In vitro studies were conducted using cell viability assays. In vivo experiments were conducted in mice inoculated with A99 or TCC-NECT-2 and treated with no agent, CDDP, CDDP+ETP (EP) or CDDP+CPT-11 (IP). TYUC-1 was the most susceptible to all agents, whereas A99 was refractory. Classical isobolograms showed synergism in both the EP and IP combinations for the three cell lines. In the TCC-NECT-2 mouse model, the IP regimen showed a significant antitumor effect, and CDDP alone showed a marginal effect compared to the control. In contrast, no effect was detected in the A99 model, probably because A99 was established from a metastatic tumor after chemotherapy with EP. Gene expression analysis of the ATP-binding cassette transporters revealed that ATP binding cassette subfamily B member1 (ABCB1) was conspicuously expressed in A99, and ABCB1 and ATP binding cassette subfamily C member2 (ABCC2) were deficient in TYUC-1, which might explain a part of different CDDP susceptibilities between cell lines. These preclinical models indicate that CDDP is a key agent, and IP regimen might be a reasonable option, although its efficacy is moderate. Our data on the platinum-based regimen will be useful as reference information in developing new agents for GEP-NEC.Gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) is a rare and devastating malignancy, and preclinical studies are needed to evaluate potential therapeutic regimens. Here, we examined the antitumor effects of cisplatin (CDDP), etoposide (ETP) and irinotecan (CPT-11) and their combinations on GEP-NEC using three small-cell GEP-NEC cell lines .  So far,g index) , 13. Recapproach , 15. Howapproach –18. Amonapproach . On the approach .in vitro and in vivo studies of GEP-NEC cell lines. We previously established A99 from a pancreatic NEC, which harbored inactivating mutations of RB1 and TP53 [et al. also established TYUC-1 from a small-cell esophageal NEC, and conducted whole-exome sequencing of the cell line, which revealed TP53, PIK3CA, KMT2D, CACNA1H, NCOR1 and HRAS mutations [in vitro and in vivo using the above small-cell GEP-NEC cell lines together with TCC-NECT-2, a cell line established from duodenum NEC.While preclinical studies are essential for understanding the cell biology of the disease and repositioning of existing agents, there have been no detailed and TP53 . We alsoand TP53 . Shimadautations . Here, w50 values determined from the curves was as follows: CDDP, 0.26 μg/mL for TYUC-1, 3.74 μg/mL for A99 and 1.06 μg/mL for TCC-NECT-2; CPT-11, 0.20 μg/mL for TYUC-1, 3.75 μg/mL for A99 and 1.85 μg/mL for TCC-NECT-2; ETP, 0.17 μg/mL for TYUC-1, 2.62 μg/mL for A99 and 2.76 μg/mL for TCC-NECT-2. Overall, TYUC-1 showed the greatest susceptibility among the three cell lines, whereas the susceptibility of A99 was generally low. In terms of combination effects, classical isobolograms for the three cell lines showed well-maintained synergism in both combinations (EP and IP) at various drug concentrations . The tumor volume on each monitoring day was expressed as relative tumor volume (RTV), which is the ratio of the volume to that on the day therapy was initiated. Similarly, the weight on each monitoring day was expressed as relative weight. The changes of RTV and relative weight on days 1–29 in each treatment group are shown in Figure P = 0.14, 0.21, 0.25 on day 22 and P = 0.57, 0.39, 0.57 on day 29). In terms of toxicities, no evident weight loss was detected in any treatment group. Hematological and non-hematological toxicities were not detected upon examination of blood samples, as shown in A99: Average tumor volume at initiation of therapy  was 196 ± 44 mm3 in the control group, 163 ± 29 mm3 in the CDDP group, 155 ± 25 mm3 in the EP group and 159 ± 20 mm3 in the IP group. As with A99, there was no significant difference in tumor volume at the start of experiments . The changes of RTV and weight on days 1–29 in each treatment group are shown in Figure P = 0.02 on day 22 and 6.46 ± 1.89 vs. 13.52 ± 2.42, P = 0.02 on day 29), and RTV in the CDDP group was marginally decreased compared with the control group . Compared with the CDDP group, the IP group showed marginal enhancement of antitumor effect on day 22 (P = 0.06). On the other hand, RTV was not significantly different between the EP group and control group (P = 0.11 on day 22 and day 29). In terms of toxicities, no evident weight loss was detected in any treatment group. Except for thrombocytopenia in one mouse in the EP group, no remarkable hematological and non-hematological toxicities were detected (Average tumor volume at initiation of therapy (with SD) was 169 ± 27 mmdetected .Histopathological images of tumors from control mice inoculated with A99, TYUC-1 and TCC-NECT-2 are shown in Figure To reveal the mechanisms causing different CDDP susceptibilities between three cell lines, we assessed the gene expressions of three representative ABC transporters (ATP binding cassette subfamily B member1 (ABCB1) termed as P-glycoprotein (P-gp); ATP binding cassette subfamily C member2 (ABCC2) as multidrug resistance-associated protein2 (MRP2); ATP binding cassette subfamily C member1 (ABCC1) as multidrug resistance-associated protein1 (MRP1)) using real-time quantitative PCR. ABCB1 expression was conspicuously elevated in A99, and deficient in TYUC-1 Figure . As for BRCA1, BRCA2, ATM) pertaining to BRCA pathway in either of the cell lines.To investigate the relationship of CDDP susceptibility to genotype, we conducted next-generation sequencing analysis for A99 and TCC-NECT-2. Since the normal counterpart of TYUC-1 (necessary for identifying somatic mutations) was not available, genomic analysis of TYUC-1 was not performed. We found no deleterious variants of representative genes  and oxaliplatin (L-OHP) using two large-cell NEC (LCNEC) cell lines derived from the gastroesophageal junction (NEC-DUE1) and large intestine (NEC-DUE2), and reported high sensitivity of NEC-DUE1 to 5-FU and low sensitivity of both cell lines to ETP, CDDP and L-OHP [in vitro data [et al. conducted targeted sequencing for 45 pulmonary LCNEC cases, and genetically classified this neoplasm into three subgroups  [et al. found that the combination of CDDP and ETP effectively reduced growth of a cervical NEC cell line in vitro [This is the first preclinical study to evaluate the effects of cytotoxic agents on small-cell type poorly differentiated GEP-NEC cell lines nd L-OHP . Althougtro data . Rekhtmarations) . Their ain vitro .in vivo study revealed significant tumor growth inhibition in the IP group and a considerable antitumor effect of CDDP single administration for TCC-NECT-2, whereas no treatment group showed marked efficacy in A99-inoculated mice. As in the case of in vitro drug susceptibility assay, the different antitumor effects on the two tumor lines could be explained by the discrepancy of potential resistance associated with the source material for cell line establishment. Our data suggest that the IP regimen might be a reasonable treatment option, and support the role of CDDP as a key agent in current treatment strategies for this disease. At the same time, dramatic tumor growth inhibition was not observed in any treated group, which implies that further new treatments are warranted for this disease. There have been no other similar studies for GEP-NEC, so it is impossible to directly compare our results with previous data. As for SCLC, Kondo et al. and Kudoh et al. independently evaluated in vivo antitumor effects of EP and IP regimens, and showed that the combination regimens had an enhanced antitumor effect [Our r effect , 29.in vitro and in vivo results. In our preliminary study, the administered dose was 1.5 times higher than in the current experiment (three treatment groups), and dose reduction was made due to the average 10% weight loss and disability in the EP group. Although blood concentration monitoring was not conducted, there is a possibility that the relatively low blood concentration of each agent resulted in an insufficient synergistic effect, especially in mice inoculated with potentially refractory A99. Although TCC-NECT-2 was also established from a patient who was receiving chemotherapy, the treatment period in this case was shorter than that in the case of A99, and moderate drug sensitivity observed in this cell line might be consistent with an antitumor effect of the IP regimen. Moreover, drug exposure time was different between the in vitro and in vivo studies , which might partly explain the difference of combination effects in the two models.There are some concerns about the discordance between BRCA mutations with CDDP sensitivity or survival [Gene expression analysis showed that ABCB1 was conspicuously expressed in chemo-refractory A99, and ABCB1 and ABCC2 were deficient in chemo-naïve TYUC-1. ABC transporters reduce uptake or enhance efflux of drugs in cancer cells, leading to decreasing the intracellular drug accumulation . ABCB1 isurvival , 40, altet al. analyzed 305 patients with advanced GEP-NEC, and reported that there was no significant difference of OS between the two platinum regimens [et al. also reviewed 258 patients with advanced GEP-NEC, and showed that RR and OS were better in the IP regimen than in the EP regimen, although the difference may partly reflect the fact that the IP regimen is mainly employed for GI-NEC, and the EP regimen for hepato-biliary-pancreatic NEC [In contrast to the availability of phase III trials or meta-analysis between EP and IP regimens for SCLC, most of the clinical data for GEP-NEC derive from retrospective studies –44. Prevregimens . Yamagucatic NEC . As mentin vitro and in vivo data using three types of GEP-NEC cell lines would be underpowered for drawing some conclusion about a clinical position of each regimen. Actually, reports about the establishment of GEP-NEC cell lines are limited, and GEP-NEC cell lines are almost commercially unavailable. Second, CDDP resistance is caused by multiple mechanisms such as increased inactivation by reactive oxygen species, mismatch repair deficiency, increased nucleotide excision repair, increased homologous recombination proficiency and over expression of antiapoptotic BCL-2 as well as ABC transporters [Some limitations should be clarified in this study. First, sporters , 54–55. In conclusion, our preclinical findings support the idea that CDDP is a key agent in treatment strategies for GEP-NEC, and the addiction of CPT-11 reasonably strengthens anti-tumor effect of CDDP. However, our study also suggests that CDDP-based chemotherapy for GEP-NEC could induce drug resistance partly owing to increased drug efflux by ABC transporters, as observed in the A99 cell line, which was established from a recurrent lesion after EP treatment. We believe that these findings will be helpful to interpret the results of prospective clinical trials. These models should also be useful in developing new agents for this intractable disease, with our data for the platinum-based