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


It is indisputable that every day it is demonstrated that natural products present diverse therapeutic benefits, which has boosted their incorporation within various products for clinical use. However, this must be accompanied by knowledge of their effect on cell lines to ensure their use is safe. The objective of this study was to evaluate the cytotoxic effect of two ethanolic extracts based on Peruvian natural products, on three human cell lines. Cervical cancer cell lines (HeLa), human gingival fibroblasts (HGF-1 - ATCC CRL-2014) (HGF-1) and peripheral blood mononuclear cells (PBMCs) were cultured and subsequently treated with preparations of ethanolic extracts of propolis (EEP) and Psidium guajava (EEG) from a concentration of 50 mg/mL to 0.024 mg/mL, by the 3--2,5-diphenyltetrazole bromide reduction assay. At a concentration of 0.24 mg/mL EEG, viability of 99.7±1.24%, 99.8±2.2% and 99.7±2.7% was observed in HeLa, HGF-1 and PBMCs, respectively; >90% cell viability values were observed with EPP at 0.024 mg/mL, with HGF-1 showing the highest viability (96.9±1.15%). A dose-dependent effect was observed for both extracts with a decrease in cell viability as concentrations increased (up to 50 mg/mL). EEP and EEG extracts at low concentrations do not show cytotoxicity in human cell lines, these findings are an advance in the preclinical evaluation on their safety and open a continuity to further studies for their potential applications in dentistry and medicine. Even so, man in the 21st century has not been able to avoid death by limiting himself to mitigating symptoms of diseases and avoiding the development of others.,Since time immemorial, man has tried to mitigate his ailments and prolong his life. This fact has been observed since there have been historical records, from civilization to civilization, until today.,In times when man only had at his disposal the resources that the planet gave him, he sought in these the tools to reduce physical pain and avoid death. Among the resources most exploited by different cultures throughout history are mineral, animal and vegetable resources. Until the middle of the 20th century, these were the therapeutic resources par excellence. Plants, thanks to their marvelous and complex metabolism, constitute a true chemical arsenal. Of which only a third is currently known, considering the variety of existing species worldwide, without considering those species already extinct.,Among the kingdoms of nature that contribute, to this day, to reducing symptoms and preventing diseases, the plant kingdom stands out. Over time, these local characteristic therapies came to form the so-called traditional medicine and, when being preserved by the native peoples, is sometimes called aboriginal or autochthonous medicine, as well as traditional or autochthonous recipes that group together uses, forms of preparation, administration, dosage, among other modern pharmacological parameters. This is because our therapeutic reality today is governed by synthetic chemistry, but what few people know is that these successful molecules that cure are nothing more than improved copies of chemical substances that nature spontaneously created.Each region of the world developed its own way of healing from medicinal plants, which is unique and characteristic since species endemic to regions were used. which is composed of approximately 50% to 55% resins and balsams, 30% to 40% wax, 10% to 15% essential oils, 5% pollen and 5% minerals. In its components we can mention that it has phenolic compounds: Flavonoids, flavones, isoflavones and flavonones in 50%, which inhibit bacteria and fungi. The amount of flavonoids confers the antibacterial power to propolis. This quantity depends on the flora surrounding the bee hives. Its antibacterial action mechanism is given by the inhibition of cell division, DNA disruption, disorganization of the cytoplasmic membrane and inhibition of cell wall synthesis, causing partial bacteriolysis and inhibiting protein synthesis.,One of the most studied products is propolis,Psidium guajava) is a fruit native to Central America and the Caribbean, belonging to the Myrtaceae family, distributed in the tropics and subtropics around the world. Guava fruits stand out among tropical fruits not only because of their good organoleptic characteristics (flavor and aroma) but also nutritionally, they are a source of vitamin A, B1, B3 and C, fiber, minerals such as potassium, calcium, iron and phosphorus. The guava also has a relevant content of lycopene, an important carotenoid with therapeutic properties, so it has been widely studied.,Guava  and peripheral blood mononuclear (PBMCs) cells.Cytotoxic evaluation, as the main factor of biocompatibility, is determined by the cell cultures to be selected forP. guajava samples were collected by researchers in the Oxapampa valley, Pasco, Peru following the methodology