APH) ligand with Cu(II) (1), Co(II) (2), Ni(II) (3) and Zn(II) (4), have been synthesized and characterized using different physicochemical methods. The structure of the ligand APH and its copper complex 1 have been established by single-crystal X-ray diffraction direct methods, which reveal that complex 1 has distorted square-pyramidal geometry. Complexes 1–4 are screened against seven human cancer cell lines namely, breast cancer cell lines , prostate cancer cell lines  and the colorectal cancer cell line Coco-2, for their antiproliferative activities. Complex 1 has shown a promising anticancer activity compared to the other ones. The structural and spectroscopic analysis of APH and its complexes are confirmed by DFT calculations.Four new complexes derived from adamantly containing hydrazone ( In adhe 1960s ,6,7.d-orbitals, are the main factors in the activities and selectivities of the designed complexes. Another factor that also effects both selectivity and the activities of complexes is the existence of biologically relevant moieties as a counter ligand to the metal of interest.The ability of complexes to adopt numerous three-dimensional structures allows them to have a diverse formation of functionalized targets of unique molecular structures, geometries and kinetic properties , which mOne main strategy adopted in molecular design of the complexes is to change the central metal ion/atom/oxidation state that leads to change in the physiochemical, thermodynamic and kinetics properties, hence, making the complex scaffolds potential candidates for many pharmaceutical applications ,9,10. InOwing to their rapid access, availability and diversity, hydrazones are one of the most studied classes of compounds. Acylhydrazones have an additional donor site, namely C=O, which determines the versatility and flexibility of these compounds. Such molecules have attracted considerable attention for their antibacterial ,15,16,17In this contest, adamantane derivatives are known to exhibit considerable biological significance, which includes antiviral , antibacQuite recently, we had reported on the synthesis of new adamantane containing hydrazone compounds, where pyridine was one of the arms within the hydrazine-adamantyl compounds ,34, wherThe selected metal ions in this study are of proven record as an essential element that plays a crucial rules in various biological settings ,6,7. TheAPH) was obtained via condensation of 2-pyridinecarbaldehyde with adamantane-1-carboxylic acid hydrazide according to published procedures , as shown in In the ligand omplexes . A molecC4v) with CShM = 1.545, in comparison with the closest value for a vacant octahedron (a regular polyhedron with one or two vertices removed) with CShM = 3.186.Applying continuous shape measures (CShM) on Cu(II) ion by comparing with all reference standard 5-coordinate polyhedrons were calculated using SHAPE 2.0 program . This fiAdamantane derivatives are found to have numerous biological activities, angiogenesis inhibition , antivir1), Co(II) (2), Ni(II) (3) and Zn(II) (4) containing adamantne-1-carboxylic acid pyridin-2-ylmethylene hydrazide APH are studied.In this work, the cytotoxicity of four different complexes Cu(II)  are used to assess the effects of those compounds on cell proliferation. Two different concentrations of the test compounds are used: 12.5 μM and 50 μM. The % of cell growth inhibition is assessed by an MTT assay. The obtained results are compared with the ligand se cells ), is useAPH at both treatment doses of 12.5 and 50 µM. Compounds 1 and 2 are the most cytotoxic of them all. Compound 1 has shown activities of 93.30 ± 1.20%, 88.60 ± 1.17%, 90.26 ± 0.55%, 89.88 ± 0.39%, 89.85 ± 0.73%, 87.06 ± 1.57% of cell viability inhibition in MCF7, T47D, MDA-MB-23, PC3, DU145 and Caco-2 cell lines, respectively. Compound 2, with a concentration of 50 µM  complexes with 1-adamantoyl hydrazone bearing pyridine rings are found to induce apoptosis and inhibit agiogenesis in some tumor cell lines ; Anal. Calcd for C17H21N3O·H2O: C, 67.87; H, 7.84; N, 14.12; O, 10.17. Found: C, 67.75; H, 7.69; N, 13.94; O, 10.62%.APH. The resultant solution was refluxed for 2–5 h. The produced solid complexes were filtered off, washed with cold ethanol, dried under vacuum and recrystallized from an appropriate solvent.The complexes were prepared by adding equimolar amounts of the appropriate metal (II) chloride hydrate to a magnetically stirred ethanol solution of 3. Green crystals ). Selected FTIR (KBr): max (cm−1) = (C=O) 1588 s, (C=N) 1527 s; (C–N)py 684 m. HRMS : m/z 381.06681 ; Anal. Calcd for C17H21Cl2CuN3O: C, 48.87; H, 5.07; N, 10.06. Found: C, 48.95; H, 5.12; N, 10.13.Recrystallized from CHCl3. Green crystals .Recrystallized from CHClmax (cm−1) = (C=N) 1615 s, (C=N) 1529 s; (C–O) 1146 s; (C–N)py 677 m. HRMS : m/z 377.06950 [[M–Cl]+, Calcd for C17H21ClCoN3O: 377.06997]; Anal. Calcd for C17H21Cl2CoN3O: C, 49.53; H, 4.89; N, 10.19. Found: C, 49.75; H, 4.93; N, 10.25%.Selected FTIR (KBr): Green powder ).max (cm−1) = (C=O) 1606 s, (C=N) 1523 s; (C–N) py 670 m. HRMS : m/z 376.07218 .Selected FTIR (KBr): White powder ).1H NMR : δ 1.73 ; 1.81 ; 1.89 ; 7.40 d; 7.87 m (2H); 8.39 s (N=CH); 8.54 d (1H); 11.36 s . 13C NMR : δ 26.1 ; 27.6 ; 38.3 ; 146.3 (C=N); 120.9, 124.8, 137.5, 149.5, 153.0 (C-pyridine); 174.4 (C=O). Selected FTIR (KBr): max (cm−1) = (C=O) 1615 s, (C=N) 1529 s; (C–N) py 677 m. HRMS : m/z 416.02835 ; Anal. Calcd for C17H21Cl2N3OZn: C, 48.65; H, 5.04; N, 10.01. Found: C, 48.45; H, 5.12; N, 10.09.APH and complex 1 were prepared using relevant crystallographic data. The structure refinement parameters for APH and complex 1 are summarized in APH and 1 are listed in APH are listed in APH) and CCDC-1558283 (for 1) contain the http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; or by e-mail: deposit@ccdc.cam.ac.uk.Single crystal X-ray diffraction data were measured using Oxford diffraction Xcalibur (Mo) X-ray source (λ = 0.71073 Å) at 293(2) K. Data collection, reduction and cell refinement were performed using CrysAlisPro . The str1, seesaw geometry on complex 2, distorted square pyramidal on complex 3 and distorted tetrahedral geometry on complex 4. The optimized structure of complex 1 is in complete agreement with the obtained single crystal structure one. In order to validate the calculations on molecules using the previously mentioned methods, another DFT calculation was conducted on complex 1 with Ahlrich valence triple zeta basis set with added polarization functions DefTZVP. Results from both basis sets eluded the same frequencies as assigned in Structures of all complexes were optimized in silico gas phase using DFT methods with the Pople valence triple zeta basis set with added diffuse and polarization functions 6–311 + G . No negative or imaginary frequencies were obtained for any structures. All calculations were performed on G16 running HPC cluster. Calculations were on conducted on distorted square pyramidal on complex Three selected human breast cancer cell lines were used in this study namely: the Michigan Cancer Foundation-7 (MCF7), human breast cancer cell line, commonly used in biomedical research, involving the hormonal expression of cancer cells (T-47D), and M.D. Anderson and MB Metastasis Breast cancer (MDA-MB-231). Two human prostate cancer cell lines: the prostate cancer 3 cell line (PC3) the human androgen-independent prostate cancer cell line (DU145); and one human colorectal cancer cell line (Caco-2).2 incubator, and were harvested using trypsin-EDTA solution.The MCF7, T47D, MDA-MB-231, DU145 and PC3 cell lines were cultured using RPMI 1640 medium, whereas DEMEM medium was used for the Caco-2 cell line. The media were supplemented with 10% fetal bovine serum (FBS) , 1% of 2 mM L-glutamine, 50 IU/mL penicillin and 50 μg/mL streptomycin . All cells were grown at 37 °C in a 5% CO3 cells/well in 96-well plates and allowed to attach overnight. Cells were treated with two different concentrations (12.5 μM and 50 μM) of the tested compounds 1, 2, 3, 4 and doxorubicin, which was used as a positive control. The experiment was done in 4 replicas. After 72 h treatment, MTT -2,5-diphenyl-tetrazolium bromide) assay was performed according to the cell proliferation assay kit . Absorbance (OD) was measured at 570 nm, with background subtraction at 630 nm. DMSO was used as a negative control, and the percentage of viability inhibition was measured as an indication of the cytotoxicity of the tested compounds.Cells from each cell line were seeded at a density of 10 × 101) was analyzed using its X-ray single structure analysis, and it was found that it exists in a square pyramidal geometry. Complex 2 exists in the form of Co(III) ions. Further investigations of the structure activity-relationship studies of our new complexes are in progress in our laboratory, and the results will be reported in due course.In the present work, we have reported the synthesis, characterization and anticancer activities of four new adamantylpyridylhydrazone complexes. All prepared complexes contain the new tridentate N,N,O-donor Schiff base, which is derived from theadamantylpyridylhydrazone nucleus. One crystalline complex (


Tobacco smoking is the leading preventable cause of cancer. Moreover, continued smoking during cancer therapy reduces overall survival. Aware of the negative consequences of tobacco smoking and the challenges of smoking cessation, cancer patients are inquiring whether they should switch to electronic cigarettes (e-cigarettes). To obtain evidence-based data to inform this decision, we examined the effects of e-cigarette aerosol exposure on cisplatin resistance in head and neck cancer cells. Our results show that cancer cells exposed to e-cigarette aerosol extracts and treated with cisplatin have a significant decrease in cell death, increase in viability, and increase in clonogenic survival when compared to non-exposed cells. Moreover, exposure to e-cigarette aerosol extracts increased the concentration of cisplatin needed to induce a 50% reduction in cell growth (IC50) in a nicotine-independent manner. Tobacco smoke extracts induced similar increases in cisplatin resistance. Changes in the expression of drug influx and efflux transporters, rather than activation of cell growth-promoting pathways or DNA damage repair, contribute to e-cigarette induced cisplatin resistance. These results suggest that like combustible tobacco, e-cigarette use might increase chemotherapy resistance, and emphasize the urgent need for rigorous evaluation of e-cigarettes health effects to ensure evidence-based public health policies. Despite improved treatment options, including targeted therapies, the prognosis of HNSCC patients remains poor. The average 5-year survival rate has remained practically unchanged over the last 40 years: at 75–83% for patients with localized HNSCC, 45–65% for those whose cancer spread to regional lymph nodes, and only 33–40% for those with distant metastasis2. Only 29% of oral cavity and pharynx cancers are diagnosed at the local stage2. Thus, most head and neck tumors present as advanced disease, requiring combined modality approaches that frequently include radiotherapy and platinum-based chemotherapy, with or without surgery4. Platinum-based chemotherapy consisting of either cisplatin or carboplatin is the usual first-line treatment for inoperable recurrent or metastatic HNSCC5. In addition to late diagnosis, therapy resistance is one of the main factors contributing to poor cancer prognosis. Cisplatin resistance can emerge due to a host of environmental factors as well as genetic or epigenetic alterations in cancer cells6.