Despite a range of emerging immunotherapeutic strategies, the aggressive nature of DLBCL poses an ongoing clinical challenge. Therefore, there is a need to pursue more effective treatment modalities based on monoclonal antibodies, antibody-drug conjugates and CAR-T cell therapy. Up to now, the above-mentioned immunotherapeutic options have been extensively studied with the aid of established cell line models, which have significantly facilitated proceeding from preclinical to clinical investigations and led to improvement of DLBCL treatment. However, there are still several important challenges associated with faithful recapitulation of the aggressive nature of DLBCL. Therefore, the current review discusses means in which cell line models fulfill an essential tool leading to greater understanding DLBCL biology and development of novel immunotherapeutic strategies.Despite the high incidence of diffuse large B-cell lymphoma (DLBCL), its management constitutes an ongoing challenge. The most common DLBCL variants include activated B-cell (ABC) and germinal center B-cell-like (GCB) subtypes including DLBCL with MYC and BCL2/BCL6 rearrangements which vary among each other with sensitivity to standard rituximab (RTX)-based chemoimmunotherapy regimens and lead to distinct clinical outcomes. However, as first line therapies lead to resistance/relapse (r/r) in about half of treated patients, there is an unmet clinical need to identify novel therapeutic strategies tailored for these patients. In particular, immunotherapy constitutes an attractive option largely explored in preclinical and clinical studies. Patient-derived cell lines that model primary tumor are indispensable tools that facilitate preclinical research. The current review provides an overview of available DLBCL cell line models and their utility in designing novel immunotherapeutic strategies. The most common non-Hodgkin lymphoma (NHL) subtype, diffuse large B-cell lymphoma (DLBCL), accounts for up to 40% of all lymphoid tumors . Its manIn 2000, Alizadeh et al. identified two main subtypes: germinal center B-cell (GCB)-like and activated B-cell (ABC)-like subtype, with the latter being characterized with significantly worse prognosis . DiscoveA subgroup of DLBCL with MYC, BCL2 and BCL6 rearrangement can be identified which is linked to worse prognosis ,8. DevelSince establishment of the first leukemia-lymphoma (LL) cell lines in 1964  a vast nImmortalized LL cell lines offer a repeatable and reliable diagnostic tool to model primary tumor in the clinical arena around the world . HoweverNoteworthy, cell line models often arise from patients with end-stage, non-nodal, and leukemic phase lymphomas that possess a wide-ranging repertoire of biased mutational sequences . TherefoIt is also common to perform experiments using cell line models representative of other B-cell malignancies and extrapolating them to DLBCL as certain features overlap between them ,27. The DLBCL biology is studied with highly heterogeneous models representative of specific subtypes . Cell liABC-DLBCL, which constitutes around 30% of DLBCL cases, is characterized with a clinically unfavorable outcome as compared to GCB-DLBCL . SeveralUnique features of the available cell lines allow for the choice of tailored preclinical models. For instance, RC-K8 with dysregulated Rel/NF-κB pathway is an appealing candidate for testing novel immunotherapeutic agents for ABC subtype . MoreoveGCB-DLBCL constitutes up to 50% of DLBCL cases and has a GC phenotype defined as CD10+, BCL6+ . AlthougInterestingly, Karpas422, bearing both t and t along with several other abnormalities, is an attractive model to study chemotherapy-resistant NHL as it originated from DLBCL patient unresponsive to consecutive chemotherapeutic schemes .One of the hallmarks of GCB-DLBCL, present in up to 30% of cases, is t translocation, which results in Bcl-2 overexpression ,40. AddiUnique cell line representatives of RS-DLBCL formed on CLL background and secondary DLBCL formed on Hodgkin lymphoma (HL) background have also been developed. RS-DLBCL represents a group of mainly CD5+ and CD23+ large blast-like neoplastic B cells with small nucleoli with diffuse growth pattern and high proliferative potential . Up to 9Generally, aggressive NHL has a worse prognosis in comparison to HL ,47. HoweDespite being an indispensable tool within cancer research, most of the cell lines fail to reliably recapitulate the importance of genetic and microenvironmental factors of tumors in disease progression . Thus, 3Duś-Szachniewicz et al. established 3D spheroid models based on Ri-1 and Raji cells, which were used to test cytotoxicity of doxorubicin (DOX) and ibrutinib (IBR) on B-NHLs . Co-cultYears of RTX employment as well as recent findings on mechanisms of insensitivity to a range of immunotherapies indicate that developing resistance is an unavoidable side effect of the regimens . GeneratRTX-resistant cell lines (RRCLs) were generated by Czuczman et al. from Raji, SU-DHL-4, RL and U-2932 . The celThe mechanisms of potential insensitivity to CD37-targeting agents can also be investigated in cell line models. Accordingly, Arribas et al. generated two DLBCL cell line models (SU-DHL-2 and SU-DHL-4), which were resistant to IMGN529/DEBIO1562 (an anti-CD37 ADC) and demonstrated different phenotypic changes within the models . SU-DHL-The availability of various lymphoma cellular models is vital for testing novel antibodies in preclinical trials. Several B-cell antigens e.g., CD19, CD22, CD37, CD40, CD47, CD79b, and CD80 have been proposed as targets for mAbs mainly for r/r patients .Gehlert et al. used B-ALL cell lines  to test efficacy of CD19-targeting mAb, tafasitamab . The mAbAs demonstrated in a range of preclinical studies with cellular models, the CD37 antigen is widely expressed across multiple types of B-cell lymphoid neoplasms , which pSince it has been shown that CD38 is an important prognostic marker also in DLBCL , preclinFurthermore, Bouwstra et al. demonstrated that high expression of CD47, the so-called “don’t eat me” immune checkpoint, correlated with detrimental effect on OS in non-GCB DLBCL patients after R-CHOP therapy . FurtherBy now, it has been discovered that PD-L1 is aberrantly expressed on HLs and little is known about its role in NHLs . AnalysiAntibody-drug conjugates (ADCs) are monoclonal antibodies bound to cytotoxic payload that is delivered directly to the tumor cells . ImmunolApproval of polatuzumab vedotin (Pola) in 2019, CD79b-targeting ADC, benefited a significant percentage of r/r DLBCL patients . NonetheMoreover, sensitivity of 27 commonly used DLBCL cell lines of ABC and GCB subtypes to MMAE-conjugated ADCs: anti-CD22 (pinatuzumab vedotin) and anti-CD79B ADCs (Pola) was assessed . AlthougFurthermore, a potent in vitro activity against multiple B cell lines  via ADCC, ADCP, and CDC was exerted by naratuximab emtansine (IMGN529), an investigational CD37-targeting ADC conjugated to DM1 . The resIntroduction of T cells modified with chimeric antigen receptors (CARs) has led to a significant advancement in management of multiple malignancies, including lymphoid neoplasms . By now,The great success of anti-CD19 CAR-T cells in B-ALL encouraged preclinical studies on their utility against DLBCL. Interestingly, improved killing of B-cell lymphoma lines, including OCI-Ly2 and OCI-Ly19, Raji, Daudi, DEL (anaplastic large cell lymphoma model), Granta-519 and Jeko-1 (MCL), was triggered by the combination of anti-CD19-CAR-T cells with an anti-CD20-IFN fusion protein indicating that antibody-targeted IFN could improve the CAR-T cell therapy . NoteworFurthermore, promising findings on anti-CD37 mAbs also encouraged exploration of CD37 as a target for CAR-T cells. The panel of several B-cell lymphoma models  and two xenograft mice models were constructed to analyze the cytotoxic effect of CD37CAR .Despite the promising results on CD19 CAR-T cells, around half of high-grade lymphoma patients develops resistance after receiving such treatment . UnfortuBoth Animal models are a powerful tool to test novel immunotherapeutic options prior to clinical investigations . In esseMurine models are commonly utilized in testing efficacy and safety of mAbs, ADCs and CAR-T cells. Xenograft models derived from different cell lines injected subcutaneously  demonstrated significant lymphoma inhibition by XmAb5574, an anti-CD19 mAb with an Fc-engineered domain for effector function improvement. . FurtherThe current review has summarized available cell lines used as powerful tools in investigating DLBCL biology as well as in evaluating therapeutic efficacy of antitumor agents. Despite the wide arsenal of cellular DLBCL models representative of ABC and GCB subtypes, only several are repeatedly used, including SU-DHL-4, SU-DHL-6, OCI-Ly-3, TDM8, or U-2932, to optimize available immunotherapies. To ensure reliable modeling of the primary tumor it is crucial to analyze a genetic background of each model and ensure it remains uncontaminated with foreign material. Importantly, several lines have been authenticated and included in the LL-100 panel .Effective employment of cell line models encourages other research teams to utilize the same model systems, which on the one hand facilitates the reproducibility of the generated data among research centers yet limits the scope of investigations. After all, DLBCL remains one of the most heterogenic lymphoid malignancies and requires multiple cellular models to study its biology. Simultaneously, it is crucial to report failures associated with culturing of specific cell lines to allow for verification and perhaps modifications of the models to overcome culturing challenges. Surprisingly, U-2932 remains a frequently used cell line in DLBCL research regardless of the existing controversies on its complex phenotype, which might perhaps serve as an advantage in development of more universal immunotherapeutics. Furthermore, it remains a common strategy to use other B-cell lymphoma models, especially BT cell lines  to yield results and extrapolate them to DLBCL.Importantly, the wide arsenal of the existing cellular models contributed significantly to the acceleration of the preclinical testing of various immunotherapeutics, which has been observed in the past decade along with approval of a range of mAbs, ADCs, and CAR T. However, years of immunotherapy employment demonstrated that it inevitably leads to development of resistance, which remains an ongoing challenge to overcome. Consequently, an urge to expand even wider arsenal of commonly available patient-derived cell lines and to develop agent-resistant cell lines persists. Furthermore, investment in 3D systems is required to provide insight into efficacy of anti-tumor molecules before proceeding with animal testing. The attempts aiming at optimizing modeling of the primary tumor will benefit in improved optimization of (immuno)therapeutic strategies.