described by Milloneset al., and subsequently refrigerated until processed. After they were removed from refrigeration they were left for two hours to allow for them to reach room temperature. Once they had reached room temperature they were macerated Once room temperature was reached, they were macerated with a volume of 100 ml of absolute ethanol for every 10 grams of propolis sample, it was then left at room temperature for 24 hours. Then, the macerate was filtered using a 20 cm diameter glass funnel with sterile cotton; the filtered sample was collected in a glass refractory to finally be taken to an extraction hood so that the ethanol present in the extract evaporates completely and only a pasty mass remains. This step was performed two more times until the samples were observed to be discolored. Finally, they were stored in glass containers covered with aluminum foil to avoid degradation.Propolis andThe evaluation of cytotoxicity was performed considering oral cavity constitutive cell lines, human gingival fibroblasts (HGF-1); immune constitutive cells, peripheral blood mononuclear cells (PBMCs); and tumour constitutive cells, HeLa cell lines.(Gibco) with 4% PBS  at 37°C in 5% CO2 and 95% air. The cells were incubated for 30 min in 1 mL of dye solution in Hank’s Balanced Salt Solution ) (100 nM Mitotracker Red FM) at 37°C, 5% CO2. After incubation, the cells were washed three times with HBSS buffer.The HeLa cell line  (RRID:CVCL_0058) was cultured in a Petri dish with a 35 mm diameter glass bottom . The cell line was cultured in Eagle’s Minimum Essential Medium2 atmosphere in an incubator , and were fed every 48 hours and subcultured every 5 days at a 1:3 ratio using trypsin-EDTA  for 3 minutes at 37°C.Human gingival fibroblasts (HGF-1 - ATCC CRL-2014) (HGF-1) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Dulbecco’s medium containing glucose  supplemented with 4 mM L-glutamine (Sigma-Aldrich), 1% penicillin, streptomycin (Sigma-Aldrich) and 10% (vol/vol) heat-inactivated fetal bovine serum . Cells were incubated at 37°C in a 5% CO2 incubator, 311, USA) at 37°C, 5% CO2 and 95% humidity. The medium was changed once every 24 hours.–PBMC cells were isolated by Ficoll density gradient centrifugation  and cultured in RPMI-1640 medium with 10% fetal bovine serum and placed in a humidified incubator  contained in 25 cm2 flasks were started, the medium was discarded using a 5mL serological pipette, two washes were performed to the cell layer with PBS solution (0.004% Ethylene diamine tetracetic acid), 1 mL of Trypsin-EDTA (0.05%) was immediately added and incubated at 37°C for 15 min. A 20 μL sample was taken and 20 μL of 0.4% trypan blue  was added to perform a cell count in a Neubauer chamber  to adjust the cell concentration to 5×104 cells per reaction with Dulbecco’s Modified Eagle’s medium (d-MEM) supplemented with 10% fetal bovine serum. Then 50 μL of the cell suspension was seeded, incubated at 37° C, 5% CO2 in an incubator  until confluence was obtained (after 48 hours), then different concentrations previously evaluated in the study by Milloneset al. (50 to 0.024 mg/mL) were then applied and the plates were incubated for 48 hours. Only d-MEM culture medium was used as a negative control and as a positive control.To evaluate the cytotoxic effect, cell viability methodology was performed in microplate with 3--2,5-diphenyltetrazole bromide (MTT).The medium used was then discarded and the cell layer was washed with PBS solution (0.004% EDTA) and 50 μL of d-MEM culture medium was added.To each well, 20 μL of MTT (5 mg/mL) was added to each culture and then the cultures were incubated for four hours. After this time, the medium was aspirated. 200 μL of dimethyl sulfoxide was added to dissolve the formazan crystals; the plate was left in agitation for 15 minutes microplate shaker  and shaken at 120 revolutions per minute to ensure complete dissolution. Finally, the plate was read at 570 nm on a Smart Spectophotometer plus reader .The experimental data were analyzed using nonlinear regression with the Gompertz model to evaluate the effect according to the concentration of the doses used, whose equation is given by:P. guajava was performed using analysis of covariance, which in addition to the product includes the concentration administered. Graphical presentation was prioritized to highlight some analyses. The analyses were performed with Excel  (RRID:SCR_016137) and SPSS version 26  (RRID:SCR_016479).Where, y is the cell viability, x the administered concentration of each product (mg/mL), and α, β, and k are the parameters of the model. Comparison of the cytotoxic effect of propolis andThe cytotoxic effect of Peruvian propolis is shown inP. guajava is shown inOn the other hand, the cytotoxic effect of Peruvian2), this value is above 90% for all curves.The goodness of fit of the Gompertz curves to the cytotoxic effect on the cell lines of both products is shown by the coefficient