Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices.We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices , porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties.OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG.We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.The online version contains supplementary material available at 10.1186/s12885-023-11275-6. Although 2D culture settings on plastic are simple and convenient to use, they have many critical limitations when aiming to replicate the complexity and pathophysiology of a tumour. Under such simplified conditions, tissue-specific architecture, mechanical and biochemical signals and cell-cell and cell-matrix interactions are lost . Cell adCancer research, including drug testing, has traditionally focused mostly on cancer cells originating from primary tumours. However, in many solid cancers, metastases are the main cause of cancer deaths, and the availability of efficient drugs to prevent or treat metastatic tumours is very limited . DespiteWe formerly developed the first human tumour-derived matrix, Myogel . It is aOf head and neck cancers, oral tongue squamous cell carcinoma (OTSCC) is the most prevalent cancer in the oral cavity, and despite improvements in treatment, it remains characterized by poor survival, mostly because of its tendency for LN metastasis . HoweverThe experimental protocol for use of OTSCC patient samples was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 31/2016). Four primary  and three metastatic  tumour cell lines were isolated from OTSCC patient samples at Oulu University Hospital after informed consent. Clinical characteristics of OU-OTSCC cell lines are described in Additional file 1: Table 2 and 95% humidity. All the cell lines used were at passage number under 30.UT-SCC-24A , UT-SCC-24B , UT-SCC-42A  and UT-SCC-42B  have been established at the Department of Otorhinolaryngology – Head and Neck Surgery, Turku University Hospital, Finland and were kindly provided by Professor Reidar Grénman. UT-SCC and the commercial human OTSCC HSC-3 (Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB0623) cell lines were cultured in DMEM/F-12 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin , 0.4 µg/ml hydrocortisone (Merck), 50 µg/ml ascorbic acid  and 250 ng/ml amphotericin B (Merck). All cell lines were maintained at 37 °C with 5% COExperimental protocols on human leiomyoma tissue were approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 2/2017). Clinically, non-metastatic human cervical lymph node tissues  were collected from three oral or hypopharyngeal cancer patients undergoing neck dissection surgery. Based on the clinical evaluation, the patients had more than two metastases. The collected fresh tissue was evaluated to be macroscopically non-metastatic by the expert pathologist. The study was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 31/2016). All patient samples were collected after informed consent was obtained.Sus scrofa domesticus) sacrificed for other experimental study at the University of Oulu Laboratory Animal Centre KEKS . The animals were purchased for the experimental study from a contract breeder  providing pigs for Oulu Laboratory Animal Centre. The original experimental study protocol was reviewed and approved by the National Animal Experiment Board of Finland (ESAVI/6941/04.10.07/2016) and animals were sedated with midazolam 0.2 mg/kg  and ketamine 10 mg/kg  combination. Anesthesia was induced with fentanyl (50 µg)  and maintained with sevoflurane (1-1.5%)  and infusion of fentanyl and midazolam. After surgical operations and experiment, the animals were euthanized with pentobarbital-Na 60 mg/kg .Normal anterior cervical lymph nodes were dissected from healthy domestic pigs . The next day, the discs were transferred onto steel grids in 12-well plates with 1 ml of medium. Cells were cultured on top of the myoma disc for 18–20 days. The tissues were fixed in 4% neutral-buffered formalin, dehydrated and embedded in paraffin. Then 6 μm sections were prepared for immunohistochemistry with monoclonal pan-cytokeratin AE1/AE3 antibody  as described previously -fibrinopeptide B  in 50% acetonitrile/0.1% formic acid solution at a flow rate of 0.3 µl/min was applied every 30 s.The samples were run in ion mobility assisted in data-independent analysis mode (HDMSE), in a Synapt G2-S mass spectrometer (Waters), by alternating between low collision energy (6 V) and high collision energy ramp in the transfer compartment (20 to 45 V) and using a 1-sec cycle time. The separated peptides were detected online with a mass spectrometer, operated in positive resolution mode in the range of Protein identification relative quantification between samples using precursor ion intensities was performed with Progenesis QI for Proteomics™ Informatics for Proteomics software . MSE parameters were set as follows: low energy threshold of 135 counts, elevated energy threshold of 30 counts, and intensity threshold of precursor/fragment ion cluster 750 counts.Database searches were carried out against Human Proteome UniProtKB-SwissProt database  with Ion Accounting algorithm and using the following parameters: peptide and fragment tolerance: automatic. Maximum protein mass: 750 kDa. Minimum fragment ions matches per protein ≥ 7. Minimum fragment ions matches per peptide ≥ 3. Minimum peptide matches per protein ≥ 1. Primary digest reagent: trypsin. Missed cleavages allowed: 2. Fixed modification: carbamidomethylation C. Variable modifications: deamidation of NQ residues. Oxidation of Methionine and false discovery rate (FDR) < 4%.Statistical analyses were carried out with SPSS Statistics 27 . To determine the statistical significance, we performed with one-way analysis of variance (ANOVA) followed by Bonferroni correction or independent samples T-test. P-value < 0.05 was considered statistically significant. Figures were created with Origin 2020 graphing software  and Adobe Photoshop CC 2019 .https://metascape.org/) and can be used to assess e.g., enrichment of biological and cellular pathways, drug targets and diseases of a user-provided list of genes/proteins. The total proteomes of each matrix were compared to identify common and unique proteins.Three technical replicates for each matrix were produced by proteomic analysis. To study the proteomes of each matrix, the data from replicates were combined, and duplicates were removed based on the protein accession number. Next, uncharacterized proteins along with proteins with duplicated descriptions (Names) were removed for each matrix. Proteins found in each matrix were analysed with Metascape  to identThe proteins (by protein entry) and intensities (by matched product) identified by