Nanoparticle treatment on tumor cells is proposed for its potential radiosensitizing properties, increasing the radiation effect on tumor cells and reducing the adverse effects on healthy tissues. The present study evaluates, on two cell lines derived from colon and breast adenocarcinomas, the impact of irradiation in the presence of specifically targeted gold nanoparticles. Cells were irradiated in the absence and in the presence of non-functionalized or specifically functionalized gold nanoparticles. The results pointed out that actively targeting gold nanoparticles has a clear radiosensitizing effect in both cell lines.Radiation therapy is widely used as an anti-neoplastic treatment despite the adverse effects it can cause in non-tumoral tissues. Radiosensitizing agents, which can increase the effect of radiation in tumor cells, such as gold nanoparticles (GNPs), have been described. To evaluate the radiosensitizing effect of 50 nm GNPs, we carried out a series of studies in two neoplastic cell lines, Caco2 (colon adenocarcinoma) and SKBR3 (breast adenocarcinoma), qualitatively evaluating the internalization of the particles, determining with immunofluorescence the number of γ-H2AX foci after irradiation with ionizing radiation (3 Gy) and evaluating the viability rate of both cell lines after treatment by means of an MTT assay. Nanoparticle internalization varied between cell lines, though they both showed higher internalization degrees for functionalized GNPs. The γ-H2AX foci counts for the different times analyzed showed remarkable differences between cell lines, although they were always significantly higher for functionalized GNPs in both lines. Regarding cell viability, in most cases a statistically significant decreasing tendency was observed when treated with GNPs, especially those that were functionalized. Our results led us to conclude that, while 50 nm GNPs induce a clear radiosensitizing effect, it is highly difficult to describe the magnitude of this effect as universal because of the heterogeneity found between cell lines. Radiation therapy is one of the most common treatments when fighting neoplasms and almost 60% of patients receiving anti-neoplasic treatment undergo radiation therapy , be it aWith the aim of either diminishing the appearance of undesired side effects or increasing the radiation effect in tumoral cells, radioprotective and radiosensitizing agents can be used. A radioprotective agent reduces the damage produced in healthy cells by limiting the action on DNA of reactive radicals such as reactive oxygen species (ROS), mainly generated by the interaction of ionizing radiations and water . A pioneAn important limitation of some radiosensitizing treatments can be attributed to the unspecific distribution of the radiosensitizer throughout the body. One strategy for overcoming this limitation is the use of micro- or nanoparticles as radiosensitizers because they can naturally accumulate in tumor tissues due to the enhanced permeability and retention effect resulting from the imperfect endothelium of blood vessels formed during tumoral angiogenesis . To incrThe effect of nanoparticles as radiosensitizers has been extensively reviewed ,14,15,162 conditions. Doubling times were determined by counting stained cells with trypan blue  and visualizing by optical microscopy with the help of a hemocytometer (Sigma-Aldrich). Doubling times were 68 h for Caco2 cells and 63 h for SKBR3 cells.In the present study, two different cell lines were used: Caco2 derived from colon adenocarcinoma cells  and SKBR3 derived from breast adenocarcinoma cells (ATCC). Both cell lines were cultured with Eagle’s modified enriched medium  and McCoy’s 5A medium (ATCC), respectively, at 37 °C and in 5% CO® 488  and Goat Anti-Mouse IgG H&L Alexa Fluor® 488 . First, cells were centrifuged and rinsed with 1 × PBS, then 35.000 cells were centrifuged using a Cytospin centrifuge  and fixed to a polysine slide by applying 2% paraformaldehyde onto the sample. After 10 min, the slides were rinsed in 1 × PBS, and then fixed cells were permeabilized using 1 × PBS/0.5% Triton-100 at 4 °C. After 15 min, slides were again rinsed with 1 × PBS before applying a blocking solution (1 × PBS/0.1% Tween20/2% FCS) for 30 min. Then, cells were incubated with the primary antibody overnight in a humid chamber at 4 °C. Once incubation was complete, the slides were washed thrice for 5 min in a 1 × PBS/0.1% Tween20 solution to eliminate a possible primary antibody excess, then the secondary antibody was applied, and the slides were incubated for 1 h in a humid chamber at room temperature. After that, the slides were washed in 1 × PBS/0.1% Tween20 solution as previously described. Finally, 5 μL of Vectashield Mounting Medium  with 4,6-diamidino-2-phenylindole (DAPI) as counterstain at a 1.5 μg·mL−1 concentration was applied.To ensure that both cell lines behaved as expected and expressed the specific receptors (EpCam and TfR), an immunofluorescence staining was carried out. The primary antibodies used were Rabbit Anti-EpCam  and Mouse Anti-TfR , and the secondary antibodies were Goat Anti-Rabbit IgG H&L Alexa Fluor−1 for 4 h. Then, cultures were centrifuged and treated with a hypotonic solution (KCl 0.30 M) for 10 min, then cells were fixed with Carnoy’s solution (1 acetic acid:3 ethanol) and dropped onto slides. For the in situ hybridization, slides were firstly treated with pepsin/HCl (Sigma-Aldrich) (50 μg·mL−1) for 10 min at room temperature, washed twice with 1 × PBS for 5 min, fixed with formaldehyde 4%–PBS1×, and washed again with PBS . After that, slides were dehydrated in ethanol series  and air-dried. Then, hybridization with FITC-labeled pancentromeric and Cy3-labeled pantelomeric PNA probes  was performed. Shortly after, 10 μL of hybridizing solution with both PNA probes, 0.6 μM of the pancentromeric probe, and 0.4 μM of the pantelomeric probe were applied to the slides and covered with a coverslip. Cells and probes were denatured for 3 min at 80 °C and hybridized for 2 h at room temperature using a Vysis HYBrite Hybridization System . After hybridization, slides were washed twice, 15 min each, in 70% formamide–PBS1× and three times, 5 min each, in Tris-buffered saline solution, 1 × TBS/5% Tween20 (both from Sigma-Aldrich). Slides were then dehydrated with ethanol series and dried out, and 20 μL of the previously described DAPI solution were applied as counterstain and slides were observed on an epifluorescence microscope .To know the genomic stability of the cell lines used in the present study, first we analyzed the karyotypes of one hundred cells for each cell line. Fluorescence in situ hybridization techniques using pancentromeric and pantelomeric PNA probes were used to establish the modal karyotypes and to check for the presence of unstable chromosome aberrations. For this purpose, exponentially growing cell cultures were treated with colcemid at a final concentration of 0.3 μg·mLIn the present study, three types of GNPs were used. Citric acid stabilized 50 nm GNPs , from now on, non-functionalized gold nanoparticles (NF-GNPs), commercially Tf functionalized gold nanoparticles (TfGNPs)  and anti-EpCam antibody functionalized gold particles (AntiEpCamGNPs) functionalized with a conjugation kit  according to the manufacturer’s protocol. To measure the ζ-potential, functionalized and non-functionalized GNPs were separately resuspended in water or in EMEM (anti-EpCamGNP) and McCoy’s 5A (TfGNP) culture mediums and sonicated for 5 min  to achieve a monodispersed sample. The ζ-potential was then measured with a Zetasizer Nano ZS .5 nanoparticles·mL−1. Nanoparticle internalization was evaluated 24 h after treatment of the cell cultures with a broadband confocal microscope  located at the Microscopy Service of the Universitat Autònoma de Barcelona. CellMask™ Deep Red  was used to stain cell membranes and Hoechst 33342  to stain cell nuclei. Staining was applied 5 min prior to observation. Due to their reflective nature, no additional staining was required to visualize GNPs.Cell cultures of both cell lines were treated with 50 nm NF-GNPs. Moreover, Caco2 and SKBR3 cell cultures were treated with AntiEpCamGNPs and TfGNPs, respectively. In all cases, functionalized and non-functionalized GNPs, diluted in 1 × PBS, were added to the cell cultures at a final concentration of 7 × 10137Cs irradiator  located at the Technical Unit for Radiation Protection of the Universitat Autònoma de Barcelona. Dose rate was 5.02 Gy·min−1 and the energy peak was 662 keV. Cell cultures were irradiated with gamma-rays 24 h after incubation in the absence or in the presence of nanoparticles (NF-GNPs for both cell lines and TfGNPs for SKBR3 cells and AntiEpCamGNPs for Caco2 cells). Irradiations were carried out at 3 Gy with a To evaluate kinetics of γ-H2AX foci, five different cultures for each cell line were used, one to determine the basal frequency and four at different postirradiation times: 30 min, 2, 4 and 24 h. At each time, three irradiations were carried out for each cell line, one without GNP treatment, another in the presence of NF-GNPs and another with functionalized GNPs . To detect γ-H2AX foci, an immunofluorescence staining was conducted following the same protocol described in the γ-H2AX foci analyses were carried out using an automated scanning fluorescence microscope system  and processed using the MetaCyte software, version 3.10.2,  coupled to a motorized z-stage Zeiss Axio Imager.Z2 microscope (MetaSystems) The images were captured using a 63_PlanApo objective. The foci signals in the selected nuclei were captured using the SpOr filter (red channel). All the SpOr signals were acquired as a z-stack with a total of 10 focal planes and a z-step size of 0.35 μm between planes. A unique classifier was used to count foci in about 2000 nuclei for each experimental condition.4 cells·mL−1 concentration on a 96-well plate and cultured for 48 h before being treated with NF-GNPs, functionalized GNPs , or not treated at all in the case of our control cells. Twenty-four hours after GNP treatment, the culture medium was changed and then cells were irradiated at a 3 Gy dose. To determine the impact of treatments on cell viability, a 3--2,5-diphenyltetrazolium bromide tetrazolium reduction assay (MTT cell viability assay) was conducted following standard procedures 24 h after irradiation . Viability was calculated in relation to that observed in non-treated cells. Three replicates were carried out for each experimental condition.Cells were seeded at 4 × 10p values lower than 0.05 were considered statistically significant.Statistical analysis was conducted with IBM SPSS Statistics 24. Normality was tested in all cases using a Kolmogorov–Smirnov test with the Lilliefors correction. Since all cases showed a non-normal distribution, the statistical test chosen to compare foci frequencies was the Mann–Whitney U test. Viability comparisons were carried out with ANOVA with Tukey’s multiple comparison test. As expected, both cell lines expressed the cell membrane receptors (EpCam for Caco2 and TfR for SKBR3) described in the literature , confirmThe cytogenetic analysis by pancentromeric and pantelomeric fluorescence in situ hybridization  was carrThese results indicate that instead of the variability in the number of chromosomes, the cell lines are stable regarding the formation of unstable structural