness/EMT phenotype. A decreased Wnt activity can be found also by cSRC inhibition, but CDH3 silencing has an additional effect on morphology indicating reversal of EMT. Breast cancer is one of the most frequently occurring cancers in the world and, with an estimated 1.7 million new cases diagnosed worldwide each year, it remains a global health challenge. Although intensive screening and improved treatment options have increased the 5 year survival rate, still roughly 30% of woman with breast cancer eventually die of their disease due to metastases resistant to therapy . Not onlDuring recent years, spontaneous mammary cancer in dogs has shown great value for analysis and developing treatment methods for EMT involved signaling mechanisms . The incMetastases can be formed from epithelial cells that undergo epithelial-mesenchymal transition (EMT) resulting in cells with phenotypically stromal cell characteristics. During EMT cells detach from neighboring cells and the basal membrane, obtain a rounded morphology and acquire migratory capacity. This process can also occur in cancer cells at the invasive front of the tumors . Among oAs the question remained unanswered why our cell lines show this highly basal activated Wnt activity and a rounded morphology despite high E-cadherin expression, we decided to perform a whole exome sequencing analysis to find mutations unique for the high basal Wnt active canine mammary tumor (CMT) cell lines and to relate candidate genes to upregulated Wnt activity. These candidate genes were further investigated using mRNA silencing experiments. At the end we found that the candidate gene P-cadherin, with three extracellular domain missense mutations, was causing both Wnt activity and responsible for the morphology changes. Silencing of these P-cadherin mutations in EC1 showed that the Wnt activity is reduced and rounded cells got their spindle cell morphology back. The mutations may result in a possible enhanced sensitivity for proteolytic cleavage of the extracellular domain of P-cadherin and so stimulate Wnt signaling and morphological changes.ALDH1A1 and LGR5 were expressed at significantly higher in the rounded Wnt active cells. Expression of the epithelial marker CDH1 was higher, whereas the expression of mesenchymal markers INTα5, INTβ1 and VIM was significantly lower in the cells with rounded morphology. FOSL1 expression, involved in migration and invasion was lower. The most striking differences in the miR expression profile related to Wnt signaling and stemness were the low expression of miR-34a, -146b and -196a in the high active Wnt canine mammary tumor cell line CMT-U27, and the high expression of miR-200b, -205 and 200c compared to the control cell line CIPm . Most of the mutations were G>A or A>G  score ≤0.1. CAMK2A, CSNK2A2 and RAC1 had a splice donor (SD) or splice acceptor (SA) mutation. CSNK1D was selected although it was only mutated in CMT-U27 and CMT9 (Supplementary Table 1). CDH3 had more mutations in both the high and low active Wnt cell lines but also three specific mutations in the high active Wnt cell lines in the extracellular domain (EC). The mRNA expression levels of these selected target genes were measured. Differences in expression levels were found among the cell lines  relative to the same construct with inactivation mutations in the TCF binding site (FOPflash)   and 3D. CDH3 gene. Screening the exome sequencing data showed 3 mutations in CDH3 with high SIFT scores, meaning that the impact of these individual missense mutations on protein activity is predicted to be low. Nevertheless, the influence of these three single amino acid substitutions was further investigated. The Integrative Genomics Viewer (IGV) showed for all three missense mutations more than two reads  of the published cDNA was tested on mRNA level, protein expression, cell proliferation and Wnt activity and on CDH3 target genes in CMT-U27 cells. Silencing CDH3 had a significant knock down on mRNA of 50% at 48 h and 85% at 72 h normalized to the MOCK  resulted in decreased P-cadherin protein expression  Wnt pathway  2,5-diphenyltetrazolium bromide (MTT) assay . Briefly, 15,000 CMT-U27 cells were seeded in 96-well Primaria plates (Corning) in DMEM/F12 with 10% FBS. After 24 h of incubation cell adhesion and spreading, the cells were treated with a siRNA either or both with dasatinib , or with GI 254023X both dissolved in DMSO to a final stock concentration of 10 mM. Dasatinib was added in a concentration range from 1 μM to 50 μM diluted in medium to a final concentration of 0.2% DMSO and incubated for 24, 48 and 72 h . 5 μM GI 254023X was added and incubated for 48 h in 0.1% DMSO. The absorbance was measured at a wavelength of 595 nm by an spectrophotometer Anthos Multimode Detector . IC50 curves were plotted with Sigma-plot version 12.5.CMT-U27 cells were seeded at a density of 600,000 cells in 6-well Primaria plates (Corning) and after 24 h transient transfected with the siRNA’s. After 24, 48 and 72 h cells were washed with Hank’s balanced salt solution (Invitrogen) and lysed and scraped with RIPA buffer . Twenty t-test for two groups  or analyzed by one-way ANOVA for multiple groups. A value of P < 0.05 was considered statistically significant.Statistical analysis was conducted using IBM SPSS Statistics version 24.0.0.1 . All values were expressed as means ± SD. Statistical differences were evaluated for normal distribution, using a Student’s


