The detection of volatile organic compounds (VOCs) exhaled by human body fluids is a recent and promising method to reveal tumor formations. In this feasibility study, a patented device, based on nanostructured chemoresistive gas sensors, was employed to explore the gaseous exhalations of tumoral, immortalized, and healthy cell lines, with the aim of distinguishing their VOC patterns. The analysis of the device output to the cell VOCs, emanated at different incubation times and initial plating concentrations, was performed to evaluate the device suitability to identify the cell types and to monitor their growth. The sensors ST25 (based on tin and titanium oxides), STN , and TiTaV  used here, gave progressively increasing responses upon the cell density increase and incubation time; the sensor W11 (based on tungsten oxide) gave instead unreliable responses to all cell lines. All sensors (except for W11) gave large and consistent responses to RKO and HEK293 cells, while they were less responsive to CHO, A549, and CACO-2 ones. The encouraging results presented here, although preliminary, foresee the development of sensor arrays capable of identifying tumor presence and its type. Tumor detection and monitoring are fundamental topics in medicine worldwide. According to American Cancer Society statistics, one out of three people will be diagnosed with cancer in their lifetime ,2. For tTumor gaseous biomarkers are volatile organic compounds (VOCs) produced by cancer cells mainly as a result of cell metabolic process alterations  and cell2 molecules that are quickly released into the atmosphere , entirely designed, produced, and assembled in the “Laboratorio Sensori” of the University of Ferrara, Italy. The working principle of these sensors is based on the chemoresistivity, i.e., the capability of varying the film conductivity as a consequence of the chemical reactions occurring between the surface of the grains and the gaseous molecules of the surrounding atmosphere. The chemoresistivity process requires the thermal activation of the sensor that consists in maximizing the number of electrons (in conduction band) able to overcome the potential barrier  in water or alcohol solution , during which several changes in the gel structure and properties occur as polycondensation, lead to an increase in thickness of the colloidal particles and to the reduction of their porosity.The last step is the drying and calcination: the GEL is dried first (at about 100 °C), and then calcinated at higher temperatures (at about 400–800 °C) to completely remove the residuals of solvents and of other chemical additives. In this process, the temperature and the relative humidity greatly influence the quality of the final MOX powder.To optimize the performances of a chemoresistive MOX gas sensor, it is primarily necessary to increase the surface–volume ratio of its sensitive porous material as much as possible, to maximize its interaction with the surrounding gases. With this aim, the sol-gel chemical process was employed to synthesize networks of MOX nanometric spherical grains, precisely interconnected ,43,44,45The MOX nanopowder, synthesized through the sol–gel technique ,42,43, wST25, based on tin oxides and titanium (25%) and gold nanoparticles (1%);W11, based on tungsten oxide;STN, based on tin, titanium, and niobium oxides;TiTaV, based on titanium, tantalum and vanadium oxides.The four MOX sensors hosted in SCENT B1, listed below, were selected on the basis of previous laboratory tests carried out on pure standard gases, on VOCs exhaled by blood, feces, biopsies, and preliminary tests on cultured cells ,32,33,34All sensors were thermally treated with a firing temperature of 650 °C and were heated to the working temperature of 450 °C by the platinum coil ,32,33,34uctivity ,32. The In order to assess the reliability of the sensor responses on the cell samples presented in the following, four different STN sensors were tested, as an example, in both wet , but the human skin fibroblasts were:A549, explant culture of lung carcinomatous tissue;CACO-2, human colorectal adenocarcinoma cell line;CHO, epithelial cell line derived from the ovary of the Chinese hamster;HEK293, specific immortalized cell line originally derived from human embryonic kidney;RKO, human colorectal cancer cells.The cell types employed here, purchased from the American Type Culture Collection  taken out from the incubator were quickly cooled to room temperature on a copper plate. Room temperature was monitored and kept approximately constant at 22.0 ± 1.5 °C.4, 2.5 × 105, 5 × 105, 1 × 106 cells per dish (3.5 cm of diameter); all dishes were kept in the same incubator at 37 °C in a humidified atmosphere containing 5% of CO2.To maximize the sample exhaling surface, in order to improve the signal-to-noise ratio, three petri dishes were simultaneously measured by placing them in the sample chamber stacked in an holding tray . Each roSince the cells were kept and grown in media rich in organic compounds, it was necessary to measure 2O at 37 °C  upon the cell density increase and withdrawal time from incubator .However, all sensors poorly discriminated among the 80, 250, and 500 K cells/dish plated at 24 h, except for the 1 M one. To check that the responses to the first three cell concentrations were small, they were averaged  and are shown in Sensor responses to CHO cells, an epithelial line derived from the ovary of the Chinese hamster, and A549 cells, an explant culture of lung carcinomatous tissue, were negligible after 24 h of plating, regardless of the plating concentration. In particular, the A549 responses were smaller than one, i.e., they were smaller than the sensor response to culture medium only. Both these cell types grew quite fast, and at a concentration of 500 K, cells/dish were already confluent after 48 h of plating , insert;Lastly, the cell lines CACO-2 and human skin fibroblasts gave negligible responses . This waIn summary, it is possible that the VOCs emitted by CHO, A549, and CACO-2 cell lines, although they are expected to be quite sustained since these cells are fast growing and therefore have a high metabolism, are poorly detected by the sensors. If this is the case, it may become advantageous, since the sensors here employed, giving different response patterns with different cancer cells, could be used in the future not only to detect them (hopefully to an early stage), but also to identify their type. To further explore this idea, as shown in The same sensor gave different response amplitudes to different cell lines a, or difThe sensors ST25, STN, and TiTaV used in this work gave progressively increasing responses upon increasing the cell number, no matter if it was attained by the initial seeding concentration or by the cell growth. The sensor W11 gave instead unreliable responses to all the cell lines here employed, and therefore, they were discarded in the final analysis. The sensors gave large responses to the RKO and HEK-293 cell lines, while they were progressively less responsive to the CHO, A549, and CACO-2 cell lines. This suggests that a particular cell type could be identified by properly combining the response to its VOCs via a different sensor, allowing for the future development of sensor sets that are able to identify the presence of a tumor and even its type. The novelty of this approach, in respect to the current methods in development for tumor identification, is the employment of a portable, compact, and fast (the outcome is attained within 10–15 min) device, not requiring expensive consumables or complex and expensive analysis of the samples (that may require days), and that can be operated by untrained people.Malagù, C.; Gherardi, S.; Zonta, G.; Landini, N.; Giberti, A.; Fabbri, B.; Gaiardo, A.; Anania, G.; Rispoli, G.; Scagliarini, L. Combinazione di materiali semiconduttori nanoparticolati per uso nel distinguere cellule normali da cellule tumorali (2015), National #:102015000057717 .


