Osteosarcoma (OS) is a malignant tumour affecting mainly children and elderly people. Despite significant advances in cancer medicine, osteosarcoma patients’ survival is not improving. The primary treatment methods are established using in vitro models that rely upon the application of well-established cell lines, including U-2 OS, Saos-2 and MG-63. The molecular phenotype of these cell lines is still not fully outlined. Therefore, our study aimed to establish the expression profile of molecular markers related to osteosarcoma survival, progression and metastasis. Non-bone-related cells were used as a reference, i.e. HeLa cell line and human adipose-derived stromal cells (hASCs). Evaluated osteosarcoma cell lines showed characteristic phenotypes with unique patterns related to upregulation of MMP-7, MMP-14, BMP-7, miR-21-5p, miR-124-3p and downregulation of lncRNA MEG3. Our findings may facilitate the selection of the most reliable cellular model for pre-clinical investigations focused on developing new and satisfying methods of osteosarcoma therapy.Osteosarcoma (OS) is a bone tumour affecting adolescents and elderly people. Unfortunately, basic treatment methods are still underdeveloped, which has a high impact on the poor survivability of the patients. Studies designed to understand the underlying mechanisms of osteosarcoma development, as well as preclinical investigations aimed at establishing novel therapeutic strategies, rely significantly upon in vitro models, which apply well-established cell lines such as U-2 OS, Saos-2 and MG-63. In this study, the expression of chosen markers associated with tumour progression, metastasis and survival were identified using RT-qPCR. Levels of several onco-miRs  and long non-coding RNA MEG3 were established. The mRNA expression of bone morphogenetic proteins (BMPs), including BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, as well as their receptors: BMPR-IA, BMPR-IB and BMPR-II was also determined. Other tested markers included metalloproteinases, i.e., MMP-7 and MMP-14 and survivin (BIRC5), C-MYC, as well as CYCLIN D (CCND1). The analysis included comparing obtained profiles with transcript levels established for the osteogenic HeLa cell line and human adipose-derived stromal cells (hASCs). The tested OS cell lines were characterised by a cancer-related phenotype, such as increased expression of mRNA for BMP-7, as well as MMP-7 and MMP-14. Osteosarcoma cells differ considerably in miR-21-5p and miR-124-3p levels, which can be related to uncontrolled tumour growth. The comprehensive examination of osteosarcoma transcriptome profiles may facilitate the selection of appropriate cell models for preclinical investigations aimed at the development of new strategies for OS treatment. Osteosarcoma (OS) is a primary malignant bone tumour formed by cells producing osteoid and immature bone tissue. It mainly affects children and young people (aged 15–19 years) . To understand molecular mechanisms underlying osteosarcoma development and to determine the potential usefulness of newly developed therapeutic strategies, scientists design preclinical assays that rely significantly on in vitro models, applying well-established cell lines, including U-2 OS, Saos-2 and MG-63. Thus, this study analysed the molecular characterisation of those acknowledged human osteosarcoma cell lines. The cells are considered good models for functional osteosarcoma studies. However, in the literature, discrepancies in the characteristics of the molecular phenotype of those cells are noted. Pautke et al. profoundly characterized the osteogenic properties of the osteosarcoma cell lines Saos-2, MG-63 and U-2 OS [Furthermore, Fromigue et al. indicated that the highest migratory capacity characterizes Saos-2, while MG-63 cells have the lowest . In turnGiven the differences in the molecular phenotype of OS cell lines, the main objective of this paper was to investigate the expression profiles of several non-coding RNAs and mRNAs of genes involved in bone biology and tumour progression. The expression profiles of selected biomarkers determined in OS cell lines were compared with the HeLa cell line, known for its osteoinductive properties mediated by bone morphogenetic proteins (BMPs) ,7, as weWe have tested levels of so-called onco-microRNAs (onco-miRs), including miR-21-5p, miR-124-3p, miR-223-3p and miR-320a-3p, and long non-coding RNA—MEG3. The onco-miRs regulate multiple cellular processes of cancer cells, including their division, differentiation and viability. For example, functional in vitro studies showed that miR-21-directly targets the tumour suppressor phosphatase and tensin homologue (PTEN), regulating the proliferation of osteosarcoma cells (MG 63 and U-2 OS) and head and neck cancers (HNC) cell lines ,11. AddiPlethora of onco-miRs also regulate the expression of bone morphogenic proteins . Thus, iMoreover, given that miRNAs regulate genes controlling apoptosis and cell cycle arrest, we decided to determine mRNA levels of several pro-survival markers highly expressed in various malignant tumour tissues and tumour cell lines, including osteosarcomas. The prominent anti-apoptotic molecule, i.e., survivin (BIRC5), was identified as a gene highly expressed in osteosarcoma tissues and involved in osteosarcoma development . C-MYC, A functional characteristic of osteosarcoma based on ncRNA and mRNA profiles is crucial for the proper selection of representative models and in seeking suitable candidates for future therapeutic targets ,18. The The analysis of non-coding RNAs  revealedAnalysis of mRNA levels for particular BMPs showed common gene expression profiles of BMP-2 and BMP-6 molecules a,d. IncrBMP-7 transcripts compared to the HeLa cell line and hASC population  activity. Moreover, miR-124 targets the expression of osterix (SP7/OSX), inhibiting the differentiation of progenitor cells into osteoblasts . It was The tested reference cell lines, i.e., HeLa and hASC, showed a different pattern of miR-124 expression. We noted significantly decreased levels of miR-124 in the HeLa cell line, which is consistent with the study by Zhang et al., who indicated that miR-124 levels are reduced in HeLa and other human cervical cancer (CC), namely SiHa. The study by Zhang and his group also demonstrated that the overexpression of miR-124 might suppress proliferation and decrease the migratory and invasive properties of the tested CC cell lines. Increased expression of miR-124 induced in HeLa also inhibited the epithelial-mesenchymal transition process in vitro .Further, we demonstrated that miR-223 levels are comparable between U-2 OS, Saos-2 and MG-63 cells. MiR-223 is proposed as a good prognostic and diagnostic marker in osteosarcoma. Significantly lowered expression of miR-223 was noted in osteosarcoma patients compared to the healthy controls . The stuThis observation also corresponds with the expression pattern of miR-320, which is an essential modulator of cancer metastasis and proliferation. Increased cell viability with simultaneous downregulation of miR-320a was noted in various cancers, e.g., breast cancer , cervicaThe ASC may promote osteosarcoma growth, invasion and migratory properties by releasing exosomes that carry proteins, mRNA and various non-coding RNAs . ASCs maThe analysis of mRNA expression for BMP-2 showed that its levels are similar in U-2 OS, Saos-2 and HeLa cells. A recent study by Tian et al. revealed that BMP-2 activates the Wnt/β-catenin signalling pathway and promotes the EMT of osteosarcoma . AdditioBMPs are highly expressed in the HeLa cell line, which confirmed the study performed by Kochanowska et al. ,66. HoweWe have also determined the mRNA expression profile of BMP receptors (BMPRs), including BMPR-IA, BMPR-IB and BMPR-II, which initiate intracellular pathways through SMAD proteins . However, it may also act via the JNK and p38 MAP kinase pathway ,68. The Because non-coding RNAs regulate BMPs’ signalling cascade, their correlation has started to be intensively studied. The correlation between miRNA and BMPs can disclose the molecular aetiology of different diseases. For example, it was shown that miR-22 and BMP-7/6 form a regulatory feedback circuit in the course of renal fibrosis . In thisIn our study, we have also determined the expression of genes strictly related to cancer development and progression. The obtained results indicated that the expression of C-MYC in all OS cell lines is lowered compared to HeLa cells. C-MYC is a transcription factor significant for cancer cell proliferation and invasion. The depletion of C-MYC in HeLa cells and MDA-MB-231 cells (human breast adenocarcinoma) strongly impaired their proliferative and migratory activity, which was also accompanied by decreased accumulation of β-catenin . Moreove0/G1 phase and resulted in a shift toward rapid proliferation [Moreover, we showed that hASCs were characterised by lowered expression of both C-MYC and CYCLIN D. The study by Melnik et al. demonstrated that the expression of c-MYC protein correlates with increased proliferative activity of ASCs and may significantly decrease during its cultivation . Paula eferation .Another molecule regulating the division of cells is the survivin belonging to the inhibitor of apoptosis (IAPs) family. Gene coding of the survivin (BIRC5) is upregulated in various malignancies, including osteosarcoma . These rFinally, our study showed that all tested OS cell lines expressed significantly increased mRNA levels for metalloproteinase 7 and 14 . The results confirmed the invasive phenotype of osteosarcoma cells. Moreover, it was revealed that mRNA levels for MMP-14 differ significantly among the tested OS cell lines, suggesting that it may be an important marker of osteosarcoma development. The highest expression of MMP-14 was noted for MG-63, confirming the high invasiveness of this cell line. A potential mechanism explaining the activation of MMP-14 in MG-63 and its role during migration and invasion was presented by Wang et al. . MMP-14 The presented gene expression profile may be a valuable complement to studies related to osteosarcomas’ cancer- and bone-related phenotypes. The classification of OS cell lines based on their transcriptome profile determined using RT-qPCR should provide support to various groups commonly applying this technique in their studies. This knowledge may be beneficial in terms of preclinical studies and the identification of a relevant model. Furthermore, an analysis of the molecular mechanism underlying osteosarcoma progression is necessary. Identifying promising therapeutic candidates may facilitate osteosarcoma diagnosis, restore drug sensitivity and improve therapeutic effects by developing more effective targeted therapies to overcome the poor prognosis of this cancer.2 incubator. The cells were cultured in a complete growth media consisting of basal media, i.e., McCoy’s 5A for U-2 OS and Saos-2, Eagles’ Minimum Essential Medium for MG-63 and HeLa and Dulbecco’s Modified Eagle Medium with low glucose for hASC. All media were supplemented with 10% of foetal bovine serum (FBS) and 1% of the antibiotic solution containing 10,000 units of penicillin and 10 mg of streptomycin/mL . Cultures were maintained in T-75 flasks and harvested at logarithmic phase at confluence established at 90%. Cultures were washed in Hank’s Balanced Salt Solution without calcium and magnesium  and trypsinized for approximately 10 min at 37 °C (StableCell™ Trypsin Solution). Cells were centrifuged , washed again, counted and homogenized with Extrazol . The cell lines, including three osteosarcoma models  and human cervix epithelioid carcinoma (HeLa) were derived from the European Collection of Authenticated Cell Cultures , while and human mesenchymal stromal cells (hASC) were purchased from Millipore . The cells were propagated at constant conditions  in sterile conditions using a CO6 were homogenised using 1 mL of Extrazol . The chloroform, isopropanol, ethanol and DEPC-treated water used during total RNA isolation were derived from Sigma-Aldrich . Detailed protocols used to determine transcript levels were published previously [®&Fluorescein Kit  in a total volume equal to 10 μL. The final concentration of primers used in the reaction was 400 nM. The primers were synthesised by Sigma-Aldrich, Poznan, Poland and purified using HPLC technology. The RT-qPCR thermal cyclers from Bio-Rad  were used, i.e., T100 during RNA purification and cDNA synthesis and CFX Connect Real-Time PCR Detection System for transcript levels measurements.All reagents used for the RT-qPCR were molecular biology grade. RNase-free plastic pipette tips, PCR tubes, microcentrifuge tubes, and conical tubes were used during the experiment to avoid degradation of the matrices. Total RNA was isolated from cell homogenates using the phenol-chloroform method published by Chomczynski and Sacchi . Monolayeviously ,45,92. IThe primers used for the reaction are presented in p < 0.05, ** for p < 0.01, *** p for < 0.001 and **** p < 0.0001. The results are presented as mean values obtained in at least three independent reactions, while whiskers represent standard deviation (±SD).The obtained results were verified using analysis of variance (ANOVA) with the Bonferroni correction (Bonferroni post hoc test). The analysis was performed using GraphPad Prism 8 . Statistically significant differences were marked with * for In summary, we used the RT-qPCR technique to determine expression patterns of several non-coding RNAs and genes associated with OS development. We compared them with profiles obtained for the HeLa cell line and human adipose-derived stromal cells (hASCs).We showed that miR-21-5p and BMP-7 could be relevant markers of osteosarcoma cell lines. mRNA levels for BMP receptors showed a cell-specific expression pattern. The results confirmed the molecular phenotype of the osteosarcoma cells, associated with their invasiveness and manifested by low expression of MEG3 and high mRNA levels for metalloproteinases, especially for MMP-14. Moreover, we established that the levels of miR-223 and miR-320 noted in OS cell lines and hASC are comparable. These onco-miRs could be significant players in OS development associated with the activity of ASCs in the tumour microenvironment.The presented gene expression profile may prove to be a valuable complement to studies aimed at the characterization of osteosarcoma and its cancer- and bone-related phenotypes. Comprehensive molecular identification of OS cell lines provides helpful information that may benefit the proper selection of a model for functional analysis aimed at developing new methods of osteosarcoma treatment.