regimen as a reference.In vivo experimental protocols were approved by the Experimental Animal Care Committee of the National Cancer Center Research Institute.This study was reviewed and approved by the institutional review board of National Cancer Center, and conducted in accordance with the precepts established by the Declaration of Helsinki and Laboratory Animal Welfare. Details of the three small-cell type GEP-NEC cell lines  used in this study are as follows. A99 was established from an autopsied liver specimen of a 60-year-old Caucasian woman with metastatic pancreas NEC, who had received six courses of EP regimen as first-line treatment and BCL-2 agonist as second-line treatment. The cells were propagated in DMEM medium  with 20% fetal bovine serum , penicillin 100U/mL and streptomycin 100 μg/mL, and passaged at 30% to 50% concentration using 0.25% trypsin every 3–5 days. A99 cells lacked the ability to grow on uncoated culture dishes, and were cultivated on the poly-L-lysine-coated dishes . TYUC-1 was derived from a surgical specimen of esophageal NEC in a 56-year-old Japanese woman who had not received chemotherapy, and was purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Cell number: JCRB 1512) . TYUC-1 In vitro antitumor effects were analyzed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit . Briefly, 3 × 103 A99 cells or 1 × 104 TYUC-1 or TCC-NECT-2 cells were seeded per well in 96-well plates . On the next day, CDDP , ETP (Wako) and CPT-11  were administered either as single agents or as combinations . After incubation for a further 96 hours, 20 μL of Cell Tier 96® AQueous One Solution Reagent (Promega) was added to each well, and the number of viable cells was measured in terms of the absorbance at 490 nm after three hours. The antitumor effect of each drug was calculated as the 50% inhibitory concentration (IC50) determined from the dose-response curves. This experiment was performed at least in duplicate for each cell line. In vitro combination effects with CDDP/ETP and CDDP/CPT-11 were evaluated using the combination index (CI) proposed by Chou et al. and classical isobolograms [olograms . The com6 A99 cells, 5 × 106 TYUC-1 cells or 5 × 106 TCC-NECT-2 cells per mouse into the back. When the tumor volume reached 130–250 mm3, the mice inoculated with A99 and TCC-NECT-2 were randomized into four groups  group, and 4. IP (CDDP/CPT-11) group), and the anticancer drugs were injected intraperitoneally according to a designated schedule. Drug administration in each group was performed in one cycle with a 28-day interval based on the modified JCOG 1213 protocol as follows (2)/2, where “a” is tumor length and “b” is tumor breadth. After scheduled treatments were completed, mice were sacrificed and blood samples were collected from the inferior vena cava. For assessment of hematological and non-hematological toxicities in each regimen, complete blood counts and biochemistry were evaluated.Female nude mice with the BALB/c background at the age of five weeks were purchased from Charles River Laboratories Japan . They were inoculated subcutaneously with 2.5 × 10 follows : CDDP grAfter treatment, resected tumor tissues were formalin-fixed and paraffin-embedded, and paraffin blocks were sectioned at 4 μm for hematoxylin and eosin staining (HE staining), and for Ki-67, chromogranin A and synaptophysin immunohistochemical staining. Primary rabbit antibodies to Ki-67 , chromogranin A  and synaptophysin  were used at dilutions of 1:5000, 1:1 and 1:10000, respectively. The Ki-67 labeling index was assessed by counting over 2,000 cells, and expressed as the percentage of positive cells in the most highly labeled regions.® Mini Kit  according to the manufacture's protocol. Quantified 1μg of total RNA was used to reverse transcribe cDNA with SuperScript™ VILO™ cDNA Synthesis Kit . The generated cDNA was amplified using MiniOpticon™ real-time polymerase chain reaction (PCR) detection system (Bio-Rad), and detected with SYBR® Green Master Mix . Each target gene expression was normalized through setting GAPDH as a reference, and described as relative value to that in TCC-NECT-2. This experiment was conducted in independent duplicate samples. Primers for amplification  were as follows: ABCB1 forward: 5′-GAGAGATCCTCACCAAGCGG-3′, ABCB1 reverse: 5′-CGAGCCTGGTAGTCAATGCT-3′; ABCC1 forward: 5′-CTACCTCCTGTGGCTGAATCTG-3′, ABCC1 reverse: 5′-CATCAGCTTGATCCGATTGTCT-3′; ABCC2 forward: 5′-TAATGGTCCTAGACAACGGG-3′, ABCC2 reverse: 5′-GGGCCTTCTGCTAGAATTT-3′, GAPDH forward: 5′-GCTCTCTGCTCCTCCTGTTC-3′; GAPDH reverse: 5′-ACGACCAAATCCGTTGACTC-3′.Total RNA was extracted using RNeasyU test for in vivo weight and tumor volume, and unpaired t-test for gene expression analysis. Here, two-sided P values below 0.05 were considered statistically significant. All statistical analyses were performed with Prism version 7.0a software .The statistical significance of differences between two groups was analyzed by using Mann-Whitney's


Although significant advances have been made in the biologic understanding of graft-versus-host disease (GVHD) and its treatment options, GVHD remains the single most challenging obstacle to the success of allogeneic hematopoietic cell transplantation (HCT) due to high risk of disabling morbidity and mortality. Extracorporeal photopheresis (ECP) has promising effects in controlling steroid-refractory GVHD, both acute and chronic, and it has been studied extensively. Its putative immunomodulatory mechanisms, while not immunosuppressive, position ECP as an attractive treatment strategy for GVHD patients who are already receiving global immunosuppression. However, ECP is relatively underutilized due in part to limited access and time commitment. Here, we review the recent findings on the ECP efficacy in both acute and chronic GVHD, primarily for steroid-refractory status, and we critically appraise its benefits. We also explore salient considerations on the optimal use of ECP in the treatment of refractory GVHD. Allogeneic hematopoietic cell transplantation (HCT) remains the only curative treatment option for many patients with various malignant and benign hematologic disorders . With thPathophysiology of GVHD is complex and beyond the scope of this review; however, acute GVHD is mediated by tissue injury from preparative regimen, cytokines and alloreactive cytotoxic T cell responses, and chronic GVHD results from complex dysregulation of both adaptive and innate immune responses, resulting in a variety of autoimmune-like conditions. Corticosteroid remains the established standard first line therapy for GVHD, and multiple second-line therapies have been explored to regain responses in those patients with steroid-refractory or steroid-intolerant GVHD . Major oECP exerts its immunomodulatory properties by extracorporeal exposure of peripheral blood mononuclear cells to 8-methoxypsolaren and ultraviolet (UV) A light in an apheresis procedure and subsequent reinfusion of the treated cells back into the patient’s circulation . ECP hasp < 0.0001) [In a study of 59 patients with acute steroid-refractory GVHD (grades II to IV) treated with ECP given on two consecutive days at 1 to 2-week intervals until improvement and then every 2 to 4 weeks until maximum response, Greinix et al. noted complete response (CR) rate of 82% in cutaneous GVHD, 61% in liver GVHD, and 61% in gut GVHD [n = 51) or without ECP (n = 30) [p = 0.34) [+ and CD8+ cells and higher number of regulatory T-cells [Using an adaptive Bayesian design, Alousi et al. conducted a phase II, randomized study of 81 newly diagnosed acute GVHD patients who received <72 h of steroids, and they were randomized to receive 2 mg/kg of methylprednisolone with ((n = 30) . ECP was(n = 30) . ECP was(n = 30) . By day  = 0.34) . ECP was T-cells . Resultsp = 0.1) [Several reports regarding the efficacy of ECP in acute GVHD are available. In a single-center study of 27 patients with steroid-resistant GVHD who received ECP (for 6 courses in the first 3 weeks followed by cessation if patients achieved CR or weekly maintenance therapy until CR), Garban et al. found that 9 out of 12 patients with acute GVHD responded to treatment . There ap = 0.001; 54% versus 20%, p = 0.001, respectively) [p = 0.016) [A retrospective multicenter comparative analysis of ECP versus anticytokine therapy (inolimomab or etanercept) as a second-line treatment for steroid-refractory acute GVHD were reported by Jagasia et al. . Both ovctively) . In mult= 0.016) . ECP sch= 0.016) . Table 1Abu-Dalle et al. conducted a systematic review of 9 prospective studies  for both acute and chronic GVHD treatment with ECP and reported pooled analyses of 54 subjects (6 studies) with acute GVHD  Table 1Table 1. The American Society of Blood and Marrow Transplantation (ASBMT) developed recommendations on the second-line systemic treatment of acute GVHD based on results of 29 studies (including 2 studies on ECP) which evaluated various treatment modalities for acute GVHD ,12,15. BThe Italian Society of Hemapheresis and Cell Manipulation (SIdEM) and the Italian Group for Bone Marrow Transplantation (GITMO) reviewed 11 published reports on 293 patients and recommended the use of ECP for acute GVHD not responding to steroid and calcineurin inhibitors . Better UK and Scandinavian Photopheresis Expert Group reviewed and summarized currently published guidelines . In theiBased on the available data, authors recommend considering ECP as second line therapy for acute GVHD patients who are steroid refractory or dependent. ECP should be started at minimum of 2 days per week  and be continued for 2 to 3 months on a weekly basis, or until the resolution of GVHD symptoms. ECP would also be helpful in those patients who may not be able to tolerate systemic steroids.n = 48) or immunosuppression alone (n = 47) [p = 0.48) [Reports on the efficacy of ECP for the treatment of chronic GVHD date back to the early 1990s when several case reports and small studies demonstrated encouraging results ,23,24,25(n = 47) . The stu = 0.48) . However = 0.48) . In the  = 0.48) . Clinica = 0.48) . The hig = 0.48) .In a prospective study of 25 patients with extensive, steroid-refractory chronic GVHD treated with ECP (2 consecutive days every 2 weeks in 17 patients and once a week in 8 patients) by Foss et al. , 20 patip = 0.038) [There have been multiple retrospective studies on ECP treatment for chronic GVHD ,30. We h= 0.038) . Three lAs noted previously, Abu-Dalle et al. conducted a systematic review and reported pooled analyses of 87 subjects (5 studies) with chronic GVHD . ORR forA systematic review and meta-analysis of 18 studies (both prospective and retrospective) evaluating the efficacy of ECP in steroid-refractory chronic GVHD with 595 patients was conducted by Malik et al. . The pooSome groups have published consensus statements on the use of ECP for the treatment of chronic GVHD. The SIdEM and the GITMO jointly developed recommendations on the appropriate applications of ECP