chromosome aberrations.In A qualitative assessment of GNP internalization through confocal microscopy imaging allowed us to detect certain differences between the cell lines . As can p < 0.001).The cytogenetic characterization did not show structural chromosome aberrations, but due to the variability in the number of chromosomes, about 2000 cells were analyzed for each different postirradiation time and GNP treatment. Microscope images of γ-H2AX foci in Caco2 and SKBR3 cells are shown in p < 0.01). For SKBR3 , the difTo know if the presence of GNPs could have any effect on the rate of foci disappearance with postirradiation time, foci decay with postirradiation time has been adjusted to a one-phase decay function, obtaining the constant of the decay rate and the foci half-life estimation for each experimental condition . For Cacp < 0.05). Moreover, for both cell lines the decrease in viability was more pronounced for cells irradiated in the presence of functionalized GNPs but was only significant for Caco2 cells (p < 0.05). After irradiation, SKBR3 cells always showed significantly lower viabilities than Caco2 cells (p < 0.05). The sensitization enhancement ratio for viability was also higher than one for functionalized particles  ,35 can dThe clonogenic assay has been used to measure the radioinduced cell death or loss of reproductive potential, though it has limitations such as the need for cell differentiation and the intrinsic variations in survivability between different cell types ,49. On tIn our study, working with two different tumoral cell lines has helped us to achieve a better knowledge of the nanoparticles’ radiosensitizing effect in different cell models. First, to know if the cells used were genetically unstable, we evaluated the chromosome stability of both cell lines, and although the chromosome number was variable in both cell lines, no cells with chromosomes lacking telomere signals were observed, indicating no structural chromosome instability and hence no influence in the posterior foci counts. The radiosensitizing effect of GNPs is attributed, in part, to the increase in ROS production with irradiation, but in the absence of radiation, GNPs increase the intracellular ROS level ,25,57,58In our study, we chose nanoparticles of 50 nm in diameter because previous studies indicated an increased cell uptake for GNPs of 50 nm in diameter in relation to 14 nm and 74 nm  and it hAnother factor, different to nanoparticles properties, to consider in studies of GNP radiosensitizing effect, is the intrinsic uptake capacities and intrinsic radiosensitivity of the cell lines. Not all cell types show a similar nano- or microparticle uptake capacity. SKBR3 cells can phagocytose 3 µm polysilicon–chromium–gold microparticles , 3 µm poRegarding the effect of GNPs in SKBR3 and Caco2 cell cultures without irradiation, we observed that the presence of NF-GNPs or functionalized GNPs did not significantly change the basal frequency of γ-H2AX foci or the cell viability when compared to cells grown without GNPs. This does not mean that the presence of GNPs does not have any effect on cells, because a slight decrease in cell viability, though not statistically significant, was observed when GNPs  were present in the cell culture. This agrees with studies describing an increase in ROS by the presence of GNPs without irradiation ,25,57,58After irradiation, the γ-H2AX foci frequency was evaluated at four different postirradiation times . For Caco2 cells, significant increases in foci frequencies were observed for cells irradiated in the presence of AntiEpCamGNPs when compared with cells irradiated in the presence of NF-GNPs or in the absence of GNPs. For these cells, the sensitization enhancement ratios (SERs) for foci obtained for AntiEpCamGNPs when compared with NF-GNPs or with irradiation without GNPs were, in general, close to the values described in the literature . MorFinally, when foci frequencies of both cell lines are compared, in all experimental conditions lower frequencies were observed in SKBR3 cells. This could be related to an elevated resistance to oxidative stress, as described for cells from breast cancer patients . HoweverAlthough high Z metallic nanoparticles are currently the most used for radiosensitizing, other non-metallic nanoparticles are showing promising results. In a study by Yamaguchi et al. , siliconDespite the radiosensitizing effect of gold nanoparticles previously described , the resEven with the differences in the results of the present study between the two cell lines, gold nanoparticles remain an interesting radiosensitizing agent and a good candidate for future in vivo application in the treatment of certain types of tumors. Active targeting of GNPs has demonstrated its effectiveness to increase the genetic damage and to decrease the cell viability. This GNP radiosensitizer effect should be explored in further studies to find more effective radiation therapies against different tumor types.


Medicinal plants are regarded as a large source of phytochemicals that may have anticancer properties. This could lead to the development of innovative drugs or alternative therapy against cancer.Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea.This study was designed to determine the antioxidant and cytotoxicity effect of 5 selected indigenous South African medicinal plants namely; Phytochemical extracts namely; methanol, 50%, 100% ethanol, and water extracts were prepared from the root and shoot of the plants. The antioxidant effect of methanol extracts of the plant materials was performed using a DPPH assay. A preliminary cytotoxicity screening of the phytochemical extracts in the human colon (Caco-2), cervical (HeLa), and hepatocellular (HepG2) cell lines were determined followed by the half-maximal inhibitory concentration (IC50) using MTT assay.B. natalensis and B. frutescens (33.20% and 26.33% respectively) and shoot extract of K. uvaria (17.10%) showed the highest antioxidant. Out of the 5 plants, only 100% ethanol extract of C. comosum, K. uvaria, and T. violacea caused more than 80% cytotoxicity in HepG2 and Caco-2 cell lines. The shoot of B. frutescens (10.43 µg/ml), K. uvaria (23.0 µg/ml), and root of C. comosum (23.77 µg/ml) were the most active with the highest cytotoxicity.The methanol root extract of C. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon and liver cancers. Our results provided new pieces of evidence for antioxidant and cytotoxic activities of these plants which could be useful for developing new anticancer therapies. Cancer is a life-threatening disease that severely affects the human populationSouthern Africa has over 30000 species of higher plants with most of them possessing medicinal properties which are promising for further development and commercializationThese South African indigenous plant species come from different families such as Asphodelaceae, Alliaceae, Hypo xidaceae, Agavaceae and Asteraceae. For centuries, these indigenous medicinal plants have been adopted for primary health care and treatmentsSecondary metabolites that are derived from plants are being investigated for the development of new clinical drugs to treat cancerMedicinal plants commonly known as herbs, pharmacologically active plants remain the dominant form of medicine in most countries.These plants are regarded as a large source of therapeutic phytochemicals (phenolic and flavonoids) that may lead to the development of innovative drugs against cancerBulbine frutescens, Bulbine na talensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea were selected and tested for antioxidant and cytotoxicity activities. These selected medicinal plants have been used as traditional medicines for decades against diseasesBulbine frutescens, Bulbine natalensis, and Chlorophytum comosum cytotoxicity activities in human colon (Caco-2) and cervical (HeLa) cancer cell lines. Similarly, more information is needed regarding cytotoxicity activities of Kniphofia uvaria and Tulbaghia violacea, particularly, against hepatocellular (HepG2) cancer cell lines. Hopefully, our results may provide new information for anticancer drug development from these plants, particularly, against the aforementioned cancer cell line.In this present study, 5 indigenous South African medicinal plants, namely; Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea were obtained from Shadowlands Wholesale Nursery at Kuilsriver, Cape Town, South Africa. Plants were transplanted in potting soil and kept in an environmentally controlled greenhouse for steady and healthy growth. After 12 weeks, the shoot and root of the plants were removed individually from all selected plants. The plant materials were washed thoroughly with distilled water to ensure the removal of all the dirt, air-dried at 28°C for 4–8 days and then oven-dried at 48°C to remove residual moisture content.The dried plant materials were blended to a fine powder and stored at room temperature. Extracts were prepared as per methods described by Sousa et al.2 humidified incubator . The cells were seeded at 1 x 105 cells/ml density in a 96-well plate and incubated at 37 °C for 24 hrs. The viability of cells was evaluated by direct observation under the EVOS XL core imaging system .The human Caco-2, HeLa, and HepG2 cancer cell lines were obtained from Biolabels Node, University of Western Cape, Cape Town, South Africa. The cell cultures were prepared as previously described by Matsushita et al.2.All cell lines were maintained in a humidified incubator at 37°C in an atmosphere of 5% COThe antioxidant activity of the selected plants was measured in terms of radical scavenging ability using the stable radical DPPH as previously described by Kedare and SinghThe mitochondrial activity of cells is measured using a spectrophotometer through the reduction of a yellow-coloured MTT salt to purple formazan crystals by the enzyme succinate tetrazolium reductase or succinate dehydrogenase, present only in metabolically active cells. The greater the purple colour of the reaction, the less the extent to which a particular compound (crude fraction) induces cell death in the specific reaction. The cytotoxicity of the plant extracts in the cell lines was measured by two rounds of screening procedures in succession using MTT assay as previously described by Liang et al.An extract showing more than 80% cell cytotoxicity on at least 1 human cancer cell line in the preliminary screening was put into the secondary screening to assay its dose-dependent cytotoxic effects on the human cancer cell line. The half-maximal cytotoxic concentration of the active compounds that caused 50% cell growth inhibition (IC50) was investigated was performed based on the method previously described by Liang et al. B. frutescens, B. natalensis, C. comosum, K. uvaria, and T. violacea was determined through the percentage of DDPH inhibition by the plant extracts in the assay. The antioxidant activity through DPPH inhibiton assay of the 900 µg/ml methanol plant extracts is shown in B. natalensis (33.20%) and B. frutescens (26.33%) were found to exhibit the highest antioxidant in comparison to the root extract of other plants. Both C. comosum (10.33%) and K. uvaria (9.60%) showed a low level of antioxidant index, although it was significantly higher (p<0.05) when compared with T. violacea extracts (5.06%). In the shoot extract, the result showed