Human papillomavirus positive tonsillar and base of tongue cancers are rising in incidence but chemoradiotherapy has not improved survival, so novel treatments are needed. Therefore, targeted therapy with phosphoinositide 3-kinase, poly-ADP-ribose-polymerase, and WEE1 inhibitors was pursued alone or combined with/without radiotherapy or using APR-246 and their effects were analyzed by WST-1 viability, proliferation, and cytotoxicity assays on tonsillar and base of tongue cancer cell lines. All three inhibitors induced dose dependent inhibition of viability and proliferation and their effects were enhanced by adding irradiation or by combining the inhibitors. However, adding irradiation to already synergistic inhibitor combinations did generally not induce additional inhibitory effects. Only very high APR-246 doses decreased viability, so its use was limited in this context. To conclude, the effects of the inhibitors were frequently enhanced by adding radiotherapy or by combining them, but adding radiotherapy to the latter did not generally induce further inhibition. +) tonsillar and base of tongue cancer (TSCC/BOTSCC) is rising in incidence, but chemoradiotherapy is not curative for all. Therefore, targeted therapy with PI3K (BYL719), PARP (BMN-673), and WEE1 (MK-1775) inhibitors alone or combined was pursued with or without 10 Gy and their effects were analyzed by viability, proliferation, and cytotoxicity assays on the TSCC/BOTSCC cell lines HPV+ UPCI-SCC-154 and HPV− UT-SCC-60A. Effective single drug/10 Gy combinations were validated on additional TSCC lines. Finally, APR-246 was assessed on several TSCC/BOTSCC cell lines. BYL719, BMN-673, and MK-1775 treatments induced dose dependent responses in HPV+ UPCI-SCC-154 and HPV− UT-SCC-60A and when combined with 10 Gy, synergistic effects were disclosed, as was also the case upon validation. Using BYL719/BMN-673, BYL719/MK-1775, or BMN-673/MK-1775 combinations on HPV+ UPCI-SCC-154 and HPV− UT-SCC-60A also induced synergy compared to single drug administrations, but adding 10 Gy to these synergistic drug combinations had no further major effects. Low APR-246 concentrations had limited usefulness. To conclude, synergistic effects were disclosed when complementing single BYL719 BMN-673 and MK-1775 administrations with 10 Gy or when combining the inhibitors, while adding 10 Gy to the latter did not further enhance their already additive/synergistic effects. APR-246 was suboptimal in the present context. Human papillomavirus positive (HPV Morecurrence ,39,40,41ical use ,43. + UM-SCC-47, UPCI-SCC-154, and HPV-UT-SCC-60A [PIK3CA/FGFR3 mutations, but still showed dose dependent responses and synergy [+ CU-OP-2, -3, -20, and HPV− CU-OP-17 with/without (w/wo) PIK3CA/FGFR3 mutations and could confirm dose dependent responses and synergy [+/HPV− CU-OP lines and revealed positive synergistic effects similar to that for certain breast cancer types [Due to early inhibitor studies in vitro on breast and urothelial cancer, we also initially explored the effects of some of the then available non-FDA approved PI3K and FGFR inhibitors  on TSCC/BOTSCC cell lines HPV-SCC-60A . These l synergy . We thener types . More recently, we also tested single and combined poly-ADP-ribose-polymerase (PARP) and WEE1 inhibitors on the CU-OP lines ,47. Howe+ TSCC/BOTSCC, similar to that for other cancers, especially upon failure or resistance to anti PD-1/PDL-1 therapy, often given today, but before doing so, more knowledge would be of value [Clearly, some, but not all above combinations, could be useful clinically for relapsed HPVof value ,47,48,49of value ,47,48,49+ UPCI-SCC-154 and HPV− UT-SCC-60A. More importantly, in parallel, on the same two cell lines, we investigated whether adding irradiation (IR) to the aforementioned inhibitors alone or their combinations could improve their efficacy, since IR has been shown to enhance the effect of PARP inhibitors in other systems [+/HPV− TSCC/BOTSCC cell lines. In this study, we explored the effects of PI3K, PARP, and WEE1 inhibitors  alone and the recent BMN-673 and MK-1775 combination  as well  systems . We then systems , we also systems . We therTP53 and HPV− UT-SCC-60A, a TSCC with mutant TP53, respectively, were obtained from Susan Gollin, University of Pittsburgh USA and Reidar Grénman, University of Turku, Finland, respectively, and both were kept in Dulbecco’s modified Eagle’s medium (Gibco) [+ CU-OP-2 (with a PIK3CA and FGFR3 mutation), CU-OP-3, CU-OP-20 (with a PIK3CA mutation), and HPV− CU-OP-17, all with wt TP53, were obtained from N. Powell, Cardiff University UK [2.UPCI-SCC-154, a tongue squamous cell carcinoma with wt  (Gibco) ,53. HPV+rsity UK ,54. Theyrsity UK ,54. All For analysis, UPCI-SCC-154 and UT-SCC-60A were seeded with 5000 cells/well, while the CU-OP lines (without feeders) were seeded at 7500 cells/well with all in 96-well plates in 90–200 μL media. All experiments were repeated at least three times.FDA approved phosphoinositide 3-kinase (PI3K) inhibitor BYL719, poly-ADP-ribose-polymerase (PARP) inhibitor BMN-673 as well as non-FDA approved WEE1 inhibitor MK-1775 were all purchased at Selleckhem Chemicals Munich, Germany. Stock solutions at 10 mM for all inhibitors were diluted in the presence of 1% DMSO. Further dilutions were, however, all made with PBS and stored at −20 °C. The inhibitors were diluted once again before use  and utilized in the following doses: BYL719 0.5–20 μM; PD-0332991 5–40 μM; and for BMN-673 0.1–50 μM and MK-1775 0.1–50 μM. APR-246 was kindly obtained from Prof. Klas Wiman, Karolinska Institutet, Stockholm, Sweden and stocked in DMSO and then further diluted 10–100 μM in PBS before use. IR with 10 Gray (Gy), was given with an X-Rad 225XL machine  3 hours (h) before inhibitor treatment to UPCI-SCC-154 and UT-SCC-60A, while to CU-OP cells, 10 Gy was given at the same time or 3 h after inhibitor treatment, if not stated otherwise.Viability pursued for 72 h after treatment was assessed by a WST-1 assay  in accordance with the instructions of the manufacturer and repeated three times, as previously reported in more detail .The 96-well plates with the various cell lines were placed into the IncuCyte S3 Live® Cell Analysis System utilizing the Incucyte™ Cytotox Red Reagent  and followed for proliferation (cell confluence) and cytotoxicity by taking images every 2 h, as described earlier . Of the To verify the efficacy of the single inhibitors or their combinations in comparison to the negative PBS control, a multiple t-test followed by correction for multiple comparison of the means conferring to the Holm Sidak method was performed ,46,55,56+ CU-OP-2, -3, and -20 and HPV− CU-OP-17 presented dose dependent responses to BYL719, BMN-673, and MK-1775 [Previously, we have shown that HPV MK-1775 . Here, w MK-1775 , and thep < 0.05) (p < 0.05) but not after 72 h (p < 0.05)  A. For UTter 72 h C. Upon a < 0.05) B,D. p < 0.05) (p < 0.05) (p < 0.05) when IR was not applied  E. Upon c < 0.05) F. UT-SCC applied G. Upon c applied H. p < 0.05) (p < 0.05)  I,J. For  < 0.05) K,L.Combinational indices (CIs) 48 and 72 h after treatment of BYL719, BMN-673, and MK-1775, w/wo IR, were calculated for UPCI-SCC-154 and UT-SCC-60A to determine the effect of combinations .In line with For UT-SCC-60A, again in line with the viability data, clear positive combinational effects were observed at 48 h for the BYL719 and 10 Gy and MK-1775 and 10 Gy combinations, while the 10 Gy and BMN-673 combinations mainly resulted in neutral or adverse effects D–F. To conclude, for UPCI-SCC-154, adding 10 Gy to BYL719 and BMN-673 enhanced the efficacy of single drugs, while for the 10 Gy and MK-1775 inhibitor combination, neutral effects were obtained. For UT-SCC-60A, enhanced efficacy was shown with the 10 Gy BYL719 and 10 Gy MK-1775 combinations, but not by combining 10 Gy with BMN-673. Due to the positive effects above of single drugs and IR, we decided to conduct some complementary experiments on two additional cell lines. A pilot study on CU-OP-2, with 10 Gy given at the same time or 3 h after treatment with 0.5 µM BYL719 showed mainly positive effects, irrespective of the timing of IR (data not shown). p < 0.05) (p < 0.05), while for CU-OP-20, this increase was not so pronounced  . Moreoveonounced . When caonounced .To summarize, UPCI-SCC-154 was generally more sensitive to single doses of the inhibitors than UT-SCC-60A, however, both cell lines were most sensitive to MK-1775 and more resistant to BMN-673 with the drug doses used here. Adding IR with 10 Gy frequently decreased the viability in comparison to using single drugs alone in both cell lines, especially 48–72 h after treatment. This was illustrated in detail by calculating the combinational effects ,56, show+ CU-OP-2, -3, and -20 and HPV− CU-OP-17 were shown to present synergistic responses to BMN-673 combined with MK-1775, but not as often and also to a lesser extent when compared to the BYL719 and CDK4/6 combination [+ UPCI-SCC-154 and HPV− UT-SCC-60A) and more combinations, in other words, 0.5–1 µM each of either BYL719 and MK-1775, BYL719, and BMN-673 and the recently examined combination BMN-673 and MK-1775, and now w/wo IR with 10 Gy  (p < 0.05) (All combinations of BYL719 with MK-1775 wo IR decreased the viability significantly compared to PBS in both cell lines (at least  < 0.05) A,C. Upon < 0.05) B,D. p < 0.05) (p < 0.05) and this was enhanced further upon applying 10 Gy, since significance was reached at all timepoints except for combinations with 0.5 µM BYL719 at 24 h (at least p < 0.05)  E,F. In U < 0.05) G,H. p < 0.05) (p < 0.05) (p < 0.05)  I. In UT- < 0.05) K. Combin < 0.05) J,L.Combinational indices (CIs) of BYL719, BMN-673, and MK-1775, w/wo IR, were calculated for each cell line 48 and 72 h after treatment to determine the effect of the various combinations, and these showed mainly positive or neutral effects . UPCI-SCC-154 is often more sensitive to single drug treatments than UT-SCC-60A, so we hypothesized that CIs would thereby indicate more positive effects for the latter compared to the former cell line  due to pBYL719/MK-1775 combinations resulted exclusively in positive effects in UT-SCC-60A, while for UPCI-SCC-154, the effects were more neutral. Adding 10 Gy did not change the effects at 48 h but at 72 h, giving IR was likely less positive than wo IR for both cell lines. BYL719/BMN-673 combinations showed positive effects in both cell lines w/wo IR.BMN-673/MK-1775 combinations showed positive effects in both cell lines with lower doses of MK-1775, while higher MK-1775 doses provided either a more neutral or even negative combinational effect and with positive effects being greater in UT-SCC-60A compared to UPCI-SCC-154. Adding 10 Gy had marginal effects in both cell lines.To summarize, BYL719/MK-1775, BYL719/BMN-673, or BMN-673/MK-1775 combinations were generally quite