This study compares the capabilities of normal and tumour cells to divide after receiving spatially fractionated radiotherapy. For this treatment, a minority of cells (here ∼20%) receive very large, peak doses, whereas the remaining cells are spared and receive only a small valley dose. We show that tumour and normal cells respond differently to this treatment, and that spatially fractionated radiotherapy may have a greater effect on tumour than normal cells. Our work was conducted using laboratory equipment, rather than specialized synchrotron facilities implying that the observed response is present at conventional dose rates and hence purely an effect of the spatial fractionation of the treatment.Microbeam radiotherapy (MRT) is a preclinical method of delivering spatially-fractionated radiotherapy aiming to improve the therapeutic window between normal tissue complication and tumour control. Previously, MRT was limited to ultra-high dose rate synchrotron facilities. The aim of this study was to investigate in vitro effects of MRT on tumour and normal cells at conventional dose rates produced by a bench-top X-ray source. Two normal and two tumour cell lines were exposed to homogeneous broad beam (BB) radiation, MRT, or were separately irradiated with peak or valley doses before being mixed. Clonogenic survival was assessed and compared to BB-estimated surviving fractions calculated by the linear-quadratic (LQ)-model. All cell lines showed similar BB sensitivity. BB LQ-model predictions exceeded the survival of cell lines following MRT or mixed beam irradiation. This effect was stronger in tumour compared to normal cell lines. Dose mixing experiments could reproduce MRT survival. We observed a differential response of tumour and normal cells to spatially fractionated irradiations in vitro, indicating increased tumour cell sensitivity. Importantly, this was observed at dose rates precluding the presence of FLASH effects. The LQ-model did not predict cell survival when the cell population received split irradiation doses, indicating that factors other than local dose influenced survival after irradiation. Any cancer treatment aims to eradicate the tumour target, whilst inflicting minimal toxicity in healthy tissues. In radiation therapy (RT), this aim is conventionally achieved by geometrically confining the high dose field to the tumour, e.g., by intensity-modulated RT, and thereby limiting side effects to organs at risk (OAR). However, exposure of OARs located in close proximity to the tumour, or along the beam path, is inevitable and limits the dose escalation to the tumour with potential implications on outcome. Spatially fractionated RT, such as microbeam radiation therapy (MRT) , has prePreclinical in vivo data have demonstrated a remarkable normal tissue sparing following MRT, despite peak doses in the range of hundreds of grays ,5,6,7,8.Moreover, there is little data to support the existence of the microbeam effect in vitro ,29,30, iIn this study, we evaluate the response of two tumour and two non-tumour human cell lines in response to BB and MRT to investigate and quantify differential effects of these treatments delivered with a conventional X-ray tube and optional MRT collimation . As such2. Screening for mycoplasma contamination was performed by polymerase chain reaction  and cell lines were authenticated in-house by short tandem repeat analysis using a Gene Print 10.0 kit  and a 3730xl DNA analyser .Human umbilical vein endothelial cells (HUVEC) from pooled donors were purchased from Lonza  and MRC-5 normal lung fibroblasts, from Sigma Aldrich Ltd. . For the purposes of this manuscript, we will refer to these as “normal” cells. The human non-small cell lung cancer lines A549 and NCI-H23 were obtained from The American Type Culture Collection . Tumour cell lines were cultured in Dulbecco’s minimal essential medium F12 , and MRC-5 cells were cultured in minimal essential medium , both supplemented with 10% foetal bovine serum . HUVECs were cultured in endothelial cell growth medium-2 (Lonza), including all supplements supplied by the manufacturer. Cells were maintained in a humidified incubator at 37 °C and 5% COTo ensure that all clonogenic assays were carried out on exponentially growing cells, cells were seeded at approximately 16 h prior to RT, yielding 80% confluence at the time of irradiation. Following irradiation, cells were immediately harvested by trypsinisation, counted and plated at appropriate numbers in triplicate in 6-well plates. Cells were then incubated under the specified culture conditions and allowed to form colonies for 7–14 days, depending on the cell line. Colonies were fixed in ice-cold methanol at −20 °C for 20 min, left to dry, and stained in 0.5% crystal violet solution (Sigma Aldrich Ltd.). Colonies containing 50 or more cells were counted. Plating efficiency of the cells for each condition was calculated as the ratio of colonies counted per cell number seeded. The clonogenic survival was calculated as the ratio between plating efficiencies of treated and untreated cells. For each experiment, three independent repeats were performed, and mean values and standard deviations are reported. The cell survival data obtained are listed in For all experiments, X-rays were generated from an X-ray tube  mounted in an X-ray cabinet . An acceleration voltage of 160 kV, and a tube current of 11.3 mA for BB or 5.6 mA for MRT generation was used. The use of different amperage depending on the irradiation procedure was due to the use of an optimal focal spot size/tube filament yielding a more homogeneous dose distribution for BB at a high dose rate (0.031 ± 0.002 Gy/s), and a well-collimated beam with a high dose rate for MRT. The beam was hardened by 1 mm aluminium filtration resulting in a dose rate of escribed . In shorThe MRT field was characterised following previously published procedure  using EBFor comparison with BB irradiation at two distinct dose levels, the experimental DVH was approximated by a step function resulting in the same integral dose as the MRT dose distribution  model [The dependence of clonogenic cell survival Q) model .(1)S(d)Y  using a nonlinear least square approach resulting in 017a usinStatistical analysis was performed by two-way ANOVA testing in SPSS (version 26).In order to compare the effectiveness of BB irradiation relative to MRT, we first established the sensitivity of the cell lines to standard BB radiation , Table 1Having established that the cell lines under study displayed comparable sensitivity to BB irradiation, we next evaluated MRT irradiation sensitivity, and predicted survival based upon the LQ-model with BB parameters (Equation ). FigureTo assess the importance of the spatial distribution of dose gradients for MRT on the survival of normal and tumour cells, we performed dose mixing experiments, where cells were irradiated with BB irradiation at two dose levels and then the cells were mixed post irradiation with a PVDR of 22 and 80% of cells receiving the valley dose. t-test).In this study, we aimed to address three unmet research questions in the field of MRT: (i) is there a differential response of normal and tumour cell lines to MRT in vitro? (ii) Can the cellular response observed after synchrotron MRT be recapitulated using bench-top equipment and in the absence of FLASH effects, and (iii) how to best compare cell survival following BB irradiation to survival following exposure to inhomogeneous MRT dose distributions to obtain a biologically more meaningful representation compared to plotting as a function of either peak, valley or mean  dose levels?To date, there are few reports of a normal tissue sparing effect following spatially fractionated radiation at an in vitro level: whereas Ibahim et al. were unable to demonstrate normal tissue sparing , Peng etgnalling .The spatial arrangement of the delivered dose was found here to be irrelevant for measured surviving fractions as the dose mixing experiments  demonstrTo the best of our knowledge, we are the first to examine the effect of MRT in vitro using a bench-top X-ray source. The dose rates used here (<0.2 Gy/s) fall well below the range of dose rates previously attributed to FLASH effects . PreviouThe work presented further contributes to the ongoing discussion on how to best compare the biological efficacy of MRT and BB as a function of “dose”. It is clear that due to the nonlinear dose relation of cell survival, neither the peak, valley nor integral dose alone can be used for this purpose. Instead, the full MRT dose spectrum needs to be accounted for. Here we provide two suitable methods for a direct BB and MRT comparison: 1. We included LQ-model predictions into the visualisation of our results  that accOur study is limited to the analysis of the differential response of tumour and normal cell lines to BB and MRT by clonogenic assays, and we did not further investigate the underlying mechanisms driving this differential response. We show that tumour cells were markedly more sensitive to mixed beam irradiations than normal cells, particularly when considering they showed equivalent sensitivity to BB irradiation. Importantly, we further demonstrate no significant differences in terms of clonogenic survival between MRT and co-culture experiments of ablated and surviving cell populations. Consequently, we expect the underlying mechanism responsible for the additional cell killing to be congruent with bystander signalling that was indicated by several previous analyses in co-culture experiments . We suggA second limitation of this study was the use of clonogenic assays. Despite frequent application for the characterisation of radiosensitivity, clonogenic assays represent a rather non-physiological cellular microenvironment of isolated cells plated on a rigid two dimensional (2D) substrate, limiting communication and inter-cellular contacts relative to the in vivo setting ,48. It wFinally, the experimental arrangement used in this study posed some limitations on the delivery of BB and MRT. In order to maximise the field dose homogeneity for BB, and valley dose rate for MRT, different focal spot sizes and X-ray tube filaments were required. This implied differences in tube amperage to accommodate for the recommended specifications by the manufacturer. It should be noted that also the presence of the tungsten collimator may affect the photon spectrum in the case of MRT. Differential effects due to these variations in the photon energy spectra and dose rates between MRT and BB could not be accounted for. However, by achieving comparable cell surviving fractions, using MRT and dose mixing experiments may give an indication that the relevant differences were negligible in light of the biological uncertainty of clonogenic cell survival experiments. Furthermore, despite our best efforts, the dosimetric measurements were limited by the dynamic range and resolution of the used films. Hence, we show all of our results with the relevant dose uncertainties and accounted for these during the LQ-model predictions as well.Using a bench-top X-ray source, we have demonstrated a differential in vitro response of lung cancer cell lines, endothelial cells and fibroblasts to microbeam irradiation at dose rates precluding the presence of FLASH effects. Specifically, we observed an increased tumour cell sensitivity to MRT, whereas normal cell survival following MRT was comparable to survival after BB irradiation. Cell survival after MRT was replicated by mixing populations of cells irradiated separately with high and low BB doses. These results provide a strong motivation for further investigations of the underlying mechanisms to confirm a potential role of bystander signalling.