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy, with over 657,000 newly diagnosed cases and 330,000 deaths per year worldwide12. Smoking at the time of diagnosis has been shown to increase resistance to radio- and chemo-therapy13 and tumor recurrence15. Alarmingly, data show that about 60% of lung and head and neck cancer patients, who were cigarette smokers before their diagnosis, continued to smoke during treatment16. Concerned about the negative consequences of smoking but unable or unwilling to quit due to nicotine addiction, cancer patients, and consumers in general, are increasingly seeking other methods of nicotine delivery.Tobacco smoking is the leading preventable cause of death and the main risk factor for lung and head and neck cancers. It is also well established that cigarette smoking has negative implications on cancer outcomes, including increasing disease progression, incidence of second primary tumors, and treatment-related toxicity, and reducing patient survival19, free-radicals20, and heavy metals 25. Some e-cigarette aerosol components are carcinogenic or potentially carcinogenic 26. Curiously, some of the individual constituents present in e-cigarette aerosols have been shown to increase chemotherapy sensitivity 27, while others have been shown to increase chemotherapy resistance 29. Yet, thanks to few restrictions and a lack of regulatory guidelines, e-cigarettes are aggressively marketed both as a safer alternative to combustible tobacco and as a smoking cessation aid. Consequently, the use of e-cigarettes has increased exponentially since introduction into the U.S. market in 2007. More than 5.3 million American high and middle school students reported using e-cigs in 201930. Among adults, current smokers had the highest rate of use, with 52.5% reporting to ever used an e-cigarette and 10.8% currently using e-cigarettes31. The rate of e-cigarette use reported in lung and head and neck cancer patients was about 33% in 201332, and is currently the highest (~ 80%) among cancer patients who are younger than 50 years old and current smokers33. These alarming numbers and the presence of harmful and potentially harmful chemicals in e-cigarette aerosols24, coupled with the paucity of short- and long-term studies on the safety of e-cigarettes, all stress the urgent need to investigate the potential effects of e-cigarettes on cancer treatment thoroughly.Electronic cigarettes (e-cigarettes) are battery-operated devices that heat a solution of chemicals  with or without nicotine, to produce an inhalable aerosol. Studies have shown that e-cigarette aerosols contain harmful and potentially harmful toxicants such as volatile organic compounds 34. Nicotine, a main component of e-cigarette aerosol, has been shown to reduce cisplatin-induced cell death28. Other toxicants present in e-cigarette aerosols have also been shown to positively or negatively impact chemotherapy sensitivity. Hence, we hypothesized that exposure to e-cigarette aerosol might alter cisplatin resistance. Here we show that exposure of oral cancer cells to e-cigarette aerosol extracts increases cisplatin resistance through nicotine-dependent and independent mechanisms.E-cigarettes can deliver nicotine levels similar to those observed in smokers13. Moreover, in vitro studies have demonstrated that cigarette smoke condensate and nicotine can increase cancer therapy resistance in diverse cancer cell lines, including those derived from HNSCC35. Hence, it is essential to determine whether exposure to e-cigarette aerosols might modify cancer response to chemotherapy. To explore this important clinical question, we began by evaluating the effect of e-cigarette exposure on cell viability after cisplatin treatment. Three distinct HNSCC cell lines  were exposed to e-cigarette aerosol extracts for 48 h, followed by 48 h of treatment with cisplatin (10 µM) in the presence of e-cigarette aerosol extracts. Nicotine is the only tobacco smoke component with a well-documented role in cisplatin resistance28. To dissect the contribution of nicotine, within the complex e-cigarette aerosol mixture, to cisplatin resistance our study design included an extract without nicotine (E0). In total, we used five different e-cigarette extracts  obtained from two distinct brands of e-cigarettes with varying e-liquid nicotine content . All extracts were diluted to a dose of 10 puffs/5 L, yielding exposures that encompass nicotine concentrations between 0 and 39 ng/ml  extracts. Cell viability was determined by MTT  assay. Exposure to e-cigarette aerosol extracts led to a significant increase in cell viability after cisplatin treatment (p < 0.01) in WSU-HN6 and UM-SCC-1 cells for all extracts compared to the vehicle control  increase in cell viability after cisplatin treatment  in all cell lines, indicating that nicotine-independent mechanisms contribute to the observed results  increase in cell viability across all cell lines after cisplatin treatment.Concurrent smoking during cancer therapy has been reported to increase chemo and/or radiotherapy resistanceml Table , a range36, exposure to e-cigarette aerosol or tobacco smoke extracts alone, at the doses used in this study, did not increase cell growth  reduced the cell death induced by cisplatin in all cell lines and for all extracts  reduction in cisplatin-induced cell death for each e-cigarette aerosol exposure across all cell lines. Collectively, these data show that in vitro exposure to e-cigarette aerosol does not change cell proliferation but significantly reduces cell death induced by cisplatin. Remarkably, our data suggest that nicotine-independent mechanisms play a key role in the observed decrease in cisplatin-induced cell death.To rule out the possibility that the observed increase in cell viability reflected an effect of the extracts alone on cell growth, we exposed UM-SCC-1, WSU-HN6, and WSU-HN30 cells to e-cigarette aerosol extracts alone for 96 h and measured cell proliferation by MTT assay. Consistent with previous reportsh Figure a–c. Thes37, and has been reported to be predictive of cancer chemoresistance in patients38. Hence, to further investigate the potential clinical significance of our observations, we examined whether the presence of e-cigarette aerosol extracts altered the dose of cisplatin needed to induce a 50% reduction in head and neck cancer cell growth. HNSCC cells were exposed to e-cigarette aerosol extracts for 48 h and treated for an additional 48 h with extracts and cisplatin (0.01–100 μM). Compared to vehicle-exposed cells (control), exposure to e-cigarette aerosol extracts significantly increased the cisplatin IC50 values for all extracts and cell lines  is an in vitro measure essential for understanding the pharmacological potency of a drug under specific conditionslatin 0.0–100 μM. 39. Cell viability after cisplatin treatment provides a measure of cisplatin effects on a short time interval , but does not inform about the indefinite reproductive viability of the surviving cells40. To further elucidate the potential clinical implications of e-cigarette use during cisplatin treatment, we used the clonogenic survival assay to test whether cancer cells surviving cisplatin treatment retained their indefinite reproductive ability. Cells were exposed to e-cigarette aerosol extracts for 48 h, followed by 48 h treatment with cisplatin in the presence of e-cigarette aerosol extracts. Cells were then collected and plated for clonogenic survival assessment. The formation of large colonies visible by eye after 1–2 weeks  higher increase in clonogenic survival after cisplatin treatment than cells exposed to e-cigarette extracts  in WSU-HN6 and UM-SCC-1 for all e-cigarette aerosol extracts, and in WSU-HN30 for E18 , and thus are nicotine-independent  reduction in MMS19 and ERCC1 expression in all three cell lines 44. Nonetheless, the following ATP-binding cassette (ABC) ATPase genes ABCG2, ABCC2, ABCA1, and ABCC1 have been associated with cisplatin resistance in diverse cancers47 Thus, we investigated the mRNA expression levels of CTR1, ATP7A, ATP7B, ABCG2, ABCC2, ABCA1, and ABCC1 after exposure to e-cigarette extracts alone without cisplatin. Following 48 h of e-cigarette exposure, we observed that the expression of the copper transporter CTR1, which mediates the uptake of cisplatin, was down-regulated at the mRNA level in WSU-HN6 for all e-cigarette extracts tested, but remained unchanged in the other cell lines  for all e-cigarette extracts in WSU-HN6 and UM-SCC-1  increase in ABCG2 protein expression in all cell lines and for all extracts . A considerable increase in ATP7A and a decrease in CTR1 protein expression following exposure to e-cigarette aerosol were also observed for all cell lines tested  for all cell lines following exposure to E18. The decrease in CTR1 expression was significant for all extracts in cell line WSU-HN30  and increased ATP7A (p < 0.05) and ABCG2 (p ≤ 0.001) expression. Our data clearly shows that exposure to e-cigarette aerosol alters the expression of the main known cisplatin active membrane transporters, which might lead to reduced cisplatin influx and increased efflux and consequently increase in cisplatin resistance. Supporting the clinical relevance of these findings, the expression of CTR1, ATP7A and ABCG2 has been reported to have predictive value in the response to platinum-based chemotherapy and patient prognosis in tobacco-associated cancers50.In addition to faster repairing of DNA lesions, reduced cisplatin accumulation inside cancer cells is one of the primary mechanisms responsible for cisplatin resistances Figure a. The co1 Figure b. Among e Figure c–f, whilt Figure g. Of thee p < 0.0 for all e p < 0.0 for all e p < 0.0 for all 33, 33% of head and neck cancer patients32, and 80% of cancer patients younger than 50 years old and current smokers33. Moreover, nearly 50% of all cancer patients, and the vast majority of head and neck cancer patients, are treated with cisplatin44. In the present study we investigate whether exposure to e-cigarette aerosol alters cisplatin response in cancer cell lines established from oral and pharyngeal mucosa, a primary site for e-cigarette aerosol deposition51. Our data show that, at least in vitro, e-cigarette exposure during cisplatin treatment reduces cancer cell death and increases the dose of cisplatin required to induce a 50% reduction in cell growth (IC50) in head and neck cancer cells. Consistent with previous reports35, we also observed that exposure to tobacco smoke extracts increased cisplatin resistance in oral cancer cell lines. These data are clinically relevant as in vitro cisplatin resistance has been reported to be predictive of cancer chemoresistance in patients38, and resistance to platinum-based chemotherapy is one of the main factors contributing to poor patient prognosis48.We have uncovered a novel effect of e-cigarette aerosol, the induction of cisplatin resistance in cancer