Understanding the impact of spatial heterogeneities that are known to occur in large‐scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0–7.5 and DOT 10%–30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale‐up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large‐scale bioreactors. Small‐scale bioreactor models allow for time and cost‐efficient development and optimization of cell culture processes. During initial cell line selection, miniature bioreactors within the microliter and milliliter scales are used for high‐throughput experimentation to reduce development time and costs.3 vessel and Lara et al3 stirred tank reactor (STR). Others have shown that in vessels up to 8000 L poor mixing can lead to high pH excursions after base addition.In the case of microbial fermentations, a number of publications have described the effects of heterogeneous conditions caused by poor mixing.Despite the few examples of large‐scale bioreactor studies in literature, detailed and reliable data is still missing.A number of scale‐down approaches have been developed to study the effects of heterogeneities on mammalian cell culture experimentally. In cell culture the addition of base, typically sodium hydroxide or sodium bicarbonate, is used for pH control and these additions can give rise to regions of high pH around the “addition zone.”Saccharomyces cerevisiae fermentation in a 300 m3 STR. Others suggest this to be higher, around 1%Several groups have developed scale‐down compartment models to investigate heterogeneity effects.Traditional compartment models using STRs and PFRs can be complex to set up and are not amenable to modern high throughput experimentation. This study presents a new approach to creation of a single‐compartment model using the ambr®15 automated miniature bioreactor system. The aim is to mimic the conditions within a small zone of a large‐scale vessel where the cells would be exposed to heterogeneities. The utility of this approach is that it will enable rapid identification of cell lines which might be vulnerable to fluctuations in pH and/or DOT. This study describes the development of a fluctuation methodology in ambr®15 bioreactors and initial evaluation of a number of cell lines with different growth characteristics. The impact of pH and DOT fluctuations on growth and productivity as well as product quality was assessed. The data obtained from these scaled‐down studies could be fed into computational population balance models which could then predict overall behavior of the cell lines at large‐scale as has been demonstrated with microbial fermentations.22.11 MAbs. Prior to bioreactor inoculation a vial of the cell line stock was thawed and cultured aseptically in a 250 ml shake flask in a shaken incubator  at 140 rpm, 5% vol/vol CO2, and 36.5°C. The culture was passaged every 3–4 days until used for bioreactor inoculation.Four recombinant GS‐CHO cell lines were used in this study: AZCL_1, AZCL_3, AZCL_4, and AZCL_5 from AstraZeneca. All cell lines were developed in proprietary media and produce monoclonal antibodies (MAbs). AZCL_1 produces a bispecific fab fragment fusion antibody and the other cell lines produce different IgG2.26 cells ml−1 in an initial volume of 13.5 ml. The temperature of each vessel was controlled at 35.5 ± 0.5°C. The pH was controlled to a set point of 7.0 ± 0.1 using CO2 (max flowrate 1.23 ml min−1) and 1 M sodium bicarbonate. DOT was controlled at 50% of air saturation using oxygen. These set points were used for all experiments unless otherwise stated, that is, when conducting cultures with fluctuating pH or DOT conditions as discussed later. Pure nitrogen was sparged at a constant rate of 0.15 or 1.00 ml min−1. A solution of 0.5% vol/vol Antifoam C  was added to each vessel at an average rate of 20 μl per day. Proprietary media and feeds were used. Nutrient feeds were added periodically and glucose was maintained with daily additions of 500 g L−1 glucose stock solution.The ambr®15 system  is widely used in industry as a tool for high‐throughput process development and is described in detail elsewhere.2.32 and pCO2 measurements were also taken every 2 days using an ABL90 Flex Plus  blood gas analyzer. Beginning on day 2, samples were also taken for antibody titre measurements. Supernatant was analyzed using the Octet  which uses a protein A‐based quantification method.Daily measurements of metabolite concentrations (glucose and lactate) were made using a YSI 2900 analyzer . All other measurements were made every 2 days. Viable cell density and viability measurements were made using a ViCell XR  which uses the trypan blue dye exclusion method. Offline pH, pO2.4Harvested cell culture broth (cultures end on day 14) was centrifuged and then filtered through a 0.22 μm SteriFlip vacuum filter  before being purified using a TECAN automated liquid handling system  equipped with RoboColumns containing MabSelect SuRe Protein A resin (Sigma‐Aldrich). Antibody purity and aggregation was determined using a standard HPLC‐SEC method using an Agilent 1200/1100 HPLC system  with a TSKgel column (Sigma‐Aldrich). Protein fragmentation was determined using a standard capillary electrophoresis (CE‐SDS) method with a Beckman PA800 Plus instrument (Beckman Coulter). Glycan profiles were obtained using a semi‐quantitative method of reversed phase HPLC (RP‐HPLC) combined with quadruple time‐of‐flight (QTOF) mass spectrometry (MS). Samples were reduced with dithiothreitol (DTT) and glycans released using N‐Glycosidase F . Samples were injected onto an Acquity UPLC BEH300 column , from which the eluate was directly fed into the SYNAPT mass spectrometer (Waters UK) with electrospray ionization (ESI). The mass to charge raw data was deconvoluted to a relative mass; the largest peak in each chromatogram is always the G0F glycoform and all other glycoforms are inferred based on their mass relative to the G0F peak.2.5Gluc), lactate production (qLac) and protein production (qp) rates were determined by linear regression of cumulative production (or consumption) against the cumulative integral of viable cell density (cIVC), using the following equations:IVCt is the cumulative integral of viable density at time t (cells‐day ml−1), VCD is the viable cell density (cells ml−1), and t is time (days). The maximum specific growth rate was determined from the highest specific growth rate of the culture calculated between measured time points using the equation below:Average cell‐specific glucose consumption . Conditions yielding 33.1Two‐compartment scale‐down models used to study large‐scale bioreactor heterogeneities have complex setups and required liter quantities of media making them time consuming and expensive to implement. The current work addresses this problem by using an automated miniature bioreactor platform to mimic just a portion of a larger scale vessel, representing the heterogeneous environment that a small volume of cells is exposed to. The simulation of heterogeneity is achieved by introducing programmed pH and DOT fluctuations in a well‐mixed miniature bioreactor. The pH and DOT set points are changed at fixed regular intervals and durations, similar to the approach taken by Osman et al.Initial work focused on establishing a robust experimental methodology for creating fluctuations in the ambr®15. Figure 2 or O2 stripping and therefore drop pH or DOT more rapidly. Fluctuations were mainly driven by manipulating the controller settings; increasing both proportional (P) and integral (I) components to induce a more aggressive response from the controller, hence creating the fluctuations. Due to the nature of PI control in the ambr®15, the pH and DOT signals exhibited over and under‐shoot as can be seen in Figure The method for achieving fluctuations in the ambr®15 was transferred from preliminary studies using another miniature bioreactor system; the micro24®  (data not shown).Figure 2 flow into the bioreactor, allowing the nitrogen to strip the CO2 and adding bicarbonate base to raise the pH. Longer durations give the system more time to raise the pH. This difference between the programmed and actual range is likely due to the nature of PI controls of the ambr®15, the buffering capacity of the cell culture media and lactate accumulation within the medium.The amplitude of the fluctuations achieved at the three frequencies were lower than programmed and many cultures struggled to reach pH 7.5. Table 2 was being sparged into the system. This effect is more dramatic in AZCL_3 cultures and increases with fluctuations of longer duration  the pH fluctuations tapered off and the pH remained at the lower  set point for several hours or days before the fluctuations restarted. During this period no COn Figure . This is2 flow rate; the oxygen consumption rate of the cells caused the DOT to drop even quicker. As a result, the response is much quicker than with pH and hence better fluctuations are achieved. AZCL_3 cultures grew to higher densities and therefore exhibited more noise in their DOT profiles and the fluctuation amplitudes were higher due to PI controller overshoot. In general, the pH and DOT fluctuations achieved were stable. Specific control of DOT was easier to achieve than pH. This initial work demonstrates that it is possible to programme and maintain fluctuations in the ambr®15 bioreactors throughout the course of a CHO fed‐batch culture.In contrast, stable DOT fluctuations were achieved with all cell lines across all frequencies. This is because the DOT control system does not rely on addition of liquid and is not limited by the buffering capacity of the media, which inherently makes it more difficult to maintain a stable pH set point. The DOT fluctuations were driven by the manipulation of the O3.26 cells ml−1) and the other with a “high” peak VCD (~60 x 106 cells ml−1). This section describes the investigation into the “low” growing cell line (AZCL_1).Preliminary studies using the micro24® bioreactor indicated that the effects of fluctuations are likely to be cell line dependent.