efficient and superior to using one inhibitor alone, with UPCI-SCC-154 generally being more sensitive to most combined treatments compared to UT-SCC-60A. However, adding 10 Gy to already efficient combinations did not usually improve the inhibition of viability further, and although IR tended to possibly boost the effect of all inhibitor combinations after 48 h , this wa+ UPCI-SCC-154, CU-OP-2, -3, -20, and HPV− UT-SCC-60A and CU-OP-17 , while this was still not the case for HPV+ CU-OP-3 and HPV− UT-SCC-60A (All cell lines were relatively resistant to APR-246. Only the highest dose (70 µM) was able to decrease the viability significantly after 72 h for HPV-SCC-60A . These f+ UPCI-SCC-154 and HPV− UT-SCC-60A. To validate the data, with some very efficient combinations in the viability tests, the same doses of BYL719 and MK-1775 w/wo 10 Gy were used to follow their effects on the proliferation of the HPV+ CU-OP-2 and -20 cell lines. Due to the positive effects with a decrease in viability using single administrations of 0.5 µM or 1.0 µM of either BYL719, BMN-673 and MK-1775 together with 10 Gy, we here examined the effects of these treatments and corresponding doses on cell confluence and cytotoxicity on HPV− UT-SCC-60A lines. Compared to PBS, low doses of BYL719 and BMN-673 had marginal effects on the cell confluence on both cell lines, while the effects of MK-1775 were slightly more pronounced, but when adding 10 Gy to the single inhibitors, a prominent decease was observed in cell confluence, especially for the UT-SCC-60A cell line with BYL719 and MK-1775, but not BMN-673, in line with the viability data  with the same doses used as in the viability tests for HPVd BYL719 . Cytotox− UT-SCC-60A. Compared to PBS, low doses of the inhibitors had virtually no or marginal effects on cytotoxicity in either cell line, however, adding 10 Gy enhanced the effect of MK-1775, but not for BYL719 and BMN-673 on both cell lines  alone or in different combinations w/wo IR (10 Gy) on two well-established TSCC/BOTSCC cell lines HPVell line ,45,54. F+ UPCI-SCC-154 and HPV− UT-SCC-60A, with the former being generally more sensitive than the latter and by adding 10 Gy, mainly positive or synergistic effects were obtained. Similar data were seen with BYL719, BMN-673, and MK-1775 w/wo 10 Gy on CU-OP-2, while these effects were not at all pronounced for the more sensitive cell line CU-OP-20. Dose dependent responses to single BYL719, BMN-673, and MK-1775 treatments were found in HPV+ UPCI-SCC-154 and HPV− UT-SCC-60A generally decreased the viability much more than using one drug alone in both cell lines, however, adding 10 Gy did not markedly improve this already efficient decrease in the viability in any of the cell lines. Combining BYL719/BMN-673, BYL719/MK-1775, or BMN-673/MK-1775 in HPVFinally, only relatively high concentrations of APR-246 exhibited a prominent decrease on all of the tested cell lines. The dose dependent responses found in UPCI-SCC-154 and UT-SCC-60A with single BYL719, BMN-673, and MK-1775 administrations was expected, since this has been demonstrated before with corresponding inhibitors on other tumor cell lines by others and us ,45,46,48+ UPCI-SCC-154 and CU-OP-2 as well as HPV− UT-SCC-60A, but not so much on CU-OP-20, irrespective of whether they had PIK3CA mutations or not. We presently have no direct explanation for this positive finding, but PI3K inhibitors have been shown to interact with IR in other systems [Adding 10 Gy to BYL719 posed additive effects on HPV systems . For exa systems . Likewis systems . Consequ+ UPCI-SCC-154 showed positive responses to that combination, this was not the case for UT-SCC-60A, despite the two cell lines being relatively equally sensitive to 10 Gy. Similarly, HPV+ CU-OP-2 and CU-OP-20 showed differing responses, with the former being sensitive and the latter insensitive to the BMN-673 10 Gy combination.Combining 10 Gy with BMN-673 showed different patterns. While HPVThe fact that both UPCI-SCC-154 and UT-SCC-60A were relatively insensitive to treatment with BMN alone is, however, not surprising. We have previously shown that the TSCC/BOTSCC cell lines, independent of HPV status, can be either sensitive or resistant to the PARP inhibitor olaparib . FurtherBRCA-1 and 2 mutations, which would be sensitive to PARP inhibitors by using different biomarkers, was pointed out as an important issue and discussed in some detail [In contrast, in other systems, it has been shown that PARP inhibitors, together with IR, have very good effects such as in BRCA1(+/−) lymphoblastoid cells, hepatocellular cancer and lung and breast cancer xenografts ,61,62. Me detail . Clearly+ UPCI-SCC-154 and HPV− UT-SCC-60A were responsive to low doses of MK-1775, similar to what we have shown before (and here) for the CU-OP-cell lines and in line with data by others on the HPV+/HPV− head and neck cancer cell lines [TP53 mutation, it is more sensitive to MK-1775 than to BYL719 and BMN-673, while instead, UPCI-SCC-154 with no TP53 mutation and being HPV+ also exhibited positive effects to PARP inhibitors, as previously shown for other HPV+ cell lines [Notably, both HPVll lines ,65. Furtll lines  (for revll lines ). + head and neck cancer cell lines, but there was no investigation with regard to RT/IR [+ cell lines [− cell lines, whereas Chk1 inhibitors were more efficient in HPV+ tumor models [Combining WEE1 inhibitors with IR has positive effects, as has been shown before for other cancer types such as leukemic T-cells, osteosarcoma, or cells with TP53 mutations such as lung and breast cancer cell lines ,67,68. Tto RT/IR . In anotll lines . In a moll lines . In thatr models . TP53 mutations or various BRCA-1 or 2 mutations or others, may affect the different responses to the various combinations, irrespective of the HPV status of the tumors. It is likely that identifying HPV status alone will be insufficient and one will have to characterize the tumors in more detail before applying specific individualized therapies [Clearly, there are some data using various inhibitors in combination with IR, however, these are not always concordant, suggesting that these issues need to be further clarified. It is most likely more complicated, since the mutational profiles of the individual tumors vary, and harboring, for example, herapies ,69,70. Combining BYL719/BMN-673, BYL719/MK-1775, and BMN-673/MK-1775 mainly elicited positive effects on both cell lines and decreased the viability with low doses. This was of note, but not entirely unexpected, although the latter combination (BMN-673/MK-1775) in our hands had not previously shown the most optimal synergy on the CU-OP cell lines . ObviousThe BYL719 and BMN-673 combination seemed useful in both cell lines in this study. This would then be in line with other studies, where PARP inhibitors have been combined with PI3K inhibitors for high grade serous ovarian and breast cancer with some antitumor effects . The autThe final inhibitor combination in this report was using PI3K together with a WEE1 inhibitor. Here, an additive/synergistic effect was also mainly observed on UT-SCC-60A, most likely due to it being the most resistant cell line, while for UPCI-SCC-154, already sensitive to MK-1775, the additive/synergistic effect was not as pronounced. We attempted to compare our data to similar reports, but were not able to find larger studies using this approach in head and neck cancer, instead, both were often combined separately (e.g. with PARP inhibitors) ,72,73,74Adding IR to the three BYL719/BMN-673, BYL719/MK-1775, and BMN-673/MK-1775 beneficial combinations did not really improve their effects further in a pronounced way. However, in some cases, an earlier response and more marked decrease in viability was observed already after 48 h with regard to the UT-SCC-60A cell line, but this was not markedly reflected when calculating the CIs. Nevertheless, it was of interest to follow the effects of single inhibitors alone w/wo IR as well as to follow the effects of combined inhibitors w/wo IR. The data suggest that depending on the tumor and the situation, one could design a treatment that would include a specific inhibitor together with IR or use two inhibitors in combination. For these, further studies would be of interest, however, the decisive choice would then be either upon recurrence, if the patient tumor does not respond to the possibilities used today, or in the future for primary treatment. However, for the latter, further studies are of importance.TP53 mutations in tumors sensitive to WEE1 are not similar to the ones where APR-246 has its best effects, since the UT-SCC-60A line has a p.Arg342Ter (c.1024C > T), which initiates a premature stop codon in exon 9 of TP53 and results in a truncated TP53 protein [Finally, in this report, we also explored the effect of APR-246 on quite a few cell lines with the anticipation that it would possibly show some effects because high risk HPV E6 downregulates TP53 or because UT-SCC-60A has a documented TP53 mutation ,76. Howe protein ,78,79. I protein ,81. FurtThere were limitations in this study, in that we had a small number of inhibitors and cell lines. However, we did validate some of the most important combinations, and it is obvious that the variability between the different cell lines, irrespective of HPV status, poses additional issues. Obviously, it is not possible to generalize on the sensitivity of the tumors to specific inhibitors or their combinations w/wo IR, solely by HPV status. In the future, a profound molecular analysis of the various cancers should be conducted before the selection of treatment, and experiments carried out to follow if the selection was optimal.In summary, combining 10 Gy with single BYL719 BMN-673 and MK1775 inhibitors can potentially be of use for TSCC/BOTSCC, but is likely not needed when already combining inhibitors exhibiting synergy, while APR-246 was not optimal in this context. Nevertheless, without more detailed analysis of the individual tumors, it may not be possible to predict which combination is the best. +/HPV− TSCC/ BOTSCC cell lines, and when the inhibitors were combined with 10 Gy, or used in combination, mainly positive/synergistic effects were obtained. However, adding 10 Gy to the already efficient postive/synergistic inhibitor combinations had no major further effects. Furthermore, low APR-246 concentrations had limited effects. In this study, single PI3K, PARP, and WEE1 inhibitors BYL719, BMN-673, and MK-1775 induced dose dependent inhibition of viability and proliferation in the HPVTo conclude, adding 10 Gy to single BYL719, BMN-673, and MK-1775 low dose administrations or combining these inhibitors notably enhanced their efficacy, while adding 10 Gy to the latter did not induce any major further improvement of their already enhanced effects. APR-246 was suboptimal in this context.