This paper examines the complexation of anti-cancer small interfering RNAs (siRNAs) by cationic carbosilane dendrimers, and the interaction of the formed complexes with HeLa and HL-60 cancer cells. Stepwise formation of the complexes accompanied by the evolution of their properties has been observed through the increase of the charge ratio (dendrimer/siRNA). The complexes decrease the viability of both “easy-to-transfect” cells (HeLa) and “hard-to transfect” ones (HL-60), indicating a high potential of the cationic carbosilane dendrimers for siRNA delivery into tumor cells. Therapeutic nucleic acids hold great potential for anti-tumor therapy. However, the natural barriers of a cell form considerable obstacles against the efficient transport of nucleic acids (NA) into cytosol or target compartments ,2,3,4. NAmong many types of dendrimers described so far, cationic carbosilane dendritic systems gain considerable attention as potential NA-based drug transporters, due to their good biocompatibility, chemical stability, highly controlled size, inert inner branches and high charge density on the surface. To date, their utility has been demonstrated for the efficient delivery of model antisense oligonucleotide-targeted mRNA of the Renilla luciferase gene , pro-apoHere we report on a detailed physico-chemical study of the formation and stability of dendriplexes formed by cationic carbosilane dendrimers and pro-apoptotic siRNAs. Also, we evaluated the cell cytotoxicity of the dendriplexes towards cancer cell lines of different permeability, namely, the suspension human acute promyelocytic leukemia cell line (HL-60) and the adherent human cervical cancer cell line (HeLa).Cationic carbosilane dendrimers with peripheral quaternary ammonium groups were prepared and characterized according to the reported method . The molO-tert-butyldimethylsilyl-protected RNA phosphoramidites (5-(ethylthio)-1H-tetrazole as an activator; the coupling time was 5 min) and with automated procedures being optimized for the synthesizer. Cleavage from a solid support, and the removal of protecting groups of oligoribonucleotides were performed under the conditions described in 12 (BDEF32) and its fluorescein isothiocyanate (FITC)-labeled analogue G2O3[SNMe3I]11-FITC, for the delivery of the specific anti-HIV-1 siRNA Nef to interfere with HIV-infectivity in human primary astrocytes. The work indicates that this type of transporters is a promising alternative to achieving very high transfection levels in “hard-to-transfect” astrocytes without causing cytotoxicity. The biodistribution of the dendriplex formed by the siRNA and the dendrimer loaded with FITC was studied in vivo in BALB/c mice. The dendrimer efficiently transferred the siRNA to mouse brains and it crossed the bloodαbrain barrier, showing its great potential as a drug transporter for the therapy of neurological disorders. Motivated by these inspiring results we have chosen this family of cationic dendrimers for the binding and transport of pro-apoptotic siRNAs to “easy-to-transfect” (HeLa) and “hard-to transfect” (HL-60) tumor cell lines. The cationic carbosilane dendrimers of the second and the third generations GnO3[SNMe3I]m ; n = 3, m = 24 (BDEF33)) were prepared according to the convenient procedure described in [1H- and 13C-NMR spectroscopy and mass spectrometry.The study  demonstrribed in . In brieCationic carbosilane dendrimers can bind siRNAs, mainly by the means of electrostatic interactions. The properties of the dendriplexes formed depend on their composition, i.e., on the charge ratio of the components taken. To study the ability of the dendrimers BDEF32 and BDEF33 to bind siRNAs, and the properties of the formed dendriplexes, and to find a ratio that could be considered optimal for the further cell experiments, the profiles of siRNA binding by cationic dendrimers were obtained by the means of several physico-chemical methods. The use of independent indirect methods to study siRNA binding is required, since each of these methods provides information on only on an exact property of the dendriplexes  [The early stage of the siRNA binding to the dendrimers was studied by means of the ethidium bromide intercalation assay (EBIA). The method is based on the intercalation of the fluorescent dye EB into a double strand siRNA (a binding site of 2–4 base pairs). These interactions result in a significant increase of EB fluorescence intensity, and they cause a blue shift