Serinicoccus sp., seriniquinone (SQ1) has been characterized by its selective activity in melanoma cell lines marked by its modulation of human dermcidin and induction of autophagy and apoptosis. While an active lead, the lack of solubility of SQ1 in both organic and aqueous media has complicated its preclinical evaluation. In response, our team turned its effort to explore analogues with the goal of returning synthetically accessible materials with comparable selectivity and activity. The analogue SQ2 showed improved solubility and reached a 30–40-fold greater selectivity for melanoma cells. Here, we report a detailed comparison of the activity of SQ1 and SQ2 in SK-MEL-28 and SK-MEL-147 cell lines, carrying the top melanoma-associated mutations, BRAFV600E and NRASQ61R, respectively. These studies provide a definitive report on the activity, viability, clonogenicity, dermcidin expression, autophagy, and apoptosis induction following exposure to SQ1 or SQ2. Overall, these studies showed that SQ1 and SQ2 demonstrated comparable activity and modulation of dermcidin expression. These studies are further supported through the evaluation of a panel of basal expression of key-genes related to autophagy and apoptosis, providing further insight into the role of these mutations. To explore this rather as a survival or death mechanism, autophagy inhibition sensibilized BRAF mutants to SQ1 and SQ2, whereas the opposite happened to NRAS mutants. These data suggest that the seriniquinones remain active, independently of the melanoma mutation, and suggest the future combination of their application with inhibitors of autophagy to treat BRAF-mutated tumors.Isolated from the marine bacteria  SQ1, Serinicoccus, displays potent cytotoxic activity against a panel of tumor cell lines with particular selectivity towards melanoma [SQ1 was shown to target dermcidin [SQ1 analogues, illustrated by SQ2–SQ5, Seriniquinone SQ1, A, a natuV600E inhibitors [Melanoma, a cancer originated from melanocytes, frequently presents mutations in the MAPK pathway, mainly in BRAF and NRAS proteins, at incidences up to 52% and 28%, respectively, according to The Cancer Genome Atlas Network . This dihibitors . Thus, tDCD) gene. This full-length protein is known to be the source of peptides, including the proteolysis-inducing factor (PIF) and antimicrobial peptides constitutively produced by eccrine sweat glands [DCD in breast and lung cancer cell lines repressed cell proliferation and tumorigenesis, therefore reinforcing its relevance for cancer progression [SQ1) and its analogues (SQ2–SQ5) as agents that target cancer’s bearing point mutations on BRAF and NRAS genes.Human dermcidin is a 110 amino acid protein encoded by the dermcidin  value for SQ1 in melanoma cells ranged from 0.06 µM in SK-MEL-19 to 1.4 µM for MM200 cell lines, while it reached 3.3 µM in breast adenocarcinoma (MCF7 cell line) and 1.0 µM in colorectal carcinoma (HCT-116 cell line). For the synthetic analogues, cytotoxicity varied from 0.04 to 1.8 µM for melanoma cells and from 0.15 to 1.9 µM for MCF7 and HCT-116. The most prominent effects were obtained for melanoma cell lines . For fibroblast cells (MRC-5 and FDH cell lines), SQ1 had an activity of 0.6 and 1.2 µM, respectively. In comparable assays, the synthetic analogues ranged between 0.6 and 3.5 µM for MRC-5 and FDH cells, respectively. Doxorubicin, a positive control, was generally less toxic than seriniquinone and its analogues in melanoma cells, with the exception of WM293A and MM200 cell lines. Nonetheless, it was far more toxic to breast carcinoma (MCF-7) and colon carcinoma (HCT-116) cells, and nontumor cell lines (MRC-5 and FDH).Seriniquinone SQ1, A and its SQ2–SQ5 A display SQ2–SQ5 . The IC550 value for the respective tumor cell line to the IC50 for MRC-5 or FDH, the non-tumor cell lines) were improved mainly for SQ2 and SQ3, relative to those of SQ1  and NRASQ61R mutated (SK-MEL-147) cell lines.Selectivity indices  and long exposure (72 h) periods , 24-h treatments induced toxicity by significantly reducing the number of viable cells and increasing non-viable ones in either cell lines , for which SQ1 reached an expression twofold higher than the control and SQ2 induced overexpression almost eight times  or rapamycin (an autophagy inducer) to further explore cell death mechanisms elicited by seriniquinones. We complemented this study with a similar approach to understand the role of the seriquinones on apoptosis using pre-treatments with Z-VAD-FMK (an apoptosis inhibitor) or doxorubicin (apoptosis inducer). In both cell lines, it was possible to demonstrate the difference of cell death triggered by iability , corroboiability B. In coniability . Also, fobserved . IC50 vaV600E has prompted a clinical exploration for drugs that selectively inhibit the mutated protein, such as vemurafenib and dabrafenib. However, most patients develop resistant tumors after 5–6 months of treatment [The main therapeutic barrier for melanoma is tumor aggressiveness and propensity to recur. Both features can be related to development of resistance to available treatments, due, in part, to the malleable metabolism within the tumor microenvironment ,14. The reatment ,16,17. Wreatment , no succreatment .SQ1) have advanced analogues with improved solubility, cytotoxicity, and tumor selectivity [SQ1, which displayed higher solubility and similar or improved toxicity [Considering these limited treatments, alternative treatments are necessary. Medicinal chemical studies on the natural product seriniquinone SQ have advtoxicity , represeSQ2, SQ3, SQ4, and SQ5) maintained or improved the biological activity of the natural compound SQ1. Further studies showed that SQ2, SQ3, SQ4, and SQ5 provided effective selectivity indices (≥twofold) to SK-Mel cells in relation to non-tumor cell lines. Interestingly, SQ2 showed 30–40-times greater selectivity toward melanoma cell lines. Such selectivity is important to minimize side effects and has been widely explored for the development of novel anticancer drugs [Our studies demonstrated that synthetic analogues . For the BRAFV600E and NRASQ61R melanoma cells, SQ1 also displayed a time-dependent activity, increasing its activity between 24 and 72 h. After the initial 24-h exposure, SQ2 caused a higher decrease in cell viability than SQ1. Here, we found that viable SK-MEL-28 cells were able to adhere and proliferate in fresh, drug-free medium, in contrast to those exposed to SQ1. For SK-MEL-147, the parameters of colony number and area were also decreased by exposure to either SQ1 or SQ2 with cell proliferation, expressed by colony area, decreased only 50%. These data suggest that SQ1 may produce an irreversible scenario for cell survival in SK-MEL-28. Additionally, these studies provided the first evidence that BRAFV600E and NRASQ61R melanoma cells may have different responses to SQ1 and SQ2. Supporting this observation, we found that the gene expression of dermcidin, the target of SQ1, was modulated at different levels within these cell lines.According to Trzoss et al. , the cytDCD expression varied among the biological triplicates from both SK-MEL-28 and SK-MEL-147 cells with enhanced expression in the NRAS mutant cell line. This observation may suggest why SK-MEL-147 cells are more resistant to SQ1 and SQ2 treatments during short periods (24 and 48 h) where DCD can act as an oncogene. Previous studies have shown that SQ1 and its analogues modulated DCD mRNA expression in HCT-116 , PC-3M (prostate carcinoma), MALME-3M (melanoma) and SK-MEL-28 (melanoma) cell lines [SQ1 and SQ2 also up-regulated the expression of such transcripts in SK-MEL-28 and SK-MEL-147 cells in a dose-dependent manner, suggesting that analogs such as SQ2 also act by targeting dermcidin.Besides the well-established role for the dermcidin in skin defense, this protein has been described as a cancer survival factor ,22,23,24SQ1 and SQ2 induced caspase-mediated apoptosis; nonetheless, the cell lines responded differently through autophagy inhibition. The BRAFV600E mutant cells were sensitized by pre-treatment with an autophagy inducer, while the opposite happened to the NRASQ61R bearing cells. Other groups reported a higher basal autophagy level for BRAF mutant cells, which corroborates with our gene panel data, and abolition of this process also caused a decrease in cell viability due to its relation to the maintenance of functional mitochondria [Q61R cells also trigger cytotoxic autophagy that, when inhibited, may cause a restoration of NRAS protein levels, which could explain the increase in cell proliferation. Studies are now underway to explore this phenomenon.Autophagy plays an important role in cellular homeostasis, such as regular intracellular maintenance, tissue development and regeneration, aging and cell survival through starvation, and other stress conditions . Howeverchondria ,31,32. Hchondria ,34 despichondria . These schondria . These dSQ1 and SQ2, may be key for melanoma treatment [V600E mutant cells. Furthermore, after triggered, autophagy may turn into a cytotoxic process in cells that have this mechanism suppressed. Here, small molecule modulation provided a response that can be detrimental to their basal maintenance, as evident in NRASQ61R mutant cells. Further studies are needed to elucidate the relation between dermcidin modulation and autophagy activation. Moreover, additional effort is required to understand the role of dermcidin in tumor cell survival and resistance. Additionally, to advance with seriniquinone into preclinical studies using animal models, it is necessary to overcome its poor water-solubility. As discussed by Apolinario et al. [Challenges remain in clinical treatment of melanoma. When addressed with the conventional therapy, such as the alkylating agent dacarbazine, melanoma easily turns into a refractory tumor. There are some available options of molecular targeted inhibitors for this type of cancer, like BRAF and MEK inhibitors. However, the fast tumor recurrence is still problematic. Immunotherapies (anti-CTLA-4 and anti-PD-1 immune checkpoint inhibitor) have shown promising outcomes; however, these extensive treatments have shown to be non-responsive in patient cohorts . Consequreatment . Here, wo et al. , the devV600E, NRASWT), SK-MEL-28 , SK-MEL-147 , MRC-5 (fibroblasts), and primary fibroblast (FDH) were kindly provided by Professor Glaucia Santelli . MCF7 , and HCT-116  cells were obtained from American Type Culture Collection (ATCC) and deposited in the Cell Bank of Rio de Janeiro, Brazil. WM293A , 501mel , and MM200  were kindly provided by Professor Sharon Prince, University of Cape Town, South Africa. All human melanoma and breast cell lines were cultivated with Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 . MM200 and HCT-116 were maintained with Roswell Park Memorial Institute (RPMI) medium . Unless stated otherwise, media was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin . Cells were incubated with 5% CO2 at 37 °C. When required, cells were detached from flask using trypsin-EDTA 0.05% .Human cell lines were used throughout this program. SK-MEL-19 (melanoma BRAFSQ1) and its synthetic analogues SQ2, SQ3, SQ4, and SQ5 . Seriniquinone ( and SQ5 A were ob4 cells/well) and, 24 h later, exposed to serial dilutions of the test compounds (from 0.00032 to 5.0 µM) for 24, 48, and 72 h. For each analysis, negative controls received 0.5% DMSO and positive control was delivered by doxorubicin . At the end of the incubation periods, the medium was replaced with fresh medium containing 0.5 mg/mL of MTT and supernatant was removed 3 h later. The plates were dried at 50 °C for 30 min and formazan was solubilized in DMSO (150 µL/well) for the absorbance measure at 570 nm on a plate reader . IC50 values  along with 95% CI  were calculated by