proteomic analysis for three technical replicates of Myogel and h-LG were combined. For quality control and to compare levels of proteins present in both matrices, the proteins present in only one technical replicate or in only one matrix were filtered out. Missing single values were replaced by the average intensity of the two other technical replicates. The intensity values for each protein entry were log2-transformed and normalized with quantile normalization method. The mean transformed and normalized intensity values for each protein entry were compared between matrices, and a fold-change of 2 was used as the threshold to identify differentially expressed proteins between matrices. R version 4.2.2 with “dplyr”,”data.table” and ”zoo” packages were utilized for this analysis.Cell adhesion-related proteins common and unique to each matrix were extracted from the total proteome of each matrix by a comparison with proteins associated with the GO-term GO:0007155 (Cell Adhesion) in the GeneOntology (GO) database. Shared proteins and adhesion proteins with similar names and different accession numbers between human and porcine matrices were compared based on sequence identity on the BLAST tool from NCBI . Information on the cellular location for adhesion-related proteins were collected from UniProt via UniProt ID mapping tool .OU-OTSCC cell lines established from primary and metastatic tumours showed pancytokeratin immunostaining similar to HSC-3 cells, confirming that all cell lines are epithelial in origin and considered as carcinoma cell lines  and h-LG the largest (74) Fig. . Myogel Results of cancer progression, metastatic processes and drug testing may often be misleading due to the lack of biologically relevant study matrices. Particularly, a LN-derived matrix that would be suitable for metastatic cell lines remains unavailable on the market. In this study, we established three tongue cancer cell line pairs: OU-OTSCC-7, -9, -18 including primary tumour (A) and their corresponding lymph node metastases (B) cells. In addition, we established one primary tumour cell line, OU-SCC-15, and used a commercial human metastatic OTSCC cell line, HSC-3, as a control for aggressive tumour behaviour. We used these cell lines to assess the effect of three different TME matrices, human uterine leiomyoma-derived Myogel and normal porcine neck LN- and human HNSCC patients’ pre-metastatic neck LN-derived Lymphogels, on carcinoma cells’ proliferation, adhesion, migration and invasion. We also investigated the protein composition of Myogel and both Lymphogels and compared them with each other to elucidate the differences in cell behaviour.Primary and metastatic OTSCC cell lines showed similar cell viability on Myogel and both Lymphogels, confirming that all matrices had a protein composition that supports cell growth. In all primary-metastatic pairs, metastatic cells adhered more on human pre-metastatic LN-derived Lymphogel (h-LG). Although this difference between metastatic and primary cell adhesion on h-LG was clear, the same effect was not present on healthy porcine LN-derived Lymphogel (p-LG). When we studied OTSCC cells’ migration and invasion abilities on these different matrices by spheroid and wound healing assays, no clear matrix preference for primary or metastatic cell lines emerged. However, there was a non-significant trend that metastatic cells also invaded fastest on Myogel. This suggests that the protein composition, which induces the cell adhesion, may not support cell invasion. These results are in line with our previous findings that Myogel, compared with various other matrices, induces the invasion of cancer cells the most .We performed the pathway and process enrichment analysis by Metascape to study the most active pathways in each matrix. Common enriched clusters of functional characteristics in Myogel and h-LG were cytoskeleton-related pathways and processes. Processes unique to Myogel seemingly associated with ECM and cell adhesion such as ECM reorganization, matrix metalloproteases (MMPs) and Rho GTPases. MMPs and Rho GTPases are linked to numerous key cell functions such as cell cycle progression, cell migration, malignant transformation, cell polarity, invasion and metastasis , 29. In To focus on adhesion molecules for each matrix, a Gene Ontology functional annotation was applied to identify adhesion-related proteins in each matrix. p-LG contained the least adhesion proteins relative to Myogel and h-LG and had only a few proteins in common with these matrices. This might explain why a low number of cells attached to p-LG compared with other matrices. Myogel and h-LG shared 42 proteins, however, 31 adhesion proteins were only found in h-LG, in which we saw a difference between primary tumour and metastatic cell adhesion. Among unique adhesion proteins in h-LG were 12 proteins reported to localize outside the cell, in cell junctions or in extracellular matrix. These proteins included, for example, leucine-rich alpha-2-glycoprotein, annexin A1, annexin A2, galectin-3-binding protein, laminin subunit gamma-2 and plakophilin-2. These proteins have recently been linked to cancer progression. Overexpression of leucine-rich alpha-2-glycoprotein in malignant tumours has been shown to promote angiogenesis and EMT and inhibit apoptosis of cancer cells . The expThis study has potential limitations. Use of several more cell lines would have strengthened our conclusions on the effects of matrix on OTSCC cell adhesion and invasion patterns. However, due to unavailability of resources we were not able to include more than three primary-metastatic cancer cell lines to our experiments. Furthermore, since h-LG was done by combining material from more than one patient the variation in protein expression between patients was lost. In the future, a set of h-LGs produced from one patient could be analysed to find common proteins that might affect OTSCC cell behaviour. The wide range of standard deviations can be explained by high biological variation, which is natural to aggressive cancer cells, especially since the patient-derived cell lines we have used are not fully established. Furthermore, we report the combined averages of three or more experiments, which evidently increases the SD. Finally, the matrices could have been compared to widely used MatriGel, but since we have done this in our previous publications , 17, 18 To increase the reliability of cancer cell experiments in vitro, it is critical to study the cells under biologically relevant conditions, understand the effects of TME and choose the appropriate matrix based on the cell lines and the type of assays. Here, we demonstrated that human HNSCC patients’ lymph node-derived matrix is suitable for studying metastatic oral tongue squamous cell carcinoma cells. It could be used for various 3D assays, e.g. spheroid invasion, in scratch wound migration and in invasion assays. In the future, it should be investigated whether this novel h-LG matrix could be useful in LN metastatic cancer’s drug screening and for personalized cancer medicine in vitro assays.Below is the link to the electronic supplementary material.Additional file 1: Excel TablesAdditional file 2: Supplementary Figures