in EB fluorescence emission maximum. Strong electrostatic interactions between cationic dendrimers and siRNAs lead to a higher affinity of the dendrimers to siRNAs. The following structural distortions of siRNA causes the displacement of intercalated EB from the duplex and subsequent quenching of EB fluorescence. Thus, EBIA is a sensitive method for monitoring even subtle changes in double-stranded siRNA structure . We obseThe reversible nature of the binding was confirmed by recovery of EB fluorescence intensity upon the treatment of the saturated dendriplexes with heparin, a natural polyanion . When thFurther evolution of the double-stranded RNA structure upon the binding with dendrimers was studied by CD spectroscopy. The CD spectra of the naked siRNAs indicate the typical curve of an A-form helix geometry, with a strong positive band at ~260–265 nm, and a negative band at ~210 nm a,b. SincIn addition, the complexation was studied by the means of an agarose gel retardation assay. This method is based on the mobility of the charged macromolecules (or complexes of molecules) in an electric field through a porous lattice formed by agarose gel, with the retardation of the molecules depending on their charge and mass. Herein, it allowed us to observe the non-complexed siRNA in samples. The electrophoresis data show that the siRNA was fully complexed by cationic dendrimers, being in 2-fold charge excess, which corresponded to a 6.7-fold molar excess (BDEF32) or a 3.3-fold molar excess (BDEF33), respectively . The comIn the experiments, we observed the initial moment when stabile supramolecular associates were formed. The increase of the dendrimer/siRNA ratio led to the further evolution of the dendriplex structure, with the reorganization of the particles and the increase of their size and surface charge. The changes could be monitored by the means of electrophoretic light scattering. The technique was based on the scattering of the laser light by nanoscale objects moving in the electric field. The value of the potential on the border of the solvate shell  calculated from the scattering data gives the idea of the surface charge of nanoparticles. The profiles had a sigmoidal shape; negative charges of siRNAs were compensated at the 12-fold excess of the G2 dendrimer, and at the 4-fold excess of the G3 one. A further increase of the dendrimer concentration led to a charge saturation at the 20-fold (G2) or 10-fold (G3) molar excesses of dendrimers .The results obtained from the biophysical assays described above are summarized in the 50, CR50) corresponding to the charge or molar ratio of the half-binding; and (2) the saturation point  corresponding to the charge or molar ratio where the measured parameter is no longer changed Whereas the former point is convenient for observing dendritic effects, the latter represents the properties of dendriplexes at the end of the complexation.In the complexation profiles, two points are of special interest: (1) half-effect point . In contrast, the zeta potential values continue to evolve even after the saturation achieved, as shown by other assays  or 2.4 (for G3), a strong positive dendritic effect is observed; CRsat is 6 for both generations).Analyzing the data from the assays studying the dendriplexes’ formation, one could find an interesting regularity in the results obtained: the binding and saturation values that were obtained by CD spectroscopy, EBIA, and agarose gel electrophoresis were quite close one to another . Howobserved  analyzesIt should be noted that the stepwise formation of dendriplexes have never been observed upon the binding of NAs to dendrimer-based nanoparticles. The surface charge of dendriplex nanoparticles is stabilized at relatively low CR/MR values, corresponding to the full NA binding. However, similar behaviors has been observed for amphiphilic PAMAM dendrons ,39 and dsat/MRsat obtained from the zeta potential profiles for the preparation of dendriplexes is likely preferential for the biological experiments . The AFM images of the dendriplexes prepared at the CR value of 5, which is close to the saturation region, are given in the One can speculate whether the dendriplex composition  is optimal for biological experiments. Indeed, the initial intention is to keep the dendrimer content in samples as low as possible, to avoid unwanted side effects, for instance, increased cytotoxicity. However, the use of higher dendrimer excess likely leads to the decrease of the particle size ,41,42, wAs target cell lines, HeLa  and HL60 (human myeloid leukemia cells) were chosen to study the influence of the cells’ characteristics on the transfection efficiency. The lines under study represented two major types of the human cancer cell lines: adherent (HeLa) and suspension (HL60) lines. Due to the peculiarities of the cell membrane composition, HL60 cells are known as hard-to-transfect , whereasBoth types of dendrimers exhibited dose-response