−1) as encountered in the cited works. For this reason, the pH fluctuations reported here were induced by changing controller set points and PI settings, reducing excessive base addition. The ambr®15 automatically added base if the pH fell below 6.9. In the case of AZCL_1 lactate build‐up during the culture did not exceed the buffering capacity of the medium hence no additional base was used. However, without base addition, the amplitude of the pH fluctuations was limited.Previous studies investigating the effects of pH heterogeneity have typically introduced fluctuations by adding base either on a fixed daily basis2 purge gas flow rate and PI settings. Controls 1 and 2 have the “new” set up; the former using all the new conditions, that is, higher N2 flowrate and PI values and the latter keeps the original PI values but has a higher N2 flow rate. All vessels were cultured with the standard (lower) PI and N2 flowrates until day 2, at which point fluctuations were initiated.The pH and DOT fluctuations achieved experimentally are shown in the left column of Figure 6 cells ml−1. Control 3 appears to grow 22% less than the other two controls with higher gas flowrate, peaking at only 15 x 106 cells ml−1. This is also reflected in the lower lactate accumulation, suggesting that the lower N2 flowrate may be the cause of lower growth and lactate production (qlac) in AZCL_1 cultures. Interestingly however, the titre was not affected in the same way, meaning the qp of Control 3 was higher than the two other controls  below 2 gl−1 compared to the pH and DOT fluctuated vessels that peaked around 2.3 gl−1. However, this difference was found not to be statistically significant. All cultures experience a metabolic shift into lactate consumption around day 10. A slight difference in viable cell density between the 15, 30, and 60 min pH fluctuated cultures was observed; the cultures with longer fluctuation frequencies (60 min) had lower growth, however this difference was also not significant (p > 0.05). This difference is not seen in cultures with DOT fluctuation.Comparing across the rows in Figure gluc) was observed. Fluctuated vessels did, however, have higher initial lactate production (qlac), particularly in pH‐fluctuated vessels which had lactate production rates twice as high as controls between day 0 and 6. Fluctuations appeared to also slightly increase the growth rate (μmax), as seen in Table Looking at the derived culture parameters in Table 3.3Higher growing cell lines require better bioreactor control as the high‐cell densities achieved can lead to high‐oxygen uptake rates and increased lactate accumulation. For this reason, high‐cell density cultures performed at large scale could be more susceptible to spatial gradients and heterogeneities.6 cells ml−1, which again match the growth profiles seen in previous 5 L cultures. The 60 min pH fluctuations did appeared to have slightly lower growth and product titre on average, compared to the other fluctuation frequencies. However, the 15 and 30 min pH‐fluctuated vessels had higher peak product titre (5.5 gL−1) compared to the control vessels (4.9 gL−1). This was a slightly surprising result although these differences were not statistically significant.As with the low‐growing cell line (AZCL_1), pH fluctuations are seen to have little effect on growth, productivity or lactate levels in AZCL_3 cultures. The control and pH‐fluctuated vessels had very similar growth profiles peaking around 62 × 10−1 on day 6 when the metabolic shift occurred and lactate consumption began. Lactate increased toward the end of the culture as the cells died. Control 2 had a slightly different profile to the other controls, particularly with regard to lactate. This control was set at 30% DOT to test whether the effects of fluctuating DOT between 10% and 30% were not simply due to the overall lower DOT (compared to 50% DOT). Control 2 had a higher initial lactate production, qlac, and the culture switched to consumption on day 5; but had a much lower consumption rate than the other controls . The specific productivity, qp, was not affected however (Table lac [day 0−6]) than controls, as seen in Table gluc, was also affected by DOT fluctuations; all DO‐fluctuated vessels exhibited higher qgluc than controls, as seen in Table In contrast, cultures with fluctuating DOT conditions demonstrated significant differences to control cultures in the case of the “high” growing AZCL_3 cell line. DOT‐fluctuated cultures showed a 35% decrease in growth compared to controls. This decrease impacts the productivity by the same degree; DOT‐fluctuated cultures had peak antibody titres between 2.5 and 3.1 gler Table . The DOTA previous study has found that fluctuating DOT in a hybridoma cell line caused higher lactate yield.3.4All cultures shown in Figures 3.4.1p = 0.004). There was no fragmentation observed in AZCL_3 cultures. The AZCL_1 cell line produces a bispecific fusion product, so upon reduction fragmentation is expected, although there were no significant differences between fluctuated vessels and controls. However, in both cell lines, an increase in LMW species before reduction was observed in DOT‐fluctuated vessels compared to controls. Upon reduction a similar increase was observed in the levels of the light chain (LC) fragment, suggesting that DOT‐fluctuated cultures over‐produced free‐LC. As well as the increased level of LC these cultures also exhibited a decreased level of heavy chain (HC) species.As discussed above, pH fluctuations had little impact on the growth or productivity of both cell lines and this was also seen in product quality data. With regard to product aggregation and fragmentation, there appeared to be more variation between triplicate vessels with fluctuated conditions however pH fluctuations did not show significant differences in either AZCL_1 or AZCL_3 cultures.3.4.2Glycan analysis was focused on AZCL_3 cultures due to the observed impact of fluctuations on growth and productivity. The glycan profiles of all the cell lines used in this study are dominated by the bi‐antennary glycan with no terminal galactose (G0F), which makes up >68% of the overall profile. The next largest peaks are typically the galactosylated glycans G1F and G2F, which have been reported grouped and make up between 15% and 30% of the profile. High‐mannose structures (Man5) are another key glycan that is typically monitored and the cell lines had <5% of these species present.Figure While DOT fluctuations had a pronounced impact on cell growth Figure , they ha3.52 flowrate was set to 1.00 ml min−1 in AZCL_5 cultures and 0.15 ml min−1 in AZCL_4 cultures to assess if DOT fluctuations could be achieved with standard flow rates. Stable DOT fluctuations were achieved with both N2 flowrates. AZCL_5 and AZCL_4 cultures peaked at around 35 × 106 cells ml−1 and 40 × 106 cells ml−1, respectively, so they are not as low growing as AZCL_1 but not as high as AZCL_3. In both additional cell lines DO‐fluctuated cultures had lower peak VCD values and higher lactate accumulation compared to controls, but these differences were less substantial than ones observed in AZCL_3 cultures. There was no discernible difference between the negative control and DOT‐fluctuated vessels, suggesting the effects may be due to the overall lower DOT. The results with these additional cell lines support the hypothesis that higher growing cell lines are more susceptible to DOT fluctuations (or shifts in DOT set points). The effects also appear to be highly cell line specific, which is in agreement with the mixed results seen in other published studies.Two additional cell lines (AZCL_4 and AZCL_5) were used to further explore the effects of DOT fluctuations. Similar experiments were set up with AZCL_5 and AZCL_4 where DOT was fluctuated between 10% and 30% in the same manner. A positive (30% DOT) and negative (10% DOT) control were used with the higher PI settings, as described in Table Figure 4A novel methodology using an automated miniature bioreactor system to investigate the effects of pH and DOT fluctuations on CHO cell culture performance is presented. This provides a high throughput platform for assessing the potential impact of large‐scale bioreactor heterogeneities on different cell lines and antibody products. In the two different cell lines used to exemplify the utility of the platform, fluctuations in pH had minimal impact on growth and productivity. An increase in lactate production was observed which matched observations from similar literature studies.Overall, this work suggests that the impact of pH and DOT fluctuations are cell line and product specific and are more likely to affect higher growing cell lines. The automated miniature bioreactor therefore provides a platform for rapid investigation of potential large‐scale culture heterogeneities on candidate cell lines and antibody products. Data from fluctuating and non‐fluctuating bioreactor studies is currently being used to try and predict overall culture performance at large scale using population balance approaches.Roman Zakrzewski: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); writing – original draft (lead); writing ‐ review and editing . Gary J. Lye: Conceptualization (supporting); supervision (supporting); writing – review and editing . Kenneth Lee: Conceptualization (supporting); data curation (supporting); supervision (supporting); writing – review and editing (supporting).https://publons.com/publon/10.1002/btpr.3264.The peer review history for this article is available at