Veronica species  and to test their antiproliferative and apoptotic activities, according to the authors’ knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34–6.49%), linalool (0.34–6.61%), (E)-caryophyllene (0.97–7.55%), allo-aromadendrene (0.18–2.21%), caryophyllene oxide (1.42–23.83%), benzene acetaldehyde (0.26–13.34%), and β-ionone (1.08–16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41–42.05%) compared to EOs (IC50 158.1–970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line  and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control  on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different  Veronica, which has recently been studied because of its characteristic specialized metabolites, formerly a member of the Scrophulariaceae, was subsequently transferred to the Plantaginaceae family after phylogenetic and chemotaxonomic studies [Veronica has mainly focused on iridoid glycosides, flavonoids, and saponins [Veronica species are used in traditional medicine worldwide for the treatment of different disorders such as the following: as an antiscorbutic, for wound healing, in respiratory diseases for cough, or as an expectorant [Veronica species has shown that because of their specialized metabolites, they possess antioxidant, antimicrobial, cytotoxic, and antitumor activities [Veronica species against two tumor cell lines, KB and B16 cells. Their results showed that the MeOH extracts possessed anti-inflammatory and cytotoxic activities. EOs have been demonstrated to possess anticancer properties through different mechanisms, including cancer prevention mechanisms, as well as the impact of the established tumor cell itself and the interaction with the microenvironment [Veronica extracts having been used in traditional medicine for cancer treatment, only a few species have been studied for their cytotoxic and anticancer activity [Veronica were selected for this study.Throughout the long development of civilization, people have selected plants as food or/and medicine, primarily based on organoleptic evaluations. Scientific confirmation of the use of medicinal plants began with the development of analytical methods, especially chromatography. Chromatographic techniques enabled the identification and quantification of components of specialized metabolites from plant extracts and stimulated interest in studying the effects of these natural products on long-term health and preventive treatment . Prior t studies . To datesaponins ,13,14, wsaponins ,16,17. Sectorant . Previoutivities ,20,21. Htivities  demonstrironment . Key feaironment . In mediironment . Despiteactivity . Defectsactivity . TherefoVeronica species distributed in Croatia, V. agrestis L., V. anagalloides Guss., V. austriaca ssp. jacquinii L., V. beccabunga L., V. cymbalaria Bodard, and V. officinalis L., and determination of their antiproliferative and apoptotic activities against two human cancer cell lines: breast cancer cell line MDA-MB-231, and bladder cancer cell line T24. Consequently, the aim of this study was the phytochemical identification of FVCs in EOs and HYs isolated by microwave-assisted extraction (MAE) from six different Veronica species collected in 2022 (Extraction of volatiles from the six Croatian  in 2022  was perfE)-caryophyllene, allo-aromadendrene, caryophyllene oxide, hexahydrofarnesyl acetone, phytol, β-ionone, hexadecanoic acid, docosane, tricosane, tetracosane, and octacosane were detected in six studied Veronica EOs ; in V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%). In addition, in the composition of V. austriaca ssp. jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) are the predominant compounds. Phytol and hexadecanoic acid are also the predominant constituents in the species V. beccabunga, with 28.08% and 17.06%, respectively. In addition to these two compounds already mentioned, the compound caryophyllene oxide (23.83%) is significantly present in the species V. cymbalaria. Together with phytol and hexadecanoic acid, which are also significantly present in the EO composition of V. officinalis, heptacosane is the most abundant compound (17.21%) (Peculiarities in the EO composition for each species were investigated. In the composition of (17.21%) .4-ol, linalool, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, benzaldehyde, benzene acetaldehyde, and β-ionone were detected in the six studied Veronica HYs , (E)-β-damascenone (12.42%), benzene acetaldehyde (11.56%), and β-ionone (10.32%). In V. anagalloides HY, β-ionone (16.53%), benzaldehyde (13.56%), methyl eugenol (13.57%), and (E)-β-damascenone (11.55%) dominate. In addition, methyl eugenol accounts for 37.01%, more than one-third of all identified components in the HY composition of V. austriaca ssp. jacquinii. The HY composition of V. beccabunga differs significantly from the other Veronica species studied, as the HY composition is dominated by α-pinene and piperitone with 17.11% and 19.54%, respectively. The HY of V. cymbalaria is rich in the phenolic constituents methyl eugenol (38.61%) and (Z)-methyl isoeugenol (31.32%). Methyl eugenol also predominates in the HY of V. officinalis with 22.01% and is the most abundant compound, followed by the compounds (E)-β-damascenone and β-ionone with an identification percentage of more than 14%  after MAE for 4, 24, 48, and 72 h. After MTT cell proliferation assay, obtained results were expressed as % of metabolically active cells . In this work, only results for the times at which the samples showed antiproliferative activity in tested concentrations and dilutions are presented. In Cell viability and proliferation after treatment with essential oils (EOs) and hydrosols (HYs) of six ve cells  and IC50V. agrestis EO on MDA-MB-231 after 48 and 72 h was IC50 572.1 µg/mL and 586.9 µg/mL  and IC50 340.6 µg/mL on T24 after 72 h   a,b. On t and 50% c.V. anagalloides EO showed antiproliferative activity after 48 and 72 h on the MDA-MB-231 cancer cell line with IC50 180.1 µg/mL and 243.4 µg/mL  and T24 cancer cell line was IC50 389.9 µg/mL and 158.1 µg/mL   and 33.70%, 26.96%, and 31.76% after 24, 48, and 72 h on T24 cancer cell lines  (0 µg/mL) a,b. HY oand 50%) c,d.V. austriaca ssp. jacquinii EO showed antiproliferative activity