cytotoxicities towards HeLa and HL60 cells. The cytotoxicity profiles looked similar for both cell lines, thus suggesting that leukemia cells (HL60) are principally permeable by dendrimers a,c. As eComplexes of carbosilane dendrimers and siRNAs were shown to penetrate efficiently across the cell membrane . Herein,Upon treating target cells with anti-cancer dendriplexes, several consecutive processes take place: dendriplexes’ endocytosis, endosomal escape, siRNA release and functioning, apoptosis induction . A deficDendriplexes containing pro-apoptotic siRNAs (at 10-fold cation excess) have been shown to decrease the target cell viability, with the cytotoxic effect being the dose-response . The modIt is worth noting that, unlike the expected result, scramble siRNA appeared to exhibit some cytotoxic effect. Such an artifact is explained by the partial pairing of the scramble siRNA strands with mRNAs of the genes that are essential for normal cell functioning , resulting in an inhibition of their translation . These eTo use dendrimers as carriers for therapeutic nucleic acids, their compositions should be optimized to achieve better efficiency. Along with other factors, the dendrimer:NA ratio appears to have a considerable effect on the biological activities of the dendriplexes. Using cationic carbosilane dendrimers and anti-cancer siRNAs as a convenient model, we have summarized the data on the siRNA complexation obtained by several indirect methods, to find the optimal composition for dendriplexes for the siRNA delivery. Based on our findings, we have suggested a scheme of stepwise dendriplexes formation consisting the NA binding step, and further rearrangements of dendriplexes upon the increase of the dendrimer excess. The dendriplexes containing pro-apoptotic siRNAs Bcl-2, Bcl-xL, and Mcl-1 induced the cell death in both HeLa and HL-60 cell lines. This evidences that cationic carbosilane dendrimers are versatile agents to transfect both adherent and suspension cell lines, with the latter being known to be resistant to common synthetic vectors. The results obtained provide useful practical data for the design of dendrimer-based gene therapy tools.


For that purpose, 1H-NMR spectra of both cellular extracts and culture media were combined with multivariate data analysis, bringing to light no less than 20 discriminant metabolites able to separate the metastatic metabolomes. The supervised approach succeeded in classifying the metastatic cell lines depending on their glucose metabolism, more glycolysis-oriented in the BRAF proto-oncogene mutated cell lines compared to the others. Other adaptive metabolic features also contributed to the classification, such as the increased total choline content (tCho), UDP-GlcNAc detection, and various changes in the glucose-related metabolites tree, giving additional information about the metastatic metabolome status and direction. Finally, common metabolic features detected via 1H-NMR in the studied cancer cell lines are discussed, identifying the glycolytic pathway, Kennedy’s pathway, and the glutaminolysis as potential and common targets in metastasis, opening up new avenues to cure cancer.In this study, metastatic melanoma, breast, and prostate cancer cell lines were analyzed using a  Cancer is a genetic or epigenetic disease resulting in altered metabolic activities compared to normal cells, including biological hallmarks such as the Warburg effect ,2, the oMetastasis describes a phenomenon where cancer cells disseminate from a primary site to a secondary site, a critical state responsible for about 90% of deaths in cancer patients . DependiThe metabolic flexibility of metastasis complicates the handling and treatment cares, with cells adapting to their surrounding environment to support tumor growth. Therefore, identifying the preferred metabolic pathways in different types of metastatic cancer cells could open up new avenues in metastatic treatment based on metabolic interventions. Nowadays, metabonomics approaches have been successful in detaching from the one-size-fits-all treatment to adaptive treatment in the cancer field . The met1H-NMR) spectroscopy is a non-destructive technic, highly reproducible, giving an overview of the metabolome based on the simultaneous detection of a wide range of low molecular weight (LWM) metabolites. Altogether, these metabolites draw a signature that is different for each metastatic cell line. After the digitization of spectral data, individual signatures can be compared through multivariate analyses. The high demanding metabolic cell lines used in this study were selected to cover a wide range of metastatic cancer features: The MCF-7 breast cancer cell line harbors estrogen receptors whereas MDA-MB-231 was triple-negative; the bone-metastatic PC-3 cell line was TP53 mutated, whereas the LNCaP metastatic cell line was androgen-dependent; finally, the considered BRAFV600E-mutated metastatic melanoma were