Currently there is no effective treatment for UM. Multi-kinase inhibitors targeting dysregulated pro-tumorigenic signalling pathways have revolutionised anti-cancer treatment but, as yet, their efficacy in UM has not been established. Here, we identified the multi-kinase inhibitor afatinib as a highly effective agent that exerts anti-UM effects in in vitroWe assessed the anti-cancer effects of afatinib using cell viability, cell death and cell cycle assays in in vitro and ex vivo UM models. The signaling pathways involved in the anti-UM effects of afatinib were evaluated by Western blotting. The in vivo activity of afatinib was evaluated in UM xenograft models using tumour mass measurement, PET scan, immunohistochemical staining and TUNEL assays.We found that afatinib reduced cell viability and activated apoptosis and cell cycle arrest in multiple established UM cell lines and in patient tumour-derived primary cell lines. Afatinib impaired cell migration and enhanced reproductive death in these UM cell models. Afatinib-induced cell death was accompanied by activation of STAT1 expression and downregulation of Bcl-xL and cyclin D1 expression, which control cell survival and cell cycle progression. Afatinib attenuated HER2-AKT/ERK/PI3K signalling in UM cell lines. Consistent with these observations, we found that afatinib suppressed tumour growth in UM xenografted mice.Our data indicate that afatinib activates UM cell death and targets the HER2-mediated cascade, which modulates STAT1-Bcl-xL/cyclin D1 signalling. Thus, targeting HER2 with agents like afatinib may be a novel therapeutic strategy to treat UM and to prevent metastasis.The online version contains supplementary material available at 10.1007/s13402-022-00686-5. Uveal melanoma (UM) accounts for ~85% of ocular melanomas and 3% of all melanomas in humans. The National Organization for Rare Diseases estimates that the incidence of UM is ~5–7 per million in the general population , 2 AlthoCurrently, front-line treatment for UM includes radiotherapy, phototherapy and surgery, but vision impairment, blindness or eye removal are common clinical consequences following these treatments . BecauseMost UM tumours exhibit mutations in genes encoding the G protein-alpha subunits GNAQ or GNA11 that activate the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)/Akt signalling pathways . InitialThere are four isoforms in the ErbB lineage of proteins (ErbB1–4) that function as homo- and heterodimers . The dim, ex vivo and in vivo models. Thus, afatinib emerges as a new candidate for clinical evaluation in UM patients.HER2 is expressed in UM tumour cells . In the Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI-1640), Fetal Bovine Serum (FBS), Insulin-Transferrin-Selenium (ITS), Penicillin-Streptomycin (P/S) and L-Glutamine were purchased from Thermo Scientific . Giant cell tumour (GCT) conditioned medium was obtained from United Biosciences . Dimethyl sulfoxide (DMSO), L-glutamic acid monosodium salt hydrate and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich . The MKIs afatinib, bosutinib, cediranib, foretinib, lapatinib, erlotinib, gefitinib, neratinib, pelitinib, vandetanib, crizotinib, sorafenib and sunitinib were obtained from Selleck Chemicals  ; all MKI2) at 37 °C and used within 20 passages after thawing.The human Mel202 UM cell line was kindly provided by Prof. B. Ksander . The 92.1 cell line was a gift from Prof. M.J. Jager  and the OMM-1 cell line was a gift from Prof. G.P. Luyten . The C918 cell line was purchased from BioScientific  and the BeNa Culture Collection . All cell lines were authenticated in-house or by the respective commercial suppliers and routinely checked for mycoplasma contamination every 6 months using a MycoAlert Mycoplasma Detection kit . They were always negative. C918, Mel202 and 92.1 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (v/v), 1% P/S and 1% L-Glutamine . OMM-1 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS (v/v), 1% P/S and 1% L-Glutamine. All cell lines were maintained in a humidified incubator (5% CO4 cells/well) for 24 h. Subsequently, cells were treated with MKIs (10 μM in 0.1% DMSO) in RPMI-1640 or DMEM containing 1% FBS (v/v) for 24 h; 0.1% DMSO was used as the negative control. Following treatments, cells were incubated with MTT (0.5 mg/ml) in the dark for 3 h and then washed with phosphate-buffered saline . Next, the cells were treated with DMSO and the plate was shaken for 10 min at room temperature. Absorbance values were measured at 550 nm in a microplate reader   Ga-NOTA-PRGD2 tracer, fresh 68Ga activity was eluted from a 68Ge/68Ga generator with 0.05 M HCl; 1.5 ml fractions were collected. The radioactive fraction (~5.26 MBq) was added to 1 M sodium acetate buffer containing 50 μg NOTA-PRGD2. The mixture was then heated at 97 °C for 10 min and loaded onto a C18 column  using deionized water and then eluted with ethanol containing 10 mM HCl. On the day PET scans were conducted, ~3.7 MBq of 68Ga labelled tracer was administered to the nude mice under isoflurane anaesthesia via tail vein injection. PET scans were performed using an Inveon microPET scanner . Dynamic image acquisition continued for 60 min after administration. For each scan, regions of interest (ROIs) were determined using vendor software (ASI Pro 5.2.4.0) of decay-corrected whole-body coronal images. The radioactivity concentrations (accumulation) were obtained from mean pixel values within the multiple ROI volume and then converted to MBq/ml. Assuming a tissue density of 1 g/ml, these values were then divided by the administered activity to obtain an image-ROI-derived percent injected dose per gram.To prepare [Tumour tissues embedded in paraffin were sectioned (8 μm thickness) and then stained with hematoxylin and eosin . For immunohistochemical staining, the sections were incubated with an anti-Ki67 antibody  at 4 °C overnight and then incubated with HRP-conjugated secondary antibodies. The sections were visualized using a DAB substrate kit  and an Olympus light microscope .Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay in paraffin-embedded tumour sections fixed on slides. The slides were stained using a TUNEL assay kit  following the manufacturer’s instructions. Nuclei were counter-stained with hematoxylin. Staining was visualized using a Magscanner KF-PRO-120 .p < 0.05. In vivo studies were randomized, and observers didn’t know the group allocation. Statistical analyses were performed using GraphPad Prism 9.0 software with one-way ANOVA followed by Dunnett’s post-hoc test when comparing multiple independent groups. An unpaired t-test was used to analyse differences between two groups. Two-way ANOVA was used to analyse data from treatment and control groups with or without serum stimulation.Data are reported throughout as mean ± standard deviation with significance defined as 50 values for afatinib in four cell lines that represent primary and subcutis metastatic UM were in the range 3.43–5.29 μM  to decrease cell viability was assessed in Mel202, 92.1, C918 and OMM-1 UM. The Mel202, 92.1 and C918 cell lines were derived from primary UM tumours, while OMM-1 is a well-established subcutis metastatic UM cell model. The concentration of 10 μM was selected in initial screening experiments because it exceeds the reported serum trough levels in patients who were treated with the MKIs tested in the present study. This was done to ensure effective cell killing across multiple UM cell lines. The most active agent across the four UM cell lines (< 20% viability remaining) was afatinib, while pelitinib was also active in Mel202 and OMM-1 cells; cediranib, foretinib, lapatinib and neratinib were most effective in OMM-1 cells  and Bcl-xL (anti-apoptotic) - that regulate apoptosis, was assessed in UM cells Fig. . We founSTAT1 acts as a tumour suppressor in a range of cancer types and is associated with the expression of Bcl-xL and cyclin D1 . We founAfatinib has been reported to act as a dual inhibitor of EGFR and HER2 in non-small cell lung cancer and other cancer cells . In thisp < 0.01; Fig. The activity of afatinib was further evaluated in an in vivo C918 UM cell xenograft mouse model. We found that afatinib treatment (15 mg/kg daily for 16 days) markedly decreased the final weights of C918-derived UM tumours in mice Fig.  and stroSubsequently, we performed immunohistochemical staining of the tumour tissues harvested from the xenografted mice Fig. . We founEGFR mutations, ALK fusions and ERBB2/HER2-amplification, respectively [UM has a poor prognosis and, currently, there are no effective treatment options. MKI drugs have revolutionised the treatment of many cancer types by targeting kinases that drive important tumorigenic mechanisms such as proliferation, survival, motility and angiogenesis. Gefitinib, crizotinib and afatinib are kinase inhibitors that are used to treat tumours that exhibit ectively –41. Otheectively . MKIs thectively , 15.The EGFR family encompasses four ErbB members (ErbB1–4) that form homo- and hetero-dimers . Apart fConsistent with findings in the present study, it has been reported that HER2 is expressed in UM cells . ForsberAfatinib-induced apoptosis is associated with decreased expression of the anti-apoptotic Bcl-xL protein, the cell cycle progression regulator cyclin D1, and a decrease in the Bcl-xL/BAX ratio in UM cell lines. Afatinib-driven cellular apoptosis was also accompanied by an elevated expression of STAT1. STAT1 plays an important role in cell survival, viability and responses to pathogens. STAT1 induces cell cycle arrest in response to interferon-γ by interacting with D-type cyclins and cyclin-dependent kinase-4 in fibrosarcoma cells . STAT1 aWe confirmed HER2 activation upon acute afatinib treatment in all the four UM cell lines tested, which suggests that HER2 is likely the molecular target of afatinib and, thus, may be a viable anti-cancer target in UM. Unlike the EGFR inhibitors erlotinib and gefitinib that act in a reversible fashion, the HER2 inhibitors afatinib, pelitinib and neratinib also include an irreversible component in their inhibitory mechanism . These aSeveral signalling cascades, including the AKT, PI3K and ERK pathways, have been shown to be activated by HER2 , 60. Themax of afatinib is ~0.16 μM after administration of multiple daily oral doses of 50 mg in patients [Afatinib is clinically approved to treat non-small cell lung carcinoma (NSCLC) and head and neck squamous carcinoma –64. A phpatients . Brain ppatients , 68. TheIn summary, we found that afatinib has potent anti-cancer properties in in vitro, ex vivo and in vivo UM models. HER2 signalling has emerged as a likely molecular target that activates apoptosis upon afatinib treatment in UM cells. Afatinib also has the advantage of preventing UM cell migration and enhancing reproductive cell death, which may contribute to suppressing UM metastasis. Together, our data indicate that afatinib may serve as a novel candidate drug with an improved therapeutic effect and selectivity in treating UM by targeting HER2.ESM 1(DOCX 721 kb)