only after 72 h on both mentioned cancer cell lines as IC50 816.0 µg/mL on MDA-MB-231  and 617.1 µg/mL on T24  . After 48 h on MDA-MB-231, it showed an antiproliferative effect of 42.05% but pointed to the T24 cell line antiproliferative activity of 33.65%, 26.72%, and 23.58%  (Species 0 µg/mL) a,b. In gand 50%) c,d. V. beccabunga HY showed only antiproliferative effect on T24 cancer cell line, IC50 36.05%, 29.64%, and 39.29% after 24, 48, and 72 h, and metabolically active cells are presented in V. beccabunga did not show any antiproliferative effect.V. cymbalaria showed antiproliferative effect on the MDA-MB-231 cancer cell line (IC50 249.9 µg/mL and 841.3 µg/mL after 48 and 72 h)  compared to V. cymbalaria EO on the T24 cancer cell line (IC50 970.4 µg/mL after 72 h)    (EO of 0 µg/mL) a,b. HY oand 50%) c,d.V. officinalis EO did not show antiproliferative activity at all four tested times , while V. officinalis HY showed excellent antiproliferative activity on MDA-MB-231 and T24 cancer cell lines. V. officinalis HY showed on MDA-MB-231 an antiproliferative effect of the cells after 48 and 72 h   and after 24, 48, and 72 h on the T24 cancer cell line as IC50 21.83%, 13.41%, and 15.22%  (It is interesting that the and 50%) a,b. Veronica species that showed antiproliferative activity after 48 h was determined on two cancer cell lines: breast cancer cell line MDA-MB-231 and bladder cancer cell line T24. Results are expressed as the distinction between early (Annexin-V+/PI−) and late (Annexin-V+/PI+) apoptotic cells. All results are expressed as mean ± SD (n = 3). V. agrestis EO showed apoptotic activity on MDA-MB-231 of 10.47 ± 0.53% and 9.06 ± 0.74%, while V. agrestis HY showed 1.37 ± 0.33% and 0.40 ± 0.09% in early apoptosis and late apoptosis  and hydrosols (HYs) of some poptosis a,b, resppoptosis a,b, respell line . V. cymbectively a. HY of ectively b. V. offpoptosis , respectV. anagalloides EO was 6.1 ± 1.79% and 0.05 ± 0.0.5% . Twelve samples were obtained—two samples for each species  and hydrosol (HY)). All samples were analyzed by gas chromatography–mass spectrometry (GC-MS), and the obtained data are presented in Veronica species was collected in 2022  of six Croatian  in 2022 .V. agrestis were analyzed for the first time, and phytol was the dominant component in the fraction EO with 56.57% (E)-β-damascenone (12.42%), benzene acetaldehyde (11.56%), and β-ionone (10.32%)  . Veronica species studied in this paper were compared with previously published data [V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%). Comparing the composition of the oil components of this species with the data previously published in the article by Dunkić et al. [V. anagalloides was 14.33% for HD and 19.12% for MAE [β-ionone relative percentage was found in this sample obtained by MAE compared to this study: only 4.22%.The FVCs of the other five hed data ,7,28. InV. austriaca ssp. jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) were the predominant compounds (V. austriaca ssp. jacquinii in MAE extract (22.17%) [V. beccabunga, with 28.08% and 17.06%, respectively. The dominant compound in V. beccabunga collected in 2021 is oxygenated diterpene phytol with 34.54% for MAE [In the composition of ompounds . Hexadecoic acid 7.66% andoic acid 7.66% andoic acid 7.66% andV. cymbalaria with 3.71% in 2021 and 16.66% in 2022   , while iV. anagalloides species are the following: in the extracts from 2022, the compound β-ionone was 16.53% and (E)-β-damascenone was 11.55% (β-ionone with 6.07% and (E)-β-damascenone with 1.52% [V. anagalloides, V. austriaca ssp. jacquinii, V. cymbalaria, and V. officinalis, could also be related to different locations of material collection, not just the difference in the year of collection.The most significant differences in the proportions of the following compounds in the HY of s 11.55% , while iV. austriaca ssp. jacquinii from 2022, while it was not identified at all in the year before; moreover, not a single phenolic component characteristic of species of the genus Veronica was identified [V. beccabunga HY is dominated by α-pinene and piperitone at 17.11% and 19.54%  and (Z)-methyl isoeugenol (31.32%), while oxygenated sesquiterpene caryophyllene oxide is 6.26% -methyl isoeugenol was identified with less than 1% [V. officinalis with 22.01%, while in the previously published composition, the hydrosols of phenolic compounds was the only one represented by 2-methoxy-4-vinylphenol with 11.12%. The compounds (E)-β-damascenone and β-ionone for V. officinalis were identified in a similar percentage in both years compared . V. agrestis EO showed significant antiproliferative activity on MDA-MB-231 after 48 and 72 h (572.1 µg/mL and 586.9 µg/mL), and 340.6 µg/mL on T24 after 72 h. According to Nazlić et al. [V. saturejoides (Kamešnica Sample) on the HeLa, HCT116, and U2OS cell lines. EO of V. anagalloides showed the highest antiproliferative activity among all tested EOs . Nazlić et al. [V. austriaca ssp. jacquinii EO showed antiproliferative activity above 1000 µg/mL after 48 h on the tested cell lines, while in our present study, the EO of this species showed slightly better results, but after 72 h on both mentioned cancer cell lines (816.0 µg/mL on MDA-MB-231 and 617.1 µg/mL on T24). EO of V. cymbalaria showed an antiproliferative effect on the MDA-MB-231 cancer cell line , while V. cymbalaria EO showed lower activity on the T24 cancer cell line (IC50 970.4 µg/mL after 72 h). According to Nazlić et al. [V. officinalis EO showed significant antiproliferative activity on the HCT116 (IC5O > 500 µg/mL), HeLa, and U2OS cancer cell lines (IC5O > 1000 µg/mL). That could be a reason why the EOs of V. officinalis and V. beccabunga did not show antiproliferative activity in this study, as the intervals of tested concentrations were 50–1000 µg/mL. Generally, V. agrestis EO, V. anagalloides EO, and V. cymbalaria EO showed the highest antiproliferative activity of all tested Veronica species’ EOs. This could be due to the higher relative percentage of phytol, (E)-caryophyllene, and caryophyllene oxide in these species. For these compounds, significant antiproliferative and/or apoptotic activity was reported in recent studies [So far, only a few onducted ,30. In tć et al.  reported MDA-MB-2 and T24. studies ,33.V. anagalloides, V. austriaca ssp. jacquinii, and V. cymbalaria showed antiproliferative activity on MDA-MB-231 only after 48 h (17–43%). V. beccabunga HY did not show an antiproliferative effect at the tested dilutions and times on the MDA-MB-231 cancer cell line. HYs of V. anagalloides, V. austriaca ssp. jacquinii, V. cymbalaria, and V. beccabunga showed high antiproliferative activity (18–40%), especially on the T24 cancer cell line after 