either sensitive  or resistant (D10) to the standard of care BRAF/MEK combined inhibitors. This motley population of cell lines, which are gender-dependent or not, differ from one another in terms of oncomutation(s), tissue’s origin and metastatic dissemination site. Thus, all of them were considered in the present study in order to first obtain the most comprehensive view of the metabolome of those breast, prostatic, and melanoma metastatic cell lines and, secondly, to establish a new classification of metastases according to their metabolome. Thus, through this study, we hope to identify common biological pathways shared by the metastatic cell lines, but also some discriminating specific features, which all together could be used as potential targets for future therapeutic strategies. Hence, the importance of the biological pathways identified will be validated by the use of specific inhibitors in a cell viability test.Proton NMR . The separation observed among the experimental groups was perfectly highlighted on the scores plot  generated from the cellular extracts of melanoma , prostate cancer , and breast cancer  were integrated, and the numerical values exported to an Excel table. Then, a signal normalization step was performed, dividing each 0.04 ppm length descriptor by the total area under the curve (AUC) signal of their corresponding cell line. The normalized data were next integrated into the SIMCA-P+ multivariate data analysis software in order to highlight possible metabolic differences between the different cancer cell lines. No outlier possibly arising either from experimental bias or from technical issues was detected by the principal component analysis PCA-X  initiallres plot A, where ngs plot B displayN-Acetyl-Glucosamine. Furthermore, the resonances arising from both PCho and GPC were successfully separated from each other. The signature displayed in Due to overlapping or low signal intensity, some biologically relevant information can be hidden and escape the multivariate analysis. In such cases, further information can be collected directly from the spectra using the MestRenova Peak Picking tool. Thus, two extra metabolites were detected and added to the metastatic signature thanks to additional investigations, being aspartate and UDP-1H-NMR metabolites intensities was generated . MSEA is a strong tool used for data interpretation of metabolites set with relative concentration variations, indicating which biological pathway was more stimulated from one cell line to another. Thus, the analysis below highlighted the potential biological pathways related to the 13 identified metabolites A. The deFor instance, metabolic pathways such as “glutamate metabolism”, “malate-aspartate shuttle”, and the “Warburg effect” were informative on the origin of energy resources, either from glycolysis or OXPHOS. Other information provided from “arginine and proline metabolism”, “glycine and serine metabolism”, and “amino sugar metabolism” were indicative of the various usages of glucose in cancer cells and that the glucose tree structure can be quite different depending on the cancer type.1H-NMR analysis, namely lactate, myo-inositol, glycine, alanine, and UDP-N-Acetylglucosamine. This analysis was possible because of the culture media composition, uniform between the cell lines (excepting LNCaP), and containing glucose as the main carbon source. The results highlighted two main profiles between the studied cancer cell lines. In three BRAF-mutated cell lines , lactate represented more than 65% of the glucose-related metabolites AUC and was associated with high myo-inositol content and lower amounts in glycine, alanine, and UDP-N-Acetylglucosamine. Considering the remaining cell lines, the glucose was less directed towards lactate synthesis, with lactate rates reduced from 65% to 45% and with an increase in glucose-related amino acids and UDP-N-Acetyl-Glucosamine. The PC-3 glucose-related metabolism was quite different from the others, with the lesser amount of lactate between all the cell lines and intensive production in myo-inositol. A ratio B was appBMR DT, PC3, and MCF-7 cell lines that harbored a higher level of aspartate and a lower level of lactate, resulting in a small Glycolytic/OXPHOS ratio. The ratios of the D10BMR, MDA-MB-231, 451-Lu, and M229 took the opposite direction and were increased significantly, with levels >100. The 451-Lu was clearly the most glycolytic cell line based on the ratio analysis, with a value above 700. This finding is in adequacy with the PLS model described in The Warburg effect and the malate-aspartate shuttle were two pathways identified by the MSEA analysis, respectively, with the following metabolites lactate and aspartate. Lactate, the end-product of glycolysis, indicated that ATP production was favored via the glycolytic pathway, whereas aspartate, a metabolite part of the malate-aspartate shuttle used for translocating electrons across