non-linear regression using GraphPad Prism 8.0 software .Cytotoxic activity was evaluated by the transformation of 3--2,5-diphenyltetrazolium bromide  into formazan by viable cells . Cells w50 values obtained through MTT assay (72 h), a selectivity index (SI) was determined for each tumor cell line treated with SQ1 or SQ2 in relation to a non-tumor cell by the following formula:Based on the respective IC4) cells in 1 mL of media were seeded in cell culture dishes and, 24 h later, treated with SQ1 or SQ2 , vehicle (DMSO) and doxorubicin  for 24 h. Cells were detached, stained with Trypan Blue solution 0.4%  and counted in a Neubauer chamber under optical microscopy to determine concentrations of viable and non-viable cells.Fifty thousand (5 × 105) cells in 1 mL of media were seeded in cell culture dishes and, 24 h later, treated with SQ1 (0.3 µM and 0.6 µM for SK-MEL-28 and 0.9 µM and 1.8 µM for SK-MEL-147) or SQ2 (0.05 µM and 0.1 µM for SK-MEL-28 and 0.6 µM and 1.2 µM for SK-MEL-147) for 24 h. Doxorubicin  was used as positive control and 0.5% DMSO as negative control. Concentrations were based on ½ IC50 values at 24 h and IC50 values at 24 h, respectively, from each cell line.Twenty fifty thousand  and 0.5 g crystal violet . Images were acquired using G:BOX Chemi XRQ  and quantified using the ImageJ program .After the cells were submitted to the treatment scheme , adherenDCD expression, purified total RNA was quantified using an Eon Microplate Spectrophotometer . Quantitative PCR (qPCR) was conducted on a Rotor Gene 6000 real-time cycler  using GoTaq 1-Step RT-qPCR System . The cycle conditions were as follows: 45 °C for 10 min, 95 °C for 15 min and 40 cycles of 95 °C for 15 s; 60 °C for 60 s, followed by the melt. Relative expression was calculated using the comparative CT method Delta-Delta-Ct (DDCt) [DCD and RPLPO) can be consulted in Treated cells  had theit (DDCt) . The priFor the gene panel, the total RNA from three independent samples was extracted using TRIzol reagent. The samples were quantified, and their quality assessed by NanoDrop 2000 One Microvolume UV-Vis Spectrophotometer . cDNA was synthesized from 1 µg RNA using a High-Capacity cDNA Reverse Transcription Kit . qPCR was performed using a QuantStudio 3 Real-Time PCR System in conjunction with a SybrGreen System . Relative expression was calculated using the comparative CT method Delta-Delta-Ct (DDCt) . All priTreated cells  had theit-test. Data are expressed in terms of mean and standard deviation (mean ± SD).Data were analyzed in GraphPad Prism 8.0  using one-way ANOVA followed by Dunnett’s test or Student’s SQ1) and synthetic analogue SQ2 in melanoma cell lines. The resulting data provided new insights into the molecular MOA of this novel class of chemotherapeutic, as well as, revealing a further understanding of the mechanisms associated with induction of autophagy and apoptosis. Additionally, the cancer driver mutations (BRAFV600E and NRASQ61R) were found to not impact SQ1 or SQ2 cytotoxicity. The evidence herein suggests that a combination of SQ1 or SQ2 with autophagy inhibitors could be further explored to expand the efficacy of these agents and ideally enhance the targeting of BRAF mutated melanomas.This study provides an in-depth evaluation of the biological activity of the natural product seriniquinone (


Plant-derived natural products are significant resources for drug discovery and development including appreciable potentials in preventing and managing oxidative stress, making them promising candidates in cancer and other disease therapeutics. Their effects have been linked to phytochemicals such as phenolic compounds and their antioxidant activities. The abundance and complexity of these bio-constituents highlight the need for well-defined in vitro characterization and quantification of the plant extracts/preparations that can translate to in vivo effects and hopefully to clinical use. This review article seeks to provide relevant information about the applicability of cell-based assays in assessing anti-cytotoxicity of phytochemicals considering several traditional and current methods. Cancer is one of the leading causes of death worldwide. It is the first or second leading cause of death prior to age 70 in 112 of 183 countries and third or fourth leading cause in a further 23 countries, according to World Health Organization (WHO) estimates in 2019 [The most common types of cancer treatments available today are chemotherapy, surgery, and radiotherapy. Chemotherapy is curative in subsets of patients presenting with advanced disease, including Hodgkin’s and non-Hodgkin’s lymphoma, acute lymphoblastic and acute myelogenous leukemia, germ cell cancer, small cell lung cancer, ovarian cancer, and choriocarcinoma ,5. ChemoIncreased reactive oxygen species (ROS) levels have been found in almost all cancers and are thought to play an important role in the initiation and progression of cancers . These hPhytochemicals are classified as primary or secondary metabolites based on their role in plant metabolism . SecondaIn the last decades, several plants have been confirmed to contain chemo-preventive and therapeutic agents for various cancers ,18,19,20There have been studies to examine the effect of crude plant extracts or fractions containing phenolic compounds on cancer cells to test the hypothesis that potent antioxidants possess anticancer potential. Some of these studies revealed that the efficacy of phenolic compounds in inhibiting cancer activity differs based on the structure of the phenolic compound and its molecular target . PhenoliPandanus amaryllifolius extracts containing gallic acid, cinnamic acid and ferulic acid, with reported in vitro inhibition of breast cancer cell lines [Teucrium species extracts containing hydroxycinnamic acid derivatives, phenylethanoid glycosides, flavonoid glycosides, and flavonoid aglycones, with reported antiproliferative and proapoptotic activities in HCT-116 colon cancer cell lines [Baccharis trimera extracts containing gallic acid, pyrogallol, syringic acid and caffeic acid, with reported suppression of tumor cell colony formation and proliferation of SiHa cell line (isolated from a primary uterine squamous cell carcinoma); and Prunus africanus extracts, containing artraric and ferulic acids and N-butylbenzene-sulfonamide (NBBS) with reported antiproliferative effect on prostate cancer cells [Anticancer activity of phenolic compounds has been studied with the use of crude extracts containing mixtures of phenolic compounds and with isolated phenolic compounds. Some examples of crude extracts with reported anticancer activity include: ll lines . Severalll lines ,28; Baccer cells ,29. Prunus africana [Prunus africana (Hook.f.) Kalkman) or Pygeum africanum (Hook. f.), bitter almond, African prune, and red. Besides its use for timber, it is employed as a medicinal plant, whose leaves, roots and bark are used in traditional medicine in Africa [Prunus [Over 60% of currently used anti-cancer agents are estimated to be derived from natural sources, such as plants, marine organisms, and microorganisms ,17,30,31africana ,17, Afrin Africa ,33,34,35 [Prunus ,37. ManyPhytochemicals and their derivate metabolites present in plants have been shown to possess several beneficial effects in humans. Some of the more widely known phytochemicals with anticancer properties include vincristine, vinblastine, camptothecin, bleomycin, paclitaxel, and Taxol among others ,38. DiffCancer cell lines are useful tools because they provide a multifaceted model of the biological mechanisms involved in cancer development and progression. The use of cancer cell lines improved the knowledge of deregulated genes and signaling pathways involved in cancer progression; cell lines have also been used to define potential molecular markers for cancer screening and prognosis . NumerouCell lines are easy to handle and manipulate genetically/epigenetically by using demethylation agents, small interfering ribonucleic acid (siRNA), expression vectors, and they can be pharmacologically manipulated through cytostatics (cell growth inhibitors). Cell lines are homogenous, providing identical tumor cells for easier analysis unlike in heterogeneous solid tumors. However, to imitate in vivo tumor characteristics as closely as possible, a cancer cell line panel representative of the heterogeneity observed in the primary tumors can be used. Cancer cell lines are pure populations of tumor cells and have a high degree of similarity with the initial tumor. Because of the homogeneity of cell lines, results of experiments using correct conditions are easily reproducible . In addiSince the isolation of the first cell line in the 1950s , a variety of cancer cell lines have been developed for preliminary drug testing . The dif−/−/rag2−/− double knock-out mice, resulting in primary tumor growth and pulmonary metastases in 100% of LNCaP-injected mice, and detection of DNA of 266 circulating tumor cells (CTC) per mL of blood and 35 disseminated tumor cells (DTC) per mL bone marrow after Alu-PCR analysis [There are a handful of prostate cancer cell lines in use today, most of which have been established from metastatic deposits . The LNCanalysis . Throughanalysis ,50. An AI cell line, C4-2, reproducibly and consistently follows the metastatic patterns of hormone-refractory prostate cancer by producing lymph node and bone metastases when injected either subcutaneously or orthotopically in either hormonally intact or castrated hosts. This model enables the study of factors that determine the predilection of prostate cancer cells for the skeletal microenvironment . These CIn vitro anti-cancer screening has long been used by researchers as a rapid tool in screening natural and synthetic compounds for drug development . To asseIn the MTT assay, the MTT salt is bio-reduced by dehydrogenase inside living cells, using the succinate-tetrazolium reductase system, to form a colored formazan dye, while a similar bioconversion using the MTS salt and phenazine ethosulfate as an electron coupling reagent occurs in the MTS assay ,59. In aThe sulforhodamine B (SRB) assay is a rapid, sensitive, and inexpensive method for determining cell growth, utilizing a bright pink anionic dye that binds electrostatically to basic amino acids of trichloroacetic acid fixed cells. The protein-bound dye is extracted with Tris (tris (hydroxymethyl) aminomethane) base to quantify the protein content indirectly with spectrophotometry . The endA known characteristic of cancer cell growth and metastasis is the ability of the cells to escape apoptosis because of a mutation in tumor suppressor genes. Induction of apoptosis is thus used as an important indicator of the ability of chemotherapeutic agents to inhibit tumor growth and progression. The acridine orange/ethidium bromide (AO/EB) apoptosis assay is used to study changes in cellular and nuclear morphology and characteristics of apoptosis under a fluorescent microscope . Both AOThe living status of a cell can be determined by measuring the amount of ATP in the cell, since ATP is necessary for life and function of all cells, and levels of cytoplasmic ATP decrease in cases of injury and hypoxia. After a cell is lysed, ATP is free to react with luciferin and luciferase, producing high quantum chemiluminescence that is linearly proportional to the ATP concentration under optimum conditions. Compared to the MTT assay, the luciferase assay had higher sensitivity and reproducibility over several days and was able to detect the viability of cells with cell counts as low as 2000 cells/well compared to a minimum of 25,000 cells/well required for the MTT assay mentioned above. The ATP assay has been reported as sensitive compared to MTT and calcein assays, used to determine the potency of cytotoxic agents . This hiMany anticancer drugs in use today inactivate target cells by inducing apoptosis . As one Although two-dimensional (2D) cell culture systems involving the growth of a monolayer of cells on a plastic surface or in vivo animals were the standard for drug testing, data from 2D models are often misleading, resulting in difficulties with translational efficacy in vivo ,71. ThisOne of the new systems used to model the complex in vivo intercellular interactions in vitro is the Boyden chamber, consisting of two chambers containing media and partitioned by a semi-permeable membrane ,75. To sAn ideal 3D system would mimic a specific solid tumor microenvironment, where cells are able to replicate and interact with other cells, while promoting differentiation . Most 3DBioprinting allows for the creation of various models that mimic the processes that occur in the tumor microenvironment (TME) and is a method for constructing complex 3D biological structures. This is achieved by printing a bio ink composed of an extracellular matrix (ECM) or other synthetic substrate and cells layer-by-layer in a computer-designed pattern ,82. In tThree-dimensional bioprinting can also be used to design systems that simulate aberrant tumor vascularization to better understand tumor biology and for in vitro drug testing ,82. MicrWith the establishment of 3D cultures, there is a need for monitoring and recording the cultures in 3D in real time and over the time needed for progression to occur, namely, in 4D . Real-ti® system was used to investigate the killing potential of immune cells on cancer cell lines, tracking living cells labeled by a red fluorescent protein, and cell death through the green fluorescent signal generated when apoptotic pathways are activated [® system is a fully integrated incubator, with continuous live cell imaging and an automated analysis platform that combines phase-contrast microscopy and fluorescent image acquisition with an analyzer software package for the quantification of migration image data [The standard IncuCyte-FLR™ system can accommodate up to six 384-well plates and can automatically monitor cell growth, cell migration into a wounded monolayer, angiogenesis and apoptosis . In a rectivated . The Celage data ,87. Procage data . The Nikage data . The Bioage data .The ability of these systems to document real-time drug response in cells have the added advantage of making it easier to quantify transient phenotypic responses, optimize time points for endpoint studies, determine accurate dosing and scheduling regimens, identify cancer cell adaptive responses, and facilitate more robust quantitative analysis from less specimens . The comThe increasing prevalence of cancers worldwide has made the development of quicker methods to create, develop, and test new anticancer drugs a necessity. Anticancer bioassays have proven to be powerful tools in the drug discovery process and preclinical authentication. However, caution should be exercised in comparing results from the different assays, as they usually target different mechanisms. The choice of an anti-cancer bioassay to use depends, to a large extent, on the researcher’s objective, the target cancer cells and phytochemical composition of the medicinal plant, the availability of reagents and cost of reagents, and the experience of the research team. It is also important that a few known anticancer compounds of known potency are included for comparison with potential medicinal plant extracts, regardless of what method is used for cytotoxicity screening, for objectivity and relevance. Cell-based anti-cancer bioassays have much to offer prior to animal testing. Because of lower cost, the investigator has more control of confronting variables, and models can be developed to predict or approximate the phytochemical  effect in animal and, with additional research, human subjects.


Nanoparticulate systems have been extensively explored for the treatment of various diseases, including cancers. The outstanding characteristics of nanoparticles have made it possible to administer them via different routes such as intravenous or inhalation. This flexibility can improve the delivery of encapsulated drugs to the targeted cells for the treatment of lung-related diseases and cancers such as non-small cell lung cancers. The effectiveness of a treatment option needs to be validated in suitable in vitro and/or in vivo models. As the handling of in vivo models is a challenge, many researchers have turned towards in vitro models that use normal cells or specific cells from diseased tissues. This review focuses on the currently available nanoparticles for lung cancers and the type of cellular work that can be conducted to evaluate the effectiveness of a nanoparticulate system for this cancer type.Lung cancers, the number one cancer killer, can be broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC being the most commonly diagnosed type. Anticancer agents for NSCLC suffer from various limitations that can be partly overcome by the application of nanomedicines. Nanoparticles is a branch within nanomedicine that can improve the delivery of anticancer drugs, whilst ensuring the stability and sufficient bioavailability following administration. There are many publications available in the literature exploring different types of nanoparticles from different materials. The effectiveness of a treatment option needs to be validated in suitable in vitro and/or in vivo models. This includes the developed nanoparticles, to prove their safety and efficacy. Many researchers have turned towards in vitro models that use normal cells or specific cells from diseased tissues. However, in cellular works, the physiological dynamics that is available in the body could not be mimicked entirely, and hence, there is still possible development of false positive or false negative results from the in vitro models. This article provides an overview of NSCLC, the different nanoparticles available to date, and in vitro evaluation of the nanoparticles. Different types of cells suitable for in vitro study and the important precautions to limit the development of false results are also extensively discussed. After breast cancer, lung cancer (LC) is the second most diagnosed cancer in the world, with over two million new cases in 2020 . HoweverNSCLC can be further categorized into three types, namely, adenocarcinomas, squamous cell carcinomas and large cell carcinomas. Lung adenocarcinoma occurs in the lung periphery, usually related to the surface alveolar epithelium or bronchial mucosal glands, accounting for about half of all lung cancers . AlthougCurrent treatment options for NSCLC include surgery, targeted therapy, chemotherapy, and radiation therapy . SystemiOther treatment options for patients with metastatic NSCLC who progress after platinum-based chemotherapy (second line and beyond) include pemetrexed and docetaxel  as well Nanomedicine is a nanotechnology branch that employs materials between 5–200 nm, applied to health and medicine ,22. NanoAmong the challenges for progress in NDDS is the requirement for thorough physicochemical characterization and evidence of safety and efficacy in the delivery of the encapsulated drug to cancer cells . PredictBased on the data available in the literature, NDDS has a high potential for lung delivery of active pharmaceutical ingredients (API). Due to their nanosize, these nanocarriers may be capable of effectively traversing the bronchial epithelium barrier and accumulating in deep lung regions. Herein, we present an overview of the latest nanotechnological approaches for the delivery of chemo-immunotherapeutic agents against NSCLC. Types of NDDS, use of cell evaluation, and the rationale behind their applications in permeability and cell uptake studies as well as toxicity evaluation of the NDDS are also presented and discussed.Various nanocarrier platforms are being investigated as the drug delivery systems for an arsenal treatment of NSCLC. Lipid-based  and nanostructured lipid carriers (NLCs)), polymeric-based, and metal-based nanoparticles are some of the well-studied classes of nanocarriers. They are briefly discussed in the following subsections.Doxil is the first nanoliposome formulation containing doxorubicin approved by the US FDA in 1995 to treat AIDS-related Kaposis sarcoma . LiposomSolid lipid nanoparticles (SLNs) are colloidal nanocarriers developed in the 1990s to overcome the limitations of emulsions, liposomes, and polymeric systems . SLNs arNanostructured lipid carriers (NLCs) are second-generation lipid-based nanoparticles reported in the mid-1990s to mitigate the drawback associated with SLNs, as mentioned in Polymeric nanoparticles have been extensively researched for NSCLC therapy, with polymers such as poly-lactic-acid-co-glycolic acid (PLGA), poly-lactic-acid (PLA), chitosan, and polycaprolactone ,53. PolyDendrimers are a unique class of polymeric nanoparticles first reported in the late 1970s . They arPolymeric micelles (PMs) (10–100 nm) are produced by the self-assembly of copolymers or amphiphilic surfactants in water above its critical micellar concentration f. PMs coVarious classes of metal-based nanoparticles such as gold g, silverGraphene h, a carbDelivering NDDS for lung-related diseases has been challenging, but it holds significant potential. Numerous efforts have been reported in the literature to determine the best approach, route, material, and technique to achieve the desired therapeutic outcomes. In general, lung targeted delivery may be achieved through localized  or systemic (intravenous) administration. Systemic administration has been the method of choice to ease delivery, but it causes several drawbacks such as sub-optimal concentration of drug at the site of lung cancer and toxicities on healthy cells .Localized delivery is nevertheless a preferable approach as it lowers the occurrences of adverse reactions that may happen due to systemic distribution. The inhalation route of administration is not only useful for its local effect, but may also contribute to the systemic efficacy of drugs as they may also accumulate in the lymphatic circulation following administration . As NDDSThere are specific challenges that need to be considered to ensure an efficient pulmonary delivery of NDDS. The structure of the respiratory system and lung clearance mechanism may complicate the effort to ensure a sufficient amount of drug-encapsulated NDDS are accumulated at the cancer site ,77,78. TEmerging evidence has highlighted the usefulness of nanoparticles as drug carriers, especially in cancer targeting, due to their ability to deliver various drugs with diverse characteristics in nature. However, it remains a challenge to choose the most suitable approach to closely characterize and evaluate the efficacy and safety profiles of nanomedicines in conditions that closely reflect the physiological complexity. It should be noted that the findings from preclinical studies may not represent the actual clinical outcomes of the developed nanomedicines. Herein, the cellular evaluation of nanomedicines against NSCLC lung cancer cells is reviewed based on the available data in the literature. Different types of cell lines have been reported as models to aid in the understanding of nanoformulation activity and effectiveness at the cellular level. This section will describe in detail the type of cell lines and the suitability of the cells to give the information needed in the evaluation of nanoformulations.Since the 1970s, over 200 lung cancer cell lines encompassing many of the different histological subtypes of the disease have been established. Despite the fact that they have been established many years ago, they often retain characteristics of the original tumor they were derived from . The numEpithelial lung cells are valuable tools for the study of multi-stage lung cancer pathogenesis. For research, two types of culture models are available, namely primary cultured cells and immortalized cell lines.Generally, primary cell cultures retain the morphological and biochemical characteristics of the tumor from which they were originally derived . The priPrimary cell immortalization can be performed using in vivo models, usually of mice. The microenvironment of living tissue promotes cancer cells’ growth, mainly due to the high number of epithelial components and the subsequent crosstalk of the tumor . The comIn vitro cultures of immortalized cell lines isolated from tumors have been used as model systems in cancer for at least 65 years . UnderstImmortalized cell-based models can be composed of solely immortalized cells of two primary pulmonary cell types, namely bronchial and alveolar cell lines. The commonly used bronchial cell lines include Calu-3. This cell line can be cultured under different conditions, namely air–liquid and liquid–liquid interfaces, resulting in the formation of tight epithelia in both cell cultures . HoweverThe Calu-3 cell line can be used to assess formulation transportation in the lung epithelial cell such as in the evaluation of intracellular uptake and transport capability of a liposomal powder formulation loaded with ciprofloxacin and colistin. A study showed that the co-loaded liposomes resulted in a lower transport capability of both drugs across the Calu-3 cell monolayer, resulting in an accumulation in the cell. Since the treatment was aimed at respiratory tract infections, drug retention in the cells is expected to be beneficial .In utilizing alveolar cell lines, the human lung alveolar adenocarcinoma cell line (A549) was often used as a model as it mimics the morphological and biochemical properties of human lung cells and secretes surfactant protein . MonolayCo-culture of the A549 cell line with two different types of cells to create a more complex system resembling the human airways was reported in a study by Wang and co-workers . The A54The monolayers of the single- or co-cultured cell lines can be grown under different conditions, namely liquid–liquid interface (LLI) and air–liquid interface (ALI) conditions. The former is a conventional method to evaluate nanoparticles’ dissolution rate but may not be suitable for assessing nanoparticles intended for inhalation purposes due to the extremely thin aqueous layers in the lungs . DiffereCell-based in vitro assays are routinely used to determine direct cytotoxic or antiproliferative effects of test substances on various cell lines. These assays have been used in the characterization of nanoformulations for NSCLC treatment including liposomes ,101, solA number of antiproliferative assays with different principles have been developed for the cytotoxicity screening of nanomaterials, as shown in MTT assay was first described by Mosmann  and subsIt is worthwhile to note that MTT reduction is readily affected by metabolic and other factors such as the composition of cell medium, which may, in turn, substantially affect the quantitation of cell viability . MTT forXTT, including MTS and WST-1, also presents several problems associated with high-flux drug screening, including the inability of many cell lines to metabolize the tetrazolium in the absence of an added electron transfer reagent such a phenazine methosulfate . HoweverThe WST-1 substrate offers several advantages over MTT and XTT, including water-solubility and greater stability and sensitivity . HoweverSeveral studies have highlighted misleading cell viability results when using tetrazolium salts to evaluate cell viability following treatment with nanomaterials. Wörle-Knirsch and co-workers have demonstrated that single-walled carbon nanotubes bind to MTT-formazan crystals, producing false cytotoxicity results . Data frThe measurement of ATP in cytotoxicity testing is based on the luciferin–luciferase bioluminescent reaction, which requires luciferase enzyme, luciferin and ATP, magnesium , and oxygen. ATP concentration declines rapidly when cells undergo cell death; hence, ATP levels are a reliable indicator of cytotoxicity effects . ATP in 2-25) nanoparticles are also capable of adsorbing LDH molecules, affecting the LDH assay [The LDH assay principle is typically based on cell membrane integrity and monitoring the release of lactate dehydrogenase from compromised cells . LDH assDH assay . ZnO intDH assay . AdsorptDH assay .The trypan blue exclusion assay principle is similar to the LDH assay, except that the trypan blue assay involved microscopy examination of cells. This simple assay requires the use of a hemocytometer and the calculation of cells stained with the blue dye indicating dead cells. . Hence, A recent innovative strategy to measure cell viability in real time was developed to overcome such limitations. The bioluminescent assay is based on the cellular capacity to reduce a luciferase prosubstrate to a form that is then rapidly utilized by the luciferase enzyme present in the assay . Since tIn nanoparticle-based drug delivery system, parameters such as the nanoparticle size, concentration, coating, use of GRAS excipients, agglomeration, surface corona, charge, and hydrophobicity/hydrophilicity significantly affect the biological responses at the cellular level . Thus, oCytotoxicity assays are crucial preliminary assays to determine the safety profile of nanoparticles using cell lines. In fact, the EU NanoSafety Cluster group has suggested that at least four methods of determining cytotoxicity should be used in order to obtain a reliable safety profile for novel nanomaterials . As in tIndeed, the barriers involved in the administration route must be fully understood to ensure optimal delivery of the drugs to the cancerous site. In vitro techniques are imperative to assess the fate of the formulated drug in the lung including binding, transport, uptake, metabolism, and any toxicity related to pulmonary route administration. Particularly, in vitro models for predicting the permeability of drugs or nanoparticles would offer several advantages compared to the in vivo studies, as the former offer simplicity and reproducibility, are less expensive, and do not require ethical considerations . The in Vesicular transcytosis happens in the presence of caveolae  and clathrin-coated pits  . The vesIn vitro and in vivo studies suggest that active targeting nanoparticles will increase selectivity in the cellular uptake and/or cytotoxicity over the conventional chemotherapeutic drugs and non-targeted nanoparticle platform, particularly enhancing drug efficacy and safety . Ligand For a better understanding of the uptake process, quantification of the nanoparticles in the cells would be valuable. However, the small particle size and low mass of the delivery system limit the sensitivity and resolution of certain techniques. Inductively coupled plasma (ICP)-based spectroscopic techniques have been used in the quantification of the nanoparticle uptake in the NSCLC cell lines including ICP-mass spectrometry (ICP-MS) ,174 as wAnimal models have been used in the evaluation of toxicological, allergic, pro-inflammatory, and immunological properties of pharmaceutical products or formulations in pre-clinical studies. However, utilization of ex vivo human systems has gained increasing attention for high-throughput screening of potential toxicological and allergic reactions of the lung towards pharmaceutical formulations, as they can overcome some limitations related to the animal models, including high cost and time spent. One of the most common tissue models is precision-cut lung slices (PCLS), which can be cultured from the explanted healthy or diseased lung of human or animal models, with a more mechanistic resemblance to multiple regions of the lung . This moPlacke and Fisher in 1987 had successfully developed a method to obtain PCLS, which previously imposed a technical challenge for soft tissues such as lungs . The metPCLS has been used to assess the toxicity and efficacy of biological agents or materials to the respiratory tract ,185, incBeing cultured in ALI conditions, the multi-layered monocultures were demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer, offering an advantage over 3D tumor spheroids . There aTo date, there is a variety of 3D cell models available that can be employed to design and evaluate the efficiency of drug nanocarriers. These models enable the researchers to study the specific aspects of the nanomedicine behavior in vivo, although none of these models can recapitulate the exact complex tumor micro-environment. Among the common 3D multicellular in vitro tissue systems are organoid and spheroid systems . They caAnother advanced 3D cell model is the microfluidics-based organ-on-a-chip (Organ Chip), which has recently received increased attention as a promising platform as this cell model accurately replicates the microenvironments of native tissues and various tissue-tissue interactions . It simuNotwithstanding the advantages of cell lines in evaluating the efficiency of delivering nanomedicine for lung cancer treatment, questions have been raised about their reliability since not all lung cancer subtypes are well represented by these cell-based experimental models . The strThe outcome of a seemingly simple cytotoxicity assay might be highly dependent upon the respective protocol and the test substance used. Various factors are known to contribute to aberrant results, such as the physical interactions between the nanomaterials used to manufacture the nanoparticles and the components in the assays , changes in cellular activities involved in redox reactions in response to the nanomaterials, and many other unknown factors that may influence the determination of the cytotoxicity profile of nano-particulate systems . PerhapsIn addition, higher concentrations of nanoparticles (>10 mg/L) appear to have a greater probability of interfering with assay function . Hence, Pharmaceutical technology has been playing a vital role in medicine, as it allows the exploration of different materials and their combinations to prepare drug carriers, and to further endow these with better properties that enable them to reach the desired target site. Many questions arise around the topic of nanocarrier-based lung drug delivery systems. Scientists have given a plethora of responses in the attempt to address all the issues raised, finding alternatives and engineering adequate systems to fulfil requirements and needs. One of the concerns is always the fate of the drug particles once they reached the target site. In pulmonary drug delivery, the objective is sometimes to retain the drug in the lung, as happens in local delivery approaches, thus minimizing the systemic absorption. In other cases, an intravenous delivery route would aim for a systemic effect, and the carriers are engineered to avoid retention at the administration site, with a specific targeting agent attached that would eventually bring the nanocarrier to the lung for the desired therapeutic effect.In vitro cell-culture-based models have been used for permeability screening and cell uptake study of drugs or formulations. Although some cell lines have limitations, continuous improvements have been made including co-culture with other cells to produce a model that can mimic the human lung epithelial barrier as closely as possible. These models are easy to exploit and do not involve ethical considerations related to animal use, as required in a general in vivo study. In a nutshell, the basis behind each decision made on an experimental model should be aligned to the research aims and relevance to human physiology and should be pertinently translated.