In order to investigate novel AS-associated LGR4, we discarded LGR4 isoforms from GSO databases. We used siRNA to knock down the specific isoform to check the cell proliferation, migration, and invasion when treated with metformin.  First, the expression of LGR4 in HCC tumor tissues and cell lines was detected by western blotting and immunofluorescence. The ability of cell proliferation, migration, and invasion was detected with CCK8, wound-healing, and transwell assays when overexpressing LGR4 or treating with metformin. The  The level of LGR4 expression was higher in HCC cell lines and tumor tissues. The HCC cell proliferation, migration, and invasion were increased when overexpressing LGR4, which could be reduced by metformin treatment. The GEO database (GSE190076) showed that LGR4 had switching properties in HCC cell lines treated with metformin. We used siRNA to knock down the specific isoform, and the result showed that the specific isoform siRNA could promote the inhibition of cell invasion caused by metformin treatment.  LGR4 could promote the ability of cell proliferation, migration, and invasion in HCC, which could be reduced by metformin through alternative splicing. Hepatocellular carcinoma is the most common type of liver cancer all over the world, especially in China , 2, althLGR4 belongs to the GPCRs superfamily and is recognized as a trans-membrane receptor of the LGR family. Recently, a study indicated that LGR4 could interact with PrPc to promote tumorigenesis and liver metastasis by stemness of colorectal cancer stem cells . In acutIn our research, we investigated the function of LGR4 in the HCC, and we also found that metformin could reduce the expression of LGR4. We also indicated the mechanism of the regulatory effect of metformin. The study could provide evidence of LGR4 function, which could be recognized as a target for HCC treatment.2 at 37°C. The HCC cell lines were treated with metformin (20 μM) [Normal cell lines (L02) and Huh-7 and HepG3B cell lines were obtained from the American Type Culture Collection (ATCC). All cells were cultured in DMEM  with FBS (10%), in an atmosphere containing 5% CO (20 μM) . The HCCAccording to the previous procedure , the tre5 cells per well. And DMEM (10% FBS) was cultured in the bottom chambers. After 6 hours, 4% paraformaldehyde was used to fix the inserts and stain them with 0.1% crystal violet solution. Also, the images were obtained under a microscope.The treated cells were digested and added into the top chambers of transwell inserts with FBS-free DMEM. The cell density is 2 × 10μL pipette tip was used to scratch, cells were washed, and the cell was incubated with DMEM for another 12 hours. Three random areas were photographed to assess the distance of migration.The treated cells were digested and cultured in 6-well plates. After treatment, a 200 According to the previous procedure , proteinβ-catenin staining, the treated cells were washed with PBS three times and fixed, then treated with 0.1% Triton X . Subsequently, a blocking buffer was added and the primary antibody was incubated overnight at 4°C. The next day, the treated cells were incubated with a secondary antibody, then treated with DAPI. Finally, the images were captured by a microscope.For The GEO database (GSE190076) was used to indicate the different gene expressions. Then, the transcript was analyzed with an isoform switch analyser. A total of 480 genes underwent isoform switching. KEGG pathway analysis was also used to analyze the genes' function.p value < 0.05. Differences were determined using a two-way analysis of variance (ANOVA) or unpaired Student's t-test by Prism software.All measurements were presented as the mean ± standard deviations (SD) from three independent experiments. Statistical significance was defined as a First, the level of LGR4 expression was detected in HCC tissues. It showed that LGR4 expression was increased in the tumor tissues . We alsoβ-catenin pathway in many cancers. So, the immunofluorescence indicated that the β-catenin expression was higher in the LGR4 overexpressed cells  to indicate the different gene expressions. It showed the mRNA level of LGR4 had no difference between the two groups, which meant it may be regulated by alternative splicing. Then, we analyzed transcript usage with an isoform switch analyser. A total of 480 genes underwent isoform switching . KEGG paβ-catenin expression. It indicated that the specific isoform siRNA could also reduce the expression of catenin in response to metformin treatment , the use of metformin may improve disease-free survival and overall survival in CRC patients with T2DM . In bladβ-catenin axis to promote the progression of hepatocellular carcinoma [β-catenin signaling in many diseases [β-catenin.LGR4 belongs to the GPCRs superfamily and is recognized as a trans-membrane receptor of the LGR family. In this study, we found that the level of LGR4 in Huh-7 and HepG3B cell lines was much higher than that in L02 cell lines. It indicated that the LGR4 expression was increased in HCC tissues and at cell levels. LGR4 overexpression could promote the processes of proliferation, migration, and invasion. Although the study indicated that Hsa_circ_0003945 could regulate the miR-34c-5p/LGR4/arcinoma , the exadiseases –18. So, β-catenin expression. Apart from alternative splicing, the genes also focus on sulfur metabolism pathways, and the study also indicated that sulfane sulfur metabolism could be used to treat cancers. We will also analyze sulfur metabolism in the future.Alternative splicing could produce multiple transcripts of mRNA to regulate the gene expression . Then, wIn conclusion, we demonstrated that LGR4 could promote the proliferation, migration, and invasion of HCC, which could be reduced by metformin through alternative splicing.


A major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.A comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.Several compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.Our study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors. There is an urgent need for effective therapies for patients with platinum-resistant high-grade serous ovarian cancer (HGSOC). While most HGSOC patients initially respond to the standard first-line platinum–taxane combination chemotherapy, relapse within 18 months is common followed by chemoresistance . ReasonsIn vitro models are essential for identifying effective oncology compounds or drug combinations for any type of cancer, including HGSOC. Until recently, most of the in vitro studies have been conducted using publicly available ovarian cancer cell lines that may not represent the HGSOC subtype and that may have undergone a variety of in vitro alterations during extensive passaging  showed the most consistent inhibitory results. Wee1 kinase plays a crucial role in cell-cycle regulation and DNA damage identification and repair in malignant and non-malignant cells, and its inhibition has shown promising results in early phase clinical trials –14, inclTumor and ascites material and clinical information was collected from consenting patients treated at the Department of Obstetrics and Gynecology, Turku University Hospital, Turku, Finland, as described previously , 15. TheDNA/RNA sequencing and functional assessment of homologous recombination capacity). In addition to these patient-derived cell lines, two conventional HGSOC cell lines were explored: CAOV3  and OVCAR8 .Two patient-derived cell lines were established from ascites (OC002 and M022i), of which OC002 was treatment-naive and M022i was from IDS. One cell line originated from omental metastasis M048i) and was from IDS , and/or cells from the cultures. Germline reference was available for M022i and M048i, fresh frozen tumor tissue or ascites for M048i (N = 4) and OC002 (N = 1), and cultured cells for M022i and M048i. DNA/RNA was extracted from samples with AllPrep DNA/RNA Mini Kit (Qiagen). Sequencing was performed in the BGI (Beijing Genomics Institute) as whole-genome sequencing (WGS) with HiSeq X Ten or with whole exome sequencing (WES) with Agilent SureSelect Human All Exon V5 using HiSeq 2000.To genetically characterize tumors and identify patient-specific TP53 mutations, which were used to verify cell identity in the cultures , we analyzed RNA expression data of the ABC transporters (ATP-binding cassette transporters). The results are provided as reads per kilobase per million (RPKM) values, normalized to GAPDH expression. A heatmap was produced using Log2+1 values at the publicly available web software Heatmapper (heatmapper.ca) .The five cell lines were subjected to high-throughput screening (HTS) with a panel of 306 clinical FDA- and/or EMA-approved and emerging oncology drug compounds Table S4https://breeze.fimm.fi/)  was used , 10. Thiimm.fi/) .® (Promega) cell viability test. To test cells' sensitivity to cisplatin, 5,000 cells/well were plated on a 96-well plate in triplicates. Cisplatin was added in concentrations of 0.01–100 µM, and cell viability was measured after 72 h of incubation. Luminescence was detected with a Victor2 luminometer . The IC50 value of adavosertib for each cell culture was calculated, and a dose of 500 nM was selected to be used for the functional experiments. The IC50 value for cisplatin was calculated using log-transformed data and logarithmic trend line.The cytotoxic effect of adavosertib  was further validated with a CellTiter-GloThe M048i, OC002, CAOV3, and OVCAR8 cells were investigated with a BD LSRFortessa™ flow cytometer . For each measurement, 10,000–30,000 events were assessed. The flow cytometry data were analyzed with Flowing Software 2.5.1 . Duplicates of each sample were tested, and the experiment was repeated a minimum of three times.Early and late apoptoses were detected after 48 and 72 h of treatment with adavosertib with an Annexin V-FITC Apoptosis Detection Kit . Samples were processed according to the kit's protocol.Cell-cycle phases were detected with a Click-iT EdU Flow Cytometry Assay Kit Pacific Blue . Cell-cycle progression was studied in vehicle- and adavosertib-treated cells at time points 24, 48, and 72 h. Samples were collected, and the protocol was performed according to the manufacturers' instructions.Cell proliferation of the HGSOC cell lines was inspected for 72 h at 2-h intervals with an IncuCyte S3 high-content imager . Cells were plated in 96-well plates (Greiner Bio-One) to be 10% confluent and treated with 500 nM adavosertib. Cells treated with vehicle (DMSO) were used as control in all the experiments. Each sample was measured in triplicate, and the experiments were repeated a minimum of three times. Proliferation was measured by confluence area by IncuCyte software (Essen BioScience).Wound healing of the HGSOC cells was measured for 72 h at 2-h intervals with an IncuCyte S3 high-content imager . Experiments were performed on 96-well plates  with adavosertib (500 nM) or vehicle (DMSO). Each sample was measured in triplicate, and the experiments were repeated a minimum of three times. Relative wound density was analyzed by IncuCyte software (Essen BioScience). The wells were precoated with Geltrex (Gibco) for migration experiments and with Matrigel  for invasion experiments. Wells were pretreated for 24 h with adavosertib (500 nM) before wound making with a wound-maker provided with IncuCyte S3 (Essen Bioscience). In the invasion experiments after wound making, the cells were covered with 50 µl Matrigel (2 mg/ml) for 30 min in the incubator; thereafter, adavosertib was added.