in patients with GVHD . During UK and Scandinavian Photopheresis Expert Group summarized 23 studies on chronic GVHD treated with ECP in 521 patients (up to 2005) and reported that based on the 18 studies reporting the responses, the mean cutaneous chronic GVHD response rate was 68%  while that of hepatic chronic GVHD (reported in 10 studies) was 63% and 63% for mucosal GVHD (9 studies) . The 201A joint working group established by the BCSH and the BSBMT recommends that ECP may be considered as a second-line treatment in skin, oral, or liver chronic GVHD . ECP schAuthors believe that ECP would be a viable second- or third-line treatment option for chronic GVHD patients where ECP can be performed every 2 weeks for approximately 3 months to assess initial response. ECP has favorable response rates in the majority of chronic GVHD patients (ORR > 60%) with higher organ specific response rates in cutaneous, oral mucosa, ocular and GI tract.ECP plays a pivotal role mostly as a second-line therapy for both acute and chronic steroid-refractory GVHD. Its steroid-sparing benefit and facilitation of steroid taper have been demonstrated in multiple studies, and it remains an attractive option especially for those who cannot tolerate corticosteroids ,39,40,41Although numerous studies on ECP, including those with open label randomized designs, are available, the quality of evidence on the ECP as a treatment option for GVHD is somewhat limited in part due to the absence of blind studies of ECP. Many of the studies quoted in the recommendations are also retrospective in nature. Acknowledging the limitations of clinical evidence which supports the use of ECP, the procedure has benefits to GVHD patients not only based on its therapeutic efficacy but also based on its well-established safety profile. Of more than 500,000 ECP treatments performed worldwide since 1987, the incidence of reported adverse events is less than 0.003% ,36,42,43TM machine as low body mass patients would unlikely be able to tolerate the volume shifts during ECP treatment. However, the closed system CELLEXTM (Therakos) has recently replaced the UVAR-XTSTM (Therakos) system. The CELLEXTM (Therakos) system allows shorter treatment time  and treatment of lower body weight patients expanding the ECP indications to pediatric population [9/L) is considered a contraindication to ECP. Additionally, patients would need to have appropriate venous access such as photopheresis-compatible port-a-cath or central venous catheter. Physicians should discuss risks and potential complications associated with central venous access such as bleeding, thrombosis and infections. Patient access to ECP sites may be somewhat limited, though it is estimated that over 200 locations  may be available worldwide [Though ECP is generally safe, a few considerations are worth noting. First, the ECP circuit has a relatively small fluid volume . ECP would be best performed in a closely monitored outpatient unit or in the hospital, and one has to be mindful of the volume status of potentially ill GVHD patients. Secondly, intravenous 8-methoxypsolaren is only licensed in the Therakos closed system, and those patients who are photosensitive or have a sensitivity to psolaren compounds are excluded from ECP option. Additionally, weight under 40 kg is contraindicated in the ECP Therakos UVAR-XTSpulation . Both heorldwide . FurtherThe exact mechanism by which ECP induces tolerance in patients with GVHD remains an open question. There are several observations that may help to grasp current understanding of immunologic mechanisms of ECP. Data suggest that ECP-induced T-cell tolerance seems to depend on T-cell apoptosis, the presence of CD11c+ monocytes and regulatory T-cell (T-reg) induction ,45,46,47Steroid-refractory GVHD remains a major cause of disabling morbidity and mortality after allogeneic HCT. ECP is an attractive second- and third-line option for those patients with GVHD, but its use may be influenced by clinical experience, center preference and patient access due in part to the lack of high-quality randomized clinical trials comparing ECP with other modalities, and paucity of algorithms incorporating ECP over other therapeutic options. The current decisions to prescribe ECP are largely made on an individual patient basis. The procedure has an excellent safety profile and it would be an ideal option for those who are unable to tolerate higher doses of corticosteroids. Extensive literature, albeit mostly retrospective in nature, exists on the efficacy of ECP in GVHD, and clinicians should consider ECP early on as a promising therapeutic modality for those patients who require corticosteroids for the treatment of GVHD. The standardization of ECP treatment may be important in delivering consistent therapy and produce reliable outcomes. With emerging GVHD therapies modulating JAK-STAT and BTK pathways, the treatment options for GVHD patients are growing, but ECP would be a useful therapeutic armamentarium for those GVHD patients undergoing life-threatening medical conditions.


Among the therapeutic approaches employed, exon skipping using antisense oligonucleotides (AOs) is one of the most promising strategies. This strategy aims to restore the reading frame, thus producing a truncated, yet functioning dystrophin protein. In 2016, the Food and Drug Administration (FDA) conditionally approved the first AO-based drug, eteplirsen (Exondys 51), developed for DMD exon 51 skipping. An accurate and reproducible method to quantify exon skipping efficacy is essential for evaluating the therapeutic potential of different AOs sequences. However, previous in vitro screening studies have been hampered by the limited proliferative capacity and insufficient amounts of dystrophin expressed by primary muscle cell lines that have been the main system used to evaluate AOs sequences. In this paper, we illustrate the challenges associated with primary muscle cell lines and describe a novel approach that utilizes immortalized cell lines to quantitatively evaluate the exon skipping efficacy in in vitro studies.Duchenne muscular dystrophy (DMD) is a lethal genetic disorder that most commonly results from mutations disrupting the reading frame of the  Dystrophin has four domains: an actin-binding N-terminal domain, a rod domain consisting of 24 spectrin-like repeat motifs, a cysteine-rich domain, and a C-terminal domain. Dystrophin is localized to the muscle fiber plasma membrane in association with dystrophin-glycoprotein complex (DGC). The DGC anchors the sarcolemma to the outermost myofilament layer of myofiber, providing membrane stabilization during muscle contraction ,9. The cIn the absence of dystrophin, muscle fibers experience increased mechanical stress during contraction and relaxation cycles, with the sarcolemma membrane becoming fragile and susceptible to tearing and fragmentation . This maDMD exons, including golodirsen (SRP-4053) and NS-065/NCNP-01  are currently under clinical trials. The exon skipping efficacy of different AO oligonucleotide sequences needs evaluation in vitro and in vivo. Animal models such as mice and dogs have been developed for DMD, however, each presents with its own limitations [mdx mouse model for DMD exhibits a milder phenotype: their lifespan is only reduced by ~25% in contrast to humans where the disease is invariably fatal with ~75% reduction in lifespan [An active body of research continues to explore therapeutic treatments to lessen the severity of DMD . Currentitations ,31. Natuitations ,33,34. Alifespan . A celluPrimary DMD muscle cells are the most common cell type used in AO screening experiments . SimilarProlonged in vitro cultivation may lead to deviations from normal biological processes that are important for cellular differentiation in a normal in vivo environment. One problem associated with primary muscle cell cultures is the decrease in myogenicity, and subsequently enrichment for non-myogenic cells with successive passaging. S. Perie et al. showed the percentage of desmin-positive cells (a marker specific for myogenic cells) decreased significantly during primary cell cultures’ proliferative lifespan . AlthougQuantification of dystrophin expression level is the criterion by which the effectiveness of in vitro exon skipping is evaluated. Primary muscle cells prove to be challenging in this regard as they may not express sufficient amount of dystrophin mRNA and protein for quantification. Previous studies have tried to overcome this limitation by using an extra cycle of PCR to quantify the first PCR cycle’s products (nested PCR) ,43,44,45Previous studies have constructed cell models in which a plasmid harboring the native human dystrophin sequences was introduced . This arTo overcome problems with quantification, our group’s recent studies have demonstrated the feasibility of quantifying dystrophin in immortalized DMD cell lines ,48. MuscR2 of 0.89 for both exon 53 and 44. Observed dystrophin protein level also correlated well with predicted values, at an average R2 of 0.78 for exon 53 and 0.73 for exon 44. Of the parameters tested, the binding energetics of the oligonucleotide to the RNA and the distance in bases of the target site from the splice acceptor site were confirmed to be the two most predictive.In an effort to develop an in silico screening tool to predict the optimal target sites for exon skipping, our group utilized immortalized cell lines for subsequent in vitro testing to validate the predictive algorithm . These cO-methyl-phosphorothioate-based AO drisapersen, due to concerns over its therapeutic effectiveness and safety [DMD gene (hDMD mice). Exon skipping efficacy was analyzed by RT-PCR in tibialis anterior muscles two weeks after intramuscular injection of Ac0 or analog eteplirsen. Ac0 induced over 7% exon skipping, compared to less than 5% in analog eteplirsen injected mice. These experiments also demonstrated for the first time that the observed exon skipping efficacy correlated well with the amount of dystrophin rescued. Therefore, we demonstrate that immortalized cell lines have the potential to be a powerful screening tool for identifying new sequences yielding higher exon skipping efficacy, and one that overcomes many of the challenges presented by dystrophin quantifications.In 2016, the FDA approved the first AO based drug, eteplirsen, designed for DMD exon 51 skipping. The FDA’s approval of the drug remains a controversial decision; there is a dearth of evidence to support the effectiveness of eteplirsen in terms of its therapeutic effects in patients ,53,54. Td safety . Previoud safety ,42. Usind safety ,48,56. ISeveral groups, including our own, have shown immortalized cell lines to be an invaluable tool to study cellular and molecular mechanisms in a disease model ,40,48,57Although in vitro models remain an approximation of the level of complexity of a disease in a human body, immortalized cell lines prove to be a powerful tool for therapeutic preclinical studies.