Cholangiocarcinoma (CC) is an aggressive malignancy with an inferior prognosis due to limited systemic treatment options. As preclinical models such as CC cell lines are extremely rare, this manuscript reports a protocol of cholangiocarcinoma patient-derived organoid culture as well as a protocol for the transition of 3D organoid lines to 2D cell lines. Tissue samples of non-cancer bile duct and cholangiocarcinoma were obtained during surgical resection. Organoid lines were generated following a standardized protocol. 2D cell lines were generated from established organoid lines following a novel protocol. Subcutaneous and orthotopic patient-derived xenografts were generated from CC organoid lines, histologically examined, and treated using standard CC protocols. Therapeutic responses of organoids and 2D cell lines were examined using standard CC agents. Next-generation exome and RNA sequencing was performed on primary tumors and CC organoid lines. Patient-derived organoids closely recapitulated the original features of the primary tumors on multiple levels. Treatment experiments demonstrated that patient-derived organoids of cholangiocarcinoma and organoid-derived xenografts can be used for the evaluation of novel treatments and may therefore be used in personalized oncology approaches. In summary, this study establishes cholangiocarcinoma organoids and organoid-derived cell lines, thus expanding translational research resources of cholangiocarcinoma. Cholangiocarcinoma (CC) presents a heterogeneous group of malignancies originating from the biliary epithelium accounting for about 3% of all gastrointestinal cancers. According to its anatomical location, CC can be classified into distal (dCC), perihilar (pCC) and intrahepatic (iCC) tumors ,2. AlthoKRAS), tumor suppressor genes , epigenetic factors (IDH1/2), chromatin-modifying genes  [KRAS and TP53 results in a more aggressive CC phenotype [KRAS mutations are more common in pCCs , while IDH1/2 mutations are more frequently present in iCCs [Numerous somatic alterations were described in CC affecting oncogenes  ,12,13,14henotype . Furtherhenotype ,12,16. F in iCCs ,17. The  in iCCs . TestingPreclinical cancer models, such as classical 2D cell lines (henceforth referred to as CCLs) and patient-derived xenografts (PDXs), are essential tools for cancer research and therefore aid in developing potential treatment options ,20,21. HOrganoid culture is an emerging three-dimensional (3D) cell culture technique that allows for more self-organization of tumor cells than CCLs, thus better mimicking the complexity of the parental disease . Human cn = 9) could not be maintained for more than two passages. Based on a trial-and-error method following in-depth literature research, the culture medium was optimized (by adding a Rho-associated protein kinase (ROCK) inhibitor, forskolin, insulin, transferrin, and selenite), leading to the generation of stable CC organoid cultures that could be maintained long-term. Two lines (P68 and P83) were selected for more in-depth characterization in this study /20% fetal calf serum (FCS) and 1/3 Keratinocyte serum-free medium (K-SFM), and supplemented with 100 U/mL penicillin/streptomycin, was finally used as a culture medium, allowing the permanent 2D culture of the CC PDOs  Supplem.The viability, proliferation, and metabolic activity of PDO-derived CCLs were measured using PrestoBlue cell viability reagent (Invitrogen) . While Tscid Il2rgtm1WjlPrkdc/SzJ) [Next, we evaluated the tumorigenicity of the PDOs in a murine xenograft experiment. Organoids were injected subcutaneously  or orthotopically (left liver lobe) into immunodeficient NSG   mice andThe PDOs and corresponding xenograft tumors retained their primary tumors’ histological features, in particular, strong positivity for CK7 and MUC1 as well as nuclear accumulation of P53 .Furthermore, we also examined the CCLs for typical CC markers . All celduct), and non-cancer bile duct mucosa (P121mucosa)) were subjected to RNA sequencing and transcriptomic analysis. After data normalization  and negative controls , full-wall non-cancer bile duct tissue  A,B. In cTo account for the above-described factors causing transcriptomic differences upon the direct comparison of the primary tumor (T) and patient-derived organoids (PDO), differentially expressed genes between tumor and corresponding non-cancer tissue (biliary mucosa) (H) were compared to differentially expressed genes between patient-derived organoids and benign biliary mucosa-derived organoids (BBO). There were 2651 , and 1344  significantly aberrant expressing genes . Upon fuWhile over-representation analysis shows common differences, it disregards slight but concordant differences between phenotypes. The gene set enrichment analysis (GSEA) directly addresses this limitation. The GSEA was administered to validate further the PDO’s ideal reproduction of the tumor’s molecular characteristics. We first defined the P68 prominent gene set, which consists of 66 genes, and then analyzed it as a background biological term of the following GSEA . Figure Except for RNA sequencing, whole-exome sequencing of the PDOs and the corresponding tumor was applied to explore the genomic similarities. The exome sequencing data revealed a broad overlap between non-synonymous mutations between PDO and primary tumor. In the aggregate, 92.7 % (51/55) of non-synonymous variants from patient 68 primary tumors could be reproduced in the organoids . Other cThese next-generation sequencing results substantiate that the patient-derived organoids retain transcriptomic features and the mutational landscape of the parental tumor.We next investigated the responses of PDOs and corresponding CCLs to anti-cancer agents regularly used in CC  in a PrestoBlue based cell viability assay. While the inhibitory effects of gemcitabine and sorafenib on the CCLs were quite similar, our PDOs presented individual drug responses, with P68 being more resistant A–D. Not In the following in vivo drug response experiment, gemcitabine was used and compared with the control group. After 14 days, treated xenografts showed halted tumor growth while the control group kept gaining size until euthanasia E.CC represents a rare but highly malignant cancer with subtypes characterized by unique etiology, risk factors, carcinogenesis, and molecular profile . As precKRAS, TP53, IDH1, or ARID1A. After optimization of the culture medium, organoid cultures could be maintained for extended periods of time. Moreover, this finding demonstrates that it is possible to generate stable CCLs from 3D cultures and therefore gain more options for high throughput experiments. All cell lines showed diverse morphologic characteristics in 2D and organoid cultures due to the different origins in the bile duct system, indicating divergent underlying carcinogenesis mechanisms. The PDOs showed individual drug responses to established anti-cancer agents, whereas the corresponding CCLs, as less complex in vitro models, responded quite similarly, emphasizing the potential advantage of PDOs over CCLs in therapeutic experiments. Nonetheless, both PDOs and CCLs carry the inherent limitations of cell culture experiments as there are no other cell types such as stromal or immune cells present. The complex interactions between tumor cells, microenvironment, and the host immune system cannot be reproduced in either system [Patient-derived cancer organoids (PDOs) present a relatively novel and highly realistic modeling system with various applications in preclinical cancer research, enabling translational individualized treatment regimens ,56,57,58r system .Patient-derived xenografts (PDXs) retain the histopathological and genomic features of primary tumors, especially the tumor heterogeneity, and present valuable models for studying in vivo drug responses and tumor-stroma/immune interactions ,61. On tCurrently, there are limited published sequencing data comparing cholangiocarcinoma PDOs and corresponding tumors . This prTo illustrate the consistency of histologic architecture, we examined the IHC profiles of our PDOs and corresponding xenografts compared to their primary tumors. At present, no particular immunohistochemical profile exists for bile duct cancers . CytokerThe current research established a robust protocol for PDO generation and a biobank from CC samples. We present the molecular characteristics of permanent CC-PDO lines and report a protocol for the transition of PDOs to classical 2D cell lines. A major limitation is the small sample size and the lack of an organoid line of dCC. dCCs are often small tumors as occlusion of the bile duct occurs early during tumor development . The lacduct) and the isolated mucosa (P121mucosa) were used as two individual samples. We established benign bile duct organoids with tissue from P119 using the same protocol for the cholangiocarcinoma samples (more detailed information is given in Cholangiocarcinoma tissue of patients who underwent surgery (January 2015 to May 2018) at the Department of Gastrointestinal, Thoracic, and Vascular Surgery of University Hospital Carl Gustav Carus Dresden, was obtained directly after resection by a board-certified pathologist, stored on ice, and immediately processed as described below. All patients gave their written informed consent prior to inclusion into the study. Detailed information is shown in All animal experiments were carried out in strict compliance with German and European Animal Protection Acts and approved by institutional and governmental animal welfare commissions before initiation of the experiments. Mice were housed in a specific pathogen-free environment with a 12 h light/dark cycle and were given ad libitum access to standard laboratory diet and water.scid Il2rgtm1WjlPrkdc/SzJ) [NSG mice   were breg, 3 min), and then filtered through 100 μm and then 40 μm cell strainers to remove any huge tissue clusters. Next, resuspended the single cells/cell clusters fraction (1 × 105/well) in Matrigel (Corning) and seeded 30 μL per well in a pre-warmed 48-well plate. After solidifying the Matrigel domes (10 min at 37 °C), 200 µL of medium , and digested using Dispase II (Roche) 1 mg/mL and Collagenase type I (Worthington) 0.1 mg/mL at 37 °C for 2 h. During disaggregation, the samples were pipetted up and down every hour. Obtained tissue fragments were washed with PBS, differential centrifugation (200× f medium  was adde5/well) were resuspended in Matrigel and cultured using identical conditions. To augment the survival time of the organoids, we deviated from the above protocol and modified the medium as described in 2 atmosphere.Trypsinized TFK-1 cells (1 × 10After organoid lines were established stably (after six passages), organoids were transferred to two-dimensional (2D) culture and maintained as permanent cell lines. 2–4 wells of organoids were dissociated and mixed with cell recovery solution (Corning). The obtained cell pellet was resuspended in an organoid medium  and tranPlease see TM mice. Subcutaneous tumors were measured with calipers, and tumor volume was calculated using the following formula: V = (Width2 × Length)/2. The presence of orthotopic tumor growth was confirmed via necropsy and or ultrasound.For xenografting, 15 complete wells of each organoid line were harvested and resuspended in a mixture of 20 µL Organoid medium with 20 µL Matrigel. Organoids were injected either subcutaneously in both hind legs or orthotopically in the left liver lobe of NSGOrganoids were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 2.5 µm sections. Hematoxylin and eosin (H&E) staining, as well as immunohistochemistry (IHC) stainings for CK7, TP53, and MUC1, were performed. The 2D cell lines were cultured on chamber slides and fixed with 2% PFA. Immunofluorescence (IF) for CK7, CK19, EpCAM, and TP53 were carried out. All the histology checking procedures followed the standard protocols. The detailed information of antibodies mentioned in this manuscript was listed in duct, P121mucosa), and benign biliary organoids (P119). Besides the primary tumor, there are three biological repetitions for each group.This project recruits several groups for transcriptome sequencing, including primary cholangiocarcinoma (P68), corresponding patient-derived organoids (P68), TFK-1 cell lines, TFK-1 organoids, non-cancer bile duct  values are given in the relevant figures and p-value < 0.050. Doubling time and IC50 calculation were determined with non-linear regression.All experiments have been performed at least two times independently, with summary data being presented as mean ± standard deviation (SD). Sample size (We established human cholangiocarcinoma organoid and corresponding 2D cell lines using a robust protocol for CC organoid generation and culture. The organoids react to treatment and grow as subcutaneous and orthotopic tumors in mice. This methodological study thus reports novel tools to establish organoid lines, 2D cell lines, and mouse models for preclinical cholangiocarcinoma research.