cells, through nicotine-dependent and -independent mechanisms. To our knowledge, this is the first report documenting that e-cigarette aerosol has the potential to negatively impact cancer treatment and ultimately patient survival. This is highly relevant as e-cigarettes are used by over 10% of all cancer patients52, further supports the clinical relevance of these in vitro studies.Exposure to e-cigarette aerosol significantly increased cisplatin resistance in all HNSCC cell lines tested, but the amplitude of the increase varied by cell line Table . Notably40. Curiously, while the effect of e-cigarette exposure on cisplatin-induced cell death and cell growth (IC50) was independent of nicotine content, the amplitude of the increase in clonogenic survival, although still considerable, was lower in the absence of nicotine in two (UM-SCC-1 and UM-HN30) of the cell lines studied  similar to that commonly observed in the plasma of vapers and smokers34, while the studies cited above used pure nicotine at much higher nicotine concentrations (146–1460 ng/ml). So, it is possible that the proliferative and antiapoptotic effects of nicotine are only seen at much higher nicotine doses. Supporting this hypothesis, a dose- and time-dependent effect of nicotine on oral cancer cell survival has recently been reported, with low nicotine concentration (15 ng/ml) showing no effect, and 146 ng/ml increasing cancer cell survival and reducing cisplatin-induced apoptosis56. Another possibility is that some e-cigarette aerosol components counteracted the proliferative and anti-apoptotic effects of nicotine. In favor of this hypothesis, glycerol a main e-cigarette humectant, constituting 30–70% of most e-liquids, has been shown to inhibit oral cancer cell growth and enhance cisplatin-induced apoptosis27. Altogether, these data show that at the clinically relevant nicotine concentration used in our study, nicotine is not the major contributor to the observed increase in cisplatin resistance associated with e-cigarette aerosol exposure. Nonetheless, our data also shows that the presence of nicotine in the e-cigarette aerosol can increase the number of cancer cells that retain their indefinite reproductive ability  present in the aerosol. Flavorings and flavored e-liquids without nicotine have been shown to trigger inflammatory responses, mediated by ROS production, in vitro59. E-cigarette aerosols containing propylene glycol with and without nicotine have also been shown to induce the release of cytokines such as IL-6, type I interferon (IFN) and other pro-inflammatory mediators60. Metals present in the aerosol  have been shown to modulate drug transport at a posttranscriptional level61, as well as to contribute to increased ROS levels62. In turn, ROS and pro-inflammatory mediators such as IL-6 and IFN have been reported to increase chemotherapy resistance by up-regulating anti-apoptotic genes, stimulating stemness, increasing repair of damaged DNA, and increasing drug efflux63. Exposure to metals , oxidative stress, or inflammatory cytokines  can also increase metallothioneins64, a class of low-molecular-weight thiol-containing proteins that bind and inactivate cisplatin and is considered one of the most important mechanisms of cisplatin resistance44. Consistent with these mechanisms being physiologically relevant, e-cigarette users have been shown to have increased levels of systemic biomarkers of oxidative stress65 and inflammation 66, and increased levels of metals and metallothioneins in the urine67. Overall, these findings show that besides nicotine, e-cigarette users are exposed to significant levels of ROS, metals and other aerosol components which can potentially contribute to cisplatin resistance by inducing multiple non-redundant molecular or cellular events, including inflammatory responses and oxidative stress, increasing drug detoxification, and altering cisplatin drug transporters.Collectively, our data suggest that nicotine-independent mechanisms play a significant role in the observed increase in cisplatin resistance. Nicotine-independent mechanisms have been reported in several e-cigarette health effects, including induction of oxidative stress and DNA damage in oral cell lines46. Yet, cisplatin resistance stems from complex genetic and epigenetic changes occurring in almost every mechanism supporting cell survival, including cell growth-promoting pathways, DNA damage repair, cell-surface binding, and transporters for cisplatin47. As discussed above, at the dose of e-cigarette tested, no increase in cell growth was observed. Our data also show that short-term exposure (48 h) to e-cigarette extract causes a decrease in the expression of nucleotide excision repair genes essential for the removal of cisplatin-induced DNA lesions. Supporting the relevance of these findings, long-term exposure to e-cigarette aerosols consistently42 leads to a decrease in the expression of DNA repair genes  in vitro36, in mice43, and in e-cigarette users68. These data document that the increase in cisplatin resistance observed during e-cigarette aerosol exposure is not due to cell growth-promoting pathways or to an increase in DNA damage repair.The anti-tumor function of cisplatin, an intercalating agent, has been linked to its ability to crosslink with the purine bases on the DNA, causing DNA damage, interfering with DNA repair mechanisms, and subsequently inducing cell cycle arrest and apoptosis in cancer cells45. Despite, not having the dominant role in cisplatin resistance, ABC proteins, such as ABCG2, ABCC2, ABCA1, and ABCC1, transport a wide variety of chemical compounds in an ATP-dependent manner and play an important role in cisplatin resistance in diverse cancers47. Of these, ABCG2 has been characterized as the main ABC protein contributing to tobacco smoke-associated cisplatin resistance35. Consistent with the multifactorial nature of cisplatin resistance41, our data show that exposure to e-cigarette aerosol extracts significantly alters the expression of the main cisplatin drug transporters  in all cell lines studied. We also observed a significant increase in ABCC2, ABCA1, and ABCC1 mRNA, in at least one cell line, following exposure to all e-cigarette extracts. At the protein level, our linear regression analysis also consistently showed that, when adjusted for cell lines, exposure to each of the e-cigarette aerosol extracts significantly decreased the expression of CTR1 (p < 0.001), the principal active cisplatin influx transporter, and increased the expression of multidrug efflux transporters ATP7A (p < 0.05) and ABCG2 (p ≤ 0.001). The alterations in protein expression were nicotine-independent, as they were observed even after exposure to the nicotine-free e-cigarette aerosol extract -based DNA profiling and multiplex PCR by CellCheck Cell Line Authentication .Human epithelial cancer cell lines from different head and neck regions UM-SCC-1 (floor of mouth), WSU-HN6 (tongue), and WSU-HN30  were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% antibiotic–antimycotic (Gibco). Cell cultures were maintained in an incubator at 37 °C and 5% CO36. E0 corresponds to an e-cigarette aerosol without nicotine . Media was replaced every other day with freshly diluted extracts. Cisplatin was reconstituted in 0.9% w/v sodium chloride to a stock concentration of 1 mg/ml, and aliquots were kept at − 20 °C. For cell culture treatment, cisplatin was diluted to desired concentrations by serial dilution in complete media. 0.9% sodium chloride was used as a vehicle control. The amount of nicotine across extractions was measured by mass spectrometry.E-cigarette  aerosol and mainstream tobacco smoke (MS) extracts were prepared as previously describedne Table . Cells wCells were plated at optimized numbers in 96-well plates and subsequently treated with e-cigarette aerosol extracts for 48 h. At 48 h, plates were replenished with media treated with e-cigarette extracts or treated with e-cigarette extracts and cisplatin. Cell viability was evaluated by the MTT  assay . 5 mg/ml MTT was added to cells and incubated for 4 h. The resultant formazan crystals were dissolved in the solvent  overnight. MTT assay was performed at 96 h of e-cigarette exposure. OD values were obtained by spectrophotometry . Experiments were repeated at least three times, each with six replicates.To examine cisplatin chemosensitivity, we measured cell viability using the MTT assay. In brief, cells were seeded in 96-well plates using optimized cell numbers for each cell line. Cells were incubated at standard conditions for 48 h with e-cigarette extracts, followed by treatment with e-cigarette extracts and cisplatin at concentrations  for another 48 h. Cell viability was evaluated by the MTT as described above. The dose of cisplatin required to induce a 50% reduction in cell growth  was computed from fitting a symmetrical sigmoidal curve to the logarithm of the response with the logarithm of the cisplatin concentration as the predictor using GraphPad Prism software, California, USA. Experiments were repeated at least three times, each with six replicates.To assess colony survival, cell lines were exposed to e-cigarette extracts for 48 h, followed by another 48 h with e-cigarette extracts plus 2.5 μM cisplatin doses. Cells were trypsinized, counted, and seeded (at pre-optimized numbers determined from a test experiment using various cell dilutions to yield a trustable number of colonies) in six-well plates and assessed for colony formation. Cells were cultured without added extracts for 8–16 days. Media was replaced every three days to allow colony formation. Each experiment was performed three times with three technical replicates. At suitable time points, colonies were fixed with methanol followed by staining with 0.5% crystal violet in 25% methanol for 10 min. Colonies were washed with DI water and air-dried. Colonies with ≥ 50 cells were counted. Colony images were obtained using colony counter, Optronix GelCount, Abingdon, OX14 4SA, UK.Cells were seeded in 48-well plates overnight. Cells were subsequently exposed to e-cigarette aerosol extracts for 48 h followed by treatment with e-cigarette aerosol extracts and 2.5 µM cisplatin for another 48 h before analysis. To assess cell death, cells were harvested using trypsin, pelleted, and stained with 0.4% trypan blue staining at a 1:1 ratio (v/v) and counted using a Countess II automated cell counter (Invitrogen) to determine the percentage of live and dead cells. Floating cells were included.35. To determine the levels of mRNA expression, the first-strand cDNA was amplified using KAPA SYBR FAST Universal (KAPA Biosystems) on a BioRad CFX384 Real-Time System, BioRad, California, USA. The sequences of primers used are listed in Supplementary Table Total RNA from cells exposed to extracts for 48 h was extracted using TRIzol reagent (Invitrogen) and quantified using a nano-drop followed by Reverse Transcriptase PCR using random primers per manufacturer’s recommendations. The 48 h time interval has been used as standard in the tobacco field to assess the effect of tobacco exposure in diverse cellular mechanisms including cisplatin resistanceCells were exposed to e-cigarette aerosol extracts and MS smoke extracts for 48 h. Cells were trypsinized, pelleted, and washed with ice-cold phosphate-buffered saline (PBS) followed by resuspension in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1X Protein Inhibitor Cocktail and phenylmethylsulfonyl fluoride (PMSF) (Roche). Protein concentration was measured using the Bio-Rad protein assay dye reagent concentrate (Cat. #500-0006) with bovine serum albumin (BSA) as the standard. 30–50 µg of protein per lane were loaded onto 7.5–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes . After blocking with 5% non-fat milk or 5% BSA, blots were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-ATP7A , anti-CTR1 , anti-ABCG2 , and β-actin as a loading control . Membranes were washed three times with Tris-buffered saline with 0.05% Tween and incubated in secondary antibodies for 1 h at room temperature. Secondary antibodies were rabbit anti-mouse IgG  and goat anti-Rabbit IgG . The protein bands were visualized using the Clarity Western Blot enhanced chemiluminescence (ECL) Substrates . Expression was detected using the ChemiDoc™ touch imaging system  and analyzed using Image Lab software .p value < 0.05 was considered statistically significant throughout the analysis. The analysis was performed using SAS software. IC50 values were obtained by analyzing the data using GraphPad Prism 5.0 software.All data are expressed as mean ± SD unless otherwise stated. The difference in the treatment outcome  by treatment  was tested using One Way ANOVA, separately for all three cell lines. Linear regression analysis was performed to assess the effect of each treatment (exposure) on each outcome  adjusting for cell line specific effect. A Supplementary Information