the mitochondria membrane indicated ATP production through OXPHOS. Therefore, it would be advantageous to know how cancer cells used glucose for ATP synthesis. In this respect, the lactate-to-aspartate AUCs ratio was indicative. The calculated ratio shown in 1H-NMR spectra of the culture media, returning high R2 and Q2 values . Groups separation was highlighted on the scores plot  was also carried out on the res plot A, where ngs plot  and was 1H-NMR analysis. Viability results are merged in In order to verify whether the biochemical pathways identified by the metabonomics approach were indeed critical for cancer cell survival and growth, specific chemical inhibitors were tested on each cell line and cell viability measured, except for LNCaP, which were not appropriate cells for such test due to their poor adherence properties. Thus, three inhibitors named CB-839, Hemicholinium-3 (HC-3), and Oxamate were used to, respectively, impact the glutaminolysis, the choline metabolism, and the lactate production. The established mechanisms of the considered inhibitors are displayed in V600E melanoma [i/MEKi therapy redirects the ratio in the D10BMR cell lines from glycolytic-orientated to OXPHOS-orientated, meaning that BRAF inhibition may switch the ATP production metabolism. Furthermore, the consumption of extracellular glucose and its intracellular handling also reflected the two metabolic profiles, increased in BRAF-mutated cell lines thanks to glycolytic preferred activation.Based on the lactate and aspartate intracellular levels, two metastatic subclasses were identified in terms of ATP synthesis, either more glycolytic- or more OXPHOS-oriented. As expected, the BRAF-mutated cell lines presented the most glycolytic-orientated NMR signatures, with a higher amount in lactate, the end-product of glycolysis, in both intra- and extracellular compartments , and a lmelanoma , a transmelanoma . It is wmelanoma ,25. Thermelanoma . Interes1H-NMR approach revealed two metabolites related to this energetic shuttle, namely creatine and phosphocreatine. Three metastatic cell lines  displayed reduced intensities for those metabolites in their NMR-based metabolomes. This observation suggests the limited use of this metabolic shuttle in those cell lines. Therefore, this feature could be used to classify metastatic cells since some of them preferably rely on the creatine shuttle. In addition, such a metabolic singularity could be targeted by specific inhibitors of the creatine shuttle [Phosphocreatine (PCr) is synthesized from creatine (Cr) by creatine kinase (CK) , a shutt shuttle . For tho shuttle ,31,32. P shuttle ,34. Inte1H-NMR signature gave an overview of this differential glucose handling. BRAF-mutated cell lines showed a common glucose-related profile clearly separated from the others, orientated in both lactate and myo-inositol production, indicating a glucose use for ATP and phosphoinositides productions, respectively. As discussed, all metastatic cell lines produced detectable levels of lactate, indicating sustained anaerobic glycolysis. However, BRAF-mutated cell lines showed the greatest lactate levels as a potential response to MAPK overstimulation, HIF-1-α stabilization, and sustained glycolysis. In addition, BRAF-mutated metastases overproduced myo-inositol, a precursor used in cancer cells for the synthesis of phosphoinositides, messengers involved in the phosphoinositide signaling system. These lipids family are located in the inner membrane of the cells and are involved in pro-tumoral key protein regulation such as PTEN and AKT, resulting in cell survival, proliferation, and growth [BMR, LNCaP) production. Glycine is produced from glucose through serine metabolism, via the action of phosphoglycerate dehydrogenase (PHGDH) [BMR cell lines in this category, because BRAFi resistance has been incriminated in the metabolic switch of melanoma cells, with an increase in oxidative metabolism and glutamine dependency [N-Acetylglucosamine, a metabolite derived from glucose and glutamine, in LNCaP and D10BMR cell lines indicated a post-translational ability of the metastatic cells, to add a N-acetylglucosamine group on serine and threonine residues of key proteins, promoting migration and invasion [1H-NMR signature may give some clues on how glucose is preferentially handled in different metastases, for phosphoinositides synthesis in glycolytic-orientated cells, and amino acids synthesis in OXPHOS-orientated cells. In addition, the production and detection of UDP-N-Acetylglucosamine indicate an O-glycosylation of key proteins promoting metastasis invasion.Cancer cells are highly addicted to glucose uptake and metabolism, which can be consumed through different pathways depending on their own genome mutations. Here, some glucose-related metabolites detected in the metastatic d growth . Interes (PHGDH) . PHGDH w (PHGDH) . Conside (PHGDH) . It is npendency . Finallyinvasion . A receninvasion . As a