A distinct p53 target gene set predicts for response to the selective p53–HDM2 inhibitor NVP-CGM097.Published 12, May 2015We would like to issue the following corrections to our research article entitled, ‘A distinct p53 target gene set predicts for response to the selective p53–HDM2 inhibitor NVP-CGM097’. In the original version of our article, TP53 mutation calls were defined based on the integration of various sources available in 2011, including exon capture sequencing (ECS) data for the majority of cell lines. Using a more recent version of the ECS with full CCLE cell line coverage complemented by the more sensitive Raindance technology, 13 cell lines initially called p53 wt have now been found to be mutant among the 356 cell lines used in the discovery analysis. Similarly, among the 52 cell lines used for validation of the 13-gene signature , 10 cell lines initially called p53 wt have now been found to be p53 mut. A total of 55 PDX models were also used for in vivo validation purposes. Among them, two models initially called p53 wt have been found mutated for p53, and one model initially called p53 mutant has now been confirmed p53 wt.In Sonkin’s 2015 report, the reannotation of cell lines affected two of our key datasets: 29 cell lines in the 'discovery set' (CCLE panel)  in the clinic, or have explored the use of gene expression-based signatures due to the complexity required in Sonkin’s approach.i.e. 13 mutated cells in the ‘discovery set’ and 10 mutated cells in the ‘validation set’ shown in red in i.e. 29 and 12 cell lines in the ‘discovery set’ and ‘validation set’, respectively). Importantly, we continue to show that our gene expression signature with the reannotation is more predictive than p53 mutation status alone.It is for these reasons that we developed a gene expression stratification signature and used p53 mutation status as the appropriate benchmark. Importantly, in Sonkin’s report (2015), 13/29 reannotated cell lines in the ‘discovery set’ harbor p53 mutations  used in our study. This correction changes the quantitative numbers in the original paper but none of the conclusions are affected. Below are the corrected tables, figures, and text that changed.https://elifesciences.org/content/4/e06498v2.The article (text and figures) has been corrected accordingly, and 2 supplementary figures have been added. The original version of the article published on 12 May 2015 is available at The updated The corrected The corrected We have added We have added The corrected The updated WT cell lines (n=52)’ is now ‘Sensitivity prediction and sensitivity to NVP-CFC218 and NVP-CGM097 of an external set of cell lines (n=52)’. The p53 mut and p53 wt cell lines are indicated in the table.Title ‘Sensitivity prediction and sensitivity to NVP-CFC218 and NVP-CGM097 of an external set of p53Corrections to the main textIn the Introduction section, we had: ‘The analysis of sensitivity profiles across 355 cell lines led to the confirmation that p53 mutant cancer cells fail to respond to HDM2 inhibitors.’ This has been corrected to ‘The analysis of sensitivity profiles across 356 cell lines led to the confirmation that p53 mutant cancer cells fail to respond to HDM2 inhibitors.’-20) (-23)  and this association was statistically significant by Fisher’s exact test  ’ is now 0) (-23) .’‘Interestingly, among all p53 wild-type cell lines for which compound data were available, 62% scored insensitive to NVP-CFC218 .’ is nowTP53 mutation status was the feature most associated with response to NVP-CFC218 , with an odds-ratio of 0.024.’ is now ‘… to NVP-CFC218 , with an odds-ratio of 0.013.’‘The TP53 mutation status. The AUC for the 13-gene signature was higher than for the other two models: 0.947 vs. 0.850 or 0.836 . The AUC for the 13-gene signature was higher than for 215-feature set model: 0.947 vs. 0.855 (‘We compared ROC (receiver operating characteristic) curves based on the predictions made on the test sets across 5 cross-validation runs for the models based on the 215-feature set, the 13-gene signature and or 0.836 ’ is now s. 0.855 .’‘Furthermore, the precision–recall curves for the 13-gene signature showed that an 80% true sensitive prediction rate could be achieved while recalling more than 90% of the truly NVP-CFC218-sensitive cell lines ’ is now vs. 59% for the 215-feature set vs. 56% for TP53 mutation status.’ is now ‘…  : 76% fors now ‘… : 76% forTP53 mutation status and the larger signature consisting of 215 significant features.’ is now ‘… and the larger signature consisting of 215 significant features. Moreover, these results suggest that the 13-gene signature has utility in TP53 wild-type genetic backgrounds.’‘Thus, these results show that the 13-gene signature provides an improvement in response prediction to drug treatment over both the ‘The predictive value of the 13-gene signature was subsequently tested in a second, independent data set composed of only wild type p53 cell lines of multiple cancer types representative of the lineages included in the first proliferation screen.’ is now ‘The predictive value of the 13-gene signature was subsequently tested in a second, independent data set of cell lines representative of multiple cancer types representative of the lineages included in the first proliferation screen.’‘As shown in ‘Moreover, 28/55 in vivo models were predicted to be insensitive, and 20/28 were found to indeed be truly insensitive, leading to a significant NPV of 71.5% .’ is now‘As shown in In the Results section, numbers and text have been updated to reflect the revised p53 mutation calls:Additionally in a p53 wild-type background (n=68), the 13-gene signature was compared to the p53 mutation model . Since iWT cell lines in this external set greatly impaired the specificity and NPV values . Taken together, these results indicate the 13-gene signature to be a better predictive feature than TP53 mutation status in unselected cell lines. In addition, our results in both the discovery  of the p53 wild-type cell lines for which compound sensitivity data were available, scored insensitive to NVP-CFC218 .’ is nowvs. 56% and 59%, respectively)   .’ is nowctively) .’In the Discussion section, numbers and text have been updated to reflect the revised p53 mutation calls:The original article has been corrected accordingly.


HOXA9 promoter methylation status is associated with response to cisplatin-based chemotherapy in bladder cancer cell lines and in metastatic bladder cancer. Bladder cancer cells resistant to cisplatin chemotherapy can be sensitized to cisplatin by the DNA methylation inhibitor decitabine. Our data suggest that HOXA9 promoter methylation could serve as potential predictive biomarker and decitabine might sensitize resistant tumors in patients receiving cisplatin-based chemotherapy.Bladder cancer is among the five most common cancers diagnosed in the Western world and causes significant mortality and morbidity rates in affected patients. Therapeutic options to treat the disease in advanced muscle-invasive bladder cancer (MIBC) include cystectomy and chemotherapy. Neoadjuvant cisplatin-based combination chemotherapy is effective in MIBC; however, it has not been widely adopted by the community. One reason is that many patients do not respond to neoadjuvant chemotherapy, and no biomarker currently exists to identify these patients. It is also not clear whether a strategy to sensitize chemoresistant patients may exist. We sought to identify cisplatin-resistance patterns in preclinical models of bladder cancer, and test whether treatment with the epigenetic modifier decitabine is able to sensitize cisplatin-resistant bladder cancer cell lines. Using a screening approach in cisplatin-resistant bladder cancer cell lines, we identified dysregulated genes by RNA sequencing (RNAseq) and DNA methylation assays. DNA methylation analysis of tumors from 18 patients receiving cisplatin-based chemotherapy was used to confirm in vitro results. Cisplatin-resistant bladder cancer cells were treated with decitabine to investigate epigenetic sensitization of resistant cell lines. Our results show that  Bladder cancer is the second most common genitourinary cancer, with an estimated 74,000 new cases and 16,000 deaths per year in the USA . NeverthChemoresistance is due to genetic and epigenetic alterations accumulating in response to treatment ,7,8. WheHOXA9 to be specifically downregulated by promoter DNA hypermethylation in resistant cell lines. We confirmed HOXA9 promoter methylation status in both cell lines and patient tumor samples. In addition, we show that HOXA9 promoter methylation status might be used as a potential biomarker for predicting cisplatin sensitivity in patients with MIBC. Furthermore, we found that low-dose decitabine and vorinostat treatment was able to induce sensitization to cisplatin and other common chemotherapeutic agents in resistant cell lines. In a translational approach to study cisplatin-based chemotherapy resistance, we evaluated a panel of 35 bladder cancer cell lines for treatment-refractory patterns. We hypothesized that bladder cancer cell lines would show differential sensitivity responses to standard chemotherapeutic drugs. Furthermore, we expected that gene expression signatures of sensitive and resistant cells (pre-treatment) could help us to identify molecular markers associated with response to chemotherapeutic drugs. We identified a distinct gene signature associated with resistant cell lines that could predict response to cisplatin in our preclinical model. Among the gene signature, we found the homeobox gene 25/IC50) values for each of them. Distinct patterns of resistance to cisplatin, vorinostat and decitabine were observed among cell lines  of cisplatin. Based on these IC50 ranks, we assembled the cell lines into three categories, which corresponded to high, intermediate, and low cisplatin resistance (50 < average (IC50) − 1 SD (standard deviation)), whereas UMUC14, RT4, 96-1 and 97-1 showed low cisplatin sensitivity (IC50 > average (IC50) + 1 SD). There was no association between the original stages/grades of the patients’ tumors from which the cell lines derive and the resistance patterns  were differentially expressed between these two groups  to identify genes that were differentially expressed between our cisplatin resistant and sensitive cell lines see B. Our ano groups A. HOXA9,ines see . HOXA9 as a biomarker, as promoter methylation of HOXA9 has been associated with aggressive tumor features, such as more advanced tumor stage, higher grade, larger tumor size and worse prognosis in patients with non-muscle-invasive bladder cancer [HOXA9 suggested that its expression could be silenced by DNA methylation at its promoter region in resistant cell lines, which would allow for the establishment of a DNA methylation-based biomarker with several potential advantages over gene expression markers [We were especially interested in the potential application of r cancer ,13,14. T markers . HOXA9 methylation has been proposed as a urinary biomarker for bladder cancer diagnosis [HOXA9 promoter methylation in resistant cell lines. The HOXA9 methylation levels of the CpG islands in each of the cell lines of the sensitive and resistant groups were quantified by real-time quantitative methylation-specific polymerase chain reaction .iagnosis . Based oreaction B. The paHOXA9 methylation between cisplatin resistant and sensitive tumors (HOXA9 when compared to sensitive tumors (pT0) (p < 0.001). Moreover, the cut-off of 12% was accurate in all cases.Next, we aimed to further confirm these findings in selected patients with MIBC responding or not responding to chemotherapy. We confirmed the differential pattern of e tumors A,B. The We hypothesized that the DNMT inhibitor decitabine  or the HDAC inhibitor vorinostat  might be able to enhance sensitivity to cisplatin in resistant cell lines. This hypothesis was based on previous evidence showing that inhibitor-induced changes in epigenetic patterns are able to return resistant tumor cells to a baseline state of treatment susceptibility ,10. 