that the free radicals were inhibited the most by K. uvaria (17.10%) and C. comosum (15.13%) compared to T. violacea (8.43%), B. natalensis (8.16%), and B. frutescens (7.86%).The antioxidant of the root and shoot methanol extracts of Experiment I: Selection of cytotoxicity above 80% cell growth inhibition.B. frutescens, B. natalensis, C. comosum, K. uvaria and T. violacea in Caco-2, HeLa and HepG2 cell lines using MTT assay. It was observed that only 100% ethanol extract of 3 plants  caused more than 80% cytotoxicity against HepG2 and Caco-2 cell lines as shown in C. comosum (90.47%) and T. violacea (90.20%). It was only C. comosum (root) that inhibits growth (88.20%) of Caco-2 compared to other root plant extracts. For the100% ethanol (shoot) extracts, K. uvaria inhibits (85.43%) growth of Caco-2. In HepG2, T. violacea and K. uvaria caused high cytotoxic activity (85.43% and 84.43%) respectively, while that of B. frutescens (71.56%) and B. natalensis (72.87%) cytotoxicity was high but not above 80%. It was noted that none of 50% ethanol and aqueous extracts of the selected 5 plants showed above 50% cytotoxicity against the cell lines. Also, none of the plant material extracts resulted in a cytotoxic effect > 80% against the HeLa cell line. To further confirm the cytotoxicity of plant extracts showing more than 80% cytotoxicity in the cell lines, we assayed all the extracts against HepG2 which was most sensitive to the 100% ethanol plant extract to determine the dose-dependent half-maximal cytotoxic concentration and the most active plant extracts that caused 50% cell growth inhibition (IC50). The 100% ethanol extracts for all the plants showed a stronger cytotoxic effect against HepG2 cells except for B. natalensis. The 100% ethanol extracts for B. frutescens (shoot), K. uvaria (shoot) and C. comosum (root) were the most active with IC50 = 10.43 µg/ml, IC50 = 23.0 µg/ml and IC50 = 23.77 µg/ml respectively exhibiting the highest cytotoxicity as shown in B. frutescens (root) IC50 value was 32.72 µg/ml and it was the only 50% ethanol extract amongst all the plant extracts to exhibit cytotoxic effect with IC50 < 50 µg/ml. The shoot of T. violacea was shown to have significant cytotoxicity with IC50 < 50 µg/mlA preliminary assay was performed to evaluate the cytotoxicity effect above 80% cell growth inhibition of 50%, 100% ethanol, and aqueous extracts of the roots and shoots of Bulbine frutescens and Bulbine natalensis showed high antioxidant ativities compared to the extracts from the shoot of both plants and the root and shoot extracts of T. violacea with low antioxidants. This possibly means the root part of the Bulbine sp contains sensitive phytochemicals capable of scavenging free radicals. A previous study reported that B. frutescens contain secondary metabolites such as quinones and glycoside which are potential anticancer agentC. comosum showed high antioxidant activity. In contrast, a previous study reported the root methanolic extract of C. comosum exhibited a strong and significant antioxidant activityC. comosum root extract in comparison to the ethanol and aqueous leave extracts. The concentration of the methanol solvents could affect the phenolic content which is a strong antioxidant agent in plant extractsB. frutescens (root and shoot), K. uvaria (shoot), and C. comosum (root) were confirmed the most active plant extracts showing the great cytotoxic effect (> 80%) and the IC50 values were between 10.0 – 35.0 µg/ml in HepG2. However, we noticed that T. violacea recorded high cytotoxicity with IC50 = 45.61 µg/ml. 16reported that methanol extract of B. frutescens induced reactive oxygen species (ROS) by 7%n cancer cell lines when compared to the untreated cells. Although, cancer cells may develop defense mechanisms against antioxidants and anti-proliferative agents which counters the effect of elevated ROS. Excessive production of ROS and potential changes in mitochondrial membrane is associated with the initiation of cell deathB. frutescens methanol and hexane extracts induced cell cycle arrest, ROS production, apoptosis, and mitochondria membrane potential disruption in breast cancer cellsBulbine sp induced potent cytotoxicity between 70 – 72% with B. frutescens IC50 value < 50 µg/ml compared to B. natalensis > 50 µg/ml. B. natalensis and B. frutescens induced a cytotoxic effect in the human laryngeal carcinoma cell line, however, the expression of caspase-3 which indicates apoptotic pathway was induced in shoot and leaf ethanol extracts except for the root of B. frutescens. Our study corroborates with previous studies which showed root extracts of C. comosum possess anti-proliferative activity against many human cancer cell lines. C. comosum. Saponins isolated from roots of C. comosum exhibited cytotoxic and antitumour promoter activity through induced apoptosis in selected cancer cell linesK. uvaria. The shoot extract of K. uvaria presented the highest antioxidant value compared to the other plants while the root extracts showed moderate antioxidant activity. K. uvaria is one of the most traditional medicinal herbs used for antimalaria and anti-inflammation but so far there is no research on the bioactivities related to its chemical components. We noticed there is little or no literature report on K. uvaria, however, K. foliosa through its derivative compounds, knipholone and knipholone anthrone demonstrated poor and potent antioxidant activity respectivelyK. foliosa contain anthraquinones, flavonoids, and alkaloids which have anticancer activitiesT. violacea induced a high cytotoxic effect in HepG2 and Caco-2 when compared to HeLa cell lines. T. violacea inhibited the growth of HeLa and HT-29 (colon cancer) cell lines. This was similar to our result which also showed a moderate degree of inhibition in both HeLa and Caco-2 cell lines. However, our study showed that the inhibition was high in the Caco-2 compared to HeLa as opposed 13 report showing high inhibition in HeLa in comparison to HT-2922. This could plausibly be the effect of the different solvent types used to extract the phytochemicals. Phytochemical compounds present in different parts of T. violacea may vary and these include tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins41In this present study, we evaluated the antioxidant effect of the phytochemicals that scavenged free radicals in the DPPH assay. The root methanolic extract of T. violacea exhibited apoptotic effect, although through a compound moiety-methyl-α-D-glucopyranoside which serves as a potential anticancer agent by interfering with hexokinase to inhibit the induction of ROST. violacea in HepG2 compared to other plants or cell lines could suggest that cytotoxicity was induced by an apoptotic pathway that could be caspase-3 dependentT. violacea extracts and cell lines to text its cytotoxicity is an important factor to always consider for future studiesWe noticed the aqueous extract has little or no cytotoxic effect on the selected cell lines. Previous studies showed that the aqueous extract from B. natalensis, C. comosum, K. uvaria and T. violacea against the selected human cancer cell lines implies the potential therapeutic source which can be utilized towards the development of new anticancer drugs. It is evident from this study that C. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon (Caco-2) and liver (HepG2) cancers. This study provides new insight into the potential bioactive properties, most importantly the antioxidant activity of K. uvaria. In general, different cell lines respond differently to the same compound. So it remains unclear whether similar results would emerge if other cell lines were used alongside the Caco-2, HeLa, and Hep-2 cell lines, hence further studies will be needed to unveil it. The report of Singh and ReddyBulbine spp. fractions that could. be used to further validate their findings. It is believed that our study has provided more information in addition to the existing data regarding the cytotoxicity of these plants. Phytochemical screening of the fractions may be required to identify the cytotoxic compounds in the fractions before this result can be extrapolated on animal models to study the in vivo effects. This ethnomedicinal knowledge could enhance the efficacy of the already existing or little ethnomedicinal use of these plants.The invitro antioxidant and cytotoxicity activities of B. frutescens, It is important to mention that further studies are needed to investigate the possible specific targets involved in the cytotoxic pathway of each plant extracts using molecular techniques.


Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex  in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models. Epigenetic modifications regulate gene expression and are altered by developmental and environmental cues . Strict CRISPR/Cas9 technology is a fast, effective, and easy-to-use genome engineering method and has drastically accelerated functional genomics research . Its simEpigenetic Knockout Library (EPIKOL), which targets a broader range of epigenetic modifiers and consists of more sgRNAs for each gene when compared to previously published libraries . Al. Al34]. To uncover epigenetic modifiers important for cancer cell fitness, we conducted negative selection (drop-out) screens using EPIKOL. Three different triple negative breast cancer (TNBC) cell lines MDA-MB-231, SUM149PT and SUM159PT were screened in addition to non-malignant human mammary epithelium cells (HMLE) . Similarp-values, log fold changes and gene rankings in different screens. 2 sgRNAs per gene were cloned individually into lentiGuide-puro vector and a dual-color competition assay was performed in all TNBC cell lines  is the catalytic member of the complex and acetylates histone lysine residues [SS18 gene, which is associated with chromosomal translocation characteristics of synovial sarcoma. However, the exact role of SS18L2 in synovial sarcoma or any other cancer is not known [To further delineate the effects of novel epigenetic modifiers that regulate cell fitness, four of the TNBC specific genes  were selected based on their strong depletion scores in MDA-MB-231 Fig. . Three oresidues . SS18L2 ot known . NSL comTo identify the mechanism through which cancer cell fitness is reduced, we first investigated whether knockout of the hit genes result in apoptosis. Extensive cleavage of PARP was observed with all four gene knockouts, indicating induction of apoptosis Fig. . SimilarTo assess the performance of EPIKOL in vivo, we performed an in vivo screen by using Firefly Luciferase (Fluc) expressing MDA-MB-231-Cas9 cell line. Following transduction and puromycin selection, cells were subcutaneously injected into Nude mice, and tumors were collected at weeks 2 and 4 post-implantation Fig. . Gene-leTo gain a mechanistic understanding for the essential role of SS18L2 in TNBC cells, we analyzed the transcriptomic changes caused by the knockout of SS18L2. We performed RNA sequencing comparing MDA-MB-231-Cas9 cells expressing control and SS18L2 sgRNAs. SS18L2 knockout resulted in 1283 upregulated and 255 downregulated genes Fig. . AverageIn this study, we present a focused epigenetic knockout library (EPIKOL) that can be utilized to investigate chromatin-based vulnerabilities in different biological contexts. We performed eight in vitro screens in two cancer types and identified novel chromatin modifiers