® TGX™ Precast Protein Gels (Bio-Rad) and transferred to the 0.2-µm PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% BSA  in Tris-buffered saline with 0.05% Tween 20 (TBST) and probed with primary antibodies diluted in the same solution. Primary antibodies used in the Western blotting were rabbit monoclonal anti-Cyclin B1 , rabbit monoclonal anti-Cyclin E1 , mouse anti-PCNA , and mouse monoclonal anti-γH2AX . GAPDH-HRP-conjugated  or mouse monoclonal anti-α-Tubulin  was used as control for protein loading. The secondary antibodies were HRP-conjugated swine anti-rabbit and rabbit anti-mouse immunoglobulins  diluted in a blocking solution. Membranes were washed three times with TBST between antibody incubations. Bound proteins were detected by enhanced chemiluminescence using ChemiDoc™ Gel Imaging System (Bio-Rad), and the signals were quantified using ImageJ.The effect of adavosertib on the cell cycle was investigated in the cell cultures treated for 72 h. Cells were grown in a DMEM/F-12 or RPMI medium and harvested and lysed with a RIPA Buffer supplemented with protease inhibitors. Protein concentrations were measured using a Bio-Rad protein assay kit according to the manufacturer's instructions. Equal amounts of proteins in the Laemmli buffer were separated in 4%–20% polyacrylamide PROTEANCells were grown on glass slides with a Geltrex (Gibco) coating and a modified OCMI or RPMI medium. After 72 h of incubation with 500 nM adavosertib, the cells were fixed for 10 min in 4% paraformaldehyde and washed with PBS. Blocking was performed with 5% BSA and 0.5% Triton X-100 in PBS for 30 min. Slides were incubated at room temperature for 60 min with a primary antibody diluted in a blocking buffer and thereafter for 60 min with a secondary antibody. The slides were washed twice after the antibody incubations with PBS and embedded in a mounting medium containing DAPI for staining nuclei . The following primary antibodies were diluted in the blocking buffer: mouse monoclonal anti-α-Tubulin  and mouse monoclonal anti-γH2AX . Alexa Fluor 555 Donkey anti-Mouse  was used as a secondary antibody together with Alexa Fluor 488-conjugated phalloidin  for actin filament visualization.http://rsbweb.nih.gov/ij/).Nuclear morphology was assessed after 72 h after treatment with vehicle or 500 nM adavosertib by staining the tubulin for cell structure and embedding in mounting medium containing DAPI for the cell nucleus staining. At least 100 cells were counted in each sample, and the nuclei were categorized as normal, abnormal, or multinuclear. Images were taken with a Nikon Eclipse Ni fluorescence microscope, and different channels were merged using ImageJ v1.53a software . P values ≤0.05 were considered statistically significant.The IC50 values for the adavosertib cytotoxicity validation were acquired using a sigmoidal dose–response curve. The differences in proliferation, wound healing, cell cycle, apoptosis, nuclear abnormalities frequencies, and protein levels between vehicle- and adavosertib-treated cells were compared using the two-sided The drug sensitivity testing was performed under two growth conditions, i.e., conventional adherent and spheroid conditions inducing stemness features and mimicking the environment of the malignant cells in ascitic fluid. We employed a correlation plot analysis for sDSS to assess the condition-related variations in drug sensitivity Table S6Ten clinically interesting drug compounds from the initial screen were selected for further investigation according to two criteria: literature search and ongoing clinical studies in HGSOC or other cancers Table S8To study whether adavosertib induces apoptosis, HGSOC cells were treated for 48–72 h before labeling with Annexin V. Adavosertib-induced apoptosis was evident in all of the examined cell lines, surprisingly also including M048i, the most resistant cell line in viability tests   was detected with Western blotting to confirm the confluence area-based proliferation result. The amount of PCNA decreased after 72 h of adavosertib treatment in the tested HGSOC lines  in the HGSOC lines  capacity using both genomic Table S2Despite the advances in understanding the molecular background of HGSOC, patients who do not initially respond or acquire resistance to platinum compounds or PARP inhibitors have limited treatment options. Based on the high-throughput screen of HGSOC cell lines, and with a focus on compounds in the clinical development, we identified two compound groups cytotoxic to HGSOC independent of the cell culture method: Wee1 inhibitor  and Hsp90 inhibitors . In a further validation using three patient-derived and two conventionally available HGSOC cell lines, adavosertib provided the best cytotoxicity result. Wee1 has already proven to be a potential target in genomically unstable cancers, including HGSOC, due to its role in cell-cycle control and DNA-damage response (DDR) pathways [reviewed in –28]. OurWe also noticed generally similar effects on the three HRP and two HRD cell lines. Additionally, adavosertib inhibited equally well patient-derived HGSOC cells and the publicly available HGSOC cell lines. One of the patient-derived HGSOC cell lines, M048i, was less responsive than the other cell lines in the high-throughput cytotoxicity assay to most of the 306 drugs including adavosertib for reasons that remain unclear. No significant difference was found in the DNA sequencing or RNA expression data of the multidrug resistance (MDR) ABC transporters (ATP-binding cassette transporters) compared to other cell lines , 30. In via inhibiting CDK1 and delays the mitosis entry of cells with DNA damage. Its inhibition in cells with DNA damage allows these cells to enter mitosis prematurely, which leads to mitotic catastrophe and apoptosis. Wee1 is also involved in regulating the G1/S checkpoint via CDK2 and thereby DNA replication by phosphorylating CDK2-bound cyclin A/E during the S phase  found that adavosertib impairs HR and may work as a combination therapy with PARP inhibitor olaparib in BRCA1/2-mutant leukemias . AnotherOur findings lay the basis for treatment of HRP patients, with whom treatment strategies are scarce. It is especially important to find an effective DNA-damaging agent to be used in combination with adavosertib for the platinum-resistant HGSOC patients. In this regard, gemcitabine has already shown promising results.In conclusion, our study suggests Wee1 inhibitor adavosertib as a candidate compound to treat HGSOC patients independent of the HR status of the tumor.The raw sequencing data are available from the European Genome-Phenome Archive (EGA). RNA data is under accession number EGAD00001006456 and DNA data is available under same DAC (EGAC00001001760).The studies involving human participants were reviewed and approved by Ethics Committee of the Hospital District of Southwest Finland (ETMK): ETMK 53/180/2009 §238 and ETMK 69/180/2010 and The Finnish National Supervisory Authority for Welfare and Health in Finland : DNRO 6550/05.01.06/2010 and STH507A.The patients/participants provided their written informed consent to participate in this study.PR, AA, and OC conceived the project. JH, SG, and AA contributed to the material acquisition. KK and PR established and maintained patient-derived HGSOC cell lines. PR, AS, VH, SaP, and KK designed the experiments. PR, AS, VH, PM, SaP, and SwP carried out the experiments. PR, AS, VH, KH, SaP, YL, and JO analyzed the data. PR, AS, and VH generated the figures. PR prepared the original draft. All authors reviewed and commented on the article. All authors contributed to the article and approved the submitted version.The project has received funding from EU Horizon 2020 (667403), The Academy of Finland (292606), Finnish Cancer Society, Sigrid Juselius Foundation, Finska Läkaresällskapet, and Helsinki University Hospital Research Funds. PR has received research grants from K. Albin Johanssons Stiftelse, Varsinais-Suomen rahasto (85211878), Orion Research Foundation, Lounais-Suomen Syöpäyhdistys, University of Turku Postgraduation Education Unit, and Ida Montinin Säätiö.We thank the DDCB core facility (FIMM HTB unit) supported by the University of Helsinki and Biocenter Finland. We also thank CSC – IT Center for Science Ltd. for computational resources.AA reports receiving honoraria and travel support from GSK and is a member in Nordic Society of Gynecologic Oncology NSGO-CTU Foundation Board. KW reports receiving payment for attending and presenting at meetings organized by Pfizer and Amgen. JH has received funding from EU Horizon 2020 (965193).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prostate cancer (PC) is a common malignancy and is one of the leading causes of cancer-related death in men worldwide. Osteosarcoma (OS) is the most common bone cancer, representing 20–40% of all bone malignancy cases. Cancer metastasis is a process by which malignant tumor cells detach from the primary tumor site via a cascade of processes and migrate to secondary sites through the blood circulation or lymphatic system to colonize and form secondary tumors. PC has a specific affinity to the bone based on the “seed and soil” theory; once PC reach the bone, it becomes incurable. Several studies have identified long noncoding RNAs (lncRNAs) as potential targets for cancer therapy or as diagnostic and prognostic biomarkers. The dysregulation of various lncRNAs has been found in various cancer types, including PC, OS, and metastasis. However, the mechanisms underlying lncRNA oncogenic activity in tumor progression and metastasis are extremely complex and remain incompletely understood. Therefore, understanding oncogenic lncRNAs and their role in OS, PC, and metastasis and the underlying mechanism may help better manage and treat this malignancy. The aim of this review is to summarize current knowledge of oncogenic lncRNAs and their involvement in PC, OS, and bone metastasis. Prostate cancer (PC) is a common malignancy and is one of the leading causes of cancer-related death in men worldwide. Wang et al. categorized PC as the fifth-most commonly detected malignancy, with an estimated 1,414,000 new cases and 375,304 deaths in men globally . While pMore than 90% of patients diagnosed with advanced PC show evidence of bone metastasis . GeneralMost human RNA transcripts are now known to be noncoding. While 70–90% of the human genome is transcribed into RNA, only ~2% encodes proteins . TranscrScientists long believed that lncRNAs were just RNA polymerase II transcription byproducts that represented transcriptional noise and had no biological function . They reLncRNAs are necessary for diverse gene expression events and mRNA processing stages, particularly splicing, editing, transcription regulation, and chromatin remodeling. Furthermore, they affect other molecular processes, such as being the precursors of small RNAs, affecting other ncRNA processing, and targeting proteins at specific gene loci to directly affect transcription patterns . DespiteLncRNAs comprise many introns and function as sponges, specifically for micro RNAs (miRNAs), leading to indirect mRNA regulation ,29. MiRNConsequently, several studies have identified lncRNAs as potential cancer therapy targets or diagnostic and prognostic biomarkers ,24,33,34As mentioned above, abnormal lncRNA expression exists in many tumorigeneses. Recent studies have shown that PC-associated lncRNAs regulate diverse cellular processes that induce (oncogenes) or suppress (tumor suppressors) tumorigenesis . AdditioDANCR is highly upregulated in PC, with PC invasion and migration promoted by DANCR in vitro [DANCR overexpression in a xenograft prostate tumor mouse model [Differentiation antagonizing non-protein coding RNA (DANCR) is an oncogenic lncRNA with a crucial role in the progression of many cancer types . Jia et in vitro . Moreovese model . Therefose model . Anotherse model .DANCR overexpression led to miRNA 33b-5p (miR-33b-5p) downregulation via sponging, promoting glycolysis in a Taxol-resistant cell line (PC3-TXR) . DANCR kAXL) expression, increasing the activity of downstream targets of the AXL/protein kinase B (AKT) axis [ZNF750 mutations or deletions showed upregulated DANCR sponging of miR-4707-3p, increasing forkhead box C2 (FOXC2) expression and worsening prognosis [DANCR overexpression promoted OS cell line proliferation, migration, and invasion and promoted tumor growth and metastasis in vivo . ThroughKT) axis . TherefoKT) axis . NotablyKT) axis . In metaKT) axis . Metastarognosis .Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) overexpression has been found in various cancer cell lines and tumors . MALAT1 RET) upregulation due to MALAT1 sponging of miR-129-5p, activating the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway [MALAT1 overexpression was also found in OS samples and different human OS cell lines [MET) and SRY-box transcription factor 4 (SOX4) expression through its sponging of miR-34a), miR-34c-5p, miR-449a, and miR-449b [A recent study showed that MALAT1 upregulation was significantly associated with increased tumor progression and metastases in patients with OS . MALAT1  pathway . MALAT1 ll lines . In addill lines . MALAT1 ll lines . MALAT1 miR-449b . MALAT1 NEAT1 suppression inhibited PC cell proliferation, invasion, and migration and stimulated apoptosis and cell cycle arrest [Nuclear enriched abundant transcript 1 (NEAT1) expression is upregulated in various human malignancies, including PC . NEAT1 ie arrest .STAT3) expression, enhancing migration, invasion, and EMT in OS cell lines, particularly U2OS and MG-63 [NEAT1 knockdown in an OS cell line (U2OS) reduced OS-associated metastases when these cells were transplanted into nude mice [NEAT1 silencing suppressed OS MET at metastasis sites [NEAT1 knockdown suppressed OS proliferation, migration, and invasion in vitro [A recent study found that miR-483 was downregulated by NEAT1 sponging in both OS cell lines and patient tissues. This downregulation increased the signal transducer and activator of transcription 3  gene as a novel HOTAIR target crucial for promoting PC invasiveness, which inhibits mitogen-activated protein kinase (MAPK) signaling [hepaCAM promoter, reducing its expression [HOTAIR expression; however, its expression was upregulated in PC patients after androgen deprivation therapies (ADT) and in castration-resistant PC (CRPC) tumors [HOTAIR decreases androgen receptor (AR) ubiquitination by binding directly to the N-terminal domain (NTD) of the AR protein to prevent mouse double minute 2 (MDM2), which is an E3 ubiquitin ligase, from binding to that particular NTD; as a result, AR protein degradation is blocked, and AR activity increased without the presence of androgen [HOTAIR in LNCaP cells activated several genes that were also induced by androgen and promoted PC cell proliferation and invasion [HOTAIR knockdown led to a reduction of CRPC cell line (C4-2B) proliferation and invasion, and the presence of enzalutamide along with HOTAIR knockdown was more effective to reduce C4-2B cell proliferation [HOTAIR overexpression is critical for CRPC progression, and it could be an enzalutamide resistance biomarker for CRPC patients [HOTAIR was the most significantly downregulated lncRNA during their investigation of bufalin anticancer effects on PC cell lines (PC3 and DU145) [HOTAIR abolished the bufalin effects on these cells, and it increased their migration and invasion activities via direct sponging of miR520b, leading to an increased expression of FGFR1, which is the miR520b target gene [HOTAIR and bone metabolic markers  levels were higher in the serum of PC patients with bone metastasis in comparison to PC patients without bone metastasis [HOTAIR overexpression promoted PC-associated bone metastasis through the HOTAIR/miR-520b/FGFR1 network [HOX transcript antisense RNA (HOTAIR) is an oncogenic lncRNA believed to be a significant diagnostic and prognostic biomarker for several tumors . It was ignaling . HOTAIR pression . Zhang e) tumors . HOTAIR androgen . The oveinvasion . HOTAIR feration . Thus, Hpatients . In a red DU145) . Bufalinget gene . Moreoveget gene . HOTAIR tastasis . This st network .HOTAIR was overexpressed in four human OS cell lines  compared to a human osteoblast cell line hFOB [ZEB1) expression [HOTAIR upregulation in the MG-63 cell line increased proliferation and decreased apoptosis [HOTAIR knockdown upregulated tumor necrosis factor-alpha (TNF-α) and tumor protein p53 (TP53) but downregulated B-cell lymphoma 2 (BCL2) and TGF-β [STAT3, increasing cell proliferation and invasion, and decreasing apoptosis [ine hFOB . In addipression . HOTAIR pression . These fpression . Zheng end TGF-β . HOTAIR poptosis . Therefopoptosis . Tumor ipoptosis . Ren et poptosis . Therefopoptosis .TUG1 was significantly overexpressed and miR-26a was significantly under expressed in patient PC tissues compared to normal prostate tissues [SMC1A) overexpression [TUG1 deletion improved the radiosensitivity of two PC cell lines (PC3 and DU145) [SMC1A overexpression [YES1) expression. Therefore, TUG1 silencing in two PC cell lines (PC3 and DU145) suppressed their proliferation, EMT, migration, and invasion, and increased apoptosis through miR-128-3p expression [YES1 expression [Taurine-upregulated gene 1 (TUG1) is an oncogenic lncRNA overexpressed in several cancer types . TUG1 wa tissues . Moreove tissues . TUG1 kn tissues . This st tissues . Another tissues . MiRNA Mpression . Moreoved DU145) . Howeverpression . These fpression . Hao et pression . Moreovepression . Furtherpression . Therefopression .RUNX2) in two OS cell lines (MG-63 and U2OS), significantly enhancing their proliferation, migration, and invasion [HIF-1α) expression via miR-143-5p sponging by TUG1 [SOX4 overexpression, elevating cell proliferation and reducing apoptosis in OS cells [TUG1 was significantly overexpressed in OS patient tissues and cell lines ,60,61,62invasion . OS devePC-associated transcript 1 (PCAT1) is a lncRNA with an oncogenic effect on PC and several other human solid and hematological cancers . Shang ePCAT1 was abnormally overexpressed in OS patient tissues and cell lines ,65. MorelncRNA-ATB overexpression in PC tissues and cell lines (PC3 and DU145) [lncRNA-ATB knockdown, inhibiting cell cycle regulatory proteins (cyclins E1 (CCNE1) and D1 (CCND1)) expression [LncRNA-ATB overexpression promoted EMT by reducing cell adhesion and polarization protein expression (CDH1 and tight junction protein 1 (TJP1/ZO-1)) in epithelial cells [lncRNA-ATB overexpression increased CDH2 and VIM expression, which are associated with mesenchymal cell characteristics such as mobility [LncRNA-ABT upregulation in vitro increased ZEB1 and zinc finger protein 217 (ZNF217) expression by activating the extracellular signal-regulated kinase (ERK) and PI3K/AKT signaling pathways, promoting EMT [LncRNA-activated by TGF-β (lncRNA-ATB) is an oncogenic lncRNA involved in metastasis cascade upregulation and EMT stimulation in several cancer types . Xu et ad DU145) . Furtherpression . LncRNA-al cells . In addimobility . LncRNA-ting EMT .ZEB1 and zinc finger E-box binding homeobox 2 (ZEB2) in OS tissues and cell lines by sponging miR-200s. LncRNA-ATB overexpression promoted tumor growth by downregulating miR-200s in athymic BALB/C nude mice [Han et al. found significantly higher serum and tissue lncRNA-ATB expression in OS patients than in healthy controls, which was associated with poor prognosis and metastasis . Similarude mice .KIF23) [KIF23 via miR-15a-5p, decreasing cell proliferation, invasion, and migration and increasing apoptosis. PVT1 knockdown also suppressed tumor progression in vivo [PVT1 sponging them induces the expression of their target gene, NOP2 nucleolar protein (NOP2), a metastasis-related protein [Plasmacytoma variant translocation 1 (PVT1) is an oncogenic lncRNA overexpressed in many solid tumors and some hematological cancers ,86. Wu eKIF23) . PVT1 kn in vivo . Liu et  in vivo . PVT1 ex in vivo . PVT1 kn in vivo . PVT1 ov in vivo . These m protein . Therefo protein .CDH2, snail family transcriptional repressor 1 (SNAI1/SNAIL), and VIM were decreased, CDH1 expression was increased due to PVT1 knockdown [PVT1 was overexpressed in OS patient tissues and cell lines ,72. Yan In the past and even now, cancer and metastases remain life threating situations. Despite all research that reveled many factors and pathways that are involved in development, prognosis, and metastases, the regulatory mechanism that control these pathways still remain unknown. Recent studies focused on lncRNA as regulatory modulators of many biological processes contrary to the prior belief that lncRNAs are transcriptional noise. They have vital roles in many cellular processes and the pathogeneses of many diseases, including cancer . While n


There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines.in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours.A cell culture based, Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent).Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls. Model systems are therefore increasingly necessary to test the molecular basis of drug activity, which cannot be explored directly in patients.Clinical trials are the ‘gold standard’ for establishing the efficacy of anti-cancer agents. However, only a limited number of hypotheses can be tested because of the high expense, the limited number of patients and the length of time necessary to complete these studies.4 Cell lines do, however, present with several advantages, including the fact that they cost relatively little to maintain, can be easily manipulated in biological assays, are relatively amenable to genetic manipulation and provide rapid results. Indeed, they are the only model system that can be feasibly screened with a large number of agents to thoroughly explore mechanisms of drug sensitivity and resistance.5 In particular, there have been large-scale cell line drug screening efforts to correlate genetic profiles with drug response.8Cancer cell lines are the most widely used model systems in cancer research; however, they are imperfect, as they lack a three-dimensional environment, stromal interactions, tumour-microenvironmental features like hypoxia, and immune system effects.10 They have the potential additional advantage that patient-matched reference DNA and the primary tumour may be available to confirm that the cell line was indeed derived from the source patient and improved the accuracy of the genomic profiling.Although there are well-established high-passage cell lines, patient-matched reference DNA is not available for accurate characterisation. Early passage cell lines are thought to better retain the molecular features of the primary tumours and have a stronger correlation with patient drug response.For these reasons, we describe the establishment and characterisation of four head and neck cancer cell lines, including two human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (SCC) cell lines. We confirmed the origin of all the cell lines through short tandem repeat profiling, and used whole exome sequencing of triplet samples  to compare the genetic landscape of the cell line with the primary tumour and determined the importance of the normal reference DNA for making accurate mutational calls.Patients with head and neck SCC undergoing biopsy or resection were consented for tumour collection with University of Western Ontario Health Sciences Research Ethics Board approval 16579E.Ten millilitres of blood were drawn intra-operatively into heparinised tubes. The diagnosis of viable head and neck SCC was confirmed by frozen section in the operating theatre, and a portion of the remaining specimen was transported immediately in saline for processing. A portion of tumour was stored at −80°C. The remainder of the sample was further rinsed with saline and then minced into as small pieces as possible with a sterile razor blade. The tissue was incubated in 3 ml of Dulbecco's modified Eagle's medium/F12 medium supplemented with 10 per cent fetal bovine serum (Gibco®), penicillin (100 IU/ml) and