50 reduction for cisplatin in 96-1, RT4 and also SW1710 cells compared to IC50 values without sensitization treatment  cell lines and an intermediate (SW1710) cell line with the two epigenetic inhibitors alone and in combination at non-toxic doses (100 nM) has an effect on cisplatin sensitivity. We first confirmed that a concentration of 100 nM applied over 120 h (a time period comprising 3–4 population doublings with passive demethylation/acetylation due to inhibitors) does not have an effect on cell proliferation in these cell lines . We chosreatment . No signines see . Notewor50 values for a selection of other common chemotherapeutics in urogenital cancers  would decrease after pre-treatment with decitabine, vorinostat or a combination of both. As illustrated in 50 values for vinblastine in 96-1 cells and for doxorubicin, etoposide and vinblastine in RT4 cells. As observed for cisplatin resistance, single vorinostat pre-treatment did not have a pronounced effect on drug sensitization.We chose the two cisplatin resistant cell lines, 96-1 and RT4, that had shown the most significant increase in cisplatin sensitivity after epigenetic sensitization to test whether ICHOXA9 could help predict resistance or response to cisplatin-based chemotherapy in patients with bladder cancer. HOX genes belong to a group of transcription factors that regulate limb development during embryogenesis and are involved in tissue differentiation. HOXA9 is part of the A cluster on chromosome 7, and has been shown to act as an oncogene in acute myeloid leukemia, whereas silencing of HOXA9 by promoter methylation has been detected in several solid tumors such as breast, lung, ovarian and oral cancer [HOXA9 was reported to regulate the expression of the tumor suppressor BRCA1, and re-expression of HOXA9 led to reduced proliferation and migration of malignant cells [HOXA9 has been associated with detection, aggressiveness and prognostication [HOXA9 in this disease. In general, silencing of HOX genes by DNA methylation has been detected in a range of cancer types and has been associated with an epigenetic stem cell signature. In this cancer-specific signature, DNA methylation replaces repressive histone methylation marks found at genes important for development and differentiation in embryonic stem cells [In the present study, we found in a translational stepwise approach that promoter DNA methylation of the homeobox (HOX) gene l cancer ,19,20,21nt cells . In bladtication ,13,14. Hem cells ,24. ThusHOXA9 methylation levels in tumors) might be necessary for successful outcome in bladder cancer patients [50 value for decitabine compared to cell lines 96-1, RT4 and SW1710, and could—in contrast to the other three—not be sensitized by the DNA methylation inhibitor. After validation of these findings, eligible patients with low HOXA9 methylation levels could be offered cisplatin-based combination chemotherapy, while those with high levels will be recommended to undergo immediate radical cystectomy. Alternatively, patients with high levels of HOXA9 methylation could receive several days of low doses of decitabine before chemotherapy for sensitization of chemoresistant tumors. Furthermore, HOXA9 promoter methylation could be used as read-out for treatment response as a monitoring biomarker [HOXA9 promoter methylation, in a correlative biomarker experiment, to assess its association with treatment outcomes.We also have demonstrated that sensitization of cisplatin resistant bladder cancer cell lines can be achieved in vitro by pre-treatment using the Food and Drug Administration (FDA)-approved DNMT inhibitor decitabine alone and in combination with the HDAC inhibitor vorinostat, confirming a previous in vitro finding on cisplatin and decitabine co-treatment in bladder cancer cells by Shang et al. . Thus, dpatients ,11,30. Siomarker . In ordeMycoplasma sp. and cell identification by Single Nucleotide Polymorphism (SNP) using the MycoSEQ detection kit .Bladder cancer cell lines 5637, HT1197, HT1376, J82, RT4, SCaBER, SW780, T24, TCCSUP and UM-UC3 were purchased from ATCC . 647V, BC-3C, BFTC-905, CAL-29, JMSU-1, KU-19-19, RT-112, SW1710 and VM-CUB1 were purchased from DSMZ . 253J, SV-HUC, TSU-PR1, UM-UC14 and WH were obtained from Dr. Jer-Tsong Hsieh . Bladder cell lines 94-10, 96-1, 97-1, 97-7, 97-18, 97-24, DSH1, JO’N, SD, VM-CUB2 and VM-CUB3 were obtained from Dr. Margaret A. Knowles . Cell lines were re-authenticated via short tandem repeat (STR) analysis using the Cell-ID-system , and products analyzed using an Applied Biosystems 3130 Genetic Analyzer . Cell lines were cultured according to the manufacturer recommendations. We performed tests for Doxorubicin , 5-aza-2′-deoxycytidine , azacitidine , cisplatin (Sigma), gemcitabine (Sigma), etoposide (Sigma), docetaxel (Sigma), paclitaxel (Sigma), vinblastine (Sigma), panobinostat (Sigma), vorinostat (Sigma) and bortezomib (Sigma) were added from a stock solution to the 10% serum-containing RPMI medium . Doxorubicin, etoposide, docetaxel, paclitaxel, vinblastine, panobinostat, vorinostat and bortezomib were dissolved in dimethyl sulfoxide , cisplatin was dissolved in 10% DMSO (Sigma) in 10% serum-containing RPMI, and 5-AZA-CdR, azacitidine and gemcitabine were dissolved in distilled water. Dose calculation was based on human dosages and previous publications [50) were determined. Data are presented as the mean IC50 or IC25 with a 95% confidence interval for triplicate experiments.Cell viability was determined using a fluorometric resazurin reduction method  and trypan blue automatic method . Fluorescence (Ex560 nm/Em590 nm) was determined with the Synergy4 microplate reader . The number of viable cells was calculated by using the linear least-squares regression of the standard curve. Fluorescence was determined for three replicates and normalized to respective controls. To plot dose–effect curves CompuSyn  and GraphPad Prism software 6.0  were used, and drug concentrations inhibiting the growth of the cell lines by 50% compared to control (IC50. The 35 cell lines were classified into two groups: one group of extreme sensitivity (average (IC50) − one SD (IC50)) and one group of extreme resistance to cisplatin (average (IC50) + one SD (IC50)). Results were aligned by the Z-score method.To determine phenotypes of sensitivity to cisplatin, we exposed cells to a range of 11 concentrations of cisplatin (0.0025–150 μM) . Cell lines were categorized based on the ICUsing the PureLink RNA mini kit , we extracted RNA from the cell lines. We used TruSeq RNA Library Preparation Kit  to make sequening libraries. RNA was sequenced using an Illumina HiSeq platform . We obtained 60–70 million paired-end reads (51bp × 2) per cell line. After alignment using TopHat , we estimated transcript levels using CuffLinks  with upper-quartile and GC content normalization.50 values for cisplatin, etoposide, vinblastine or doxorubicin were measured after 48 h of exposure and compared to IC50 values without sensitization treatment using the method described above. Experiments were performed in triplicates for each drug.We exposed 97-1, SW1710, RT4 and 96-1 cells to decitabine at 100 nmol/L for 120 h with or without vorinostat at the same dose and then treated the cell lines with a range of ten different concentrations of cisplatin (0.3–3000 μM), etoposide, vinblastine or doxorubicin (0.1–1000 nM). ICTotal genomic DNA was extracted from eight bladder cancer cells using the PureLink mini kit (Invitrogen). EpiTYPER assays  were performed on bisulfite-converted DNA. Bisulfite conversion was performed using the EZ DNA Methylation kit from Zymo Research . EpiTYPER (Sequenom) primers were designed to cover CpG islands associated with the respective HpaII amplifiable fragments. All primers were designed using Sequenom EpiDesigner beta software. Primer sequences are shown in The study population included 18 patients who were identified retrospectively in a departmental cystectomy database  and had received a complete course of neoadjuvant cisplatin-based combination chemotherapy before radical cystectomy in the time from November 2006 to November 2010. The 15 males and three females had a mean age of 69 years at the time of cystectomy . All had histologically confirmed T2 or higher MIBC tumors in previous transurethral resection. Neoadjuvant chemotherapy consisted of the standard gemcitabine–cisplatin regimen administered for 3–4 cycles. Response to chemotherapy was defined histopathologically as the absence of residual tumor (pT0 stage) on the radical cystectomy specimen and resistance to cisplatin was defined as locally advanced disease (stage pT3–pT4). Bladder specimens from transurethral resection for DNA isolation were obtained after approval by the institutional review board and informed consent.Tissue DNA was extracted using the DNA Purification Kit . One microgram of DNA was bisulfite-modified using EpiTect (Qiagen) for the EpiTYPER assay and DNA methylation levels were determined as described above for cell lines. t-test or LIMMA on normalized arrays (after filtering for genes with a standard deviation greater than 0.5). R Bioconductor LIMMA Voom package was used  . We filtered by adjusted p value.Analysis was performed using the R open source Statistical software version 2.11.1 and Prism . To determine the differentially expressed and methylated genes we computed a Methylation differences were evaluated by a non-parametric Wilcoxon–Mann–Whitney-test . A cut-off methylation level of 12% was defined as predictive of cisplatin resistance.