of determination  and for normal human embryonic epithelial kidney (Hek-293), also reporting a higher IC50 value for Hek-293 compared to the tumor cell lines.Despite the high cytotoxicity shown by most of the propolis samples against the cell lines studied, the samples also showed toxicity to HGF-1 culture. Ling– Despite this, our preliminary data suggest that both products could have a significant biological effect on the three cell lines tested, which opens up prospects for further research in this field.Although both propolis and guava seem to exert a potential on PBMCs cells, only a few studies have been performed in this field of research.EEP and EEG at low concentrations do not show cytotoxicity in human cell lines and their effect is dose dependent. Our findings are an advance in the preclinical evaluation of natural extracts from Peru on their safety and open a continuity to further studies for their potential applications in dentistry and medicine. Despite our positive data, further study is required to evaluate the usefulness of these extracts.https://doi.org/10.17632/yt4h7h9cvv.1Mendeley: Cytotoxicity database.-citotoxicidad.sav (raw data)This project contains the following underlying data:https://doi.org/10.17632/yt4h7h9cvv.1Mendeley: Cytotoxicity database.-database.xlsx (Processed data)This project contains the following extended data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The document is interesting and presents two compounds of natural origin (Propolis and Psidium guajava) with potential for medical and dental application.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology and Immunogenetics, microbiology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Natural products and microbiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. P. guajava on HELA cell lines, human gingival fibroblasts (HGF-1), and peripheral blood mononuclear cells (PBMC), finding cell viability more significant than 90% at concentrations equal to or less than 0.24 mg/mL and 0.024 mg/mL for EEG and EPP, respectively. Within this perspective, the authors infer potential applications in medicine and dentistry.In this article, the authors evaluated the cytotoxic effect of ethanolic extracts of propolis andMain comments:In the study methodology, the authors use the HELA cell lines, human gingival fibroblasts (HGF-1), and peripheral blood mononuclear cells (PBMC); however, the reason for their selection they not mentioned in the text. I suggest the authors include some selection criteria for these cell lines used in work.P. guajava at the concentration range of 0.024 to 50 mg/mL. What were the selection criteria for this range of concentrations?To determine the cytotoxicity, the authors use the ethanolic extracts of propolis andAlthough the document achieves the objective of determining the highest concentrations of the two ethanolic extracts at which cell viabilities more significant than 90% are observed, it would also be interesting to calculate the IC50 of each extract on each cell line.Minor comment:In the last paragraph of the discussion, the authors state that their preliminary data suggest that both products could have a modulating action on the immune response. I suggest rethinking this inference because within the document's text, and mainly, neither in the objective nor in the methodology, and even less in the results, are not elements that allow this insinuation in the present study.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology and Immunogenetics, microbiology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Title: Its title is adequate, it consigns the most interesting part of the study referred to the cytotoxicity of propolis, using scientific terms keeping the coherence and sufficiency of the article's content.Summary: Although it is true that natural products are beneficial, there are few studies that mention the safety of the clinical use of the product. The article emphasizes the importance of knowledge of cell toxicity with a clear and precise explanation of the adequate concentration to avoid the cytotoxic effect.Content: The methods section is well explained, the cytotoxic assay of this study establishes which was the adequate procedure that provided the percentage of safe and reliable cell viability, being coherent with the results in Figure 1. I consider that establishing the detailed procedure as it is done in this study is important so that it is reproducible in future studies of regenerative medicine or tissue engineering.Introduction: I appreciate the effort of the background search and its respective analysis, the introduction is very good, it makes a brief summary of the background making reference to the relevant medical literature, in the last paragraph it mentions the safety of the use of propolis without deterioration of the cells, I suggest, so that the reader can make a comparison and as interesting reading, to cite references 