The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has generated a significant repercussion on the administration of adoptive cell therapies, including chimeric antigen receptor (CAR) T-cells. The closing of borders, the reduction of people transit and the confinement of the population has affected the supply chains of these life-saving medical products. The aim of this mini-review is to focus on how the COVID-19 pandemic has affected CAR T-cell therapy and taking into consideration the differences between the large-scale centralized productions for the pharmaceutical industry versus product manufacturing in the academic/hospital environment. We also review different aspects of CAR T-cell therapy and our managerial experience of patient selection, resource prioritization and some practical aspects to consider for safe administration. Although hospitals have been forced to change their usual workflows to cope with the saturation of health services by hospitalized patients, we recommend centers to continue offering this potentially curative treatment for patients with relapsed/refractory hematologic malignancies. Consequently, we propose appropriate selection criteria, early intervention to attenuate neurotoxicity or cytokine release syndrome with tocilizumab and prophylactic/preventive strategies to prevent infection. These considerations may apply to other emerging adoptive cell treatments and the corresponding manufacturing processes. The SARS-CoV-2 coronavirus has generated an unprecedented global impact on multiple aspects of society, economy and health. SARS-CoV-2 was reported as a new emerging zoonotic pathogen in Wuhan (China) in December 2019, and declared as a pandemic by the World Health Organization in March 2020 .1 are being carried out testing CAR-based products. Some of them are financed by the industry and others are being developed in the academic ambit. The cohabitation of both is inevitable and necessary. Industry production could supply CAR T-cell therapies to reach the whole population using good manufacturing practice (GMP) accredited large facilities to centralize the manufacturing process. On the other hand, academic centers should be dedicated to develop CAR T-cell therapies against less frequent diseases. Independently, both have been seriously affected by the COVID-19 pandemic despite the differences they present in product manufacturing and management processes (Chimeric antigen receptor (CAR) T-cell therapy is a life-saving bioengineered cell replacement therapy against leukemia. This adoptive antitumor immunotherapy, based on autologous T-cells transduced with a genetically engineered receptor for CD19 to redirect their cytotoxicity against native CD19 surface antigens expressed in tumor cells, has completely changed the management of patients with hematologic malignancies such as acute lymphoblastic leukemia (ALL) or non-Hodking lymphoma (NHL) –6. Tisagrocesses . The manrocesses –10.Owing to the doubtless relevance of CAR T-cell therapy for hematologic patients, it is important to have in mind some considerations within the context of the COVID-19 pandemic. Therefore, the aim of this mini-review is to focus on how the effects caused by the pandemic have affected this therapy taking into consideration the differences between the large-scale centralized production of CAR T-cells by the pharmaceutical industry versus the product manufacturing processes employed by the academic/hospital environment. We also review different aspects of CAR T-cell therapy, including patient selection and resource prioritization performed in our center during the COVID-19 pandemic.Chimeric antigen receptor T-cell therapy has elicited an unprecedented response against B-cell malignancies, but it is associated with significant toxicity, including prolonged cytopenia, cytokine release syndrome (CRS), and neurotoxicity –13. Toxiin vivo modified T-cell expansion and survival  activated and expanded in bioreactors and the resulting product might be (iv) adequately prepared and cryopreserve in infusible media for the final (v) product re-infusion into the patient  obtaining the starting patient’s cell population by leukapheresis followed by the (ii)  patient , 25. All patient .The main difference between academic and pharmaceutical CAR T-cells, regarding the timeline of the manufacturing process, is obtaining the “living drug.” As for academic products, commercial CART19 are based on autologous T-cells and each patient requires his own T-cells. In the academic CAR T-cell manufacturing, all steps are carried out in the hospital facilities enabling a more flexible, personalized and coordinated “patient-friendly process.” It allows short transfer times from the leukapheresis site to the production facilities and from the production center to the patient’s bed, both being less than 1 hour . In commHowever, COVID-19 has rapidly constrained travel and mobility, extending the delivery times of commercial CAR T-cell products. Another side effect of reduced mobility are the potential disruptions in the resources supply line essential to the manufacturing process. This may affect both academic CAR T-cell products and those produced by industry. For example, the parallel process to obtain vectors with the CAR transgene like lentiviruses is a compendium of GMP skilled steps where different reagents are needed. Similarly, other specialized products are essential for the activation and expansion of T-cells in a bioreactor like CliniMACS Prodigy , 29. TheThe reduction of personnel due to possible contagions among the staff, the restructuring of the workforce to treat infected patients and the limited resources and protective equipment has been a cause for concern . All of The staff and the reagent shortage can affect the leukapheresis process and cell processing in the laboratory. There exist strict regulations associateds with cell therapy where personnel who develop “living drugs” must work under sterile and GMP conditions. For this reason, a contagion that could spread between qualified personnel and the consequent imposing quarantine that would be imposed on the rest of the personnel can become a bottleneck in the global production of the adoptive cell therapy. The stringent and immobile regulation has not been amended in the context of the COVID-19 pandemic to ensure the safety of patients, medical staff and laboratory personnel.Similarly, within the associated legislation, a series of quality controls are imposed and must be complied with before CAR T-cell product infusion. Among them, the confirmation of the product sterility is essential. In addition, it must be ensured that the product is free of endotoxins, adventitious viruses and mycoplasma. There are other non-microbiological controls that must be carried out as the number of copies of the CAR per genome, flow cytometry experiments to determine the percentage of cells with CAR, among others. Having these controls in place for each cell product requires precious time in COVID-19 pandemic setting and is highly dependent on human work. Therefore, any delay in verifying all of these control points may delay the infusion.Once the manufactured CAR T-cell product is infused, a resulting CRS is the widest described associated side effect. A compilation of different symptoms from hypotension to fever due to a massive pro-inflammatory cytokine release  have been reported . Serum IThe management of CRS is extremely relevant given that viral infections (not only COVID-19) may promote CRS . CRS reqIn fact, the current pandemic has exposed us to an ethical dilemma regarding the prioritization of TCZ. Each case must be thoroughly evaluated to ensure the availability of this drug. Tocilizumab could decrease the duration and severity of COVID-19 symptoms allowing the weaning of the ventilatory support . On the CAR T-cell therapy has been consolidated as a potential curative therapy for patients with refractory/relapsed hematologic malignancies. The COVID-19 pandemic represents an unprecedented challenge to continue safely treating patients with this adoptive cell therapy. Nevertheless, centers should continue offering this potentially curative treatment with CAR T-cell therapy for critical patients using appropriate selection criteria, early intervention to attenuate side effects like CRS with standardized TCZ protocols, and prophylactic/preventive strategies to prevent infection. For those heavily treated patients, time is crucial. Mobility and personnel restrictions add obstacles to a truly personalized therapy in which multidisciplinary teams intervenes. This has affected both academic CAR T-cell therapies and those commercially available. There are several limitations related to delivery and the time-consuming manufacturing processes that create new concepts concerning the benefit of academic point of care proposals. Also, some regulatory rules should be re-evaluated to become more flexible during this pandemic.MJ and MP proposed, directed, discussed, and revised the development of the manuscript. GM-S, NE, and IOL had equally contributed to the data gathering and writing of the review. VO-M reviewed and collaborated particularly on aspects related to the hematological issue. VB and CG had been in charge of analyzing the technical aspects related to the product manufacturing. All authors contributed to the article and approved the submitted version.VO-M is a recipient of a research grant from Fundación Española de Hematología y Hemoterapia (FEHH), also declares travel grants , consultant or advisory fees  and honoraria (Kite). The remaining 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.


Uveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available. This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.Six UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions. 3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas. PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation. Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid. In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.All six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages. PUM spheroids were genetically identical to the original sampled tumor. In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells. Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.UM cell lines and PUM samples can successfully generate uniform 3D spheroids. PUM spheroids retain histological and genetic characteristics of the primary tumor. 3D spheroids are an important system for use in future high-throughput drug testing.The use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients. The increasing use of three-dimensional (3D) in vitro cell cultures in cancer is contributing to the development of more physiologically relevant models of tumor biology than standard two-dimensional (2D) cultures. 3D spheroids more closely recapitulate the physiology of the tumor microenvironment (TME), allowing cell-to-cell contact and cell-to-matrix synthesis,–,To date, in vitro studies examining uveal melanoma (UM) biology, such as cancer cell invasion and migration, TME signaling and crosstalk, and responses to anticancer therapies, have generally been conducted using 2D cultures of UM cell lines.for the first time, to the best of our knowledge, human primary UM (PUM) samples. We describe the UM spheroid growth, their phenotype, use in drug screening assays, and the effect of two therapeutic agents (doxorubicin and selumetinib) used previously for metastatic UM on these spheroids.In this study, we provide detailed methods for cultivating 3D spheroids using a range of UM cell lines and, –2) in RPMI with GlutaMAX  supplemented with 10% fetal calf serum , hereafter referred to as UM medium A. For all studies, UM cell lines were harvested at ∼70% confluence with 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA), counted and resuspended at varying cell densities  for 3D culture, as indicated below.The following UM cell lines (stock passage number) were used in the studies: 92.1 (P57), OMM2.5 (P20), MM66 (P44), MP41 (P177), OMM1 (P+3), and MP46 (P26).3, was finely minced with a sterile scalpel blade and digested with 500-U/ml type I collagenase  at 37°C for approximately 1 hour with occasional agitation, as previously described.l-glutamine (Sigma-Aldrich), and Gibco 1% penicillin–streptomycin . Cells were maintained in a humidified incubator (5% CO2) at 37°C and harvested at 60% to 70% confluence with 0.05% trypsin–EDTA solution (Thermo Fisher Scientific); they were then counted and resuspended for 3D culture at varying cell densities , as indicated below.This study was approved by the Health Research Authority (HRA reference: 15/SS/0097), and all UM patients provided informed written consent for the use of their material and data in research. Fresh PUM specimens from patients undergoing enucleation or local resection were obtained from the Liverpool Ocular Oncology Biobank (LOOB) (HRA reference: 16/NW/0380). A small piece of fresh PUM tissue, approximately 3mmUM cell lines and PUM cells were harvested as described above and plated into ultra-low attachment (ULA), 96-well, clear, round-bottomed plates (Corning) at varying densities in 100-µl volumes of UM medium A or medium B for cell lines and PUM cells, respectively. Cells were imaged between days 1 and 10 of incubation with the EVOS M5000 Imaging System (Thermo Fisher Scientific) or a Zeiss Axio Observer Z1 microscope .Spheroid images were analyzed for growth determination using ImageJ/Fiji software  and an open-source macro developed by Ivanov et al.PUM spheroids were removed from the ULA plates using a cut 200-µl pipette tip at various time points and fixed in 10% neutral buffered formalin for 15 minutes. Spheroids were then suspended in 2% agar before being processed using the Bayer Tissue-Tek VIP E300 tissue processor . Processed spheroids were subsequently embedded in paraffin blocks and sectioned at 4 µm onto X-tra adhesive slides , for immunohistochemical (IHC) staining.IHC staining was performed as previously describedClinical, histopathological, and genetic information for the PUM samples used in 3D culture was provided by the LOOB and is shown in UM cell lines 92.1 and MM66 were seeded at 5000, 7500, and 10,000 cells/well in ULA plates and grown for 4 days to form compact spheroids. The 92.1 and MM66 UM cells were also plated into 2D, flat-bottomed, 96-well plates at a density of 10,000 and 15,000 cells/well, respectively, for 24 hours to reach 70% confluence. For drug testing in both 2D and 3D, either doxorubicin or the MEK inhibitor selumetinib was added at doses between 0.5 and 10 µg/ml and between 0.03 and 30 µM, respectively, 24 hours after plating the cells in 2D, as well as on day 4 of spheroid formation, following imaging of each spheroid. The spheroids were imaged and both 2D and 3D cultures were assessed for cell viability 48 hours after drug addition, as described below.Cell viability was assessed in both 2D and 3D cultures using the CellTiter-Glo 3D Cell Viability Assay (Promega) according to the manufacturer's instructions using black opaque-walled multi-well plates suitable for luminescence measurements. In brief, 50 µl medium was removed from each well, and 50 µl CellTiter-Glo 3D was added. All procedures were performed at room temperature. The mixture was pipetted up and down for 30 seconds and assessed microscopically for complete lysis of the spheroid/cells before transferring to a black opaque-walled multi-well plate and incubating on a shaker in the dark for 5 minutes. After a further 25-minute incubation in the dark, luminescence was recorded using a Spark plate reader .Use of the ULA plate methodology resulted in both PUM and UM cell lines successfully forming spheroids. Spheroids that formed from different PUM and UM cell lines were of varying compactness, size, and density, as described below.P < 0.01, t-test). MM66 and MP41 spheroids demonstrated an initial growth phase up to day 7, which was followed by a reduction or stabilization of spheroid size by day 10, reflective of compaction occurring at this later time point. Statistically significant growth between day 4 and day 10 was only observed when MM66 and MP41 cells were plated at 5000 cells/well. MP46 cells showed no significant alteration in spheroid size between days 4 and 10 at any of the plating densities , possibly due to the long doubling time of these cells (∼110 hours).All UM cell lines tested successfully formed spheroids . Cell liThese spheroid characteristics were not associated with cell line tissue of origin  nor were there associations with cell morphology  or the genetic profile of UM cells.All PUM samples were successfully cultured in 2D for a single passage; however, three out of nine samples  then failed to grow and form 3D spheroids when added to ULA plates at varying cell densities. The histological and genetic characteristics of the nine samples are shown in 2, 227550 ± 7050 µm2, and 376890.5 ± 29881.3 µm2, respectively. Of note is the clear necrotic core observed in S093. This may be due to the proliferative nature of the cells in this sample as evidenced by numerous Ki67-positive cells at the periphery, which are largely absent in S121 and S104.To determine whether there was genetic drift between the spheroids and the original sampled tumor tissue, MLPA was undertaken from both the original patient tissue and pooled PUM spheroids from S121, S093, S084, S104, and S119 at day 9. MLPA showed that the PUM spheroids retained the same CNVs compared to the PUM, with the exception of S121 in which chr6p and chr8q status differed from its counterpart PUM . UM cell–,Studies have previously shown that differing drugs will induce changes in spheroid morphology characteristic of the drug and its mechanism of action.P < 0.01, t-test) decreased adenosine triphosphate (ATP) levels and hence spheroid cell viability. For 92.1, this was first seen at 0.5 µg/ml with a 31% reduction in viability; for MM66, at 1.0µg/ml with a 54% reduction in viability when compared with the untreated control . This resulted in dispersion and disaggregation of the cells with increased cross-sectional area of the spheroids in both cell lines A, 4C. In control A, 4C. Th control A, 4C.P < 0.01, t-test) decrease in ATP measurements and hence spheroid cell viability at these drug concentrations when compared with the 0.1% dimethyl sulfoxide (DMSO) control. For 92.1, there was a 45% reduction in spheroid cell viability and for MM66 a 50% reduction in spheroid cell viability when compared with the 0.1% DMSO control. At the highest concentration of selumetinib tested, 92.1 spheroid cell viability was reduced by 81.5% and MM66 by 62.5% when compared with the 0.1% DMSO control. The effect of selumetinib on 2D-cultured 92.1 cells were similar to those observed in 3D; however, for MM66 cells, selumetinib had no effect on reducing cell viability in 2D culture  to determine this. Ultimately, this may require the slower growing cells  or cells that compact less rapidly  to be plated at higher densities and/or tested at later time points.Traditionally, modeling of cancer cell biology in an in vitro setting has been confined to 2D cell culture models, which have been used to analyze a range of cell behaviors  in both drug-treated and untreated cells. However, more recently, researchers have been developing 3D cell culture models that incorporate the physiological TME, allowing them to more closely recapitulate tumor characteristics, with the aim of providing more translatable results.,.Most of the UM cell lines could be removed from the ULA plates for downstream analyses, with the exception of MP41 and OMM1 spheroids, which disaggregated with pipetting. Such tumor spheroid disaggregation on removal from ULA wells has been reported by other investigators.Also novel in the current study is the description of the successful generation of UM spheroids derived from PUM samples, with morphological, immunohistochemical, and genetic characteristics representative of the original patient tumor. In general, the PUM spheroids generated had a low proliferative index, apart from S093, which also showed central necrosis and contained admixed macrophages, which are present in both PUM,in vivo.Two other interlinked features of the UM spheroids that we examined in this study were drug penetrance and efficacy, which we investigated through the use of two drugs previously administered in the context of metastatic UM—namely doxorubicin and selumetinib. The two drugs have differing modes of action; doxorubicin acts by intercalating with DNA to cause DNA damage, and selumetinib is an ATP-independent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2.+ laser.The aspect of drug penetration was specifically assessed by profiling doxorubicin, which is known to autofluoresce and act as a biocompatible fluorophore with an absorption peak around 488 nm of ArWith respect to drug efficacy, studies have shown that in 2D cell culture systems potency is enhanced when compared with 3D models,Goncalves et al.,In conclusion, we have described optimal conditions for the reproducible production of 3D spheroids of uniform size from UM cell lines and PUM samples, as well as their use in drug screening assays. We are of the opinion that the panel of UM cell lines examined in this study can be used to mimic many aspects of the heterogeneous in vivo tumor cell population, such as proliferation, oxygen and nutrient gradients, and cellular interactions when grown in a 3D culture as compared with monolayer cultures. In particular, monitoring of 3D phenotypic parameters, such as spheroid size, shape, and internal features, combined with a variety of endpoint assays, will allow the outcomes of drug action at a multicellular level to be more accurately evaluated. Further development of 3D culture methods may involve incorporation of multiple cell types that mimic the metastatic TME and the use of hydrogels as scaffolds to encapsulate the cells or to serve as mechanical and biochemical cues that more accurately represent the composition of the tumor extracellular matrix and the sequestration of biomolecules within.


Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans’ lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and ′for information only′ characterization methods with release criteria for the establishment of clinical-grade hPSC lines. Human pluripotent stem cells (hPSCs) have held great promise for regenerative medicine since derivation of human embryonic stem cells by Thompson in 1998 . Both hEThe main difference between hESCs and hiPSCs is in the way they are created. While hESCs are derived from the inner cell mass of human preimplantation blastocysts which are not suitable for in vitro fertilization (IVF) , hiPSCs Treatment with hiPSCs in an autologous setting is still associated with a high medical cost  as it waWhile some discuss the use of undifferentiated hiPSCs as a therapy product ,19, the Since hESCs were the first type of hPSCs established, it is not surprising that the first hPSCs-in-humans clinical trial was conducted using hESCs-derived cells. More specifically, Geron conducted a safety study of hESCs-derived oligodendrocyte progenitor cells for spinal injury treatment in 2010 . ClinicaSome clinical studies focused on the utilization of hPSCs in the treatment of eye disorders, more specifically macular degenerations. The first in-human hiPSCs clinical study (even if it was stopped) used an implanted autologous hiPSCs-derived sheet of retinal pigment epithelial cells in a patient with neovascular age-related macular dystrophy . In 2012As described above, multiple clinical trials using hPSC-derived cells have been launched, and data from some of them are already available. The therapeutic potential of hPSCs is tremendous and great progress has been made from bench to bedside since their first derivation, but there are still some hurdles that need to be overcome. One of them is a need for standardization. The most feasible way to benefit from hPSCs is to make a bank or multiple banks of clinical-grade hPSC lines available for further processing in specific application. Since we need to be able to compare the banked cell lines, coming to a consensus on their quality assessment is of utmost importance. On that account, we present an overview of characterization strategies used so far in the derivation of clinical-grade hPSCs and propose a set of mandatory tests including release criteria and ′for information only′ tests.https://www.ema.europa.eu/en) in the European Union and by the US Food and Drug Administration  in the USA. Briefly, cGMP require that medicines a) are of consistent high quality; b) are appropriate for their intended use; c) meet the requirements of the marketing authorization or clinical trial authorization [In order to translate hPSCs into clinical practice, it is necessary to manufacture and perform quality control of hPSCs according to current good manufacturing practices (cGMP). cGMP are defined by the European Medicines Agency  testing and distribution. Every step of this process has to be validated and executed according to standard operating procedures (SOPs). SOPs ensure that the reproducibility of the final product is in accordance with established specifications . The usehPSC culture also has some specifics that need to be implemented into quality control. It is well-known that hPSCs acquire various genetic changes connected with culture conditions and time in culture ,30. As tAs hPSCs derivates should be used in replacement therapies, i.e., in direct contact with a human body, it is essential to ensure that animal-derived molecules do not cause an immune reaction to a patient. It was found that hESCs express culture-acquired immunogenic nonhuman sialic acid (Neu5Gc), a component of animal-derived serum replacement and animal feeder cells . As manyhttps://www.eurostemcell.org. However, there are directives and guidelines in force for all EU countries that specify good manufacturing practices relevant for the establishment of hPSCs that must be obeyed. The main directives and guidelines relevant to the establishment of clinical-grade hPSC lines are summarized in The establishment of hPSC lines must be done in accordance with the relevant laws and policies of the country where the derivation is performed. Information on the current legal position, ethical and regulatory oversight for EU countries can be found on Similarly, hPSC lines derived in the USA must be compliant with federal regulations and FDA regulations. Because stem cell research and translation to clinical practice are moving very fast, it is recommended to search for the current regulations and directions relevant to your country.http://www.stem-cell-forum.net/) and the International Society for Stem Cell Research . The ISSCR released guidelines for stem cell research and clinical translation in 2016, that can provide useful information about the ethics, research and clinical translation of hPSCs.Worldwide, there are two international organizations focusing on the field of human pluripotent stem cells: The International Stem Cell Forum  are easier to detect as antigens are accessible for antibodies. The protocol for the detection of intracellular markers  has an additional step enriched with fixation. A combination of surface markers and intracellular markers is favored. This method is fast and robust, and the results are quantitative; therefore, it is easy to compare them in time and amongst different laboratories.The characterization of intracellular markers has been used by many authors for the establishment of clinical-grade hESC lines ,37,38 anImmunocytochemistry is a laboratory assay for the detection and localization of various cell components. It is based on primary and secondary antibody binding. The secondary antibody is connected to a fluorophore that allows detecting the signal under a fluorescence microscope. The advantage of this method is that in addition to the presence of the antigen, we can also evaluate its localization within the cell. On the other hand, interpretation is difficult; thus, comparability within other results can be problematic. Other points to consider are that this technique requires trained personnel, negative controls and strict evaluation criteria.Both surface  and intracellular markers of pluripotency  can be detected by immunocytochemistry. A combination of surface markers and intracellular markers is favored, although immunocytochemistry is recommended to be used only for informative purposes according to The International Stem Cell Initiative for hESCs  and guidImmunocytochemistry was used for the detection of pluripotent markers of clinical-grade hESC lines by some authors. The determination of thresholds is difficult; nevertheless, the cited authors published their release criteria as you can see in As an early marker of the successful establishment of a hPSC line, alkaline phosphatase (AP) staining is often performed . hPSCs eThe distinction of various hPSC lines is crucial not only for experimental purposes but mainly in clinical usage. There is a risk of unwanted switch or cross-contamination between cultured hPSC lines that can be life-threatening in the case of directed differentiation and subsequent transplantation to humans . IdentifThe STR profile should match the cell donor in the case of the establishment of a hiPSC line, but this connection is impossible to establish for hESC lines as there are not many cells at the time of derivation. However, it is recommended to establish the STR profile in early passages, as it can help to recognize unwanted switch or cross-contamination.At least 8 core STR loci with an 80% threshold match should be used according to ANSI/ATCC ASN-0002-2011 standard for the authentication of human cell lines. However, 15 STR loci may be required for the unambiguous identification for autologous usage of hPSCs .Eight specific loci plus amelogenin were used for the STR profiling of clinical-grade hESCs by De Sousa , and 16 Karyotype analysis is a standard technique for the determination of genome stability. hPSCs are often unstable during long-time culture ,51; therGiemsa-banding (G-banding) should be used for chromosome counts of 20 metaphases, and further banding patterns analysis in a minimum of 8 metaphases . This teWhole-genome or exome sequencing has been proposed for hPSCs quality control ,53, but Single nucleotide polymorphism (SNP) arrays are based on probe binding and can detect changes in a genome with higher resolution (kb) than G-banding (3–20 Mb). This method enables us to study copy number variations (CNVs), >20% mosaicism and loss of heterozygosity (LOH) but on the other hand, it is unable to detect <20% mosaicism, balanced chromosomal translocations, and inversions . TherefoArray comparative genomic hybridization (array CGH) compares two genomes while one of them serves as a reference. Array CGH allows detecting unbalanced genomic changes, including CNVs, with kb resolution. However, this method is unable to detect balanced chromosomal aberrations and low-level mosaicism . On one Ye et al. used CytoSure™ ISCAv2 8×60K Comparative Genomic Hybridization (CGH) microarrays (Oxford Gene Technology) to perform array CGH for the genetic screening of clinical-grade hESC lines and identified approximately 1 Mb gain of chromosome 20q11.21 in two hESC lines. No plausibly pathogenic genetic variations were identified in the other 5 tested hESC lines .TP53, can be transferred to a patient during cell therapy [There is a serious risk that potentially harmful mutations in cancer-associated genes, for example in  therapy . These m therapy ,58, and Some reprogramming methods use foreign DNA during derivation of hiPSCs. In these cases, vector clearance should be tested. Some of the reprogramming factors have tumorigenic potential, therefore it is desirable from a safety perspective to ensure that the hiPSCs are free of introduced sequences. A quantitative PCR assay with primers detecting vector sequences is a convenient method for vector clearance testing .Human Leukocyte Antigen (HLA) typing provides useful information about polymorphisms in human genome associated with immune response during transplantations. The results of this assay can connect specific donors with compatible recipients in a way which minimizes the risk of transplant rejection. The histocompatibility of hPSC lines can be compared with a public HLA allele frequency database  to find There is an effort to create HLA-haplotype banks for hiPSCs and hESCs, as they would provide HLA-compatible cells for clinical usage. It was estimated that a bank with 50 HLA homozygous lines would cover 90.7% of the Japanese population for a three-locus match . SimilarServices of external laboratory  or in-hoTo benefit from hPSCs, it is crucial to ensure that the cells are able to leave the stem cell state and form differentiated cell types. There are different approaches to assess the differentiation capacity of a hPSC line.The most straightforward method is spontaneous differentiation. When cultured as suspension in absence of FGF, hPSCs tend