Its anti-proliferative and cell death inducing capacity is largely due to its main derivative Taurultam (TRLT). In this study it could be demonstrated, that substance 2250 - a newly defined innovative structural analogue of TRLT - exhibits an anti-neoplastic effect on malignant pancreatic carcinoma in vitro and in vivo.Former studies already revealed the anti-neoplastic properties of the anti-infective agent Taurolidine (TRD) against many tumor species in vitro and in vivoThe anti-neoplastic potential of substance 2250 as well as its mode of action was demonstrated in extensive in vitro analysis, followed by successful and effective in vivo testings, using xenograft models derived from established pancreatic cancer cell lines as well as patient derived tissue.Our functional analysis regarding the role of oxidative stress (ROS) and caspase activated apoptosis showed, that ROS driven programmed cell death (PCD) is the major mechanisms induced by substance 2250 in pancreatic carcinoma. What is strongly relevant towards clinical practice is especially the observed inhibition of patient derived pancreatic cancer tumor growth in mice treated with this new substance in combination with its sharply higher metabolic stability.These encouraging results provide new therapeutical opportunities in pancreatic cancer treatment and build the basis for further functional analysis as well as first clinical studies for this promising agent. Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer, usually diagnosed at an advanced stage when curative therapy is almost impossible . It is t. This anti-neoplastic and apoptosis inducing effect could also be verified by other research groups , similar in potency to TRD. The anti-neoplastic activity of the parent compound TRD has been demonstrated in many studies on different cancer entities in vitro as well as in vivo , 20, 21 proportional or sigmoidal dose effect where increasing concentrations of substance 2250 led to an increase of cell death after 24 and 48 h. This pattern could be observed for the cytotoxicity of the substance via MTT assay, as well as for the anti-proliferative effect by BrdU analysis. This proportional dose effect pattern, observed during this study, is analog to those described in several previous studies for the treatment with TRD [In the first part of this study we determined dose-response characteristics and analyzed the relative contribution of apoptosis and necrosis during substance 2250 induced cell death. In all five different malignant pancreatic cancer cell lines the dose response effects of substance 2250 were nearly homogenous. We found a pattern which is characterized by a with TRD –26.Three pancreatic cancer cell lines  also displayed a dose-response relationship concerning the relative distribution of viable, apoptotic and necrotic cells determined by FACS analysis, where the amount of viable cells decreased with increasing concentration of substance 2250. The incubation with 1000 μmol/l of substance 2250 was characterized by apoptotic as well as necrotic cell death whereby the apoptotic effect was the predominant characteristic. In contrast, the incubation with 1500 μmol/l of substance 2250 was characterized by a pronounced necrotic effect in BxPC-3. This effect became more obvious in AsPC-1. Opposite to this, in PancTuI cells treated with 1500 μmol/l the amount of apoptotic cells prevailed. This dose depending ratio of viable, apoptotic and necrotic cells is according to previous studies with TRD in our group , 26. RegIn our in vitro studies there is no possibility of phagocytosis by inflammatory cells, because they are not present in our cell culture model. The marker, used for necrosis (PI) could ultimately not differentiate between a substance-induced primary necrosis and a secondary necrosis - due to the lack of phagocytosis. Another possible explanation might be the induction of a programmed necrosis by substance 2250 itself, as shown previously for Taurolidine . ProgramThe second part of this study deals with the contribution of reactive oxygen species (ROS) to substance 2250 induced cell death and the analysis of another major cell death associated pathway, the caspase pathway. Previous studies with TRD revealed an involvement of ROS 12, 40 , triggerWe also analyzed another major cell death associated pathway mentioned in the literature referring TRD induced cell death - the caspase pathway , 25, 33., we analyzed the anti-neoplastic effects of substance 2250 in vivo. Prior to the in vivo studies the metabolic half-life of the new substance 2250 was determined, due to the fact that Taurolidine shows a very short metabolic half-life of only 1–2 h in healthy human volunteers [On the basis of this foregone promising in vitro resultslunteers , 38–45. lunteers , mesothelunteers , malignalunteers , 45 and lunteers , 44. It lunteers , 41, 46.lunteers , whereaslunteers , 47 and lunteers  was inhiTherefore our experimental in vivo study was based on the hypothesis that substance 2250 could also inhibit the growth of pancreatic cancer after ip administration in an established cell line model and a xenograft model with patient tissue in nude mice. We observed a significant reduction of subcutaneous tumor growth in tumors induced by established pancreatic cancer cell lines  as well as in xenograft models from patient tissue . In tumors caused by established pancreatic cell lines, the ip application led to a significant reduction by approximately 50% of relative tumor volume in comparison to the controls. These results were confirmed by the xenograft models derived from patient tissue, where a reduction of relative subcutaneous tumor volume by 30–40% was observed after treatment with substance 2250.. Substance 2250 is characterized by a clear dose response relationship due to the fact that all analyzed cells lines were susceptible to substance 2250 induced cell death. Functional analysis of the involvement of ROS driven and caspase activated PCD showed, that ROS plays a fundamental role inducing cell death in pancreatic carcinoma. In contrast, PCD by caspase activation was less important. Furthermore substance 2250 seems to effectively inhibit pancreatic cancer tumor growth in mice, simultaneously displaying a higher metabolic stability, which will be a strong benefit towards clinical practice. These encouraging results are the basis for further functional analysis and initial clinical studies for this promising agent. It remains to be seen whether 2250, in analogy to TRD, also exerts anti-inflammatory activity by reducing pro-inflammatory cytokines.In conclusion, this is the first study providing an evaluation of substance 2250 induced cell death among several pancreatic cancer cell lines in vitro and inhibition of pancreatic tumor growth in vivo


Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the “go” (8MGBA-TMZres) or “grow” (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ  Over the past 33 years – 1980–2013 – 570 clinical trials were conducted where almost 33,000 patients were treated with different novel therapeutics to better understand and treat GBM2. From these extensive studies one chemotherapeutic agent – temozolomide (TMZ) – was found to moderately improve overall survival3. In the last decade there has been little advancement in treatment, with the standard of care being radiotherapy and surgery, followed by TMZ4. However, resistance to TMZ is rapid, and a broadly effective second line of treatment has not yet been established5. For these reasons, we need better models to understand mechanisms of TMZ resistance and how to develop improved therapies for the future.Glioblastoma (GBM) is the most common glioma among adults and confers an abysmally low overall survival with only 5% of patients surviving at the 5-year markN7-guanine, N3-adenine, and O6-guanine positions. The cytotoxic effect was shown to be mainly through the O6-guanine adduct, which can be reversed by the O6-methylguanine methyl transferase (MGMT). MGMT is a suicide repair protein which removes the O6-guanine adduct and allows for proper DNA repair6. Even with this information, targeting MGMT with both small molecule drugs and mimetics has been unsuccessful, and its expression does not always correlate with resistance to TMZ11. Therefore, new models of resistance need to be developed in order to better define molecular mechanisms of GBM resistance to TMZ.Cell line models have been invaluable in elucidating the molecular mechanisms behind the uncontrolled growth of cancer cells. As resistance to TMZ is rapid in clinical models, cell lines were used to better understand the mechanism behind the initial efficacy of TMZ sensitivity. TMZ is a prodrug that is preferentially activated in a more alkaline environment, which the brain provides, that spontaneously breaks down to highly reactive methyldiazonium cations. These byproducts preferentially methylate DNA bases at the 12. After continual exposure to TMZ in culture, both cell lines no longer undergo TMZ-mediated G2/M arrest. As the cell lines acquired resistance to TMZ both cell lines gained expression of MGMT, showed an increase in nuclear size and chromosome number, and an increase of early endosomes and extracellular lipids. However, as they differed in their growth and migratory capacity, these cell lines may give us insight into the “go or grow” model for GBM recurrence13. The 42MGBA-TMZ resistant cell line had an increase in growth capturing the “go” of the “go or grow” model for resistance. By contrast, the 8MBGA-TMZ resistant cell line had an increase in migration, encompassing the “grow” aspect of resistance. These models will be useful tools for the field to better define essential drug resistance mechanisms in GBM. These two TMZ-resistant (TMZres) models from both a male and female patient have evolved distinct resistant phenotypes and therefore serve as valuable resources to investigate the heterogeneity of TMZ-resistant GBM.In this study, we present two unique TMZ-resistant GBM cell lines derived from the 42MGBA and 8MGBA parental lines which were first characterized in 1997. The 42MGBA line was derived from a temporal lobe tumor resected from a 63-year old male. The 8MGBA line was derived from a frontal lobe tumor resected from a 54-year old femaleTo better recapitulate acute temozolomide (TMZ) resistance models, we created cell lines that were resistant to 200 µM TMZ and were challenged in 100 µM TMZ for all experiments. The overall time to acquired resistance varied from approximately 2 months for the 8MBGA cell line to ~3 months for the 42MBGA cell line. Cells were defined as resistant when each no longer showed a G2/M arrest or apoptosis in response to TMZ treatment Fig. . While b14. Because BCNU can still be a component of second-line treatment for GBM18, we tested whether our TMZres cells exhibited altered responsiveness to BCNU. Since the resistant cell lines are populations selected from the wild type cell lines they have a more reproducible phenotype as the variability or heterogeneity of the cell line has decreased. Therefore, the TMZres cells still showed a statistically significant G2/M arrest upon BCNU treatment. However the biological effect is dramatically reduced, with a 74% increase in G2/M vs 8% increase in G2/M for the 42MBGA-WT vs. 42MBGA-TMZres cells compared to controls, respectively , leads to an increased sensitivity to TMZely Fig. . This isely Fig. .5. As cells have already copied their DNA at this point in the cell cycle, if a cell is to survive TMZ-treatment they may also be retaining the extra copies of chromosomes that have already been duplicated. The 42MGBA-WT cell line has a hypertetraploid karyotype (88–95 chromosomes) with 8% polyploidy, while the 8MGBA-WT cell line has a hyperdiploid karyotype with 15% polyploidy and contains 47–52 chromosomes . 