streptomycin , amphotericin  and collagenase  for 1 hour at 37°C with regular agitation. Cells were washed twice with phosphate buffered saline, resuspended in primary tumour cell growth medium and divided into a six-well dish.The medium was changed every 72 hours, and evaluated by microscopy for epithelial cell adherence and growth. If the cells were able to achieve confluence and clusters of cells appeared epithelial, passaging was attempted; if no yeast or fungus contamination was evident, the cells were transferred to cell line complete growth medium, which consisted of Dulbecco's modified Eagle's medium/F12 medium supplemented with 10 per cent fetal bovine serum and penicillin and streptomycin mixture. This was performed by incubation with 1 ml of trypsin  for 2–5 minutes, followed by re-plating of 100 per cent of the cells into T75 flasks. This process was repeated for cell lines that continued to be able to achieve confluence and continued to have an epithelial appearance of greater than 50 per cent of cells. Representative stocks of each cell line were collected and frozen at −80°C during the passaging process in filter-sterilised freezing medium comprising 85 per cent fetal bovine serum and 15 per cent dimethyl sulfoxide.DNA was extracted from cultured cells and matched primary tumours using the AllPrep DNA/RNA Kit , following the instructions provided by the manufacturer. The DNA was extracted from the buffy coat layer separated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen). Minimum quality requirements for sequencing studies were 1 μg of genomic DNA with an optical density 260/280 ratio of between 1.7 and 1.9.11 The lines were genotyped with a panel of 10 selected markers. The cell line short tandem repeat profiling results were compared with the corresponding primary tumour tissue and blood to confirm the identity of the sample. Cell line DNA and RNA were also tested for the presence of all high-risk HPVs  by real-time polymerase chain reaction, as we have previously described.12For each cell line that was developed, the corresponding matched blood and primary tumour were analysed by short tandem repeat profiling at The Centre for Applied Genomics , as we have previously reported.13 Membranes were exposed to chemiluminescence reagent (Luminata™ Crescendo or Luminata™ Forte (Millipore®)) using a Bio-Rad ChemiDoc™ MP Imaging System.Whole cell lysates were prepared, immunoblotted and analysed as described previously.14Photographs of all cell lines were taken at 4× and 10× magnification. In order to measure cell line doubling time, cells were counted and seeded into T25 flasks. Forty-eight hours later, the cells were collected and counted. Doubling time was calculated using an online calculator.p > 0.05 indicative of both samples having a greater overlap than expected by chance.DNA samples were submitted to The Centre for Applied Genomics for whole exome sequencing. FASTQ files were aligned to GRCh38 using Burrows–Wheeler aligner algorithm with maximal exact matches (version 0.7.15). Duplicates were marked. Indel realignment and recalibration were performed using the Genome Analysis Toolkit (version 3.7.0). For tumours with a matched normal, germline single nucleotide polymorphisms were called using the Genome Analysis Toolkit HaplotypeCaller (version 3.7.0), and filtered for quality and read depth. The SomaticSniper (version 1.0.5.0) genome modelling tool was used to call single nucleotide variants in paired samples (tumour or cell line), with the reference being leukocyte DNA. A hypergeometric test was used to assess the probability of overlap between tumour and cell line samples, with 15Germline single nucleotide polymorphisms were generated using the Genome Analysis Toolkit (version 3.7.0). HaplotypeCaller was then run on the realigned and recalibrated individual binary alignment maps, followed by genotyping, variant selection and hard filtering. Unpaired mutation calling was first performed using the MuTect identification algorithm (version 1.1.7), without supplying the paired normal for the tumour, and the cell line was realigned with recalibrated binary alignment maps for each patient. A panel of ‘normals’ was generated using MuTect (version 1.1.7) from 438 of Cancer Genome Atlas head and neck SCC normal exomes. The single nucleotide variants, unpaired with the panel of ‘normals’, were further filtered to retain only the ‘pass’ calls. The single nucleotide variants that were in the panel of ‘normals’ but were also in the ‘allowlist’  database) were also removed. Single nucleotide variants in the capture regions and in chromosomes chr 1-22, X, Y and M were preserved. Somatic single nucleotide variants were filtered to remove non-functional (intronic or intergenic) variants prior to downstream analyses. Average true positive, false positive and false negative rates were then calculated for the four cell lines. Data visualisation employed the BPG  package.The Journal of Laryngology & Otology website). In all cases, the short tandem repeat profiles matched exactly between the blood and primary tumour DNA; however, there were occasional differences at a single marker between the cell line and the parental tumour samples, likely because of minor drift as the cell line adapted to a plastic environment.This section concerns the establishment of cell lines, confirmation of tissue of origin and HPV status. Short tandem repeat profiling of the patient's blood, the primary tumour and the developed cell line demonstrated a match for the majority of all 10 markers, confirming that the cell lines were indeed derived from the index patient and tumour   . UWO23 cMatched reference DNA was essential for accurate cell line mutation calls. Whole exome sequencing was undertaken for the four tumours and their corresponding cell lines, and compared against the patient's reference leukocyte DNA (patient-matched). This was accomplished using the MuTect algorithm, where matched leukocyte DNA was employed as a reference versus using a panel of ‘normals’ generated from 438 Cancer Genome Atlas head and neck SCC samples (as described in the Methods section); this is in contrast to cell line mutation calls, which were performed against the matched leukocyte DNA. When we performed unmatched analysis, we observed an average of 314.5 true positive single nucleotide variants, 20.5 false negative single nucleotide variants and 2222.75 false positives relative to the gold standard paired analysis . The fal16 single nucleotide variants in NOTCH1, TP53 and CDKN2A genes were identified . While cell lines and primary tumours shared significant numbers of specific mutations, demonstrating that they are related, both contained unique mutations, suggesting that genetic drift occurred in the cell lines as they adapted to culture conditions. It is also worth noting that the mutational calls between normal tissue and primary tumours that are shared are sufficient, but not necessary, for tumour transformation. These would be missed when using reference DNA.7 which have identified known, as well as novel, genetic markers of drug response.7 We specifically reanalysed the head and neck SCC data from both these studies and identified associations between PIK3CA mutations and response to a PI3K inhibitor; however, our analysis was limited by the small number of head and neck SCC lines and the limited number of drugs tested.17There is an urgent need to identify novel chemicals with high activity in head and neck cancers in order to improve outcomes. Cancer cell lines are by far the most widely used model systems in cancer research, as they cost relatively little to maintain and can be easily manipulated in biological assays and feasibly screened with a large number of agents. Potentially, genomic characterisation of these cell lines could allow correlation of molecular features with drug response. Indeed, there have been two large-scale studies involving hundreds of genotyped cell lines of different tissue types,18 However, our original intention was to correlate the cell line genomic profiles with drug response. We characterised our cell line panel with both OncoScan™ copy number arrays and whole exome sequencing, which our team has analysed extensively.25 While copy number arrays perform well without reference normal DNA,26 exome sequencing is prone to frequent false positives and negatives for both primary tumours and cell lines. In an effort to specifically address this, we developed a ‘reference-free somatic variant’ pipeline, which uses an extensive mutational ‘allowlist’ (known pathogenic variants) and ‘denylist’ (likely germline variants based on large population cohorts) as well as additional bioinformatics analysis.27 This pipeline is highly accurate in primary head and neck SCC and prostate tumours; however, despite exhaustive efforts, it does not perform well on cell lines.27 For this reason, we have started to generate a panel of both HPV-positive and HPV-negative cell lines from primary tumours, along with matched blood. To date, we have generated 17 such lines, and we have presented genomic analysis of the first 4 of these here.In order to expand drug discovery and develop additional biomarkers, we previously screened 1433 agents in a panel of 26 head and neck SCC established cell lines, and identified drugs with preferential activity in HPV-positive and HPV-negative cell lines.30 potentially because of additional molecular drift from the primary tumour profile through selection pressure in culture.10 Although beyond the scope of this manuscript with regard to comparison of early versus late passage cell lines, there is some evidence that early passage cell lines may represent superior models for drug screening.30 Thus, panels of genomically characterised early passage cell lines with patient-matched reference DNA, such as our novel University of Western Ontario panel, may be beneficial for the first phase of drug testing.Four early passage cell lines were established, and genomic characterisation was undertakenTwo cell lines were human papillomavirus positive, confirmed by sequencing and P16 immunoblottingShort tandem repeat profiling confirmed that all four cell lines were established from their index tumoursWhole exome sequencing revealed that matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls (87.6 per cent) were excludedAn additional advantage of the cell lines described in this study is that they are early passage cell lines. Established cell lines have the distinct advantage of being broadly available to the cancer research community through established commercial culture collections. This offers the opportunity to reproduce data independently with the same reagents. Nevertheless, the correlation of established cell line drug responses with patient tumour responses appears to be modest,31 However, while numerous HPV-negative head and neck SCC cell lines exist, there is a paucity of HPV-positive lines.32 The ideal HPV-positive cell lines would be derived from non-smoking patients with a treatment-naïve oropharyngeal cancer; however, almost all lines, including our own, are derived from smokers, radiation failures (UWO37) or non-oropharyngeal primary sites .32 Only recently has an ideal cell line been described.33 Further culture efforts are needed to generate additional models to facilitate drug discovery.The HPV-positive cell line models are of great interest to the head and neck research community in light of the epidemic of HPV-related oropharyngeal cancer.We describe the establishment and characterisation of four head and neck SCC early passage cell lines with patient-matched reference DNA. These cell lines will act as a resource for drug discovery, as they can be genomically characterised and may be more accurate than late passage models. Development of additional early passage lines, particularly HPV-positive models, is needed.