to form dense structures composed of multiple cell types called embryoid bodies (EBs) . After gAnother option to assess the ability to form all three germ layers is the directed differentiation of hPSCs . In a reBoth EBs formation and directed differentiation can be further evaluated through expression analysis, which provides more specific data than immunocytochemistry . This asTraditionally, in the hPSCs field, the method considered most stringent was teratoma formation . The tesAny product intended for clinical use must undergo sterility testing. As the process of establishing hPSCs line includes cell manipulations and prolonged culture in vitro and there is no method of sterilization without destroying the product, testing of the product is crucial and pre-testing of the starting biological material can prevent wasting resources. Pre-testing possibilities differ in hESCs and hiPSCs. When deriving hESCs, the amount of material is limited and so is the possibility to pre-test. Parents pre-testing is routinely performed by IVF clinics, and sterility and mycoplasma testing can be done from media in the early stages of the process. Establishing hiPSCs can be advantageous, as in addition to pre-screening the donor, there is an option to take a sample from the obtained material and freeze the rest until the test results are ready.While there can be differences in local pharmacopoeias, there is a set of tests that needs to be performed on the product to ensure its safety in relation to contamination by bacteria, fungi or viruses. Bacterial contamination should be tested by culture-based or broth-based methods for both aerobic and anaerobic bacteria. Testing for mycoplasma can be performed using culture-based methods, but a PCR-based method is also accepted by some authorities . EndotoxCulture conditions may cause differences in cell and colony morphology. There are various surfaces and feeder cells that have an impact on the shape and size of colonies, therefore it is appropriate to have images from every important step of hPSC line establishment and further manipulation.Undifferentiated colonies of hPSCs have clear borders with well-defined edges. Colonies are typically flat, contain small round cells with large nuclei with prominent nucleoli ,71. ThisViability testing provides useful information about the current state of the culture, but it should not be taken as a quality indicator of hPSCs. It is necessary to validate the chosen method and to measure viability in determined timepoints. It is recommended to not measure viability immediately after thawing as it can provide misleading information.Flow cytometry was used for measurement of viability in the establishment of clinical-grade hESC lines  and for It is also recommended to record the number of passages, although the population doubling level can provide more accurate information about cell line age.The characterization of hPSCs is a complex process with many phases. After the early phase (EP) of the establishment of the hPSC line it is necessary to create master cell bank (MCB), which can then be used to make a working cell bank (WCB). During the manufacture, in-process testing is recommended in order to prevent wasting resources. Final product (WCB) is then tested for fulfilment of release criteria. We divided the test into two sections, mandatory tests with release criteria  and testhPSCs are a powerful technology with tremendous potential in the clinic. Multiple clinical-grade hPSC lines have been established and clinical trials are ongoing. Nevertheless, there are still obstacles in the translation of hPSC technology to the clinic. One of them is that consensus about the standards for quality control of clinical-grade hPSCs is still not completely reached, which limits not only the comparability of the cell lines but also the credibility of data obtained by preclinical and clinical studies. In this review, we summarize specifications that enable the easier transfer of hPSCs to clinical practice; therefore, we directly help cGMP facilities fulfill the minimal criteria for the establishment of clinical-grade hPSC.


Cisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, the benefits are limited due to the acquisition of drug resistance. The mechanisms of resistance remain unclear. Although there are some reports that some molecules are associated with cisplatin resistance in advanced BC, those reports have not been fully investigated. Therefore, we undertook a new search for cisplatin resistance-related genes targeted by tumor suppressive microRNAs as well as genes that were downregulated in cisplatin-resistant BC cells and clinical BC tissues.First, we established cisplatin-resistant BOY and T24 BC cell lines . Then, Next Generation Sequence analysis was performed with parental and cisplatin-resistant cell lines to search for the microRNAs responsible for cisplatin resistance. We conducted gain-of-function analysis of microRNAs and their effects on cisplatin resistance, and we searched target genes comprehensively using Next Generation mRNA sequences.miR-486-5p, a tumor suppressor miRNA, was negatively correlated with the TNM classification of clinical BC samples in The Cancer Genome Atlas (TCGA) database. Transfection of miRNA-486-5p significantly inhibited cancer cell proliferation, migration, and invasion, and also improved the cells’ resistance to cisplatin. Among the genes targeted by miRNA-486-5p, we focused on enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), which is involved in the degradation of fatty acids. EHHADH was directly regulated by miRNA-486-5p as determined by a dual-luciferase reporter assay. Loss-of-function study using EHHADH si-RNA showed significant inhibitions of cell proliferation, migration, invasion and the recovery of cisplatin sensitivity.A total of 28 microRNAs were significantly downregulated in both CDDP-R-BOY and CDDP-R-T24. Among them, EHHADH as a target of miRNA-486-5p provides novel insights into the potential mechanisms of cisplatin resistance in BC.Identification of  Bladder cancer (BC) can be roughly classified into non-muscle-invasive BC (NMIBC) and muscle-invasive BC (MIBC). Approximately 50% of MIBC patients develop metastasis within 2 years, and the 5-year survival rate remains under 50% . AdvanceCTR1) is involved in the intracellular uptake of cisplatin. Knockdown of CTR1 reduces the intracellular accumulation of cisplatin and induces cisplatin resistance [Pharmacologically, cisplatin goes through hydrolysis and avidly binds DNA through the N7-sites of either a guanine or adenine base. Crosslinks and damage to DNA result, and apoptosis is induced , 6. Howesistance . Moreovesistance , 10. In miRNA-214 into ovarian cancer cell lines suppressed PTEN, activated protein kinase AKT and led to cisplatin resistance [miRNA-181b was downregulated in cisplatin-resistant lung cancer lines, and that miRNA-181b modulated cisplatin resistance by targeting the anti-apoptotic gene BCL2 [MicroRNAs are endogenous small non-coding RNA molecules (19 ~ 22 bases in length) that regulate the expression of protein-coding/protein non-coding genes . Our presistance . In addiene BCL2 . In BC, In order to elucidate the mechanism of cisplatin resistance, we established cisplatin-resistant BC cell lines (CDDP-R-BOY and CDDP-R-T24). Then, small-RNA sequence analyses were performed with the parental and resistant cell lines to search for the microRNAs associated with cisplatin resistance. The candidate microRNA was transduced into cisplatin-resistant cell lines for functional analysis. Next, we searched for target genes using RNA next-generation sequence analysis. We also performed loss of function studies to assess its target gene.2 at 37 °C. To establish CDDP-R BC cell lines, we cultured BC cell lines with serial concentrations of cisplatin from 0.01 to 2.0 μg/mL for 6 months. The cells were cultured in 10 mL of medium for 24–36 h containing 1 mL of cisplatin that had been adjusted to 10-times the target concentration.We used 2 human BC cell lines: BOY was established in our laboratory from a 66-year-old Asian male patient, who was diagnosed with BC stage IV with many lung metastases. T24 was obtained from the American Type Culture Collection . Generation of CDDP-R-BOY and CDDP-R-T24 is described in Fig. 7 /mL, and 100 μL of the BC cells and 100 μL of Matrigel (BD Biosciences) were mixed. We subcutaneously injected parental cells  into the right flanks and CDDP-R cells  into the left flanks in a volume of 200 μL (5 mice per group). The measurement of mouse body weight and tumor size was started 7 days after the inoculation and conducted twice a week. Tumor dimensions were measured with calipers and sizes were calculated by the following formula: v = (length × width2) × (π/ 6). The administration of cisplatin  [P values less than 0.05 were accepted as statistically significant. There were no confounders in the animal experiments. All animal experiments were conduced by SO, HY, KK, and MT.The animal research described here was conducted in accordance with the regulations covering animal experiments at Kagoshima University, and the plan was approved by the Animal Experiment Committee at Kagoshima University MD18094). We used 10 female athymic nude mice (BALB/c-nu/nu), aged 5 weeks, that were purchased from Charles River Laboratories . Sample size was determined by the guidelines for the welfare and use of animals in cancer research [094. We ues/week)  also staimagej.nih.gov) software.For determination of the IC50 value, cells were seeded into 96-well plates at a density of 2000 cells per well in triplicate and treated with a series of dilute concentrations of cisplatin. After 96 h of incubation, cell proliferation was measured using the XTT assay method according to the manufacturer’s instructions. A probit regression model was used to calculate the IC50 value using ImageJ  and negative-control microRNA  were used in gain-of-function experiments. EHHADH si-RNA (cat No. HSS105529 and HSS105531) and negative-control si-RNA (D-001810-10) were used in loss-of-function experiments.BC cells were transfected with the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) with 10 nM miRNA and siRNAs as previously reported . Mature miRNA-486-5p. For the samples, a TruSeq Stranded mRNA Library Prep Kit was used to create libraries, and Illumina Inc’s flow cell was used for sequencing. The valid read length was 150 bp, and the analysis was performed using a Multiplex method. Candidate target genes were significantly downregulated after transfection with miRNA-486-5p compared with control microRNA (fold-change < − 1.0) in CDDP-R-BOY and CDDP-R-T24.To search for the microRNAs associated with cisplatin resistance, total RNAs extracted from BOY, CDDP-R-BOY, T24, and CDDP-R-T24 cell lines were subjected to microRNA sequencing, performed by RIKEN GENESIS CO., LTD., Tokyo, Japan. We compared parental and CDDP-R cell lines , and selected miRNAs with significantly downregulated expression in the CDDP-R