24, 48, and 72 h. In this study, HY of V. officinalis was the most effective in antiproliferative activity on the MDA-MB-231  and T24 cell lines . These results are in accordance with previous results on HYs of V. officinalis on HeLa, HCT116, and U2OS [V. agrestis did not show antiproliferative activity on the T24 cancer cell line. The reason for its biological inactivity potentially lies in the smaller amount of plant material from which the HY was obtained. The reason for the excellent antiproliferative activity of V. officinalis HY could be the presence of methyl eugenol, terpinen-4-ol, linalool, and β-ionone in higher relative percentages than in other tested HYs. For these compounds, significant antiproliferative and/or apoptotic activity was reported in recent studies [β-ionone was present in a higher percentage in V. anagalloides and V. beccabunga HYs, which could be the explanation for the great antiproliferative activity shown for these extracts, especially on the T24 cancer cell line. V. beccabunga HY also contained α-pinene in a high percentage, which could be explanation for the great antiproliferative activity on the T24 cancer cell line. There are many more studies of antiproliferative activity on various phenolic extracts of the Veronica species than on EOs and HYs. Harput et al. [V. hederifolia, V. cymbalaria, V. persica, V. pectinata var. glandulosa, and V. polita methanolic extracts showed cytotoxic, anti-inflammatory, and radical-scavenging activities. V. americana methanolic extract showed antiproliferative activity on two cancer cell lines, HF-6 (colon cancer) and PC-3 (prostate cancer) [V. persica Poir, V. anagallis-aquatica, and V. thymoides subsp. pseudocinerea showed cytostatic and cytotoxic activities on human and murine cancer cell lines, with verminoside being the most cytotoxic component. Another study on V. cuneifolia subsp. cuneifolia and V. cymbalaria aqueous extracts showed moderate antiproliferative activity on Hep-2 (human epidermoid carcinoma), RD (human rhabdomyosarcoma), and L-20B (transgenic murine L-cells) [HYs of and U2OS . HY of V studies ,37,38,39ivity 18–%, especiL-cells) . Veronica species that have been tested for apoptotic activity. In general, EOs have been shown to induce intrinsic (mitochondria-dependent) and extrinsic (or cell death receptor-dependent) pathways of apoptosis. A study on the antitumor properties of caffeine isolated from the EO of Piper cernuum in melanoma cells showed that this component could induce apoptosis by activating the caspase-3 pathway, as well as by activating endoplasmic reticulum (ER) stress signaling. Another study focused on evaluating the mechanism of the action of carvacrol found in oregano and thyme EO. Carvacrol induced apoptosis in the MDA-MB-231 breast cancer cell line via mitochondrial membrane permeabilization resulted in the release of cytochrome C, and the induction of caspases was indicated by DNA cleavage and fragmentation [Veronica sibirica (Vtfs) induced dose-dependent apoptosis in MCF-7 breast cancer cells with IC50 of 42 µg/mL. In our present study, apoptotic activity was tested on the previously mentioned Veronica species on MDA-MB-231 and the T24 cancer cell line. Mastelić et al. [V. agrestis EO on the MDA-MB-231 cancer cell line , and among the HYs, V. cymbalaria showed 9.95 ± 1.05% of early apoptotic and 3.06 ± 0.28% of late apoptotic cells, and V. anagalloides 8.29 ± 1.09% of early apoptotic and 1.95 ± 0.36% of late apoptotic cells, comparing to control  on the T24 cancer cell line. To our knowledge, no other study involved testing apoptotic activity of the Veronica species’ EOs or HYs. Induction of apoptosis is extremely desirable for cancer control . As far entation . Accordientation , flavonoć et al.  reportedć et al. , apoptotć et al. , cisplatVeronica species, and significant antiproliferative and apoptotic activities of tested extracts and their possible chemotherapeutic properties, which is why they deserve further investigation. Also, one of the most extensive and interesting fields of application of nanobiotechnology is medicine using plant extracts. Ahmadov et al. [Scutellaria baicalensis extract, and the components present in plant extract were the most important in the formation and stabilization of silver nanoparticles. These bioactive components can be connected to the surface of the silver nanoparticles and function as nanodrugs. Therefore, future research should be focused on FVCs present in the Veronica species on antiproliferative and apoptotic activities, their possible encapsulation for in vivo studies, and as potential nanodrugs in medicine.The results obtained in this research showed comparison of EO and HY composition from the above-mentioned v et al.  reportedVeronica species were collected during the flowering period in May and June 2022 at various locations in Croatia  of each  studied  was hydr−1 and maintained for 25 min . The condition for the CP Wax 52 column was a temperature of 70 °C  for 5 min, which was then increased to 240 °C at a rate of 3 °C min−1 and maintained for 25 min . The injection volume was 2 µL, and the split ratio was 1:20. The MS conditions were as follows: ion source temperature, 200 °C; ionization voltage, 70 eV; mass scan range, 40–350 mass units. The individual peaks of all samples were identified by comparing their retention indices of n-alkanes with those of authentic samples and the studies [Veronica species [n = 3 ± SD).Chromatographic analyses were performed using a GC  equipped with a flame ionization detector and a mass spectrometer , a nonpolar capillary column VF-5 ms , and a polar CP Wax 52 . The chromatographic methods and conditions for hydrosol fraction analysis were the same as described in the article by Dunkić et al. [ studies ,48 by co species ,50,51. RVeronica species  were performed against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, to determine which sample has the best antiproliferative activity. Stock solutions of EOs were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. Cell lines MDA-MB-231 and T24 were grown in Dulbecco’s modified Eagle’s medium  in a humidified incubator at 37 °C with 5% CO2. The DMEM medium contained 10% fetal bovine serum  and 1% antibiotics  and was used as a negative control. An equal number of cells (1 × 104) were transferred into 96 wells and left overnight. The cells were then treated with EOs of the above-mentioned Veronica species at concentrations of 50, 100, 250, 500, and 1000 µg/mL, while HYs were tested at different dilutions  after MAE for 4, 24, 48, and 72 h. Cell viability and proliferation were determined by measuring cellular metabolism using an MTT assay. Yellow tetrazoline MTT -2,5-diphenyltetrazoline bromide) is