reOur experimental approach also picked up some biochemical pathways shared by the considered metastatic cancer cells, such as those involving the glucose-related metabolites, glutamine, and glutamate, and choline-related metabolites. Although the relatively low sensitivity of 1H-NMR (0.1 mM) is criticizable, nevertheless its ability to pinpoint the main metabolic hallmarks of a specific cell type makes it a suitable technic for cancer investigation. In this study, the NMR results highlighted the Kennedy’s pathway, the glycolysis, and the glutaminolysis as three relevant pathways showing different relative intensities according to the genetic profiles of the metastatic cancer cells.Enhanced glycolysis enzymes have been strongly correlated as a cancer hallmark, underlying the high-dependency of cancers to glucose uptake and metabolism. Indeed, GLUT-1 and GLUT-3 transporters presenting higher expression and activity (10–12 fold higher) in cancer cells as compared to healthy tissues . The endGlutamine, one of the most blood circulating amino acids, is overused by cancer cells to produce antioxidants, lipids through the de novo lipogenesis process, as well as purines and pyrimidines . GlutamiThe choline metabolism was also incriminated in our study, with both detection of phosphocholine (PCho) and glycerophosphocholine (GPC). A correlation between the choline metabolism enzymes and the choline metabolism metabolites have already been described in cancer. Higher amounts of tCho metabolites were detected in both prostate and breast cancer tissues, with respect to healthy ones, and associated with an increase in key enzyme activity and/or expression . It appeIn this regard, a cell viability strategy was investigated to jeopardize metastatic cells by using specific inhibitors of critical cancer cell pathways. Interestingly, our findings suggest that blocking simultaneously both glutaminolysis and lactate production, using CB-839 and Oxamate, disrupts metastatic cell viability. Nowadays, these inhibitors are mainly used alone, or in combination with the standard of care in cancer treatments. However, a few promising studies investigated the synergistic combination of these inhibitors and provided encouraging results, such as the couple CB-839/3-BP that demonstrated positive synergistic effects in tuberous sclerosis mice .This study provides a novel metastatic metabolome classification, highlighting common metabolic shares and differences between prostate, breast, and melanoma metastatic cell lines. The overview of the metabolome enabled the selection of three targetable pathways, being the glycolysis, the glutaminolysis, and the Kennedy’s pathway. As it turns out, the viability test validated the combined glutaminolysis and glycolysis inhibition, opening up an interesting approach in the metastatic metabolome disruption.BMR cell lines, prostate cancer was represented by the PC-3 and LNCaP cell lines, and breast cancer by the MCF-7 and MDA-MB-231 cell lines. The melanoma cell lines used in this study  were kindly provided from the laboratory of Lipid Metabolism and Cancer (KUL-Belgium), and used in the following publication (https://www.nature.com/articles/s41467-018-04664-0). The PC-3 (ATCC® CRL-1435™), LNCaP (ATCC® CRL-1740™), MDA-MB-231 (ATCC® HTB-26™) and MCF-7 (ATCC® HTB-22™) cell lines were purchased from ATCC. All cells, except LNCaP, were grown in 75 cm2 flask using DMEM High-Glucose  supplemented with 4 mM of Glutamine (ThermoFisher), 10% of fetal bovine serum (Gibco Lot 42G8378K), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (ThermoFisher). LNCaP condition was slightly different with the use of RPMI 1640 (ATCC 30-2001) instead as a growth media. Concerning the D10BMR melanoma cell line, two conditions were included depending on the presence or absence of the Dabrafenib/Trametinib therapy (Selleckchem). Thus, the D10BMR DT condition corresponded to the cells that were grown in the previous described media with an addition of 0.4 μM Dabrafenib and 0.1 μM Trametinib dose, whereas D10BMR were grown with the therapy and then deprived of it during 72 h for metabolic changes observations. Melanoma cell lines were composed of M229, 451-Lu and D10n = 12 wells were performed for each condition, and absorbance was read at 570 nm. The following metabolic inhibitors were used in this study: CB-839  solubilized in DMSO, Hemicholinium-3 and Oxamate (Sigma-Aldrich) directly solubilized in the culture medium. For viability testing, cells were first seeded in a 96-well plate (4-6 k cells/well) and grown for 4 days in 10% FBS media. Then, the media were removed and replaced by fresh media containing the metabolic inhibitor(s) for 72 h. After incubation, cells were washed twice with PBS and fixed with glutaraldehyde 4%. The staining was then performed using a 1% crystal violet solution, and cell wall permeabilized using Triton. A number of n = 6 samples per group. Culture media were replaced by