Leontopodium alpinum Cass. (edelweiss) is recognized as a frequent constituent of anti-aging skin care products, providing increased antioxidant and anti-inflammatory defense. Considering the growing demand and the protected status of edelweiss in many countries, alternative methods of production have been developed, one of them being callus culturing. This study reports the phytochemical composition of a methanolic extract of L. alpinum callus cultures, characterized by liquid chromatography coupled to ion-mobility high resolution mass spectrometry (UPLC/IM-HRMS). The methanolic extract exhibited strong free radical scavenging activity (122.19 ± 7.28 mg AAE/g dw), while the quantitative evaluation revealed that four major constituents (phenylpropanoid derivatives) represent 57.13% (m/m) of the extract. Consequently, a screening of antiproliferative effects was performed on ten cancer cell lines, representative of prostate, colon, lung and breast cancer, showing inhibition of colony formation in all cases. These results provide a comprehensive phytochemical characterization of L. alpinum callus cultures using advanced IM-HRMS, while the in vitro explorations confirmed the potent antioxidant properties of edelweiss which are worth exploring further in cancer prevention. Leontopodium alpinum Cass. (edelweiss) is one of the most well-known members of the Asteraceae family. It grows on limestone surfaces, prefers the alpine climate and it is a protected species in many countries. In traditional medicine, edelweiss was used as remedy in digestive and respiratory illnesses as powder, infusion and tincture [tincture  or in thtincture . NowadayCallus culture  is a developing technique for the production of useful compounds, including pharmaceuticals . The mosLeontopodium alpinum phytochemical composition highlighted a number of compound classes, such as phenylpropanoid, sesquiterpene, benzofuran and lignan derivatives. However, data regarding the composition of callus cultures is rather limited [Previous investigations ,10,11,12 limited ,14. So f limited , showingL. alpinum extracts effects has been focused on anti-inflammatory effect evaluation [During the last decade, the preclinical research on aluation , leukotraluation , anti-agaluation  and memoaluation . Howeveraluation , due to The aim of this study was to perform a comprehensive phytochemical characterization of edelweiss callus cultures. Following the assessment of the antioxidant capacity evaluation, two major cancer hallmarks were also investigated (proliferation and resisting cell death) using a panel of ten cancer cell lines representative of breast, prostate, lung and colon cancers.L. alpinum extract was obtained by microwave assisted extraction (MAE). In comparison to ultrasound assisted extraction (UAE), this technique was found to offer similar yield in a much shorter time (5 min for MAE compared to 45 min by UAE). The total extraction yield was calculated to be 26.4% .The Further, the methanolic extract of edelweiss callus cultures was subjected to phytochemical profiling using a reversed phase LC/IM-HRMS method, achieving a good separation of the detected compounds . ConsideL. alpinum, whereas for the untargeted approach, a diagnostic product ion (DPI) strategy was implemented [L. alpinum extract: glucaric acid derivatives and quinic acid derivatives. Therefore, glucaric acid (m/z 191.0192), peaks 2 and 3 , and quinic acid (m/z 191.0556), peak 1  were used as DPI  based on the fragmentation pattern and previous reports [Both targeted and untargeted strategies have been implemented to identify most of the compounds present in the extract. The targeted approach was based on literature reports of major phytochemical constituents in lemented . Using t DPI see . Peak 4  reports . Out of  reports  and a maL. alpinum callus cultures. These derivatives are formed by the esterification of D-glucaric acid hydroxyl groups with caffeoyl and 3-hydroxybutanyl moieties. In total, 11 glucaric derivatives have been identified. Among these, peak 17 leontopodic acid A  and peak 13 leontopodic acid B  were the most abundant and their identification was validated using standards. Isomers of both leontopodic A and B were detected as peak 18 and 15, respectively.Glucaric acid derivatives are the most abundant class of secondary metabolites identified in 24H22O14, 1.2 mDa) isomers were identified as peaks 6, 7 and 8, while peak 24 represents tetra-caffeoyl glucaric acid , a tetra-substituted derivative [26, it is most likely to be a penta-substituted glucaric acid derivative , with two 3-hydroxybutanyl and three caffeoyl moieties.A series of di-caffeoyl glucaric (Cm/z 191.0556) was used as DPI. Peaks 5 and 14 were identified by comparison with standards and annotated as chlorogenic acid  and 3,5-dicaffeoyl quinic acid , respectively. Two other di-caffeoyl quinic isomers  were identified as peaks 12 and 16.For the identification of quinic acid derivatives, quinic acid , while the in vitro antioxidant activity of the extracts expressed in ascorbic acid equivalent antioxidant capacity (AEAC) was 122.19 ± 7.28 mg AAE/g dw, as determined using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay.The total polyphenol determination revealed that the The phytochemical characterization of the LA extract highlighted high concentrations of polyphenolic derivatives with strong antioxidant activities. Consequently, we exposed a panel of diverse cancer cell lines  to LA extract and performed several in vitro screenings. Particularly, we focused of two hallmarks of cancer, namely sustaining proliferative signaling (cellular proliferation and colony formation capabilities) and resisting cell death .L. alpinum extract on cancer cell panel viability was studied by 3--2,5-diphenyltetrazolium bromide (MTT) assay. The assay involves conversion of 3--2,5-diphenyltetrazolium bromide by NADPH-dependent oxidoreductases to a purple-colored formazan product whose VIS absorbance is proportional to the number of metabolic active cells. Cells were grown in 96-well plates and treated with complete medium containing serial dilutions of extract (6.25–400 µg/mL). A blank control (complete medium only) and a positive control (100% DMSO) were included in every plate. The extract showed significant effect on cell viability at the highest tested concentration (400 µg/mL) on most cell lines : this is the case for SK-MES-1 (67.2 ± 23.8%) lung cancer cell line and MDA-MB-231 (77.21 ± 4.9%) and HS578T (67.4 ± 13.3%) TNBC representatives.Effect of ll lines , exceptiL. alpinum extract had the greatest impact on cell viability and was used in the in vitro functional assays conducted further.The 400 µg/mL dose of L. alpinum callus cultures’ methanolic extract on cellular morphology and cell distribution was evaluated by 4′,6-diamidino-2-phenylindole (DAPI) and Fluorescein phalloidin  staining, used for nuclei and actin filaments labeling. The levels of toxicity and the antiproliferative effects were assessed by comparing control and treated cells groups. A summary of observed effects is found in The effect of the A substantial reduction of cell population was seen in the human non-cancer fibroblast (BJ cell line) and MDA-MB-231 TNBC in accordance with the viability assay conducted. Reduction of cell population was also observed in the case of both HCT-116 and DLD-1 colon cell lines. Particular to BJ cell line was the absence of signs of nuclear and cytoskeletal damage.L. alpinum extract induced nuclear fragmentation on most cell lines, excepting BJ, HS578T and SK-MES-1. Furthermore, multilobed nuclei were observed for HS578T, SK-MES-1, 22RV1, and HCT116, associated with rounding of cells. This last phenomenon was found also for H1792 and LNCAP in a less extensive manner. A marked cytoskeleton fragmentation was observed in the case of prostate cancer compared to the other cell lines.The L. alpinum extract, few cells presenting nuclear fragmentation and cytoskeleton damage, with most of the cells having similar shape to the control.Compared to TNBC cell lines, MCF-7 was less sensitive to Another cancer feature is the capacity to develop secondary cancer sites, also known as metastatic sites. Colony assay was chosen to analyze this ability in vitro, by measuring the colony diameter of both control and treated cells .L. alpinum extract generated small and hardly visible colonies in the captures, the colony formation capacity of 22RV1 being half of control, while for LNCAP cells the capacity was even lower.After treatment, 22RV1 and LNCAP prostate cells colonies were considerably reduced and both cell lines exhibited a low ability to create new colonies . CompareHCT116 colon cancer cells showed higher capacity to form colonies compared to DLD-1. The extract considerably inhibited the colony formation of both cell lines, with a pronounced effect on HCT116. Lung cancer cell lines (H1792 and SK-MES-1) showed high colony formation capacity, but the treatment reduced the colony number and size. The inhibitory effect of the treatment was confirmed on all three cell lines, the colony diameter being decreased by more than half compared to that of control. Similar to lung, colon and prostate cell lines, the extract inhibited colony formation was seen in all breast cancer cell lines. Breast cancer cell lines exhibited different colony formation capacities, the most potent being MCF-7, followed by MDA-MB-468 and the other two TNBC cell lines.L. alpinum was used mostly for its anti-inflammatory properties. Today, its current use is limited due to the protected species status that the plant has in many countries. However, the growing demand coming from the cosmetic industry, due to the antioxidant properties of L. alpinum, required the development of alternative means to obtain the specific constituents, such as callus cultures of edelweiss, now a world-wide substitute.In traditional medicine, In medicine, many plant antioxidants have been shown to possess antitumoral properties . AlthougMicrowave assisted extraction (MAE) and ultrasound assisted extraction (UAE) were assessed for the targeted extraction of polyphenolic compounds from edelweiss callus cultures, using the same extraction solvent (70% methanol). MAE was found to offer a faster extraction time (5 min), compared to UAE (45 min), both having comparable yield (data not shown). The dried extract was easily soluble in aqueous solutions, even at high concentrations (4 mg/mL stock solutions).L. alpinum extract was performed using LC/IM-HRMS. Compared to conventional mass spectrometry, ion-mobility mass spectrometry offers a supplementary separation dimension. Being a gas phase separation technique, ions are resolved based on their size and shape, depending on their collisional cross section (CCS). Ion-mobility mass spectrometry proved to be useful for differentiating between isomeric species [L. alpinum extract, no co-eluting isomeric species could be identified, all of them being resolved chromatographically before detection. The CCS values of the molecular ions of the constituents detected in the extract are presented in 6, 7 and 8) and di-caffeoyl quinic acid isomers . However, their separation by ion-mobility alone would probably not be possible considering the low resolution of today’s commercially available technology.The polyphenol profiling of the  species . Still, L. alpinum) such as isomers of leontopodic acids A and B, disubstituted derivatives of both glucaric (di-caffeoyl glucaric acids) and quinic acids (di-caffeoyl quinic acids), together with two highly substituted derivatives of glucaric acids (tetra-caffeoyl glucaric acid and a penta-substituted glucaric acid derivative).The analysis of the extract revealed 27 major constituents. The two detected classes of polyphenols were derivatives of glucaric and quinic acids, the most abundant being leontopodic acids A and B, chlorogenic acid and 3,5-dicaffeoyl quinic acid. Besides these already known constituents, several other derivatives were observed  with other compounds reported in the composition of terpenes ,10,11, lterpenes , or flavterpenes .L. alpinum    revealedConsidering the high quantity of polyphenols determined