reduced in metabolically active cells to the purple formazan. After 2 h, the medium with MTT was removed, and DMSO was added. The plates were incubated for 10 min at 37 °C with shaking. The absorbance was measured at 570 nm by a HiPo MPP-96 microplate photometer . All samples were run on three different plates in triplicate per plate. Solvent control was measured: the highest concentration of DMSO was adjusted to 1% (v/v) and did not show antiproliferative activity. According to Mastelić et al. [t-test with unequal variances was performed using GraphPad Prism 9.0 statistical software  with the significance set at * p < 0.05. The calculation of IC50 values was performed with GraphPad Prism software version 9.0 , normalizing the data by three independent measurements of untreated controls.Cell viability and proliferation after treatment with essential oils (EOs) and hydrosols (HYs) of six different ć et al. , the antć et al. , cisplat4) were seeded in 6-well plates and treated with essential oils (EOs) and hydrosols (HYs) of V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis for 48 h and then analyzed for apoptosis to determine which sample has the best apoptotic activity. The antiproliferative activities of V. agrestis EO and HY on the MDA-MB-231 cancer cell line after 48 were IC50 572.1 µg/mL and 28.43%. V. anagalloides EO showed antiproliferative activity after 48 on the MDA-MB-231 and T24 cancer cell lines . HY of V. anagalloides showed antiproliferative activity of 19.82% on MDA-MB-231 and of 26.96% after 48 h on T24 cancer cell lines. V. austriaca ssp. jacquinii HY showed better antiproliferative effect, especially on the T24 cancer cell line. After 48 h on MDA-MB-231, it showed antiproliferative effect of 42.05%, but it pointed to the T24 cell line antiproliferative activity of 26.72% after 48 h. V. austriaca ssp. jacquinii HY showed on MDA-MB-231 an antiproliferative effect of 42.05% and pointed to the T24 cell line an antiproliferative activity of 26.72% after 48 h. V. beccabunga HY showed an antiproliferative effect on the T24 cancer cell line of 29.64% after 48 h. EO of V. cymbalaria also showed antiproliferative activity on the MDA-MB-231 cancer cell line (IC50 249.9 µg/mL) after 48 h. HY of V. cymbalaria showed antiproliferative effect on the MDA-MB-231 and T24 cancer cell lines . V. officinalis HY showed on the MDA-MB-231 and T24 cancer cell lines significant antiproliferative effect after 48 h . A combination of Annexin-V-FITC and propidium iodide staining allows the distinction between early (Annexin-V+/PI−) and late (Annexin-V+/PI+) apoptotic cells, necrotic cells, and live cells. After treatment with EOs and HYs, the cells were trypsinized, washed with PBS, and resuspended in 100 µL of the binding buffer containing 5 µL of Annexin-V-FITC and/or 10 µL of PI . The cells were incubated for 15 min at room temperature in the dark and, thereafter, analyzed by flow cytometry . Using the FlowLogic Software , the percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD). For statistical analyses of the apoptosis rate, a t-test with unequal variances, one-way ANOVA followed by a post hoc Tukey test or Kruskal–Wallis test, followed by Dunn’s post hoc test, were performed using GraphPad Prism 7.0 statistical software , with the significance set at p < 0.05, p < 0.01 and p < 0.001.An equal number of cells (1 × 10Veronica species were obtained by microwave-assisted extraction (MAE). The identification of free volatile compounds (FVC) from six Veronica species was analyzed using gas chromatography–mass spectrometry, and the composition of the compounds was compared. The components identified in the EO extracts of all six Veronica species studied are linalool, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, phytol, benzene acetaldehyde, β-ionone, hexadecanoic acid, docosane, tricosane, tetracosane, and octocosane. The constituents terpinen-4-ol, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, benzaldehyde, benzene acetaldehyde, and β-ionone were identified in all studied HY extracts. These volatile compounds belong to the specialized metabolites, and it is important to know their composition for further research on the pharmaceutical activity of the Veronica species. Also, the antiproliferative and apoptotic effects of essential oils and hydrosols were compared. Generally, hydrosols showed better antiproliferative activity, especially on the T24 cancer cell line. Among the tested EOs, V. anagalloides exerted the best antiproliferative effect on both the MDA-MB-231 and T24 cancer cell lines, while among the tested HYs, V. officinalis HY showed the best antiproliferative activity. The best apoptotic effect of the EOs was shown by V. agrestis EO on the MDA-MB-231 cancer cell line, and among the HYs, V. cymbalaria and V. anagalloides showed the best proapoptotic activities on the T24 cancer cell line. Future research should be focused on the discovery of drugs based on natural plant extracts, which implies testing the combinatorial effect of compounds that show biological activity.Free volatile compounds (FVCs) present in essential oils (EOs) and hydrosols (HYs) of six


Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents.The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay.Substantial variations in both the basal expression  and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry.These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology. Thanks to that, it can provide new insights into cancer development and risk factors that may also affect humans. According to the American Veterinary Medical Association, about half of the dogs aged over 10 years will suffer from cancer . With thSeveral fundamental regulatory cellular processes are frequently altered in cancer. Disturbances in the functioning of the DNA Damage Response (DDR) pathway are often connected with carcinogenesis –11 and rThe DDR is one of the pathways whose dysfunction can lead to cancer. ATR and Chk1 comprise the principal DDR pathway available to most cancer cells that lack functional p53, which is found altered in almost 50% of human cancers and has also been reported in a variety of canine cancers , 20. TheTo facilitate research into the significance of DDR pathway disturbances in cancer, as well as to inform studies on the development of new therapies targeting the DDR in dogs, we conducted a series of experiments on canine lymphoma/leukemia cell lines to assess (1): the expression of transcripts of DDR components by RNA sequencing in two selected canine cancer cell lines (2), the basal expression levels of key proteins involved in the DDR , together with checking the feasibility of using commercially available antibodies, and (3) the functionality