42MGBA-TMZres cells showed a significant increase in total nuclear area  . We observed that 96% of the 42MBGA-TMZres cells had three or more copies of the X chromosome compared to only 7% of the 42MBGA-WT cells (93% of those cells had 2 copies). In contrast, this dramatic shift was not observed in 8MBGA-TMZres cells, where only a small subpopulation of cells showed an increase in the number of chromosomes 17 (18% had 3 or more copies) compared to the parental cells (6% had 3 or more copies). Taken together, these findings tracked with the stability of TMZ-resistance, with the 42MBGA-TMZres cells showing a more stable phenotype compared to 8MBGA-TMZres cells . We observed an increase in overall chromosome number in the majority of the 42MBGA-TMZres cells in contrast to the 8MBGA-TMZres cells where only a small subpopulation of cells had an increase in overall chromosome number Fig. . We alsoSH) Fig. . The cholls Fig. .Figure 3We then determined how TMZ-resistance affected cell size and proliferative vs migratory phenotypes. 42MBGA-TMZres cell size was not changed vs 42MGBA-WT, though their basal growth rate was dramatically increased Fig. . They al19. Consistent with our observation of increased extracellular vesicles, there was an increase in palmitic acid and stearic acid lipids in the GC-TOF-MS metabolites from the 8MBGA-TMZres cell line conditioned media  are implicated in TMZ responsiveness26.The enrichment of lipid metabolites and aminomalonate identified in conditioned media from resistant cells by LC-MS and GC-TOF-MS, respectively, could suggest dysregulation of mitochondrial metabolism. We therefore determined whether the expression of electron transport chain (ETC) subunits is altered between sensitive and resistant lines. There were no changes between ETC subunits in the 42MBGA-WT and -TMZres lines. However, there was an increase in three ETC subunits in the 8MBGA-TMZres cell line as compared to its WT control: MT-CO1 (Complex IV), SDHB (Complex II), and NDUFB8 (Complex I) Fig. . Increasin vitro. True to the heterogeneous nature of clinical GBM, the phenotypes displayed by these resistant cells were similar, but not identical. TMZ treatment no longer induced G2/M arrest in either of these cell lines, and both were markedly less responsive to BCNU, the previous standard of care for GBM. Both resistant cell lines also gained expression of the MGMT protein, which is responsible for removing one of the DNA adducts caused by TMZ treatment5.In the present study, we created two new GBM cell lines that are resistant to the standard of care drug TMZ 18. These TMZres cell line pairs may therefore also prove to be valuable resources for defining contexts in which BCNU is or is not appropriate second-line treatment, and for identifying other, more appropriate therapies for TMZ-resistant GBM.An important finding of the present study is that both TMZres models have severely impaired response to BCNU. For decades BCNU was the standard of care for GBM, and it is still a component of some second-line regimens. Findings from clinical studies examining the efficacy of BCNU in recurrent GBM post-TMZ treatment are inconsistent, with some suggesting BCNU remains active while others demonstrate no benefitin vitro as opposed to isolation from a resistant tumor is the ability to directly compare them to their respective sensitive parental lines. There was an increase in nuclear size in both resistant cell lines when compared with their parental cell lines. In the 42MGBA-TMZres cell line there was also a change in nuclear morphology which has been described in other GBM cell lines that have acquired resistance to TMZ28. This may be associated with stability of the resistant phenotype, as we observed that the 42MBGA-TMZres cell line resistant phenotype can be maintained without continuous TMZ treatment. However, the 8MGBA-TMZres cell line, which has not undergone nuclear structure reorganization, and showed two distinct populations with respect to metaphase spreads, regained partial sensitivity when cultured in the absence of TMZ.A key advantage provided by creating these resistant cell lines 29. In these novel TMZ resistant models, we hypothesized that the increase in nuclear size was a function of retained chromosomes. This is consistent with the mechanism of action of TMZ, which induces a G2/M arrest in cells. Cells which escape TMZ-mediated cell cycle arrest and death have already replicated their DNA, and may therefore retain sister chromatids that were not able to separate during anaphase. Because both parental GBM cell lines were initially polyploid with distinct karyotypes, we assessed two different chromosomes – 17 for the 8MBGA-WT and –TMZres cell lines, and X for the 42MBGA-WT and –TMZres cell lines – as an indicator for chromosomal copy number alterations. Both TMZ resistant variants showed significant copy number gains. Of note, our chromosome 17 locus specific probe targets the STAT5 gene, which has already been implicated as a pro-tumorigenic factor in GBM30. The increased copy number of the STAT5 locus in the 8MBGA-TMZres cell line is consistent with previous work and suggests that the observed increase in nuclear size is at least partially attributable to an increase in chromosome number. Because chromosome 17 existed in multiple copies in the 42MBGA-WT cell line, we chose an X-centromeric probe to test chromosomal copy number changes in the 42MBGA-TMZres line. Again, we saw an increase in X-chromosome copy number in the 42MBGA-TMZres line, and an overall increase in the total number of chromosomes. While it can be energetically disadvantageous to increase ploidy, we hypothesize that, as for STAT5, there may be X chromosome genes that when duplicated give a proliferative advantage to these cells.Increases in nuclear size can be driven by increased chromosome numbers or impaired transport of mRNA or proteins out of the nucleus31. However, these phenotypes are rarely displayed by the same cells, giving rise to the “go or grow” hypothesis to explain aggressive behavior and drug resistance28. Our novel in vitro models of TMZ-resistant GBM provide examples of both phenotypes. The 42MBGA-TMZres cell line did not exhibit any significant changes in migration, cytoskeletal structure, or cell size when compared to its parental cell line, but it did proliferate significantly faster. In contrast, the 8MBGA-TMZres cell line had a significantly increased migratory capacity, F-actin thickness, and overall cell size relative to 8MGBA-WT cells, but no accompanying increase in proliferation. These divergent alterations in basal growth rate and migratory capacity provide two unique models – one adapting the “go” (8MGBA-TMZres) and the other the “grow” (42MBGA-TMZres) strategy of TMZ resistance13. There have been many hypotheses as to what determines the switch between “go or grow”, including mutational status, hypoxia, and influences of the surrounding cell secretome33. Activation of the FAK and MAPK/ERK pathways have been shown to be differentially regulated between a “go” and “grow” phenotype33, respectively. Others have also hypothesized that remaining cells from the “go” population lead to recurrence of GBM13. Therefore, more insight needs to be gained into potential common pathways between the “go or grow” phenotypes that could be used as therapeutic targets for both cell populations. Candidate strategies to revert “go” phenotypes could include inhibition of the integrin signaling axis, either through blockade of integrin engagement35 or inhibition of FAK36, while MEK inhibition could suppress the “grow” phenotype38. These four cell lines are therefore valuable tools that can be used to provide important insight into the differential mechanisms of TMZ resistance.Two major challenges in the clinical management of GBM, particularly after TMZ resistance has developed, are rapid tumor cell proliferation and widespread local migration or invasionin vitro setting, Oliveira et al. showed that the in vitro secretome can affect the “go or grow” phenotype of murine GBM cells32. Therefore is it important to identify potential in vitro mechanisms that can be further extrapolated to in vivo models in the future. The increase in early endosome marker EEA1 expression is interesting, as others have shown EEA1 to interact directly with RRAD, STAT3, and EGFR to regulate EGFR’s subcellular localization and resistance to TMZ39. Clinically, higher EEA1 mRNA expression is associated with poor survival. EGFR and its structural variants have been targeted extensively in GBM, but with limited success. We suggest further study of the mechanisms by which EEA1 contributes to the TMZ resistant phenotype could provide a new way to target EGFR by modifying its subcellular localization rather than activation40. In addition to an accumulation of early endosomes, we observed vesicles expelled into the media of the resistant cell lines. Others have shown that TMZ-resistant glioma stem cells release more exosomes than their sensitive counterparts, which was consistent with our in vitro created models41. This was reflected in the putative increase of lipids from