27 and 30, which are similar studies.Results: The results of this study are accurate, achieving the objective to avoid cytotoxicity in human cell lines, recognizing that the effect is dose-dependent. Based on the experience of the researchers evidenced in previous publications, it can be observed that a line of research continues in search of new active principles that can offer a promising pharmacological product based on natural products. Although it is true that this is a preliminary study as indicated by the authors, it is important to point out that future research could consider procedures based on flow cytometry to corroborate the results obtained in the present study.Discussion: The background information showed the therapeutic benefits of natural products, with propolis standing out from the two products, for its antimicrobial, anti-inflammatory and antioxidant effects, among others; however, complementary studies are required to verify its safety in clinical use. The article specifies its strengths and limitations by mentioning which is the ideal concentration to avoid cytotoxicity and that the extracts of Peruvian propolis and guava at lower concentrations can work safely in the fibroblast cell line; however, the authors recognize that since this is an in vitro assay, this value may vary if other types of cell lines are used, making a discussion with other studies carried out; they also suggest that both products could present a modulating action on the immune response, opening perspectives for future research in this field.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Natural products and microbiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C Chemokines are a family of small cytokines secreted by cells, and they play essential roles in cell migration, inflammation, and immune responses by binding to chemokine receptors ,2,3,4. T3Mab-3  and C3Mab-4 , were established using the Cell-Based Immunization and Screening (CBIS) method  1–38); and three extracellular loops . Previously, monoclonal antibodies (mAbs) were developed against GPCRs, including an anti-mouse CCR2 mAb , an anti) method . We enco) method ,41. Ther3Mab-3 and C3Mab-4.In this study, the epitope mapping of anti-mCCR3 mAbs was conducted using flow cytometry, utilizing the extracellular region substitution and the alanine scanning methods to clarify the features of C2. The stable transfectants were cultivated in a medium containing 0.5 mg/mL Zeocin .Chinese hamster ovary (CHO)-K1 cells were obtained from the America Type Culture Collection . The CHO/mCCR3 cells were produced in our previous study . The chiSynthesized DNA  encoding mCCR3 (Accession No.: NM_009914.4) ,29,30 an3Mab-4 was established using the CBIS method together with C3Mab-3 [3Mab-7 was established using N-terminal peptide immunization as described previously [C C3Mab-3 . C3Mab-7eviously . An antiThe cells were harvested after a brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid . After washing with 0.1% bovine serum albumin in phosphate-buffered saline, the cells were treated with primary mAbs (1 μg/mL) for 30 min at 4 °C and subsequently with Alexa Fluor 488-conjugated anti-rat IgG . Fluorescence data were obtained using an EC800 Cell Analyzer (Sony Biotechnology Inc.).3Mab-3 and C3Mab-4 were established using the CBIS method, and they are applicable to flow cytometry [3Mab-3 and C3Mab-4, we focused on four extracellular regions of mCCR3, namely, the N-terminal region (aa 1-38), ECL1 (aa 96-111), ECL2 (aa 176-207), and ECL3 (aa 269-285). The four extracellular regions of mCCR3 were substituted into the corresponding regions of mCCR8, which possesses an amino acid structure similar to that of mCCR3. As shown in 3Mab-3, C3Mab-4, and commercially available J073E5 were analyzed using flow cytometry. As shown in 3Mab-3, C3Mab-4, and J073E5 slightly reacted with mCCR8 (mCCR3p1-38). In contrast, they did not react with mCCR8 (mCCR3p96-111), mCCR8 (mCCR3p176-207), or mCCR8 (mCCR3p269-285). They reacted with mCCR3, which was stably overexpressed in CHO-K1 cells. These results show that the N-terminal region of mCCR3 is recognized by C3Mab-3, C3Mab-4, and J073E5.Cytometry . To inve3Mab-3, C3Mab-4, and J073E5 was assessed using flow cytometry. As shown in 3Mab-3 did not react with four mutants . In contrast, C3Mab-3 reacted with the other 32 mutants. These results show that four residues  of mCCR3 are important for C3Mab-3 binding  but reacted with the others . We could confirm the cell surface expression of four mutants  of mCCR3 using C3Mab-7 . These results indicate that C3Mab-6 and C3Mab-7 recognize both the naked N-terminal peptide and cell-surface-expressed mCCR3. Further studies are essential to understand the difference in mCCR3 recognition between C3Mab-3/C3Mab-4 and C3Mab-6/C3Mab-7.Other anti-mCCR3 mAbs, Cnization . It was It has been reported that a CCR3 ligand, CCL11/eotaxin, binds to the N-terminal region of CCR3 ,47. Ther