of the DDR pathway in canine model cells by assessing the DDR pathway activation after DNA damage, using the DNA damaging agent etoposide (detection of γH2AX and p-Chk1). Finally, we performed DNA combing assays to assess DNA replication dynamics in canine lymphoma/leukemia cells by directly visualizing replication fork progression rates and replication origin firing.2.2.1.Ad Hoc Committee on Animal Research and were approved by the First Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland .A panel of canine lymphoma/leukemia cell lines: CLBL-1 (B-cell lymphoma), CLB70 (B-cell chronic lymphocytic leukemia), and GL-1 (B-cell leukemia) was used in this study. The CLBL-1 cell line was a gift from Barbara Rütgen from the Institute of Immunology, Department of Pathobiology from the University of Vienna , the GL-2 and 95% humidified air, at 37°C in 25 cm2 cell culture flasks .The culture medium RPMI 1640  was used for the CLBL-1 and GL-1 cell lines, and Advanced RPMI  for the CLB70 cell line. The culture media were supplemented with 2 mM L-glutamine , 100 U/mL of penicillin, 100 μg/mL of streptomycin , and 10–20% heat-inactivated fetal bovine serum . The cells were cultured in an atmosphere of 5% CO2.2.RNA was obtained from cultures of the selected cell lines CLBL-1 and GL-1 during unperturbed growth and sequenced by Novogene (United Kingdom). The expected number of Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced (FPKM) was used to calculate relative gene expression . The DDR2.3.DNA damage was induced by treatment with etoposide  (a topoisomerase II inhibitor), at 20 μM for 2 h. Treatment conditions were selected based on literature  and prev2.4.5cells/mL were cultured in 10 mL of media in a 25 cm2 culture flask per condition. After 48 h of incubation, the samples were lysed in urea/ SDS buffer , 50 μL 20% SDS), and run in 8–12% bis-tris acrylamide gels prepared using a BioRad Mini-PROTEAN Tetra Vertical Electrophoresis Cell system. The samples were transferred to nitrocellulose membrane using BioRad Mini Trans-Blot® Cell for wet transfer and BioRad Trans-Blot® Turbo™ Transfer System device for semi-dry transfer.3 x 10The antibodies used in the study were selected based either on available literature data on reactivity with canine cells  or compa2.5.2.5.1.Canis lupus familiaris nucleotide accession number sequences for mRNA of the target genes (TGs): Atr, Claspin, and six housekeeping genes (HKGs): Actb, Ppia, and Rplp0 were taken from the Nucleotide Center for Biotechnology Information (NCBI) database . The sequences were transferred into the Universal Probe Library. The designed primers and their amplified sequences were additionally verified for their specificity in the Nucleotide Basic Local Alignment Search Tool - Nucleotide-BLAST . Gene names, primer sequences for TGs and HKGs, amplicon size, as well their respective gene accession numbers are summarized in The 2.5.2.7 cells cultured in 10 mL from the CLBL-1, CLB70, and GL-1 lymphoma cell lines were centrifuged at 300 g, 4° C, and resuspended in 500 μL of TRIzol reagent . The cells were immediately transferred to a low-temperature freezer and stored in Eppendorf tubes at −80° C for further analysis. Total RNA isolation was performed using Total RNA Zol-Out™ D  according to the protocol provided in the isolation kit. Briefly, the cells were removed from the low-temperature freezer and thawed on ice for 30 min. After that, 167 μL of ultra pure molecular biology water  were added, and the sample was mixed by inversion. Next, the cells were spun for 10 min at 10000 rpm. The supernatant was mixed with 1 volume of 96–100% ethanol  and gently agitated until a homogenous solution was obtained. The supernatants from each tube were transferred into new tubes with an RNA membrane binding column and were centrifuged through the column for 1 min at 10000 rpm and 4° C. The columns were rinsed with 700 μL washing A2W buffer for 2 min at 10000 rpm. DNA digestion for 15 min at 37° C in a thermoblock was performed using DNAse according to the manufacturer’s protocol. The enzymatic activity of the digestive buffer was inhibited by adding 700 μL of R81 buffer and centrifugation [1 min at 10000 rpm at room temperature (RT)]. The filtrate was collected and loaded again onto the column. The membranes were rinsed twice with 700 μL and 200 μL of A2W buffer, centrifuged as described above, and transferred into new Eppendorf tubes. Then, 40 μL of sterile water were added, and after 3-min incubation at RT the tubes with the membranes were centrifuged as above. RNA quality and quantity were estimated using Implen NanoPhotometer , and only the samples with a 260/280 nm absorbance coefficient between 1.8 and 2.1 were used for the final experiments. The TranScriba noGenome Kit  was used to perform reverse transcription, according to the manufacturer’s recommendations in the MJ Research PTC-100 thermocycler . First, 1 μg of total RNA was mixed with the noGenome master mix. After a 10-min incubation at 42° C, 7 μL of the mentioned mix were added to the RT master mix. The RT master mix included 4 μL of TranScriba buffer, 0.5 μL of RNAse inhibitor, 2 μL of dNTP, 1 μL of starter oligo (dT), 4 μL of TranScriba reverse transcriptase and 1.5 μL of sterile water for one reaction. The reverse transcription protocol was as follows: the first step of 60 min at 42°C, the second step of 5 min at 70°C, and the final step of 5 min at 4°C. The obtained cDNA was stored at −20°C.A total of 1×102.5.3.The real-time PCR gene expression analyzes were performed in triplicate from three independent cell cultures. The reaction mix (per well) included 5 μL of RT PCR Mix SYBR® , 0.5 μM of forward and reverse primers , and 1 μL of cDNA diluted with molecular biology water (16.65 ng cDNA per well). Real-time PCR was performed using the LightCycler 480 II  instrument under the following conditions: pre-incubation at 95°C for 10 min, 50 cycles of amplification: 10 s at 95°C for denaturation, 30 s at 60°C for annealing, and 15 s at 72°C for elongation. The gene detection analyzes and primer specificity were further improved by melting curve analysis. The gene expression was categorized using the following scale:“0” lack of gene expression, Ct values above 35.“1” very low gene expression, Ct values between 30 and 35.“2” low gene expression, Ct values between 28 and 30.“3” regular gene expression, Ct values between 22 and 28.“4” high gene expression, Ct values between 15 and 22.