The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines , in comparison to extrahepatic (E)CC lines , and HCC/hepatoblastoma cell lines . Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.The online version contains supplementary material available at 10.1186/s12885-022-10247-6. Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) are the most common primary hepatic malignancies, with 841,000 new cases worldwide in 2018, out of which 75%-85% accounted for HCC and 10%-15% for CCC [Fibroblast growth factor receptor-2 gene mutation or fusion, which only represent 10–15% of ICC patients [The prognosis for liver cancer patients is very poor. For example, in the United States of America, the 5-year survival rate for patients with HCC has remained at a low 18% . Approxipatients , 10. Indpatients , and thepatients , are apppatients , 12.A particular problem of primary liver cancer is the occurrence of combined HCC/ICC (cHCC/CC). Its incidence is reported to be highly variable. Due to great diagnostic challenge, its occurrence is likely to be massively underestimated. Of note, a single pass biopsy of a non-representative area will result in an incorrect diagnosis . BesidesTP53 is among the most frequently mutated genes [In both HCC and the small number of cHCC/CC tested, tumor protein ed genes , 16. Thied genes , 18. TP5ed genes . Oncogened genes . The PI3ed genes , compareed genes .There are a considerable number of studies and clinical trials on targeted drugs in HCC and CCC. If not mentioned otherwise, all chemicals and reagents were purchased from Sigma-Aldrich  and Merck . All inhibitors were purchased from Selleckchem . Inhibitors were: Sorafenib, Lenvatinib (multi-kinase inhibitor), Dasatinib (tyrosine-kinase inhibitor), BKM120, Wortmannin, LY294002, and CAL-101 (PI3K inhibitors), MK-2206 (AKT inhibitor), and Rapamycin (mTOR inhibitor). The inhibitors were dissolved in dimethyl sulfoxide (DMSO) . 2.The cells were grown in appropriate medium , ECC , HCC  and hepatoblastoma (HEPG2) cell lines over a course of 96 h using crystal violet proliferation assays. Nine potential inhibitors including Sorafenib and Lenvatinib, which are FDA approved drugs to treat HCC, were used. Based on literature and our previous studies [Proliferation assays were performed as described . Shortly500,000 cells per well were seeded into 6-well-culture plates, and allowed to adhere overnight at 37 °C. The cells were treated with 25 µM MK-2206 diluted in DMSO for 3, 8, and 24 h. Controls were performed with DMSO, in accordance to the highest concentration used in the test group. The wells were rinsed with washing buffer followed by lysis buffer. The wells were scraped with a cell scraper and the cell lysate was collected for Western blots.Protein extraction and Western blot were performed as described previously . Cells w*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 with 95% level of confidence.The data were analyzed using Microsoft® Excel 2019 and Graph pad Prism version 4 software . All experimental errors were calculated as SEM. Statistical significance was calculated with one-way analysis of variance (ANOVA) between treatment and control groups at all time points. Statistical level of significance was accepted at Our results show that the ICC cell lines  incubated only in cell culture media and media containing DMSO increased in number in an almost linear fashion. Highly significant inhibition was observed with MK-2206, where we noticed a significant reduction in cell proliferation in a dose dependent manner. The cell numbers were reduced up to 95% compared to initial cell number Fig. A.Fig. 1PBy Western blot analysis, distinct down-regulation of p-AKT (Ser473) was noticed at 3—24 h in all of the studied ICC cell lines after MK-2206 treatment Fig. . SimilarIn parallel to ICC cell line, our studies also showed a highly significant effect of 10 µM MK-2206 on ECC cell lines EGI1 and TFK1. However, CCC5 was less susceptible . We noticed a reduction of about 90% at 96 h treatment with 25 µM MK-2206 in comparison to respective controls  and CCC5 (ECC cell line) Fig. A.Fig. 4PAt 1 µM and 5 µM, Lenvatinib significantly inhibited growth of HCC cell lines HEP3B and HUH7, but not hepatoblastoma cell line HEPG2. We did not observe any inhibitory effects on ICC and ECC cells Fig. B.Dasatinib showed maximum reduction in cell numbers on ICC cells where the dosage of 1 µM was already very effective. The results for ECC and HCC cell lines were less impressive, and only the higher dose of 10 µM induced consistent reduction of cell proliferation Fig. C.Since tumor suppressors of the PIK3 pathway such as PTEN (phosphatase and tensin homolog) were found to be mutated in combined HCC/ICC , we tested four different PI3K inhibitors, three of them inhibit predominantly PIK3CA  and one PIK3CD (CAL-101). The effects were relatively similar. PIK3CA inhibitors BKM-120 and Wortmannin showed significant inhibitory effects at higher concentrations on all cell lines, while LY294002 at higher dose had significant effect on all cell lines but RBE (ICC cell line), EGI1 (ECC cell line), and HEPG2 (hepatoblastoma cell line). The effects of CAL-101 were moderate Figs. A-D. The Finally, we studied the mTOR inhibitor Rapamycin. At a concentration of 10 nM, Rapamycin could significantly reduce the cell numbers of CCC5 (ECC cell line), HEP3B and HUH7 (HCC cell lines), while the proliferation was unaffected in other experiments Fig. .Fig. 6Pr. These preclinical rodent models can be used to investigate tumor specific and tumor type overlapping effects of therapeutics. Our preliminary data indicate overexpression of p-AKT in the murine TAA model (data not shown). Here, we searched for targeted drugs that inhibited in-vitro cell growth either in a cell type-specific or comprehensive manner.We previously developed a preclinical ICC model in mice by oral application of thioacetamide (TAA) in drinking water. Besides typical cirrhosis we observed development of ICC after 22 weeks of TAA . InteresWe observed differential effects of nine inhibitors targeting the PI3K/AKT/mTOR pathway in ICC, ECC, and HCC cell lines. The PI3K/AKT pathway plays an important role in proliferation, angiogenesis, apoptosis, cytoskeletal organization, and cell survival. This pathway is also known to interact with other signaling pathways such as the RAS-RAF pathway. Consequently, alterations in the PI3K/AKT pathway affect other signaling pathways, leading to uncontrolled cell proliferation and survival and eventually tumorigenesis by inducing transcription of the most commonly mutated genes such as AT-Rich Interaction Domain 1A (ARID1), BRCA1 Associated Protein 1 (BAP1), and Polybromo 1 (PBRM1), which are downstream of the PI3K/AKT pathway .The four PI3-kinase inhibitors tested  showed promising results by significantly reducing proliferation, with some variation depending on the tumor cell type. CAL-101 showed good effects in ICC cell lines, BKM120 and Wortmannin inhibited proliferation in all cell lines effectively. Thereby, Wortmannin showed better results in ICC and HCC than in ECC. The effects of LY294002 were somewhat intermediate. Therefore, PI3-kinase appears to be a promising target for the treatment of primary liver cancers.The AKT inhibitor MK-2206 potently blocked proliferation of all cell lines tested. This is in line with the marked down-regulation of p-AKT in the cells, whereas the expression of p-mTOR was only weakly or not at all changed in the cells. In fact, the mTOR inhibitor Rapamycin did not reveal any anti-proliferative effects in 7 out of 9 cell lines tested. In other cell types, it has been shown that AKT phosphorylates mTORC1 at Ser2448 leading to cell survival, proliferation, and metabolic changes . Our obsWhile MK-2206 has a comprehensive effect in ICC, ECC, and HCC, Dasatinib was most effective in ICC and inhibited cell growth effectively already at 1 µM. Dasatinib inhibits tyrosine-kinases including cKIT and EPH, and is approved for the treatment of chronic myeloid leukemia and acute lymphatic leukemia , 37. UprSorafenib, which is approved for HCC, was very effective in HCC/hepatoblastoma cell lines at 5 µM, but much less effective in two of three ECC cell lines. In ICC, proliferation was decreased greatly at 5 µM, but was even increased at some time points with 1 µM. Sorafenib has a very broad spectrum of activity , 38, andLenvatinib showed inhibitory effects on HCC proliferation with both concentrations, but not in the hepatoblastoma cell line HEPG2. In ICC and ECC, however, neither the lower nor the higher concentration demonstrated any anti-tumor activity. Therefore, caution should be taken with Lenvatinib administration in cHCC/CC.In sum, our studies show that primary liver tumor cell lines show distinct but overlapping patterns of reactivity to targeted molecular therapeutics. Thereby, as in case of Sorafenib, dosage is an important matter, as insufficient quantities might even induce pro-proliferative effects. For targeted therapy, it will be important to properly diagnose the incidence of combined HCC-ICC, as ICC may not or even inversely respond to Lenvatinib and Sorafenib. Customized drug therapy may be the next step to optimize therapy, tailored according to the nature of the tumor and the individual reactivity of patient-derived tumor cells.Additional file 1: Suppl. Figure 1. Uncropped Western-blot images of 25µM MK-2206-treated HUH28 and RBE and SSP25 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 1B. Suppl. Figure 2. Uncropped Western-blot images of 25µM MK-2206-treated EGI1 and CCC5 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 2B. Suppl. Figure 3. Uncropped Western-blot images of 25µM MK-2206-treated HEP3B and HUH7 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 3B.