the LC-MS analysis. Another hit, indoleacetaldehyde, is a product of tryptophan metabolism, hyperactivation of which occurs in GBM and is thought to suppress anti-tumor immunity19. Extracellular lipids, stearic and palmitic acid, were also found and validated in the GC-TOF-MS analysis along with citric acid. Citric acid is a key regulator of metabolism, and when increased in the cerebral spinal fluid of GBM patients confers a lower overall survival23. The top hit from GC-TOF-MS was the non-aminogenic amino acid aminomalonate. Aminomalonate inhibits aminolevulinic acid synthase, the rate-limiting enzyme of the protophorphyrin IX (PpI IX)/heme synthesis pathway20. PpI IX pathway activity is exploited for intra-operative imaging in GBM, and has more recently been used for potential therapeutic gain43. Defining the drivers of aminomalonate secretion in our TMZ-resistant models may be an important future strategy to identify when PpI IX pathway-based imaging and therapeutic approaches may be warranted.Acquisition of TMZ resistance was associated with a marked increase in the number of intracellular and extracellular vesicles in our models. While there are always potential limitations to studying metabolism in an In conclusion, we present two novel TMZ-resistant GBM cell lines from both a male and female patient that have distinct proliferative and migratory capacities while sharing certain GBM resistance characteristics such as increased chromosome number, nuclear size, and vesicle formation Fig. . These mMycoplasma contamination, and were maintained in a humidified incubator with 95% air: 5% carbon dioxide. All cell lines were fingerprinted by the LCCC Tissue Culture Shared Resource to verify their authenticity using the standard 9 STR loci and Y-specific amelogenin. Both the 42MBGA-TMZres and 8MGBA-TMZres fingerprinted the same as their parental cell line. 42MGBA and 8MBGA cells were grown in DMEM with 10% FBS. 42MBGA-TMZres and 8MBGA-TMZres cells were grown in DMEM with 10% FBS and 100 µM TMZ. TMZ  was dissolved in DMSO to 130 mM and used at concentrations indicated. Bis-chloroethylnitrosourea (BCNU) was a kind gift from Dr. Esther Chang, and was dissolved in ethanol to 100 mM before use at concentrations indicated.42MBGA and 8MGBA cell lines were provided by Dr. Jeffrey Toretsky (Lombardi Comprehensive Cancer Center (LCCC), Georgetown University, Washington DC). 42MBGA-TMZres and 8MGBA-TMZres were developed by our lab with constant exposure of the parental cell lines to increasing concentrations of TMZ, from 12.5, 25, 50, 100, 200 µM, over the course of 2–3 months. All cells tested negative for On day 0, cells were seeded at 100,000–150,000 cells per well in 6-well plastic tissue culture dishes one day prior to treatment with the indicated concentrations of drug. For experiments with TMZ or BCNU cells were treated for 72 hours. After 72 hour treatments, cells were collected, ethanol-fixed, stained with propidium iodide, and analyzed for cell subG1 (fragmented) DNA content and cell cycle profile by fluorescence activated cell sorting.Cells were lysed in RIPA buffer for protein extractions and separated by a 4–12% gradient gel . They were then transferred onto Nitrocellulose membranes  with the iBlot 2 and probed with antibodies against MGMT , Beta-Tubulin , PARP , Actin , and Electron Transport Chain antibody cocktail . Proteins were detected with horseradish peroxidase-conjugated secondary antibodies  and enhanced chemiluminescent detection HyGLO Quick Spray Chemiluminescent (Denville) for dark room development.44. For metaphase chromosomal copy number determination, chromosomes were stained with 4′,6-diamidino-2-phenylindole (DAPI). FISH analysis was performed using a standard protocol47. A 17q21.1-21.2 locus-specific FISH probe (Stat5A/B locus) was used to evaluate chromosome 17 copy number change in the 8MGBA-TMZres and WT cell lines, as described earlier45. For the X chromosome enumeration in the 42MGBA-TMZres and WT cell lines, an X chromosome centromeric probe obtained from Empire Genomics  was used according a manufacturer’s protocol. Scoring of cells and digital image acquisition were performed using a Leica DMRBE microscope  equipped with the appropriate optical filters and a CCD camera45.Metaphase and interphase slide preparation was done using a standard protocol48 to determine percent closed with 0% being at 0 hr.Cells were plated at 150,000–200,000 cells/well and allowed 48 hours to create a monolayer. After monolayer formation, a P200 tip was used to make the scratch. Images were taken at 0 hr, 24 hr, and 48 hr time points. Analysis was done in ImageJ48) was used to measure the thickness of F-actin fibers. Grayscale images of phalloidin-stained cells were converted to binary using the Threshold tool (black/white). The BoneJ plugin49 was then used to measure fiber thickness in pixels. The mean thickness per cell is reported for 28-29 individual cells per cell line.FIJI  with 0.2% Triton X-100 in PBS for 5 minutes at room temperature. Three washes were performed with PBS in the 12-well plate, then coverslips were inverted onto 100 μl of primary antibody in the antibody block  on strips of parafilm and incubated for one hour. Coverslips were first stained with EEA1 , an early endosomal marker . Phalloidin , and DAPI  stains were added to the secondary antibody mixtures. Each coverslip was washed three times with PBS after primary incubation and then were inverted onto 100 µL of the appropriate secondary antibody, DAPI dihydrochloride, and (where appropriate) ActiStain-488-phalloidin  in antibody block in the dark for 20 minutes. Coverslips were again washed 3x with PBS, then gently dipped three times into molecular biology-grade water before inversion onto one drop of FLUOROGEL  then allowed to air-dry in the dark for at least 10 minutes. Slides were stored at 4 °C until image collection on the LCCC Microscopy & Imaging Shared Resource’s Leica SP8 microscope with the 63x oil objective.2. The 25 µL sample was deproteinized using cold acetonitrile (40%): methanol (25%): water (35%) (175 µL) containing internal standards . All solvents used were LC-MS grade . Samples were vortexed and incubated on ice for 10 min. Incubated samples were centrifuged at 13000 rpm for 20 min at 4 °C. The supernatant was evaporated in a speed-vac with no heat. Dried samples were kept at −80 °C until analysis. For analysis, the dried samples were reconstituted in acetonitrile (1%): methanol (5%): water (94%) (200 µL). Samples were centrifuged for 20 min at 4 °C and the supernatant transferred to a vial for LC-MS analysis. Briefly, samples were injected (2 μL) into a Waters Acquity UPLC system coupled to a Xevo® G2 QTOF-MS using Leucine enkephalin as Lockspray® for accurate mass calibration. Data was processed, aligned, and analyzed using MassLynxTM , XCMS online50, Metaboanalyst, and R51.Cell culture media (500 µL) was concentrated to 25 µL under NInternal standards (1 µM 4-nitrobenzoic acid) were added to culture media (500 μL) that was deproteinized as above and then evaporated in a speed-vac and transferred to a GC vial with a silanized insert. Derivatization and injection was performed with a Gerstel  autosampler with automated liner exchange and cooled injection system. The dry residue was derivatized with 20 μL O-Methoxyamine-Hydrochloride  at 40 °C with constant agitation (30 min), and then with 80 μL of N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA)/with 1% trimethychlorosilane (TMCS) (Thermo Scientific prod # TS48915) at 40 °C with constant agitation (30 min). Samples were incubated at 20 °C for 4 hrs before injection (1.5 μL) for GC-TOF-MS analysis.52. Electron impact ionization (70 eV) at full scan mode (m/z 40–600) was used, with an acquisition rate of 30 spectra/sec. Fatty acid methyl esters , alkanes , and quality control standards  were run to ensure reproducibility of retention indices and derivatization. Data was processed, aligned, and analyzed using ChromaTOF v. 4.51.6.0 with the statistical compare function , Metaboanalyst, and in house software MetaboLyzer53 as previously described52.Samples were injected in split mode (1:5) into an Agilent 7890B GC system  that was coupled to a Pegasus HT TOF-MS . Separation was achieved on a Rtx-5 w/Integra-Guard capillary column , with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The temperature of the inlet, transfer line, and ion source was set to 150, 270, and 230 °C, respectively. The GC temperature programming was set to 0.2 min of isothermal heating at 70 °C, followed by 10 °C/min ramp to 270 °C, a 4.0 min isothermal heating of 270 °C, 20 °C/min ramp to 320 °C, and a 2.0 min isothermal heating of 320 °C as previously describedCells were seeded at a density of 1,000 cells per well in 3, 96-well plastic tissue culture dishes per cell line on day 0. On day 1, one plate was stained with crystal violet . For staining, plates were rinsed 1 time with 1X PBS to remove excess cellular debris. After, 100 µL of 3.2% PFA was added to each well and incubated at room temperature for 5 minutes followed by three washes in 1X PBS. Then, 200 µL of 0.5% crystal violet in 25% methanol was added to each well and incubated at 4 C for 10 min. The stain was then removed and the plate was rinsed 4–6 × with diH2O to remove excess stain. The plates were left to air-dry overnight. On day 8 and 10, all plates were rehydrated with 100% methanol and read at an absorbance of 550 nm.Cell size was determined by the Countess 2 (Invitrogen) using trypan blue dye exclusion of dead cells. The Countess 2 was also used to determine number of cells for 72-hour growth assays. 100,000 cells were seeded per 6-well on day 0. 24, 48, and 72 hours post-seeding, cells were collected and total cell number was determined by trypan blue dye exclusion.51.Results are represented as mean values ± standard error (SD) and considered statistically significant where p-value < 0.05. Three independent biological replicates were done for each experiment, with the exception of metabolomics where six independent replicates were performed. Statistical significance has been calculated using GraphPad Prism version 7.00 for Mac, GraphPad Software, La Jolla California USA (www.graphpad.com) for the Student’s t-test, Mann Whitney U test, one-way ANOVA with Dunnett’s correction, or χ2 analysis of 2 × 3 contingency table. The RStudio packages survival, GGally, ggplot2, readr, and magrittr were used for violin plots and big data sets Supplementary Figures