that regulate prostate and triple-negative breast cancer cell fitness. In contrast to most currently available epigenome-focused libraries, which only target chromatin modifiers such as writers, readers, and erasers –42, EPIKAvailability of EPIKOL in LentiCRISPRv2 backbone might expedite the screening process by eliminating the need for prior Cas9 introduction especially in patient-derived xenograft models and primary cell lines, in which the culturing time of the material is limited. In such cases, smaller library size will also reduce the amount of initial material required to maintain the complexity. Another advantage of EPIKOL is the presence of sgRNAs targeting context-specific control genes from different families such as nuclear receptors, transporters and EMT-related proteins. For example, Androgen Receptor (AR) targeting sgRNAs were significantly depleted in AR-dependent prostate cancer cell lines LNCaP and 22Rv1 while no change was observed in AR-negative cell line DU145 and TNBC cell lines suggesting that EPIKOL can distinguish tissue or cell line specific hits.PRMT family, exosome complex and MLL complexes, highlighting the role of these epigenetic complexes as pan-cancer essential epigenetic modifiers. Some of these were previously identified as common essential genes in Cancer Dependency Map (DepMap) based on their significant depletion in almost 750 different cancer cell lines [ASH2L as a common essential gene in both cancer types through EPIKOL screens, but it was not classified as a common essential gene in previously performed screens. EPIKOL screen can therefore identify pan-cancer epigenetic vulnerabilities as well as cell line and cancer-specific ones. To test the performance of EPIKOL in vivo, we performed a screen using MDA-MB-231-Cas9-Fluc cells at two different timepoints. As expected, overall depletion scores and number of depleted positive control genes increased from week 2 to week 4. 9 out of 10 commonly depleted genes from both timepoints of the in vivo screen were shared with the TNBC in vitro screens. TFDP1 was identified as an in vivo specific cell fitness gene. These results indicate that EPIKOL efficiently works in vivo and can recapitulate the findings of in vitro screens.From drop-out screens in multiple cell lines, we identified novel epigenetic modifiers for cancer cell fitness as well as the previously studied ones such as PRMT5, HDAC3, FOXA1 and LSD1 , 59, 60.ll lines . NotablyWe identified a group of epigenetic modifiers that belong to NSL complex as TNBC-specific cell fitness genes. Core members KANSL2 and KANSL3 were depleted in three different TNBC cell lines but they did not show significant effects in HMLE cells. Importantly, KAT8, the catalytic subunit of the NSL complex, was also a common hit in MDA-MB-231 and SUM149PT cell lines. We showed that TNBC cells were dependent on NSL complex using several functional assays. NSL complex was thought to regulate H4K16Ac which is necessary for chromatin relaxation and present at active enhancers and promoters , 71. HowSS18L2 is a homolog of SS18, which is found in ATP-dependent chromatin remodeling complex BAF (SWI/SNF) and an uncharacterized gene [SS18L2. Knockout of SS18L2 markedly impaired tumor growth confirming that SS18L2 is required for TNBC cells in vivo. Transcriptomic analyses showed that SS18L2 regulates cell cycle-related pathways and genes that function during the late G2/M phase. For example, the DREAM complex activates late G2/M genes through its interactions with MYBL2 and FOXM1, both of which are downregulated upon SS18L2 suppression [In addition to the NSL complex, we observed SS18L2 to be significantly depleted in all TNBC EPIKOL screens. Competition assays confirmed the strong effect of SS18L2 knockout on TNBC cell survival. zed gene . Togethezed gene . The rolpression , 79. In pression . In the pression –42.In conclusion, we generated and validated a focused epigenetic sgRNA library that enables identification of critical epigenetic modifiers both in vitro and in vivo. Epigenetic modifying enzymes are promising therapeutic targets as they regulate numerous critical cellular responses including cell growth, metastasis, apoptosis and others. The relatively small library size both allows for loss-of-function screens where cell numbers are limited, and also provides a focused perspective for hit prioritization. EPIKOL is therefore a robust functional genomics platform to interrogate chromatin modifiers and can guide the discovery of cell-type specific epigenetic vulnerabilities of cancers.Co-author agreement documentSupplementary informationFULL LENGTH WESTERN BLOT IMAGESSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Reproducibility checklist file


In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition,  Liver cancer is one of the most frequent malignancies and has poor prognosis, ranked as the third leading cause of cancer-related deaths worldwide and thus remains a global health issue . The inc7G-cap structure at the mRNA ‘5’ end, whereas eIF4G acts as a scaffold protein recruiting eIF4E and eIF4A. The latter, the only component of the eIF4F complex with enzymatic activity, is an ATP-dependent DEAD-box RNA helicase that unwinds the mRNA secondary structures in the 5′ untranslated regions (5′-UTRs) to enable ribosome scanning. The helicase eIF4A is required to efficiently translate mRNAs with long and highly structured 5′-UTRs, including those with G-quadruplex structures benzofuran core that suppresses cap-dependent translation by inhibiting eIF4A activity. More than one hundred natural rocaglates have been isolated from lability ,33,34. Tlability ,36, and lability . Silvestlability ,39, and lability ,41,42,43lability . Furtherlability .In this study, we investigated the pathogenetic role of eIF4A1 in human HCC and its potential usefulness as a novel molecular target for the development of novel pharmacological strategies.eIF4A1 mRNA levels in human cancers were investigated using data obtained from The Cancer Genome Atlas (TCGA) and the UALCAN  analysis tool. eIF4A1 mRNA expression was elevated in various cancer types, including colon adenocarcinoma (COAD), glioblastoma multiforme (GBM), esophageal carcinoma (ESCA), and kidney renal clear cell carcinoma (KIRC), among others , eIF4A1 was significantly overexpressed (−10) with comparable incidences for women (P = 2.566 × 10−10) and men  compared to that in normal tissue. Maximal eIF4A1 expression was detected in tumors of young patients between 21 and 40 years of age (−02). Examination of multiple clinicopathological features revealed that HCC patients with high eIF4A1 expression had poorer prognoses. The survival time of patients with high eIF4A1 expression was significantly shorter than that of patients with low or medium eIF4A1 levels . The analysis of HCC cancer stages . Importantly, high eIF4A1 expression was found in N0 and N1 nodal metastasis status , with a clear tendency toward enhanced eIF4A1 expression in N1 specimens.First, g others A. In HCCp = 0.0002; Wilcoxon test; p = 0.0249; Wilcoxon test) and the translation initiator and rocaglate target DEAD-box protein 3 DDX3 [p = 0.0088; Wilcoxon test) in HCC lesions  by immunohistochemistry . In agreOverall, the present data indicated that eIF4A1 is highly expressed in primary HCC lesions and associated with a poor outcome. These findings suggest the usefulness of eIF4A1 as a prognostic biomarker in human HCC.Next, to evaluate the therapeutic potential of targeting eIF4A1 activity in HCC, we challenged human HCC cell lines in vitro with five distinct rocaglate derivatives, namely CR-1-31-B, FL3, Rocaglamide, Silvestrol, and Zotatifin. These drugs promote the binding affinity of eIF4A1 to mRNA species containing polypurine sequence motifs, sequentially impeding the translation initiation during protein synthesis by blocking the formation of the heterotrimeric eIF4F cap-binding complex. Prior to pharmacological treatments, we assessed the endogenous eIF4A1, eIF4A2, and DDX3 protein levels using western blot analysis in various human HCC cell lines. eIF4A1, eIF4A2, and DDX3 proteins were expressed in all HCC cell lines tested .Subsequently, to assess the cytotoxic potential of eIF4A inhibitors, the HLE, HLF, and PLC/PRF/5 tumor cell lines were randomly selected and treated with a range of concentrations between 0 and 200 nM per eIF4A inhibitor, and colorimetric MTT assays were performed 48 h after starting compound exposure. Principally, cellular metabolic activity was measured to indicate cell viability, proliferation, and cytotoxicity. Overall, all five rocaglates efficiently reduced tumor cell viability in the three cell lines in a dose-dependent manner A–C. NotaTo further elucidate the mechanisms of action of eIF4A inhibitors in HCC cell lines, we selected the two most effective drugs, i.e., CR-1-31-B and Zotatifin, for the following experiments. Zotatifin was also selected because, to date, it is the only eIF4A inhibitor in clinical trials. When evaluating cell proliferation, we found that incubation with CR-1-31-B and Zotatifin induced a marked reduction in BrdU incorporation in both HLE and HLF cell lines, with a slightly more pronounced anti-growth effect in the case of CR-1-31-B A,B.Subsequently, apoptosis was determined in the same cell lines treated with the two eIF4A inhibitors. Both CR-1-31-B and Zotatifin induced higher apoptotic cell death in the cell lines than was observed in untreated and DMSO-treated cells A,B.Next, we assessed the effects of CR-1-31-B and Zotatifin on some of the most relevant oncogenic pathways in hepatocarcinogenesis by western blot analysis . Both drInspired by the findings that eIF4A inhibitors did not reduce the activation of mTOR effectors, we tested whether mTOR suppression could synergize with eIF4A inhibitors to hamper HCC cell growth. For this purpose, the HLE and HLF cell lines were treated with the pan mTOR inhibitor, Rapalink-1 , either Subsequently, we assessed proliferation and apoptosis in the same cell lines using Rapalink-1 alone and in combination with CR-1-B-R1 or Zotatifin. Again, the combined treatments resulted in a significant reduction in proliferation A and a rFinally, western blot analysis confirmed that the mTOR inhibitor, Rapalink-1, alone and in combination with the eIF4A inhibitors, CR-1-31-B or Zotatifin, fully inhibited p-RPS6 and p-4EBP1 levels. In addition, Rapalink-1 alone resulted in the downregulation of eIF4A1, eIF4A2, and DDX3, whereas inconsistent results were obtained for these proteins when Rapalink-1 was combined with CR-1-31-B or Zotatifin . These dHCC, the most common liver primary cancer, is characterized by its late diagnosis, increasing incidence worldwide, and limited treatment options . DespiteCumulating evidence indicates that translation dysregulation is critical to tumor development and progression . TherefoFurthermore, we revealed that targeting eIF4A1 using rocaglates induced strong growth suppression of human HCC cells by inhibiting proliferation and triggering apoptosis. Notably, the growth inhibitory activity of these compounds on HCC cells occurred at low nanomolar concentrations, implying their potency, at least in vitro. These data, confirming previous findings obtained in different tumor types, support the targeting of eIF4A1 as a promising therapeutic strategy against HCC .At the molecular level, we discovered that various