Bone sarcomas have not shown a significant improvement in survival for decades, due, in part, to the development of resistance to current systemic treatments, such as doxorubicin. To better understand those mechanisms mediating drug-resistance we generated three osteosarcoma and one chondrosarcoma cell lines with a stable doxorubicin-resistant phenotype, both in vitro and in vivo. These resistant strains include a pioneer model generated from a patient-derived chondrosarcoma line. The resistant phenotype was characterized by a weaker induction of apoptosis and DNA damage after doxorubicin treatment and a lower migratory capability. In addition, all resistant lines expressed higher levels of ABC pumps; meanwhile, no clear trends were found in the expression of anti-apoptotic and stem cell-related factors. Remarkably, upon the induction of resistance, the proliferation potential was reduced in osteosarcoma lines but enhanced in the chondrosarcoma model. The exposure of resistant lines to other anti-tumor drugs revealed an increased response to cisplatin and/or methotrexate in some models. Finally, the ability to retain the resistant phenotype in vivo was confirmed in an osteosarcoma model. Altogether, this work evidenced the co-existence of common and case-dependent phenotypic traits and mechanisms associated with the development of resistance to doxorubicin in bone sarcomas. Bone sarcomas comprise a rare group of mesenchymal tumors that represent less than 0.2% of cancer diagnoses . Among tOne of the most common phenotypic traits associated with the drug-resistant phenotype in osteosarcoma is the overexpression of efflux pumps of the ATP-binding cassette (ABC) family, such as ABCG2  and ABCB1 , the expression of which is associated with a lower event-free survival rate in osteosarcoma patients ,15,16. OThis work reports the development of four DOX-resistant models of bone sarcoma (three osteosarcomas and one chondrosarcoma), including a pioneer model generated from a patient-derived chondrosarcoma cell line. We performed a comprehensive functional in vitro and in vivo characterization of these models. Altogether, we proved the utility of these drug-resistant cells as models to study the mechanisms underlying the resistance to DOX in bone sarcomas.50 values for each cell line after each round of treatment  and one primary-derived chondrosarcoma line (T-CDS-17#4)) were exposed to several rounds of stepwise increasing concentrations of DOX in order to induce/select resistant phenotypes . To monireatment A–D. Aftereatment E (Table reatment .50 two times lower) than their parental counterparts (50 three times lower), MG-63-DX-R and T-CDS-17#4-DX-R were more resistant  and no differences were found in the response of parental and resistant strains of Saos-2 cells (50′s ranging from 21 (143-B) to 51 nM (T-CDS-17#4) for MTM and from 18 (143-B) to 74 nM (Saos-2) for EC-8042). With the only exception of TCDS-17#4 for MTM, all resistant lines showed increased resistance to both drugs (IC50 for MTM between 4 and 64 times higher and IC50 for EC-8042 between 2 and more than 1000 times higher)  cells, we analyzed whether DOX-resistance induced a higher tolerance or sensitivity to other compounds with reported anti-tumor activity against sarcoma cells, such as cisplatin (CIS), methotrexate (MTX), mithramycin (MTM) and EC-8042 . First, terparts A,E. No c-2 cells B,E. Fina higher) C–E.To gain insight into the resistant phenotype, we compared the effect of DOX in the induction of DNA damage and the triggering of apoptosis in parental and resistant lines. As expected, we found that DOX was a potent inducer of DNA damage, as indicated by the time-dependent accumulation of γ-H2AX foci in parental lines. Remarkably, the level of DNA damage detected in the resistant strains of the osteosarcoma cell lines 143-B and Saos-2 was significantly lower than those of the parental cells, while no differences between parental and resistant cells were found in the chondrosarcoma line T-CDS-17#4 A,B.50 concentrations of DOX. As expected, the levels of cleaved PARP1 were barely increased, or not detected, in all resistant lines after 72 h of treatment with DOX  proliferated slower than their parental counterparts. However, the chondrosarcoma resistant line T-CDS-17#4-DX-R showed the opposite trend and grew faster than its parental line A. In addAltogether, these results showed that bone sarcoma DOX-resistant cells show some common phenotypic traits, such as their ability to tolerate the DNA damaging and pro-apoptotic effects of doxorubicin or their relatively lower migratory potential. However, other phenotypic features, such as proliferative and clonogenic potential, seem to be case- and/or tumor type-dependent.The development of drug resistance by tumor cells has often been linked to the acquisition of a stemness phenotype . TherefoTo further investigate the CSC phenotype in doxorubicin-resistant cells, we checked the protein levels of relevant factors associated with the drug resistant and/or stemness phenotypes. These analyses showed that the resistant phenotype was associated with the upregulation of the efflux pumps ABCB1 and ABCG2. Thus, while ABCB1 was not detected in any parental line, it was strongly upregulated in all resistant lines. Similarly, with the exception of MG-63-DX-R, we found increased levels of ABCG2 in most resistant cell lines J. On theTo assay whether the resistant phenotype is maintained in an in vivo setting, we generated tumor xenografts by inoculating parental and DOX-resistant 143-B cells subcutaneously in immunodeficient mice and treated them either with the drug vehicle (control) or 4 mg/Kg doxorubicin twice a week. In control conditions, parental cells were more tumorigenic and produced larger tumors than DOX-resistant cells A,B. In a50 values similar to those obtained before xenograft growth, thus, confirming the stability of the resistance phenotype  became slightly more sensitive to both CIS and MTX. In agreement with this finding, no cross-resistance to CIS or MTX was observed in other previously reported DOX-resistant osteosarcoma cell lines . AlthougImportantly, the treatment of xenograft models confirmed that the resistance phenotype of the 143-B-DX-R model is also maintained in the in vivo setting, thus allowing the use of this cell line as a drug testing model suitable for in vivo assessment of the effectiveness of therapies aimed to circumvent doxorubicin resistance.Altogether, the characterization of four DOX-resistant bone sarcoma models evidenced that there may be different phenotypes and mechanisms associated with the development of resistance to DOX. Anyway, this is an initial phenotypic characterization of these bone sarcoma resistant models. It is foreseen that further molecular characterization of these models, integrating proteomic, transcriptomic, epigenetic and/or metabolomics analyses together with other approaches, such as CRISPR screening assays, will contribute to the unravelling of the molecular mechanisms behind common and specific phenotypic traits in resistant cells. These analyses will also result in the identification of synthetic lethalities and drug combinations able to revert doxorubicin resistance in bone sarcoma and/or the identification of biomarkers useful to select those patients which will benefit most from DOX treatment. As future goals, it would also be of interest to compare early versus late stages of resistance in these step-wise generated models. These studies will help to understand the time course of events involved in the process of acquisition of drug resistance by bone sarcoma cells. Besides, this study only included one chrondrosarcoma resistant line. To better discern the resistance mechanisms associated with this type of bone sarcoma, it is necessary to develop and characterize more drug-resistant chondrosarcoma models. Finally, the development of drug resistance in patient-derived models, as reported in this work, may allow the implementation of personalized strategies capable of anticipating efficient second-line treatments in cases of relapse.v/v) fetal bovine serum , 1% (v/v) Penicillin-Streptomycin  and 1% (v/v) GlutaMAX  at 37 °C in a humidified atmosphere (5% CO2 and 95% air). All cultures were tested monthly to discard mycoplasma contamination using the Biotools Mycoplasma Gel Detection kit .Human osteosarcoma cell lines 143-B (CRL-8303), Saos-2 (HTB-85), and MG-63 (CRL-1427) were obtained from ATCC . Patient-derived chondrosarcoma model T-CDS-17#4 was stablished as previously described . All celAll experimental protocols have been performed in accordance with institutional review board guidelines and with the Declaration of Helsinki and were approved by the Institutional Ethics Committee of the Principado de Asturias (reference 255/19).DOX, MTX and CIS were purchased from Selleckchem . MTM and its analog EC-8042 were synthesized by EntreChem SL  ,49. Stoc20 of DOX . The drug was renewed every 3–4 days in fresh culture medium. After one month, the drug concentration was doubled and cells were cultured for another month. The whole process of step-by-step concentration increases and incubation times was repeated several times until we obtained a resistance level for each cell line that could not be increased with further increases in drug concentration. The conditions used to generate resistant models for each cell line are summarized in 50) and the resistance index (RI), calculated as the ratio between the IC50′s of resistant and parental lines, were determined.Sarcoma DOX-resistant (DX-R) cell lines were generated by continuous exposure to stepwise increasing concentrations of DOX. Briefly, the parental cell lines were continuously treated with their respective IC50 for each treatment was determined by non-linear regression using GraphPad Prism 9.0.1 software .Cell viability of cell lines after the treatment with increasing concentrations of different drugs was assayed using the cell proliferation reagent WST-1  after 72 h-treatments as previously described ,50. IC503 and 1.5 × 104 cells depending on the assayed cell line) were seeded in specially designed 8-well plates  that contained interdigitated gold microelectrodes sensors able to detect changes in cell impedance. In experiments designed to analyze the effect of DOX on cell proliferation, this drug was added 6 h after cell seeding. Cell impedance was monitored every two hours for 160 h and data was analyzed using the RTCA analysis software version 1.0 (ACEA Biosciences). The proliferation status of cells was expressed in terms of cell index (CI) normalized or not to the values measured 6 h after seeding.The differences in the proliferation potential between the DOX-resistant cell lines and their respective parental cells