cell lines (fold-change < − 1.0). mRNA sequence analysis was performed by RIKEN GENESIS CO., LTD. to identify the target mRNA of http://www.oncolnc.org/). To search for the miRNAs associated with cisplatin resistance, we identified miRNAs that were lower in expression in CDDP-R cells compared to parental cells in both BOY and T24, and had been reported as tumor suppressor genes. To identify possible target genes of miRNA-486-5p, we extracted genes that were reduced by transfection of miR486-5p in mRNA sequence analysis with genes that may be targeted by miRNA-486-5p based on TargetScan database Release 7.1 (http://www.targetscan.org).In order to evaluate the clinical relevance of our findings, a TCGA cohort database of 413 patients with BLCA was used. This study follows the criteria for the publication guidelines provided by TCGA. Kaplan-Meier analysis was used to analyze overall survival (OS) using data in the OncoLnc dataset  according to previously published conditions [RNU48  was used as the internal control. With regard to EHHADH, we applied a SYBR-green quantitative PCR-based array approach. The primer set used for determination of EHHADH mRNA expression level was as follows: forward, 5′-AAACTCAGACCCGGTTGAAGA-3′ and reverse, 5′-TTGCAGAGTCTACGGGATTCT − 3′. For glucuronidase β , the set was as follows: forward primer, 5′-CGTCCCACCTAGAATCTGCT-3′ and reverse primer, 5′-TTGCTCACAAAGGTCACAGG − 3′. The specificity of amplification was monitored using the dissociation curve of the amplified product.To quantify the expression of nditions . RNU48 (http://rsbweb.nih.gov/ij/index.html) as described previously [To prepare total protein lysates, we used NuPAGE LDS Sample Buffer . Immunoblotting was carried out with diluted anti-EHHADH , anti-cleaved PARP antibodies , PARP antibodies  and anti-β-actin antibodies . The secondary antibodies were peroxidase-labeled anti-rabbit IgG  or anti-mouse IgG . The protein levels were evaluated using ImageJ software . We determined the extent of cell proliferation 96 h after seeding with a Cell Proliferation Kit II  as described previously. When using cisplatin, we added 10 μL adjusted to 10-times the target concentration. Wound healing assays were used for cell migration activity. Cells (2 × 105 per well) were plated in 6-well plates, and after 48 h of incubation, the cell monolayer was scraped using a P-20 micropipette tip. The initial gap length (0 h) and the residual gap length 24 h after wounding were calculated from photomicrographs. For cell invasion assays, we used modified Boyden chambers consisting of Matrigel-coated Transwell membrane filter inserts with 8 μM pores in 24-well tissue culture plates . The cells that had passed through the pores and attached to the surface of the chamber were counted from photomicrographs. For apoptosis assays, double staining with FITC-Annexin V and propidium iodide was performed using a FITC Annexin V Apoptosis Detection Kit  by flow cytometry . We classified cells in 4 categories: viable cells, dead cells, early apoptotic cells or apoptotic cells using Summit 4.3 software (Beckman Coulter). The sums of the percentages of early apoptotic and apoptotic cells were compared. The cells treated with 2 μg/mL cycloheximide were used as a positive control.To evaluate cell proliferation, we used XTT assays. T24 and BOY cells were seeded in 96-well plates with 2 × 10EHHADH or those with a deleted miRNA-486-5p target site were inserted between the XhoI and PmeI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 vector . CDDP-R-BOY and CDDP-R-T24 cells were transfected with 50 ng of vector and 10 nM miRNA-486-5p. According to the manufacturer’s protocol , the activities of firefly and Renilla luciferases in cell lysates were determined with a dual luciferase assay system.Partial wild-type (WT) sequences of the 3′-UTR of First, we established cisplatin-resistant BC cell lines . BC cells (BOY and T24) were cultured with serial concentrations of cisplatin from 0.01 to 2.0 μg/mL  were identified as candidates  exhibited no significant difference between the high expression group and the low expression group in miRNA-486-5p and miRNA-545-5p  and there was no correlations between miRNA-545-5p and the target gene  identified 2779 possible mRNAs as candidate targets of miRNA-486-5p. Next, we narrowed the number of genes based upon the expression profiles of mRNAs of CDDP-R BC cell lines before and after transfection with miRNA-486-5p. Finally, we selected 7 candidate target genes   compared with parental BC cell lines . The molecular mechanism by which miRNA-486-5p is downregulated in BC remains unclear. However, miRNA-486-5p is located on chromosome 8p11, and loss of material from chromosome arm 8p was a frequent cytogenetic alteration in uroepithelial carcinoma [miRNA-486-5p was a tumor suppressor miRNA in many cancer types such as non-small cell lung, breast, colon and hepatocellular carcinoma [miRNA-486-5p overexpression inhibited cell proliferation, migration and invasion, and induced apoptosis in BC and CDDP-R BC cell lines. Also, miRNA-486-5p was reportedly involved in sensitivity to cisplatin in non-small cell lung cancer. Xiaoyan et al. reported that miRNA-486-5p inhibits EMT by targeting TWF1 and improves sensitivity to cisplatin [miRNA-486-5p overcomes cisplatin resistance and the additive effect on cell growth suppression. Recently, Salimian et al. showed that miRNA-486-5p had anti-tumor effects and improved CDDP sensitivity through induction of apoptosis in muscle-invasive BC [EHHADH was a directly targeted by miR-486-5p. This finding had not been reported previously. According to TargetScan database Release 7.1, it is possible that among the 28 microRNAs that are downregulated in cisplatin-resistant BC cell lines , 5 microRNAs  regulate the expression of EHHADH. Even though we focused on miR-486-5p in this study, it is possible that the other microRNAs are involved in cisplatin resistance by regulating EHHADH. Further study is necessary to elucidate the mechanism by which EHHADH contributes to cisplatin resistance through regulation by microRNA in BC.In cancer cells, abnormal expression of microRNAs can disturb normally operating RNA networks and disrupt physiologic processes . microRNarcinoma . It was arcinoma –44. As aisplatin . Similarasive BC . TherefoEHHADH gene function significantly inhibited cancer cell proliferation, migration and invasion and increased the cells’ sensitivity to cisplatin. However, we could not find a significant correlation between EHHADH expression levels and cisplatin-based chemotherapy response in TCGA database. This could be due to the limited number of samples in TCGA database. Also, mRNA expression does not always match protein expression. Furthermore, when cisplatin-based chemotherapy is used, cisplatin is combined with other anticancer agents. Because our analysis showed a tendency for upregulation of EHHADH expression in patients with cisplatin resistance in spite of the small sample size and bias, the results suggest that EHHADH could be a new molecular target and marker for progressive BC.In this study, we confirmed that the level of EHHADH protein was elevated in CDDP-R BC cell lines compared with parental cell lines, and that loss of ERCC1), a DNA repair gene [TP53 is a tumor suppressor gene that mainly induces apoptosis [TP53 gene cannot proceed to cellular apoptosis (post-target resistance) [EHHADH, it appears to contribute to pre-target resistance for the following reasons. First, Evelien et al. reported that increased fatty acid synthesis reduces intracellular unsaturated fatty acid production. Unsaturated fatty acids are a source of reactive free radicals, and reduction of reactive free radicals is involved in cisplatin resistance [EHHADH cause cisplatin resistance, further studies are necessary to elucidate the associations between the peroxisomal beta-oxidation pathway in which EHHADH is involved and cisplatin resistance.The mechanisms by which cells gain resistance to cisplatin are very complex. Thus, Galluzzi et al. classified the mechanisms of resistance into 4 categories . The firair gene , could rair gene . This ciair gene . In anotair gene . Becausepoptosis , patientistance) . Regardisistance . Second,sistance . This vaEHHADH as a novel target of miRNA-486-5p in cisplatin-resistant BCs. The expression of EHHADH was decreased in cisplatin-resistant BC cell lines. To the best of our knowledge, this is the first report demonstrating that EHHADH is involved in cisplatin resistance. The discovery of molecular targets mediated by tumour-suppressive microRNAs may lead to a better understanding of the mechanisms of cisplatin resistance in BC and the development of new therapeutic strategies to treat progressive BC.We identified Additional file 1 : Supplementary Figure 1. (a) Cell proliferation determined by XTT assay compared with parental BC cell lines and CDDP-R BC cell lines. *, P < 0.001. (b) Heatmap of miRNA-seq comparing parental and CDDP-R cell lines .Additional file 2 : Supplementary Figure 2. (a) Among the BLCA cohort of TCGA, expression levels of candidate miRNAs . We determined correlations among expression levels and pathological T categories and clinical stages. (b) Kaplan-Meier analysis using TCGA dataset revealed that the high miRNA-545-5p expression group did not have significantly lower OS than the low miRNA-486-5p expression group (P = 0.162).Additional file 3 : Supplementary Figure 3. (a) Expression levels of miRNA-486-5p quantified in miR-486-5p transfectants compared with mock or miR-control transfectants by qRT-PCR. (b) Pictures of cell invasion assays. (c) Pictures of cell migration assays.Additional file 4 : Supplementary Figure 4. (a) Apoptosis assays indicated that the number of apoptotic cells was significantly greater in the combination of miRNA-486-5p-transfection and cisplatin than single treatment in flow cytometry, *P < 0.0001.Additional file 5 : Supplementary Figure 5. (a) Heatmap of mRNA-seq comparing mock with miRNA-486-5p transfection in CDDP-R cell lines . (b) Expression levels of miRNA-486-5p quantified in miR-486-5p transfectants compared with mock or miR-control transfectants by qRT-PCR. (c) Among the BLCA cohort of TCGA, there were no significant differences in expression levels of EHHADH in pathological categories or clinical stages.Additional file 6 : Supplementary Figure 6. (a) Expression levels of miRNA-486-5p quantified in si-EHHADH transfectants compared with mock or si-control transfectants determined by qRT-PCR.  Pictures of cell invasion assays and cell migration assays in si-EHHADH transfectants compared with mock or si-control transfectants.Additional file 7 : Supplementary Figure 7. (a) Apoptosis assays indicated that the number of apoptotic cells was significantly greater in the combination of si-EHHADH-transfection and cisplatin than single treatment in flow cytometry, *P < 0.0001. (b) Correlations of the expression of miRNA-545-5p and EHHADH in bladder cancer samples in TCGA database.Additional file 8.