oncoproteins belonging to the JAK/STAT, ERK/MAPK, AKT, and Hippo/TAZ pathways are markedly downregulated by eIF4A inhibitors. On the other hand, we revealed that treatment with these compounds did not affect the activity of members of the mTOR signaling cascade. Strikingly, exposure of HCC cells to combinations of eIF4A inhibitors and the mTOR inhibitor, Rapalink-1, a third-generation mTOR inhibitor that links rapamycin and MLN0128 , resulteSer235/236 and p-4EBP1Thr37/46, even in the presence of eIF4A inhibitors. Hence, we suggest that a combinatory therapy with eIF4A inhibitors and Rapalink-1, a drug that was originally developed to overcome mTOR resistance mutations, might serve as a refinement strategy for systemic management of HCC by simultaneously and effectively targeting both mTOR signaling and eIF4A-dependent translation. Overall, the present data indicate that eIF4A is a potential prognostic biomarker in HCC patients and a promising therapeutic target in this deadly tumor type. In particular, eIF4A inhibitors displayed formidable cytostatic activity against HCC cells in vitro, further augmented in combination with an mTOR inhibitor. Although the antineoplastic effectiveness of these drugs, alone and in combination, requires validation in vivo, the present data suggest that targeting translation initiation can provide an innovative and valuable strategy in the development of novel treatments against human HCC.Moreover, this is the first study to demonstrate in the setting of HCC that Rapalink-1-induced inhibition of mTOR signaling can be achieved through suppression of two key downstream substrates, p-RPS6n = 25) and longer survival/better outcome , characterized by <3 and >3 years’ survival following partial liver resection, respectively. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Medical University of Regensburg .Forty-seven human HCC tumor tissue samples and paired surrounding non-tumorous tissues were collected at the Institute of Pathology at the University of Regensburg . Patient features are summarized in 2 humidified atmosphere. HLE, HLF, and PLC/PRF/5 cell lines were maintained in Dulbecco’s Modified Eagle Medium , and SNU182 and SNU449 cell lines were maintained in RPMI 1640 (Anprotec) supplemented with 10% (v/v) FCS , 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin/streptomycin solution . Mycoplasma-free status for all cell lines was monitored regularly using the PCR Mycoplasma Test Kit I , and cell line authentication was performed by Cell Lines Service . CR-1-31-B , FL3 [The human HCC cell lines HLE , HLF , PLC/PRF/5 , SNU182 , and SNU449  were cultured at 37 °C in a 5% CO-3), FL3 , Rapalin4 cells per well. After an overnight attachment period, cells were exposed to various concentrations of CR-1-31-B, FL3, Rocaglamide, Silvestrol, Zotatifin, and Rapalink-1 alone or CR-1-31-B-Rapalink-1 and Zotatifin-Rapalink-1 combinations for 48 h. Cells treated with DMSO and wells containing only culture medium served as negative and background controls, respectively.Cells were seeded in flat-bottom 96 well plates at a density of 10 x 102 for 2 h. The medium was fully aspirated, and 100 µL of 100% (v/v) DMSO was added per well to dissolve the formazan crystals. After 15 min of shaking at room temperature, the absorbances were measured at 570 and 630 nm using the FLUORstar Omega multiplate reader and MARS data analysis software . All experiments were performed in triplicate or quadruplicate and repeated at least twice. The average absorbance of the DMSO-treated cells was defined as 100% cell viability. IC50 values and standard deviations were calculated using GraphPad Prism software. Average cell viability values of three independent MTT assay experiments were employed to determine dose-effect curves (Fa = fraction affected) and the combination indices of two drugs using CompuSyn PC software version 1 .For the MTT assay, following treatment, 10 µL of 5 mg/mL methyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) solution was added per 96 well and incubated at 37 °C and under 5% COCell proliferation was evaluated in the cell lines at the 48-h time point using the BrdU Cell Proliferation Assay Kit . Briefly, following drug treatment, cells were incubated with 1× bromodeoxyuridine (BrdU) for 2 h and fixed for 30 min at room temperature. The fixing solution was discarded, and cells were incubated with the BrdU mouse detection antibody for 1 h at room temperature. After washing, cells were stained with HRP-conjugated anti-mouse secondary antibody for 30 min at room temperature and washed again. After incubation with TMB substrate solution for a further 30 min at room temperature, stop solution was added, and the absorbance was measured at 450 nm. The results are expressed as fold-change over DMSO control.Apoptosis was determined in the HCC cell lines using the Cell Death Detection Elisa plus Kit , following the manufacturer’s instructions. All cell line experiments were repeated at least three times in triplicate.TM lysis buffer  containing 1x HaltTM Protease and Phosphatase Inhibitor Cocktail  in the Bullet Blender Storm 24  for 3 min at speed 8, placed in the rotator mixer for 30–60 min at +4 °C and sonicated. Lysis of pellets from the cell lines was performed in the same lysis buffer for 1 h on ice. Protein concentrations were determined using the colorimetric BioRad Protein Assay Dye Reagent Concentrate  with bovine serum albumin (BSA) as the standard.Human HCC tumor and non-tumorous liver tissues were homogenized in 100–250 µL of T-PERTM sample buffer (B0007) and 1× BoltTM reducing agent  by boiling for 5 min at 95 °C. Samples with 10 µL of protein per lane were separated by SDS-PAGE on Bolt™ 4–12% Bis-Tris Mini Protein Gels (NW04125BOX) in 1× Bolt™ MES SDS Running Buffer  and transferred onto nitrocellulose membranes  by electroblotting running program P0 in the iBlot™ 2 Gel Transfer Device . Membranes were blocked in EveryBlot Blocking Buffer  and probed at +4°C overnight with specific antibodies: eIF4A1 ; eIF4A2 ; DDX3 ; p-RPS6Ser235//236 (4856), p-4EBP1Thr3747 (2885), p-STAT3Tyr705 (9145), AKT (4691),), ERK1/2 (4695), p-ERK1/2Thr202/204 (4370), SKP2 (2652), YAP/TAZ (8418), p-YAP/TAZ (13008), PHB1 (2426), PHB2 (14085) ; p-AKTSer473 ; and β-actin . Horseradish peroxidase-conjugated secondary antibody  was incubated for 1 h at room temperature, and blots were imaged with Clarity Max ECL Western Blotting Substrate  using ChemiDoc MP . Band volumes were quantified with Image LabTM software version 6.0.1 (Bio-Rad) and normalized to GAPDH or β-actin.For western blot analysis, protein lysates were denatured in 1× LDS BoltTM Plus 2, Thermo Fisher Scientific) were applied to the gels. After conventional gel electrophoresis, the gel fluorescence of SMA basic L  and SMA label  were imaged. Following blotting, the blot fluorescence of the SMA label  and the blot chemiluminescent signal of the target protein  were detected. Finally, the normalization of gel load , normalization of Smart Label (based on SMA label), and normalization of target protein signal  were conducted using the SPL-normalization template following the manufacturer’s protocol. Results were expressed as SPL normalized target volume. Visualization and quantification were performed using the Chemidoc MP Imaging System and Image LabTM software version 6.0.1 (both from Bio-Rad).High patient-to-patient variation of reference protein expression, such as GAPDH and β-actin, impedes proper quantification of western blot data. Hence, for accurate target protein quantification of human HCC samples, western blotting was performed using the Smart Protein Layers (SPL) Kit Red from NH DyeAgnostics  according to the manufacturer’s instructions. The SPL technology is based on three components: (i) red-fluorescent Smart Label (SMA label) reagent for labeling and visualization of total protein on gels and blots, (ii) bi-fluorescent sample-specific standard Smartalyzer L (SMA basic L) for normalization, standardization, and quantification of total protein per sample, and (iii) a calibrator (CAL) for comparison of fluorescence and target protein signals derived from different gels. Briefly, before gel electrophoresis, every protein sample was labeled with 2 µL of SMA basic L and 1 µL of Smart label working solution. Samples with of 10 µg of protein per sample, 12 µL of calibrator , and 5 µL of Biotinylated Protein Ladder  staining was conducted using a standard protocol on liver sections. Specifically, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by heating in a microwave on high for 10 min, followed by a 20-min cool down at room temperature. After blocking with 5% goat serum and the Avidin-Biotin blocking kit , the slides were incubated with the anti-eIF4A1 primary antibody  overnight at 4 °C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and, subsequently, the biotin-conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Reaction detection was achieved using the Vectastain ABC-Elite Peroxidase Kit  with ImmPACT DAB  as the chromogen. Slides were counterstained with Mayer’s hematoxylin.p values for TCGA data were obtained from the UALCAN analysis tool. The non-parametric Wilcoxon signed-rank test was used for paired sample comparison. Kaplan-Meier curves were evaluated using the log-rank test. For calculation of IC50s, data were transformed to log2, normalized, and non-linear regression (log)inhibitor vs. response–variable slope (four parameters) was performed. For the transformation of SEM to SD, SEM was multiplied by the square root of the sample size n. Tukey’s multiple comparison test was applied for multiple comparisons. Two-tailed values of * p < 0.5, ** p < 0.1, *** p < 0.01, and **** p < 0.001 were considered significant. Lowercase letters denoted statistical significance, as stated in the associated figure legends. All data are expressed as the mean ± SD or SEM.GraphPad Prism version 9.3.1 software  and IBM SPSS version 26 software  were employed to evaluate statistical significance. The Our results, obtained in silico and in vitro, constitute the initial step in an innovative direction to develop pharmacological strategies to treat liver cancer. This is particularly valuable owing to the lack of available tools for efficaciously treating advanced non-surgically resectable HCC. The fact that eIF4A1 was consistently upregulated in HCC in comparison with adjacent non-tumor liver tissue and the magnitude of this change was negatively correlated with the prognosis of HCC patients supports a role of eIF4A1 in HCC malignancy. This led us to evaluate the impact of eIF4A1 inhibitors on HCC cell viability. Interestingly, even at low nanomolar concentrations, these inhibitors triggered a remarkable reduction in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Moreover, the cytostatic effect of eIF4A1 inhibitors was further increased by the pan mTOR inhibitor, Rapalink-1.During the last decade, considerable efforts have been made to characterize the molecular basis for the high refractoriness of HCC to currently available first- and second-line drugs . This, tIn summary, the results of the present study highlight the pathogenic relevance of eIF4A1 in HCC and constitute an encouraging advance in developing new sensitizing strategies. Thus, the usefulness of combining eIF4A1 and mTOR inhibitors with currently used chemotherapeutic agents deserves further investigation.