were evaluated using the iCELLigence real-time cell analyzer  . Cells (3 cells) in 100 mm culture dishes and left to form colonies for 10 days. Following this period, cells were fixed in cold methanol and stained with 0.5% crystal violet. Colonies containing approximately 50 or more cells were counted using the ImageJ 2.1.0 software .The clonogenic capability of parental and DX-R cell lines was analyzed in colony formation unit (CFU) assays as described previously . Briefly4 cells in 100 µL per well for 143-B and 5 × 104 cells/well for the rest of cell lines. Then, 600 µL of medium was added to the lower chamber. After 24 h, cells were fixed in 70% ethanol for 10 min and stained with 0.5% crystal violet for 5–10 min. Then, cells on the upper side of inserts were removed with cotton-tipped swabs and the inserts were washed with distilled water , 1:1000 dilution] from Cell Signaling ; anti-ABCG2  and anti-Cleaved-PARP1  from Abcam ; anti-OCT4 , anti-SOX2 , anti-MCL1  and anti-Bcl-xL  from Proteintech ; and anti-β-Actin  from Sigma-Aldrich. IRDye infrared fluorescent secondary antibodies IRDye 800CW and IRDye-680RD from LI-COR Biosciences  were used for signal detection using an Odyssey Fc imaging system and the Image Studio software . β -actin levels were used as loading control.Cell extracts were lysed with Pierce RIPA buffer  supplemented with protease and phosphotase inhibitors. Lysates were centrifuged at 12,000 rpm at 4 °C and supernatants were collected. Protein concentration was quantified using the Bradford dye-binding method . A total of 30 µg of protein sample was loaded on 6–10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. After electrophoresis, gels were electro-transferred using the Trans-Blot turbo transfer system and the Trans-Blot turbo RTA mini 0.2 µm nitrocellulose transfer kit . The membranes were blocked with SuperBlock® Gold Antifade Mounting medium with DAPI  and analyzed by fluorescence microscopy. The number of γ-H2AX foci per nuclei was counted in at least 100 cells of each condition using the ImageJ 2.1.0 software and the Cell Profiler 4.2.1  [Cells were seeded at high density in 6-well plates containing glass coverslips. After 24 h, cells were treated with 1 µM DOX for 0 h (Control), 4 h, 8 h or 24 h. Then, cells were washed twice with phosphate-buffered saline  and fixed in 4% paraformaldehyde  in PBS for 20 min at room temperature. Cells were then washed twice with PBS plus 0.1% Tween 20 (Sigma-Aldrich), permeabilized in PBS plus 0.1% Triton X-100 (Sigma-Aldrich) for 20 min and washed again with PBS. Slides were blocked in 10% BSA  for 30 min and incubated overnight at 4 °C with the primary antibody anti-γ-H2AX  from Merck KGaA . Then, the slides were washed 3 times with PBS plus 0.1% Tween 20 and incubated with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG  from Invitrogen  for 1 h. Later, cells were washed another 4 times with PBS and, finally, coverslips were mounted in ProLongMA, USA) .5 143-B parental or DOX-resistant cells resuspended 1:1 in 50% (v/v) Matrigel® basement membrane matrix high concentration . Once tumors reached approximately 100 mm3, the mice were randomly assigned (n = 6 per group) to receive the following intravenous treatments twice a week (7 doses): vehicle  or 4 mg/Kg DOX. Tumor volume was determined using a caliper as previously described [t) and the tumor volume at the beginning of the treatment (V0). Drug efficacy was expressed as the percentage tumor growth inhibition (%TGI), calculated as follows: TGI (%) = 100 − (T/C × 100), where T is the mean RTV of the treated tumor and C is the mean RTV in the control group at day of measurement. Three weeks after starting the treatment, mice were sacrificed by cervical dislocation and tumors were extracted. Cell lines were stablished from representative tumors from mice of different experimental groups as described before [All animal research protocols were carried out in accordance with the institutional guidelines of the University of Oviedo and were approved by the Animal Research Ethical Committee of the University of Oviedo prior to the study (PROAE 34/2019). Female 6 weeks old athymic nude mice  were inoculated subcutaneously (s.c.) with 1 × 10escribed . Relativd before . WST-1 at test was performed to determine the statistical significance between groups. Multiple comparisons of the data were performed using one-way ANOVA and Tukey’s test, p ≤ 0.05 values were considered statistically significant.Statistical analysis was performed using GraphPad Prism 9.0.1 software. Unless otherwise indicated, all data are presented as the mean (±standard deviation or SEM as indicated) of at least three independent experiments. Two-sided Student’s


Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one. F5) has an approximate size of 80 kb, is located in chromosome 1q24.2 and consists of 25 exons and 24 introns. FV cDNA is 6914 bp in length. Eighty percent of circulating FV is produced in the liver by hepatocytes, while the remaining 20% resides in platelet granules [F5 gene and an ultra-rare condition with an incidence of 1–9/1,000,000 live births [Factor V (FV), also called labile factor or pro-accelerin, is a coagulation factor that plays a key role in the coagulation cascade. In humans, the FV gene  plasma or the use of Octaplasuding FV ,14,15.Gene editing by CRISPR/Cas9 has been proposed as a potential therapy for several genetic diseases, including FV deficiency. Indeed, given its ability to eliminate mutated exons and normalize the FV protein, CRISPR/Cas9-based gene editing can restore the protein’s original function ,17. ThisAs far as the patient is concerned, a familiar segregation analysis indicated that her mother was heterozygous for the already described c.2218C>T, p.Arg740* mutation  with FV To facilitate the availability of FV deficient cells, an attempt was made to mimic one of the mutations described in this patient in a reference hepatic cell line.The non-homologous end-joining precision deletion (NHEJ-PD) strategy was used to generate a KO FV cellular model in a HepG2 cell line (KO HepG2) A,B and rCells that contained this precise sequence were selected through colony-forming units (CFUs), and the DNA of these clones was analyzed by Sanger sequencing. Some clones were shown to exhibit the sought-after mutation B. For thF5 was restored in the three clones  was generated from KO HepG2 clones LO1, LO8 and LO9 through NHEJ-PD. In this case, a deletion of 49 bp (c.3242–3291del) removed the premature stop codon; the frameshift of the e clones D. Efficie clones C.The three cell lines obtained were compared and characterized. As shown in As shown in p-value = 0.00125). No significant differences were found between the KO HepG2 cell line and the negative controls , while the concentrated secretome from the KO HepG2 cell line contained 19% ± 6.5 of functional FV (sample coagulation time was ±31.2 s). The concentrated secretome of the TT HepG2 cell line contained 31.5% ± 9 functional FV (sample coagulation time: ±27.7 s), which was significantly different from the percentages obtained with the KO HepG2 cell line , previously described by our research group [This study proposes a new advanced gene editing-based therapy for the correction of a new mutation in the ch group . A specich group ,26.The study was devoted to the development of a custom cell model based on the mutation present in the patient , who hadDeveloping a model of the kind proposed in this study is extremely useful given the difficulties inherent in working with samples from a patient with a hemorrhagic condition ,28,29. TF5 in a constitutive manner in a hepatic cell line, which is advantageous considering that FV is synthetized in the liver [HepG2 was selected for this study as it is a human immortalized cell line characterized by its robustness, durability, fast proliferation and potential to generate colonies from a single cell. It also expresses the he liver ,31. Primhe liver ,33,34.Another option would have consisted in obtaining hepatocytes from induced pluripotent stem cells (iPSc) from the studied patient, but, as previously reported by the authors of this study, iPSc or mesenchymal stem cell-derived hepatocytes cannot produce FV as the process requires a tridimensional environment ,36. F5 mutations by gene editing or gene therapy using viral vectors.The purpose of this study was to obtain a simple and efficient cell model to test different in vitro strategies for correcting The cell model used was developed through gene editing by means of a pair of guides (gRNA) based on the CRISPR/Cas9 technique. These guides direct the activity of the Cas9 protein to very specific sites in the genome , performKO gRNAs allowed us to produce a highly efficient KO FV cell model with a nonsense codon stop mutation similar to the one exhibited by the studied patient, in approximately the same area of exon 13 and resulting in the same phenotype. The heterogeneous culture was purified and enriched by harvesting, selecting and expanding a series of clones until homogeneous cell lines were obtained. Finally, an analysis was made in three KO clonal FV cell lines.F5 gene with an efficiency of 41% and in the generation of a functional FV cell line. The design of the guides and the PAM orientation influences the cleavage/splicing efficacy achieved by the NHEJ-PD strategy [The same gene editing strategy, based on TT gRNAs, was used to correct the mutation in the three clones of the KO FV cell line. This resulted in the restoration of the frameshift of the strategy . The guistrategy ,11.This strategy, which involves the deletion of different regions of exon 13, is similar to the one developed for the B domain (exon 14) of factor VIII , reducinCharacterization of the three different cell lines  did not show differences in cellular morphology, cellular proliferation or membrane molecular markers. These results seem to indicate that gene editing using the CRISPR/Cas9 method does not alter genes associated with the previously mentioned characteristics. The analysis of the off-target genes did not show any modification in the genetic sequences derived from the unspecific editing of the CRISPR guides used, at least for the genes studied. The analysis of possible off-target sequences was conducted using the TT HepG2 cell line edited with the two CRISPR guides simultaneously using Sanger sequencing and database sequence comparison .Due to the nature of this cell line, it is difficult to perform an NGS analysis to detect potential DNA or chromosome rearrangements. Indeed, the HepG2 cell line exhibits an aberrant karyotype with multiple DNA sequence rearrangements, duplications, deletions, polyploidies, etc., making it impossible to evaluate the potential CRISPR-induced chromosome or DNA