fresh media with 10% FBS to standardize the NMR analysis 24 h before collecting the samples. The 24 h culture media were removed from each flask and stored at −20 °C. The remaining cells were washed twice with 7 mL cold D-PBS (Sigma-Aldrich), quenched using 3 mL of cold methanol, and then collected using a scraper. Cells metabolism was de novo quenched by immersing the cell pellet into liquid nitrogen for a few seconds before storing at −80 °C. A methanol: water: Chloroform 1:0.9:1 (3 mL) extraction was carried out on the cell pellet to separate polar metabolites from macromolecules. The polar phase was dried using a SpeedVacuum and was stored at −80 °C before analysis. Every cell line was grown to confluency before sample collection, with 2HPO4/0.04M NaH2PO4, pH 7.4) prepared in a mixture of H2O/D2O . The samples were centrifuged at 13,000 g for 10 min. 50 μL of a 7 mM solution of 3-trimethylsilyl propionic-2,2,3,3-d4 acid (TSP) reference prepared in 100% deuterium oxide was added to 650 μL of each supernatant. Finally, 700 μL of this final mixture was transferred into 5 mm NMR tubes prior to analysis. For the culture media preparation, 500 μL of each sample were mixed with 250 μL of phosphate buffer. The samples were prepared as described above, with an addition of 14 mM TSP solution. Acquisition of the 1H-NMR spectra were processed on a Bruker 600-MHz Advance spectrometer for 1H, using the NOESYPRESAT-1D pulse sequence. The same acquisition method was applied to the samples of cellular extracts and culture media using 256 scans.The intracellular dried samples were resuspended in 700 μL phosphate buffer . Phases and baseline of each spectra were corrected using the automatic and manual software tools. The water peak region ranging from 4.20 to 5.50 ppm was excluded, and spectra intensities were calibrated to the TSP chemical shift arbitrary fixed at 100. Spectra were binned in small subregions of 0.04 ppm width called descriptors, which gave rise to 220 descriptors for each spectrum. Next, the area under the curve (AUC) was calculated for each descriptor in all spectra to obtain numerical data. Each descriptor value was divided by the total area of the spectrum for normalization. Data were then processed for multivariate data analysis using the software SIMCA-P+ 12.0 . Principal Component Analysis (PCA) followed by a Partial Least Square Discriminant Analysis (PLS-DA) were carried out on the dataset. Possible group separations were observed in the scores plot and descriptors responsible for such separation on the loadings plot. Only descriptors with a VIP >1 were considered, and their corresponding metabolites were identified. To evaluate the quality of the model, two parameters were considered, R2, which corresponds to the « goodness of fit parameter » and explains variation in the data, and Q2, which is the « goodness of prediction parameter » and represents the predictive power of the model. Validation was carried out for the PLS model reliability using two tests: A permutation test followed by a cross-validation analysis of variance test (CV-ANOVA). Discriminant descriptors were correlated to metabolites using the Chenomx NMR suite software (version 8.1.1) and the Human metabolome database (HMDB). Because of the descriptor size of 0.04 ppm that can contain several metabolites chemical shifts, and also metabolites that have a low intensity, the multivariate data analysis was not able to detect all of the discriminant metabolites. A semi-quantification comparison of the spectra in the MestRenova software was then processed to find these new metabolites that can escape from our first analysis. p-values. Discriminant identified metabolites were converted to a heatmap in order to better visualize the metabolic signature between the cell lines. The heatmap was generated using the Morpheus online software. To identify the most changed pathways between the cell lines, a metabolic enrichment was performed on the discriminant metabolites using the online available software Metaboanalyst 4.0. The Homo Sapiens Pathway Library was selected as a reference, and the pathway analysis was investigated based on the p-values from pathway enrichment analysis and pathway impact values.The Peak Peaking tool of the MestRenova software was able to detect and precisely calculate the area under the curve (AUC) of all the peaks from each spectrum and was used for the metabolic signature validation. For each discriminant metabolite detected in the previous step, the AUC of one isolated chemical shift, previously normalized by the total AUC of each spectrum, was calculated using the Peak Picking tool in order to obtain numerical data. These numerical data were then processed in the R statistical software, using two non-parametric simultaneous tests on the data. Basically, a Kruskal—Wallis test followed by a Dunn test was performed. The Holm method adjustment was used in this validation process and applied to