in the edelweiss callus cultures, an in vitro screening procedure was implemented which allowed the evaluation of the extract on three cancer hallmarks. Its effects were studied on ten cancer cell lines, representative of four different cancer types. A summary of the obtained results is presented in L. alpinum callus cultures extract was evaluated in vitro using the MTT assay. Decreased cell viability was observed at the highest concentration tested (400 µg/mL) for TNBC breast cell lines MDA-MB-231 and HS578T, both colon , and in lung cancer cell lines (SK-MES-1 and H1792). In the specific case of MCF-7, an estrogen dependent cell line, pro-proliferative effects have been observed. It has already been documented that polyphenols, due to their chemical structure, can interact with estrogen receptors with different affinity towards estrogen receptors α and β. A deeper insight into the mechanisms involving polyphenols’ effects on hormone dependent cancer cell lines can be found in a comprehensive review published by Cipolletti et al. [The cytotoxic effect of i et al. .Next, other cancer hallmarks were studied in order to observe potential induced morphological changes and colony formation capabilities. Colony formation was inhibited for all cell lines, presenting substantially reduced diameters , with prThese results could be attributed to the high quantity of polyphenols which are known to modulate pathways relevant to proliferation, migration or colony formation and in some particular cases cell death could be triggered ,28,29,30Using in vitro models, polyphenols were previously described to possess anticarcinogenic properties, capable of inhibiting tumor growth, metastasis and inflammatory processes ,32,33. TL. alpinum extract, 3-5-dicaffeoylquinic acid, acts as antioxidant agent with neuroprotective effects [50 below 300 µM  [The second quinic acid derivative found in high quantities in  effects . On top, effects . The anteatment) . The autL.alpinum callus cultures, and remains a subject open to research.Unfortunately, less is known about the potential anticancer mechanisms of actions of the leontopodic acids A and B, the other two major constituents of The reference standards of chlorogenic acid, 3,5-dicaffeoylquinic acid and leontopodic acids A and B which were used in this work were acquired from Extrasynthese . The organic solvents, methanol and acetonitrile, used for mobile phases and extractions were of LC-MS purity and were purchased from Merck . Ultrapure water was produced with a Purelab water filtration system , while MS grade formic acid was produced by Carlo Erba . 2,2-Diphenyl-1-picrylhydrazyl and Folin-Ciocâlteu’s phenol reagent were purchased from Sigma , ascorbic acid was produced by International Laboratory , and sodium carbonate by Lachner .Leontopodium alpinum Cass. (www.semintelegumeflori.ro (accessed on 10 December 2021), batch Pl730/12/210/1274A, SC XTREME ANG MARKETING SRL, Bucharest, Romania), aseptically germinated, and seedlings were used to initiate the callus culture on Gamborg medium [Commercial seeds of g medium , supplemL. alpinum callus cultures from three batches were freeze-dried  using a LyoQuest -55 Plus instrument , then all samples were mixed to create a representative sample which was further used for this study. Ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) were assessed in order to find the most suitable extraction conditions.Prior to extraction, v/v), then sonicated for 45 min at 37 kHz  [v/v) [v/v) was performed 3 times , the organic phase being discarded [For UAE, 20 mg of freeze-dried sample was suspended in 1 mL of 70% methanol (Germany) . MAE (byy) [v/v) . The expy) [v/v) ,42,43. Tiscarded . The metm/z, using 2 functions. The collision energies were set to 4 eV (trap) and 2 eV (transfer) for the first function and a ramp between 20–45 eV (trap) for the second function. Leucine-encephalin was used as single point reference (m/z 554.2615).All analyses performed for the phytochemical characterization were carried out on a Waters I-Class UPLC instrument coupled with a Synapt G2-Si high resolution mass spectrometer . The separations were achieved by reversed phase using a BEH C18 (100 × 2.1 mm) stationary phase  and mobile phases formed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), being pumped at a flow rate of 0.3 mL/min at a temperature of 45 °C. Gradient elution was implemented as follows: 0 min 3%B–10 min 40%B–15 min 98%B. The mass spectrometer was set in resolution mode using negative electrospray ionization (2 kV capillary voltage), with source temperature of 130 °C. Data independent acquisition was achieved in two modes: (i) HDMSE (ion-mobility enabled) for qualitative runs and identification purposes and (ii) MSE (Q-ToF only) for quantification. Data was acquired in the range of 50 to 1200 Data processing  was carried out using Progenesis QI software version 3.0.76 . A first identification was performed on the most abundant compounds by using an in-house natural compounds library. Major compounds were putatively annotated based on their accurate mass, fragmentation pattern, available reference standards and on other previous studies.Quantitative determinations of leontopodic acids A and B, chlorogenic acid and 3-5-dicaffeoylquinic acid were made by constructing a 5-point calibration curve (0.1–40 µg/mL) for each analyte. Each standard solution was injected 2 times and the quantitative analysis was performed using the TargetLynx module of MassLynx software , by measuring the molecular peak areas of the analytes. Injection repeatability was assessed by injecting 5 times the previously mentioned standards (RSD less than 3.85%). Two replicates of the extract were prepared, then analyzed twice, after a 40-fold dilution.v/v) and shaken for 60 s. The mixtures were kept for 40 min in darkness at room temperature, then absorbance at λ = 515 nm was measured using a Clariostar Plus microplate reader . The blank sample consisted of 20 μL of water with 180 μL of methanol:water , while the control sample was 20 μL of water with 180 μL of DPPH solution. Each determination was performed in triplicate and the percentage of quenched DPPH was determined using Equation (1).Antioxidant assay was performed using a microplate protocol described by Navarro et al. . Two serBy calculating the concentrations of sample and extracts equivalent to 50% quenched DPPH, the ascorbic acid equivalent antioxidant capacity was determined.2CO3 was added, followed by incubation at room temperature and darkness for 2 h. The solution absorbance was measured at λ = 765 nm . The determination was made in triplicate. The gallic acid calibration curve was built, showing a linear range for concentrations between 10–200 μg/mL gallic acid. The total polyphenol content of extracts was expressed as mg of gallic acid equivalents (GAE)/g dry weight (dw), using the Equation (2) based on the calibration curve [T = the total phenolic content in mg/mL as GAE; C = the concentration of gallic acid from the calibration curve in mg/mL; V = the volume of the extract solution in mL; M = the weight of the extract in g.The total polyphenols determination was made according to the method described by Singleton et al.  and modion curve .(2)T=C L. alpinum extract. Non-cancer human epithelial fibroblast (BJ—ATCC CRL-2522) and double positive breast cancer cell line (MCF7—ATCC HTB-22) were cultivated in MEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. Triple negative breast cancer cell lines , lung cancer cell lines (H1792—ATCC CRL-5895 and SK-MES-1—ATCC HTB-58), colon cancer cell line DLD-1 (ATCC CCL-221) and prostate cancer cell lines (LNCAP—ATCC CRL-1740 and 22RV1 ATCC CRL-2505) were maintained in RPMI supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamine, while HCT116 colon cancer cell line (ATCC CCL-247) was cultivated in McCoy’s supplemented with 10% FBS and 1% Penicillin/Streptomycin. All cell lines were maintained in a humidified chamber, at 37 °C and 5% CO2. All the reagents needed for cell culturing were purchased from Gibco .A panel consisting of twelve human cell lines was chosen for the in vitro evaluation of L. alpinum extract stock solution was prepared at a concentration of 4 mg/mL by dissolving the necessary amount of freeze-dried extract in the appropriate culture medium for each cell line. Subsequent dilutions were prepared at specific concentrations needed for the different assays.The L. alpinum callus cultures’ methanolic extract was investigated using the MTT assay. Cells were grown at a density of 1 × 104 cells/well in a 96-well flat-bottom plate, each treatment having 3 replicates. After 24 h incubation, cells were treated with different concentrations of extract (6.25–400 μg/mL) for 48 h. To remove possible extract residues that might interfere with MTT assay, a wash step with Phosphate Buffer Saline 1× (PBS) was performed, then cell viability was determined after incubation for 3 h with 100 μL 1 mg/mL MTT. Afterwards, the solution was removed and 150 μL of DMSO were added to dissolve the crystals. The absorbance corresponding to the viable cells was measured at λ = 570 nm on a SPARK10M multiplate reader . A blank control (medium only), and a positive control (DMSO 100%), were included on every plate. The experiment was repeated three times. Statistical analysis was performed using Graphpad Prism (version 8) software . Cell viabilities were expressed as percentage of control (set as 100%) and represented as mean ± SEM. Data was analyzed by one-way ANOVA followed by Dunnett’s multiple-comparison test.The effect of L. alpinum extract (400 µg/mL) and incubated for 48 h, then the cell culture medium was replaced with treatment free medium. Cells were observed daily up to 14 days and when the colonies reached the proper size (more than 50 cells/colony) or when the colonies were too close to another, the experiment was stopped and the cells were fixed with methanol and stained with crystal violet  [t-test was applied to determine significant differences in colony numbers. *An equal number of cells (500 cells/well) were seeded in a 6-well plate, both for control and treatment. After 24 h the cells were treated with MO, USA) ,50. The L. alpinum extract (400 µg/mL), cells were analyzed in bright field with an inverted microscope. The same instrument was used in combination with a fluorescent source  for details regarding the cytoskeleton and nucleus, after staining these cell compartments with Phalloidin-FITC (for the cytoskeleton)  and DAPI (for the nucleus)  using a previously published protocol [To investigate the morphological changes induced by the 48 h treatment with L. alpinum callus cultures revealed high quantities of polyphenols, most of them being derivatives of glucaric and quinic acids. The four most abundant represent more than 57% (m/m) of the dry-frozen extract and around 15% (m/m) of the dry-frozen callus culture, demonstrating that in vitro culturing can be a useful alternative to produce natural compounds.The phytochemical profiling of L. alpinum callus cultures extract demonstrated interesting effects regarding its capacity to inhibit proliferation and colony formation. L. alpinum callus cultures extract showed considerable inhibitory effect on SK-MES-1, MDA-MB-231 and HCT116 cell lines, most probably being related to the high content in polyphenolic compounds which exhibit strong antioxidant capacity.The screening of cancer cell lines revealed that the L. alpinum as chemosensitizer might represent a perspective. To better understand the possible effects of leontopodic acids A and B, further research could also focus on evaluating the proliferation, migration or colony formation assays on cancer cells treated with these two natural products.Considering its abilities to inhibit cancer proliferation and colony formation, the use of Leontopodium alpinum Cass. (Edelweiss) and the crude extract, OSIM Romania Patent Application A/00740/17.11.2020.Cogălniceanu G. C., Mitoi E. M., Ciocan A. G., Holobiuc M. I., Maximilian R. C., Helepciuc F. E., Morosanu A. M. Biotechnological procedure for initiating and obtaining high proliferative cell mass producing bioactive compounds in