Angiogenesis is the formation of new blood vessels from pre-existing vessels. Adequate oxygen transport and waste removal are necessary for tissue homeostasis. Restrictions in blood supply can lead to ischaemia which can contribute to disease pathology. Vascular endothelial growth factor (VEGF) is essential in angiogenesis and myogenesis, making it an ideal candidate for angiogenic and myogenic stimulation in muscle. We established C2C12 mouse myoblast cell lines which stably express elevated levels of (i) human VEGF-A and (ii) dual human FGF4-VEGF-A. Both stably transfected cells secreted increased amounts of human VEGF-A compared to non-transfected cells, with the latter greater than the former. In vitro, conditioned media from engineered cells resulted in a significant increase in endothelial cell proliferation, migration, and tube formation. In vivo, this conditioned media produced a 1.5-fold increase in angiogenesis in the chick chorioallantoic membrane (CAM) assay. Delivery of the engineered myoblasts on Matrigel demonstrated continued biological activity by eliciting an almost 2-fold increase in angiogenic response when applied directly to the CAM assay. These studies qualify the use of genetically modified myoblasts in therapeutic angiogenesis for the treatment of muscle diseases associated with vascular defects. Adequate oxygen transport and waste removal are essential for tissue homeostasis. For this to occur, a vast network of well-connected blood vessels to deliver well-oxygenated blood is required, with ischaemia occurring in its absence leading to tissue deprivation and death ,2. AngioTherapeutic angiogenesis is the coordinated delivery of vascular growth factors with the aim of promoting blood vessel growth, leading to revascularisation of previously under-perfused tissues ,8. VascuVEGF-A is a key factor in vascular development and is the single most important regulator of blood vessel formation . VEGF exThe C2C12 immortal muscle cell line has a rapid proliferative and differentiation ability making it an ideal in vitro approach to studying myogenesis . VEGF-A Previous studies have reported the implantation of VEGF-expressing muscle-derived stem cells improves vascularisation and reduces fibrosis in dystrophic muscle ,30 and ihVEGF-A secreting stable cells. The chick CAM is a highly vascularised membrane used for nourishment and gaseous exchange in fertilized chicken eggs [Several angiogenic assays, both in vitro and in vivo have been used in the past to study angiogenesis. These include in vitro assays such as the use of mouse and human endothelial cell lines   and in vken eggs ,38. Withken eggs ,40. Matrken eggs ,42,43.hVEGF, both alone and as part of a dual plasmid co-expressing hFGF4 and hVEGF. We successfully characterised the hVEGF-A expression and the angiogenic potential of stably transfected C2C12 mouse myoblast cells using these models of angiogenesis. The first aim was to establish a stable C2C12 muscle cell line expressing hVEGF-A protein, confirmed through molecular analysis. Secondly, we observed the paracrine angiogenic efficacy of conditioned media taken from these cells using the CAM filter disc and endothelial cell assays. Finally, we investigated the in vivo pro-angiogenic efficacy of these cells when applied to the CAM angiogenic assay. This investigation combines elements of several previous studies, providing an insight into whether hVEGF-A-producing cells can be used as a pro-angiogenic factor with therapeutic potential in the treatment of conditions that experience decreased neovascularisation.We used the CAM and endothelial cell proliferation, migration, and morphogenesis assays to develop an ex-vivo strategy to deliver the angiogenic factor C2C12  mouse myoblast cells and C166  mouse yolk sac endothelial cells were cultured in high glucose Dulbecco’s Modified Eagle medium with sodium pyruvate, supplemented with 1% penicillin-streptomycin antibiotic and 10% fetal calf serum . Cells were maintained in the standard cell culture conditions (5% CO2 and 37 °C) and sub-cultured when necessary, using trypsin/EDTA solution.6) and high (0.2 × 106) densities and subjected to increasing concentrations (0–1000 μg/mL) of G418 to determine the minimum antibiotic concentration needed to kill all cells over the course of 3- 7 days. Cell confluency was assessed by visual inspection, while cell numbers were counted using the Countess™ II FL Automated Cell Counter (ThermoFisher Scientific) following 7 days of exposure. At 1000 µg/mL of G418, there was a visible reduction in confluency and increased cell toxicity in those seeded at both low and high concentrations  resistance gene. The kill curve experiment involved a dose-response where non-transfected C2C12 cells were seeded at low  was received from Prof Jozef Dulak, Department of Biochemistry, Biophysics, and Medical Biotechnology Jagiellonian University, Krakow, Poland. Heat shock was used to transform plasmids into DH5α competent E. coli cells, and subsequently cultivated. The DNA was then isolated using a Miniprep DNA Elution Kit  according to the standard manufacturer’s instructions. Confirmation of the plasmids was carried out by DNA restriction digests and gel electrophoresis.Transfections with pVEGF-EGFPN1, pEGFPN1, and FGF4/VEGF plasmids were carried out using Lipofectamine 2000 (Invitrogen), in a 1:2.5 ratio of DNA: Lipofectamine, with a DNA concentration of 8 µg for each plasmid. The DNA and Lipofectamine mixture was applied to plates containing C2C12 cells at 90% confluency and incubated at 37 °C overnight. In the case of pVEGF-EGFPN1 and pEGFPN1 plasmids, positive transfection was identified by observing GFP positive cells under FITC light 24 h post-transfection. Plates were trypsinised, serially diluted, and seeded in Geneticin (G418) selective media to screen for positively transfected cell colonies. Plates were screened for antibiotic resistance for four weeks, with media replenished every 3 days until isolated single colonies could be observed. Isolated colonies which showed continued antibiotic resistance were isolated using trypsin and cloning discs and expanded for further study with two clones selected for each pVEGF-EGFPN1 and pEGFPN1 plasmid, and eleven clones selected for FGF4/VEGF.hVEGF-A ELISA kit (Invitrogen) according to the manufacturer’s instructions. Conditioned media from non-transfected C2C12 myoblasts served as a control. Selected cell lines for each plasmid were designated as Myo-GFP, Myo-VEGF-GFP, and Myo-FGF4-VEGF and were each expanded for further investigation into their angiogenic potential.Non-transfected and stable transfected C2C12 cells were seeded and allowed to grow for 72 h. Media taken from each well was then centrifuged at 1000 rpm for 10 min to remove cell debris and frozen in aliquots at −80 °C. Protein levels of VEGF-A were determined using an ® Reagent (Invitrogen). The RNA samples were quantified using a NanoDrop ND 1000 spectrophotometer . A total of 500 ng total RNA was reverse transcribed into first-strand cDNA using Superscript III Reverse Transcriptase with random primers (ThermoFisher Scientific). qPCR was then performed using a StepOne Plus RealTime PCR system . Each 20 μL reaction mixture consisted of 5 μL cDNA, 5 μL SyberGreen, and 10 mmol/L of specific primers  or Myo-GFP, Myo-VEGF-GFP and Myo-FGF4-VEGF cells were applied. Scratches were imaged using EVOS M5000 at 10X magnification and then returned to the incubator at 37 °C for 8 h before being imaged again. Cell migration was quantified by the change in width of the scratch between experimental time points, with the distance measured using Image J software .Endothelial cell migration (Scratch) assay was carried out as described previously ,46. C166Endothelial cell proliferation was measured using a mitochondrial metabolic (MTT) activity assay, similarly to as described previously ,48. C1664 in triplicate in 100 μL of conditioned media taken from Myo (control), Myo-GFP, Myo-VEGF-GFP, or Myo-FGF4-VEGF cells. After 18 h, tube-like structures were observed. Each well was imaged at four non-overlapping locations, and the number of tubes, nodes, and total tube length were measured using Image J software  was greater than 45°, and whether it branched within a ring (⌀ 12 mm) encircling each filter disc. Similarly, in the case of the Matrigel-cell experiments, all blood vessels which approached the on-plant (A = 16 mm2) at an angle greater than 45° were assigned a score of 0, 1, or 2 based on whether they branched within the focal plane of the on-plant. In the case of both experiments, the total score for each on-plant was then determined by the sum of each of these individual scores.Any chicks which had perished during the course of the experiment were excluded from the analysis. With the analyst blinded to the treatment used for each on-plant, angiogenesis as a result was scored by determining the vascular index of blood vessels which grew towards the on-plant, similarly, to as described previously ,62,63. It-test was used to determine statistical significance between the two groups. A one-way analysis of variance (ANOVA) and Tukey’s post hoc test were applied to determine statistical significance between groups, while a two-way analysis of variance (ANOVA) was applied where two factors were present in an experiment. Data are expressed as mean ± S.E.M. or as a scatter plot ± S.E.M., with data considered statistically significant at * p < 0.05; ** p  <  0.01; *** p  <  0.001 and **** p  <  0.0001.All statistical analyses were carried out using Excel and GraphPad Prism software. A Student’s C2C12 muscle cells were transfected with pEGFPN1, pVEGF-EGFPN1, and FGF4/VEGF plasmids. Over the course of four weeks following transfection, G418 antibiotic selection was carried out. Two clones of each pEGFPN1 and pVEGF-EGFPN1 and eleven clones of FGF4/VEGF transfected cell colonies, which showed continued antibiotic resistance were selected and expanded to larger numbers. Based on the brightness of their GFP activity, pEGFPN1 and pVEGF-EGFPN1 clones were referred to as low and high expression pEGFPN1 and p-VEGF-EGFPN1 . Once eshVEGF-A protein expression in stably transfected cells, and to screen cells to identify those with the most potency. To achieve this, the level of hVEGF-A protein released from the stably transfected cell lines into the cell media was measured. Non-transfected C2C12 and pEGFPN1 transfected cells served as controls, where no hVEGF-A expression is expected. High expression pVEGF-EGFN1 transfected cell lines showed a high hVEGF-A secretion compared to pEGFPN1 and non-transfected C2C12 cell lines (p < 0.0001) (hVEGF-A protein expression (p < 0.0001)  and Myo-GFP cell lines, and an almost 50-fold increase compared to Myo-VEGF-GFP cell lines , and Myo-GFP A. A huma 0.0001) . While c 0.0001) . Based oll lines B.hVEGF and hFGF4 expression in the Myo (control), Myo-GFP, Myo-VEGF-GFP, and Myo-FGF4-VEGF cell lines to confirm altered gene expression. qPCR indicated a significant upregulation of hVEGF expression in Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines compared to non-transfected and Myo-GFP cell lines which resulted in undetermined products. A modest increase of hVEGF was seen in the Myo-VEGF-GFP cell line. Meanwhile, a much more apparent increase was seen with the Myo-FGF4-VEGF cell line compared to Myo (control) and Myo-GFP cell lines (p < 0.0001) , Myo-GFP, and Myo-VEGF-GFP (p < 0.0001) cells  C. hFGF4 1) cells D, confirhVEGF-A expression and secretion by Myo-VEGF-GFP and Myo-FGF4-VEGF cells, our next aim was to investigate the angiogenic potential of the conditioned media from these different cell lines. Both hVEGF-A and hFGF play important roles in mediating endothelial cell growth and promoting angiogenesis, as a result, endothelial cell angiogenic assays were employed. This involved monitoring the behaviour of C166 endothelial mouse yolk-sac cells, following treatment with conditioned DMEM taken from each cell line.Following confirmation of p < 0.05 (p < 0.0001) or Myo-GFP (p < 0.0051) cell lines. Myo-VEGF-GFP cell conditioned media elicited a significant increase in cell proliferation compared to non-transfected cell conditioned media (p < 0.05)  C. Interep < 0.05 , Myo-GFP Myo-VEGF-GFP, and Myo-FGF4-VEGF were investigated through an application on developmental day 8 (EDD8) for 72 h.Following confirmation that conditioned media from these stably transfected cells elicited pro-angiogenic effects in both endothelial cell angiogenic assays and the CAM filter disc assay, the focus of this study then moved to the investigation of the angiogenic response of the gene-modified cells directly. To examine this, Matrigel-cell mixtures were applied to the CAM. To begin, a dose-dependent curve using the CAM assay was carried out, with increasing quantities of Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines respectively applied to the CAM for 72 h to establish the optimum cell number required . For botp < 0.05 . Howeverobserved . Based op < 0.001) and Myo-GFP cells (p < 0.0001). Meanwhile, Myo-FGF4-VEGF cells elicited a similar response, resulting in a significant increase in angiogenic observed compared to on-plants containing Myo (control) and Myo-GFP cells, with p < 0.001  and Myo-GFP encapsulated on-plants respectively A. An ang < 0.001 B.VEGF is paramount in vascular development and is the single most important regulator of blood vessel formation ,64. AngihVEGF-A secreting C2C12 mouse myoblast cell lines, and quantify the angiogenic potential of these cells, which may prove beneficial in the amelioration of muscle ischaemia associated with diseases including CLI, PAD, diabetes, cachexia, COPD and muscular dystrophy [We conducted a number of studies in order to establish stable ystrophy ,6.hVEGF. Conditioned media taken from the hVEGF-A secreting cell lines proved efficacious in the induction of angiogenesis in the CAM angiogenic assay, while also resulting in pro-angiogenic behaviours in murine endothelial cell assays of angiogenesis. Finally, following confirmation of the pro-angiogenic benefits as a result of conditioned media, we then demonstrated how these cells provoked a potent angiogenic response in the CAM angiogenic assay.In the present study, we successfully established two stable muscle cell lines including expression of human VEGF-A protein, Myo-VEGF-GFP as a GFP tagged fusion protein, and Myo-FGF4-VEGF as cells dually expressing human FGF4 and hVEGF-A alone and in combination with hFGF4.The efficacy of myoblast-mediated VEGF gene delivery to mouse and rat muscle has been investigated in the past, with varying levels of success. Despite the potent angiogenic potential of VEGF, studies have indicated that the optimum dosage for VEGF is within a narrow limit, with overproduction leading to the formation of disorganized, malformed vessels with irregularly sized lumens and weakened wall permeability ,69,70,71hVEGF-A and hFGF4/VEGFA overexpressing C2C12 mouse myoblast cells, both as a result of stable and transient transfections [hVEGF-A secretions in the dual expression cell line are much higher than in previous reports, confirmed by both ELISA and qPCR [hVEGF-A protein secretion and mRNA from both cell lines, while qPCR alone confirmed the dual expression of FGF4 and VEGF in the Myo-FGF4-VEGF cell line. Myo-FGF4-VEGF cells secreted a significantly higher amount of hVEGF-A protein compared to non-transfected and Myo-GFP cell lines, while in comparison to the Myo-VEGF-GFP cell line, Myo-FGF4-VEGF cells exhibited an almost 50-fold increase in hVEGF-A protein secretion.Previous studies have generated fections ,26. In tand qPCR ,26,74,75hVEGF falls under IRES-dependent second gene expression, as a result, expression of hVEGF would be expected to be lower compared to the single VEGF expression plasmid [hFGF4 gene transcript level is higher than the hVEGF-A expression.Research to date has examined the expression and therapeutic benefits of both VEGF and FGF secretions separately . However plasmid . This cahVEGF-A protein secretion in these studies was observed to be much less potent. In previously published reports, transient transfection with this dual FGF4/VEGF plasmid reported hVEGF-A secretions of approximately 6000 pg/mL in human primary myoblasts [hVEGF secretions of around 5500 pg/mL. Additionally, this was used to ameliorate wound healing and post-ischemic blood flow recovery in the hindlimb ischemic mouse model [The dual FGF4/VEGF plasmid used in this study has been successfully transfected into myoblast cells in previous reports ,25,74. Hyoblasts  and 2800yoblasts . Howeveryoblasts . An AAV hVEGF-A protein secreted from Myo-VEGF-GFP cells compared to Myo (control) or Myo-GFP transfected cell lines. However, this expression is not as potent as concentrations seen in the Myo-FGF4-VEGF cell lines in the present study. Sun et al., through lentiviral transfection, developed a stable VEGF-165 expressing ADSCs which secreted approximately 920 pg/mL of VEGF [With the aim of therapeutic angiogenesis, VEGF-165 alone has been transfected into several different cell types including fibroblasts , endothe of VEGF . Meanwhi of VEGF . This rehVEGF and hFGF4/VEGF protein expression from Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines, we moved our focus to proving the pro-angiogenic effects these cells could elicit. Previously, various cells have been successfully modified or transfected to overexpress VEGF [hVEGF or hFGF4-VEGF expressing C2C12 cells using the chick CAM assay. In the current investigation, we chose to employ both in vitro murine endothelial cell angiogenic assays, and the in vivo chick CAM assay.Following on from the successful confirmation of ess VEGF ,80,82, Fess VEGF  or both ess VEGF ,74. HowehVEGF protein secretion in affecting the angiogenic response in vitro. The lower hVEGF-A secretion by the Myo-VEGF-GFP cell line is reflected. Consequently, a similar angiogenic response is not as clearly apparent, with Myo-VEGF-GFP conditioned media failing to induce a significant increase in cell proliferation compared to Myo-GFP conditioned media.VEGF-A and FGF have both proven to elicit pro-angiogenic behaviours in endothelial cells in vitro ,84. In tThe potent angiogenic ability of Myo-VEGF-GFP and Myo-FGF4-VEGF cell conditioned media is again seen in the tube formation assay using endothelial cells seeded on a basement media matrix. In vivo, VEGF triggers the release of proteolytic enzymes which degrade the surrounding basement membrane, allowing endothelial sprouts to invade the extracellular matrix leading to tube formation. Following this, endothelial cells migrate, forming tube-like structures which fuse to form an organised and functional blood vessel network . ConditiMeanwhile, only a modest difference in endothelial cell migration, proliferation, and tube formation is seen in the comparison of Myo-VEGF-GFP and Myo-FGF4-VEGF conditioned media, where despite the increased potency of VEGF-A secretion, little difference in these parameters is observed. This reinforces the delicate VEGF dose-responsive effects aforementioned, where overexpression of VEGF can lead to negative effects ,85.While in vitro assays such as the endothelial cell assays can elucidate pro-angiogenic potency, in this study, it was important to examine the efficacy of these cell secretions on the in vivo model of angiogenesis. We chose to use the CAM assay to investigate this pro-angiogenic potential. The CAM assay is a highly versatile, cost-effective model of angiogenesis with a vast vascular network of capillaries, veins, and arteries which can be easily manipulated and observed for experimental study ,39,40.A strong and potent angiogenic effect is seen in this study as a result of conditioned media taken from the stable transfected Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines. This is consistent with other studies where conditioned media from osteoblast-like cells which secreted elevated levels of VEGF-A also induced a positive angiogenic response in the CAM assay .hVEGF and have the potential to induce angiogenesis in vivo. Naturally, the next phase would be to examine the applicability and efficacy both these cell lines could have in a higher in vivo mouse model of disease. Myoblast transplantation is a promising area of therapeutics [The present findings are promising, confirming that Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines secrete elevated levels of apeutics ; howeverapeutics ,88. TherhVEGF protein production, as observed in other published studies [Application of Myo-VEGF-GFP and Myo-FGF4-VEGF cells encapsulated in Matrigel onto the CAM assay resulted in a significant increase in the angiogenic score, similar to the effects seen with cell conditioned media. In this study, the correct dosage of cell number applied was paramount to the most efficient angiogenic response. Higher levels of Myo-FGF4-VEGF cells per on-plant resulted in a reduction in total angiogenic score with a bell-shaped dose-response observed, possibly indicating excessive cell numbers prove to be harmful in the angiogenic potential. This reinforces the potentially deleterious effects of excessive  studies ,69,70. FhVEGF-A and hFGF4 from C2C12 mouse myoblast cells into an in vivo angiogenic system. These reveal several important findings which could potentially provide broad implications in the field of therapeutic angiogenesis in muscle diseases with defects in microcirculation such as diabetes and muscular dystrophies. We have successfully established two pro-angiogenic mouse myoblast cell lines which secrete hVEGF-A alone or in combination with hFGF4, with the latter cell line showing great efficacy. Both of these cell lines have proven to elicit a potent pro-angiogenic effect both in vitro in the murine endothelial cell angiogenic assay and in vivo in the chick CAM model of angiogenesis. Furthermore, the manipulation and application of these cells in higher-level in vivo studies may prove highly beneficial in the amelioration of vascular abnormalities in muscle disease.In summary, this current study demonstrated the ability to deliver growth factors such as