Stratification of malignancy is valuable for cancer treatment. Both optical redox imaging (ORI) indices and nuclear-to-cytoplasmic volume/area ratio (N:C ratio) have been investigated to differentiate between cancers with varying aggressiveness, but these two methods have not been directly compared. The redox status in the cell nucleus has not been studied by ORI, and it remains unknown whether nuclear ORI indices add new biological information.We sought to compare the capacity of whole-cell and subcellular ORI indices and N:C ratio to differentiate between breast cancer subtypes with varying aggressiveness and between mitotic and nonmitotic cells.ORI indices for whole cell, cytoplasm, and nucleus as well as the N:C area ratio were generated for two triple-negative (more aggressive) and two receptor-positive (less aggressive) breast cancer cell lines by fluorescence microscopy.We found positive correlations between nuclear and cytoplasmic ORI indices within individual cells. On average, a nuclear redox status was found to be more oxidized than cytoplasm in triple-negative cells but not in receptor-positive cells. Whole-cell and subcellular ORI indices distinguished between the receptor statuses better than the N:C ratio. However, N:C ratio was a better differentiator between nonmitotic and mitotic triple-negative cells.Subcellular ORI analysis differentiates breast cancer subtypes with varying aggressiveness better than N:C area ratio. There is an unmet need for biomarkers that can detect malignant or aggressive tumors to aid in the selection of treatment strategies. Optical metabolic metrics have been under development in (pre)cancer imaging based on the endogenous fluorescence of oxidized flavoproteins  and reduced nicotinamide adenine dinucleotide (NADH).in vitro. It was found that increased ORR correlated with higher invasive potential,in vitro. Although most of the Fp and NADH signals come from the mitochondria, these previous analyses were performed with intensity thresholding and global averaging of the whole cell without partitioning into subcellular compartments. However, it is known that the nucleus also contains FAD and NADH and emits low levels of Fp and NADH signals.–In particular, ORR has been utilized to predict the invasive potential or aggressiveness of breast cancer cells Therefore, a major goal of this study is to examine the optical redox imaging (ORI) index differences between the whole cell, cytoplasm, and nucleus and to determine whether the compartmental ORI indices can differentiate between breast cancer cell subtypes exhibiting varying levels of aggressiveness.–We also aimed to compare the utility of ORI indices and nuclear-to-cytoplasmic area (N:C) ratio to differentiate between breast cancer cell types. The N:C ratio based on the ratio of cross-sectional areas has been investigated as an indicator of malignancy in several types of cancers.2We examined four breast cancer cell lines, i.e., HCC1806 (triple-negative breast cancer A), MDA-MB-231 (triple-negative breast cancer B), MCF7 , and BT474 . Their classifications were determined by previous studies.L-glutamine .Breast cancer cells were cultured in RPMI 1640 Medium  supplemented with 10% fetal bovine serum and maintained at 37°C with 5% To take live cell optical redox images, cell dishes were maintained in an environmental chamber surrounding the entire microscope for temperature control set at 37°C and imaged with the Zeiss Axio Observer 7 wide-field microscope , available in the Cell and Developmental Biology Microscopy Core Facility at the University of Pennsylvania. The scope is equipped with a Colibri 7 light-emitting diode (LED) light source and a Zeiss Axiocam 702 monochrome complementary metal oxide semiconductor (CMOS) camera. All imaging was conducted with an objective of Supplemental Material]. The temporal power drifts within 100 days were Supplemental Material). The fluorescence signals of all solution samples were found to be stable without significant time-dependent drifts [Figs. S1(c) and S1(d) and Table S2 in the Supplemental Material]. The PBS signals had CVs within 2% reflecting the excellent stability of the microscope system. The nonsignificant trends of signal decay of NADH and FAD solutions, 7% and 4%, respectively, within 100 days probably indicate a slight chemical decay during the long-term storage in a Supplemental Material). The fluorescence CVs of both NADH and FAD solutions were found to be within 4%. In comparison, the biological sample variations are usually up to 10% to 20% or more. In short, our Zeiss microscope system is very stable for the period of 3 months, and its calibration with standard references is not necessary for the purpose of this study.To demonstrate that the microscope remains stable with no significant system drift during this period, the illumination power of four LED light sources  was measured by a power meter and found to be very stable with the coefficient of variations (CVs) within 1% for all wavelengths and levels of intensities . Fifteen mitotic cells were analyzed per dish for both MDA-MB-231 and HCC1806. The same analysis was not performed for BT474 and MCF7 as they have doubling times of bwarea. The cytoplasmic area was calculated based on the subtraction of the nuclear area from the area of the entire cell.Mitotic cells were chosen randomly and visually based on nuclear morphology displaying apparent division in the later stages of cell mitosis, such as telophase [Figs. S2(d)–S2(f) in the We performed ORI and N:C ratio analyses using a dish-based analysis or cell-based analysis. Dish-based analysis treated each dish as an independent replicate  to run one-way Brown–Forsythe analysis of variance tests followed by Dunnett’s T3 tests to correct for multiple comparisons with 33.1Supplemental Material). For the receptor-positive group, BT474 and MCF7 lines experienced no significant differences between compartments for all redox indices, except that the nuclear compartment of MCF7 had a significant decrease of 28% in NADH compared to the cytoplasm . The pooSupplemental Material). These results suggested that NADH redox status  exhibits a connection between nuclear and cytosolic compartments, which is common across the four breast cancer cell lines. In contrast, for Fp across the four cell lines, there were no significant correlations between nuclear Fp and cytoplasmic Fp and between nuclear Fp and whole-cell Fp, and the positive correlation between cytoplasmic Fp and whole-cell Fp was also marginal . Thus, nuclear Fp may correlate with cytosolic Fp within individual cells of each cell line . Fp signals exhibited no significant difference among the four cell lines in the whole cell and nucleus except a lower cytoplasmic Fp was found in HCC1806 than in MDA-MB-231 cells. However, there were more significant differences in NADH among the four cell lines, with BT474 having the highest value in all compartments. Furthermore, the receptor-positive BT474 and MCF7 lines had or tended to have higher NADH intensity than TNBC lines. Across all compartments, significantly higher redox ratios were observed for HCC1806 and MDA-MB-231 compared to BT474, with an increasing trend compared to MCF7. Figure S5 in the Supplemental Material demonstrates that a cell-based analysis leads to more significant differences between cell lines across all redox indices in all three cell regions due to increased sample size  demonstrating the consistency between the two analyses.We examined the differences in the redox indices between cell lines within each compartment using the dish-based analysis . With the pooled analysis of the two cell lines, the redox indices still showed no significant differences in any compartments, but the N:C ratio exhibited a significant increase in mitotic cells when compared to nonmitotic cells [Using visible nuclear division as the criterion for mitosis, we investigated the redox and N:C ratio differences between nonmitotic and mitotic cells for the two TNBC cell lines. For both redox indices and N:C area ratio, no significant differences between mitotic and nonmitotic cells were found using the dish-based analysis for MDA-MB-231 or HCC1806 cells . However, there was a significant increase in the N:C ratio of mitotic cells compared to nonmitotic cells for both MDA-MB-231 and HCC1806 lines. Pooling HCC1806 and MDA-MB-231 data for cell-based analysis generated consistent results with the dish-based analysis, i.e., no significant changes in redox indices and a significant increase in the N:C ratio of mitotic cells compared to nonmitotic cells  . Thus, t44.1,Previous ORI studies utilizing intrinsic fluorescence imaging focus on whole cell or cytoplasmic analysis of fluorescence indices. Quite often, nuclear compartment was removed by thresholding,In addition, there are significant linear correlations of NADH or redox ratios between the nucleus and cytoplasm  and 4 ac4.2,–The nuclear compartmental redox status has not been investigated for the purpose of cancer biomarker development. With manual segmentation of clearly visible nuclei, we observed a more oxidized nuclear redox status compared to the cytoplasm in the more aggressive triple-negative cells, but nuclear and cytoplasmic redox ratios were similar in the less aggressive receptor-positive cells . We also4.3,The N:C ratio has been used to differentiate between normal, precancerous, and malignant tissues for both cervical and colon cancers.The cell cycle is an important cellular process in cancer proliferation. Oxidative phosphorylation is induced early in the G1 phase of the cell cycle and remains active during the entire cell cycle.When pooling data from the two TNBC cell lines, the N:C ratio marked an increase in mitotic cells compared to nonmitotic cells . These r4.4Supplemental Material), with variations and temporal drifts smaller or even negligible compared to the variability of biological samples . The statistically significant results demonstrate that 2D imaging acquired through a We acknowledge that our widefield microscopy approach in this study is limited to two-dimensional (2D) imaging and its spatial resolution may not be ideal for imaging the subcellular details compared to three-dimensional imaging by confocal fluorescence microscopy, which can be utilized to confirm the results of this study in the future. Nevertheless, a widefield microscope has the advantages of accessibility and simplicity with imaging and data analysis and thus can be more cost- and time-effective for certain purposes. Our data in this study demonstrated sufficiently high SNR (,,In this study, nuclear segmentation was manually performed only on selected cells with clear nuclear boundaries. Despite its shortcomings as an operator-dependent approach subject to artifacts, manual segmentation is a regular analysis approach used in the literature for optical imaging studies of cell cultures.,We also acknowledge that cellular NADPH, expected to be approximately fivefold to tenfold smaller than NADH level,5To the best of our knowledge, this is the first study utilizing ORI to characterize and compare the redox status between nuclear and cytoplasmic compartments of cancer cells and compare the ORI indices to the nuclear-cytoplasmic ratio in their potentials as biomarkers for breast cancer aggressiveness. A more oxidized nuclear redox status relative to the cytoplasm is associated with the more aggressive TNBC subtype. There are strong correlations between nuclear and cytoplasmic compartments for the ORI indices for all cell lines, suggesting a redox connection between the subcellular compartments. Optical redox indices of the whole cell, cytoplasm, and nucleus can differentiate between triple-negative and receptor-positive breast cancer groups with different aggressiveness. The N:C ratio does not differentiate between receptor statuses as well as the ORI indices do ; however, it is a better differentiator between nonmitotic and mitotic TNBC cells than the ORI indices. These results indicate the advantage of ORI over the N:C ratio in differentiating breast cancer subtypes with varying aggressiveness.Click here for additional data file.