rearrangements as these cells per se harbor multiple DNA alterations .In the immunostaining and ELISA assays, no significant differences were found in the fluorescence or concentration of FV between the three cell lines studied . This could indicate that the three cell lines were responsible for the production and secretion of FV, as detected by polyclonal antibody-based immuno-staining assays and ELISA. In the case of the KO cell line, the coagulometry assay found that the FV produced was not functional. Our hypothesis is that the KO cell line can synthesize and secrete a stable fragment of FV (heavy chain), as detected by polyclonal antibody-based assays, but not a complete protein (without a light chain). This is in line with previous reports arguing for the need to use monoclonal antibodies to target the region of interest, as polyclonal antibodies are not able to differentiate between a full and a truncated protein just on the basis of an ELISA assay . This meThe FV obtained is, therefore, not functional because it lacks the light chain, which plays a very important role in the coagulation pathway as it enables FV to bind to platelet membrane phospholipids ,6.By the same token, although no significant differences were observed in FV concentrations between the TT cell line and the WT and KO cell lines, the functionality of FV in the TT cell line was found to be significantly higher than in the KO cell line. This shows that CRISPR/Cas9-based gene editing can effectively correct the pathological mutation and produce an FV protein with renewed functionality. There was no difference between the KO cell line and the negative controls in the fully functional FV production. This difference in the functionality of FV between the KO and the TT cell lines (ΔFV) was proportional to the editing efficiency obtained by using TT gRNAs.Confirmation of the results obtained by Western blot was not possible using any of the usual protocols and antibodies . Nor was it possible to find any study or protocol that produced positive Western blot results using the HepG2 cell line secretome. These persistently negative results are in line with those found by other authors, who have detected a 330 kda band in the plasma of patients with a truncated FV protein, corresponding to the whole protein, but have been unable to detect bands derived from a potentially truncated protein .F5 gene and restoring or increasing FV plasma levels in vivo. A few research groups have tried to develop deficient FV animal models, but such models have not been viable as none of the animals ever reach adulthood [There are currently no deficient FV animal models that allow the performance of preclinical studies that can be used to test the efficacy of our TT gRNAs in correcting the mutation of the dulthood ,46,47.The next steps of our ongoing research will be focused on developing an FV-deficient animal model to test in vivo CRISPR-based gene editing and gene therapy protocols using lentiviral vectors.In conclusion, a KO FV cell model with a mutation similar to the one carried by the patient studied was developed through NHEJ-PD with a CRISPR-based method. Moreover, an effective, precisely customized treatment, based on CRISPR gene editing, was developed to correct the mutation. The treatment proposed in this study succeeded in producing, at least in vitro, a functional FV protein. No molecular defect or alteration was found in the edited cells. This strategy could constitute a promising alternative for the development of custom pathologic models. This study analyzed a 14-year-old Caucasian female from Jaen, in the south of Spain, afflicted with severe FV deficiency (FV plasma level < 1%). She was a compound heterozygote presenting with two different mutations: one inherited from her father and the other from her mother. Both parents exhibited a mild FV deficiency. The ensuing cell model and CRISPR/Cas9-driven correction studies centered on a new mutation , previously described by our group .Genetic maps were developed with the SnapGene 1.1.3. software , and the genetic sequence and protein sequence (with their respective domains) were obtained from the NCBI (NM_000130.5), Ensembl (ENST00000367797.9) and HGMD databases.4 cells/cm2. Incubation conditions were 37 °C, 5% CO2 and 95% relative humidity.A HepG2 cell line ,31  as well as the so-called “treated HepG2” (TT HepG2) cells, where the KO mutation was eliminated or reversed, recourse was had to specific CRISPR guides (gRNA) in a ribonucleoprotein (RNP) format. The different crRNAs (CRISPR RNAs) were designed by applying the “Zhang lab’s” software (MIT) to the sequence between nucleotides 3100 and 3400, located at S. pyogenes, IDT) was added, and the mixture was incubated for at least 20 min. All reagents were obtained from Integrated DNA Technologies .gRNAs were formed by the combination of tracrRNA (trans-activating crRNA) and crRNA on the basis of the following protocol: 0.4 µL of 200 µM tracrRNA and 0.4 µL of 200 µM crRNA were incubated at 95 °C for 5 min. When the solution reached room temperature, 0.2 µL of 62 µM nuclease Cas9 (The guides used for correcting the mutation (TT gRNAs) were patented as an effective in vitro treatment by the Spanish Patent and Trademark Office (ES 2785323 B2). 5 cells were added to a solution comprising 2 µL of each RNP (gRNA + Cas9) and 20 µL of “SF cell line Nucleofector® solution” . The resulting mixture was electroporated with an Amaxa 4D-Nucleofector system , using a 20 µL volume under the EH-100 program.A total of 2 × 10The Sanger sequencing procedure was used to obtain the sequence of the different clones with a view to selecting (and subsequently expanding) the clone associated with the most accurate sequence. This sequencing method was used to determine the efficacy of the correction of the KO mutation and to analyze the sequences of potentially “off-target” genes as a way of establishing the degree of specificity of the guides. The Sanger sequencing procedure was carried out by the Genomics and Proteomics Unit of the Madrid Complutense University. ® Blood and Tissue kit”  in accordance with the manufacturer’s instructions. The end-point PCR test  was carried out using primers capable of amplifying 403 bp of WT HepG2; 368 bp of KO HepG2 and 319 bp of TT HepG2 of the human F5 (which contains the target region in the middle of the amplicon). For “off-target” genes, different primers with different amplicon sizes were used. F5 and the “off-target” genes, as well as the size of the amplicon.In order to sequence the HepG2 cell lines using the Sanger method, total genomic DNA was extracted from the cells using the “DNeasyThe results of the Sanger sequencing assay were analyzed using the online version of Synthego’s ICE software .A Zeiss Axiovert 40 C inverted microscope equipped with camera  was used to study the morphology of the different HepG2 cell lines. Images were captured with a 10× objective.4 cells were briefly seeded in a 12-well dish with 10% FBS DMEM. The Alamar Blue reagent was subsequently diluted at 1:10 with 10% FBS DMEM. The solution was incubated for two hours, and a measurement was made at 560 nm excitation and 590 nm emission. This procedure was repeated daily for 7 days. The proliferation index of the different HepG2 cell lines was evaluated using an Alamar blue assay  in accordance with the protocol established by the manufacturer. A total of 1 × 105 cells were used to test each molecular membrane marker; antibody concentrations were those recommended by the manufacturer. Between 2 µL and 10 µL of each antibody was added.Molecular membrane markers were evaluated through flow cytometry using the antibodies shown in Intracellular FV immunostaining was applied to HepG2 cells using Abbexa’s Coagulation Factor V (FV) antibody  and the DyLight™ 488 Donkey anti-rabbit IgG  Antibody  in accordance with the manufacturers’ instructions. Staining with 4′,6-diamidino-2-phenylindole (DAPI) was also carried out in order to identify the nuclei. Images were captured with a LeicaSP2 confocal microscope . Human adipose-derived stem cells (hASC) were used as control.® 20, membrane: 100.000 MWCO PES  and by centrifuging 11 mL of secretome at 3000× g for 25 min. A WT HepG2 cell line was used as positive control, while mouse hepatic oval cells and the SW480 cell line were used as negative controls. The assay selected was the Human Factor V AssayMax ELISA Kit , which contained a polyclonal antibody. All the manufacturer’s instructions were followed. The data is shown as ng/mL of FV ± standard deviation.An FV ELISA assay was performed with 6.5× concentrated secretome samples. The concentration of the secretome was achieved by using concentrator columns  to ensure that the function of FV and its synthesis by different HepG2 cell lines were correct. FV function was tested using a 6.5× concentrated secretome from various cell lines. The coagulation assay was conducted following the manufacturer’s instructions. A magnetic steel ball, 50 µL of FV-deficient human plasma  and 50 µL of concentrated secretome were placed on a tray. The solution was incubated for around one minute in the coagulometer’s “incubation zone”, after which 100 µL of Neoplastine Cl plus  was added. When the sample coagulated, the device calculated the clotting time and extrapolated the data to a previously prepared standard curve. The data is shown as a % of functional FV ± standard deviation. The oval and the SW480 cell line-derived concentrated secretomes were used as negative controls to evaluate coagulation.The potentially unspecific editions of the guides were tested by means of an analysis of the “off-target” genes using PCR assays and Sanger sequencing protocols. Two “off-target” sequence selection criteria were adopted to reduce the number of genes to be analyzed, thereby making the study feasible. One of the criteria involved an analysis of sequences as potential “off-target” sequences located in any of the gene’s exons (regardless of any differences in the number of nucleotides between the CRISPR guides and the gene). The second criterion consisted of regarding sequences located in any of the gene’s introns as potentially “off-target” provided that the difference in the number of nucleotides between the CRISPR guides and the gene was equal or less than 4. The analysis of the potential off-target editions was performed on the TT HepG2 cell line edited using the two CRISPR guides simultaneously (TT guide 4 and TT guide 2).p-value was below 0.05 (*), 0.01 (**) and 0.001 (***). The data were expressed as mean ± standard deviation.The data were analyzed using the Statgraphics centurion XVII software package . Statistical analyses were carried out by means of a t-test, which considered differences statistically significant when the