“5” very high gene expression, Ct values below 15.2.6.6 cells were cultured in 10 mL of media, then pulse-labeled with 5-iodo-2′-deoxyuridine (IdU) at 25 μM, followed by 5-chloro-2′-deoxyuridine (CldU) at 200 μM, for 15 min each. The cells were recovered by centrifugation at 300 g after each pulse, and the media were refreshed with each analog. Next, the cells were resuspended in cold PBS and warmed to 42°C, using 5×105 cells per agarose plug. The cells were gently mixed with 1% agarose in PBS and divided into the casting mold to generate the plugs. After treatment with proteinase K  at 50°C overnight, the DNA was stained with YOYO-1 (5 μM in TE solution for 2–5 min) to check the quality of the fibers. After melting the agarose, the extracted DNA was poured into a reservoir, where a coverslip was inserted, and the DNA fibers were stretched. The resulting fibers were visualized by immunofluorescence detecting the IdU and CldU analogs with red and green antibodies. The coverslips were incubated for 45 min with murine anti-BrdU , and rat anti-BrdU , as primary antibodies for the analogs, and for 30 min with goat anti-mouse IgG1 Alexa 564  and chicken anti-rat Alexa 488  as secondary antibodies for the analogs. The coverslips were incubated for 30 min with autoanti-ssDNA DSHB by Voss, E.W. (Hybridoma Product autoanti-ssDNA) autoanti-ssDNA DSHB by Voss, E.W.  to detect whole DNA fibers, and then for 30 min with a secondary antibody goat anti-mouse IgG2a Alexa 647 . Image acquisition was performed with a 40x objective using a confocal microscope . The fork velocity (FV) was calculated by multiplying the length of the green track of the fiber in micrometers by 2 to obtain Kb, and dividing it by 15 min (time of pulse). Fork asymmetry (FA) was calculated by dividing the long track by the short track. For this analysis, only the first analog incorporation tracks (green tracks) were considered.A total of 1.6×102.7.For the combing assay analysis, the Mann–Whitney test was performed to compare the cell lines and analyze potential differences. Scatterplots were prepared to visually represent the differences between the two cell lines.http://statistica.io.Statistical analysis was performed using TIBCO Software Inc. (2017) Statistica , version 13 3.3.1.The canine lymphoma cell line CLBL-1 and the canine leukemia cell line GL-1 expressed a total of 16,220 genes, from which 271 (~2%) are DDR pathway members. Specifically, the CLBL-1 cell line expressed 260 DDR genes, and the GL-1 cell line 266, with 255 genes in common . The rel3.2.Considering that ATR, Claspin, Chk1, and Rad51 are among the most important proteins of the DDR pathway, they are still quite uncharacterized in dogs. Thanks to the progress made in recent years in veterinary medicine, and in particular in veterinary oncology, currently there are some tools available for their study in dogs (for example antibodies and siRNAs).Our initial aim was to identify commercial antibodies against key components  of the DDR that would be suitable for use in canine cells. Western blot screening was performed to analyze the basal protein expression levels, and to determine whether the pathway activation in response to DNA damage occurs in canine cells  corresponded with the ~220 kDa molecular mass expected for this protein. ATR was readily detected in only two of the three cell lines analyzed, with expression of ATR being higher in CLB70 cells than CLBL-1 and undetectable in GL-1 cells . As ATR qPCR analysis confirmed that ATR mRNA is expressed in the three cell lines tested, with mean threshold cycle (Ct) values of 25.21 ± 0.27 for the CLBL-1 cell line, 28.15 ± 3.29 for the CLB70 cell line, and 25.72 ± 0.61 for the GL-1 cell line. Although the expression of ATR was substantially lower than the expression of the HKGs , Ct valu3.2.2.In the case of Claspin, BLAST alignment demonstrated 84.47% identity between the human and canine proteins, again supporting the possibility of cross-reactivity of human antibodies with canine proteins. The expression of Claspin was detectable using the Claspin B-6  antibody. The antibody recognized a protein of the expected molecular mass of ~180 kD, thus confirming cross-reactivity with canine Claspin. Claspin expression was observed in all three cell lines, although the expression levels varied. Claspin expression levels were substantially higher in the CLBL-1 and CLB70 cell lines than in the GL-1 cell line, similar to the expression of ATR .qPCR analysis showed that the Claspin mRNA was also expressed in the three cell lines, with Ct values of 24.95 ± 0.17 for the CLBL-1 cell line, 27.63 ± 2.70 for the CLB70 cell line, and 26.19 ± 0.74 for the GL-1 cell line. Similar to ATR, the expression of the Claspin mRNA in each case was substantially lower than that of the HKGs .3.2.3.BLAST alignment for Chk1 protein sequences between humans and dogs showed a high 96.2% identity. This analysis also confirmed that canine Chk1 contains the key regulatory site, serine 345 (S345), that is phosphorylated by ATR to activate Chk1 in response to genotoxic stress. This means that with the use of the tested antibodies Chk1 G-4  and Phospho-Chk1 (SER345) 133D3  directed against human epitopes, both the basal level expression of Chk1 kinase and its activation after DNA damage can be tested in canine cells. Indeed, both antibodies detected proteins of the expected molecular mass (~56 KD) in all three cell lines . Interes3.2.4.BLAST alignment comparing Rad51 protein sequences demonstrated 99.12% identity between human and canine protein, indicating a high probability that an antibody directed at the human protein will detect the canine homolog. As expected, using Rad51 G-9  antibody, we detected a band of ~37 kDa corresponding to the expected molecular mass of Rad51. Basal Rad51 recombinase expression was detected in all three cell lines with the highest level of expression in the CLBL-1 cell line .3.3.Next, we analyzed the expression levels and possible regulatory modifications of the canine DDR proteins after inducing activation of the DDR pathway by treating the cells with a classic DNA damaging agent, etoposide. Several studies have reported DDR activation through phosphorylation of Chk1 and an increase of Rad51 expression after treatment with the DNA damaging agent etoposide , 56. Thi3.4.Advanced techniques, such as DNA combing, can provide much greater insight into DNA replication dynamics by directly visualizing replication fork progression rates and replication origin firing. Due to the important role of ATR-Chk1 in replication, we wished to evaluate the feasibility of performing DNA combing in canine cells.The selected cell lines for this study were GL-1 and CLBL-1, since both present high expression level of Rad51 , which mp = 8.86−21) faster in the GL-1 line (1.5 Kb/min) than in the CLBL-1 line , while RAD51 exhibited the lowest relative expression  and leukemia (GL-1). After sorting the genes by Gene Ontology terms (GO) related to the DDR , we founpression , in blueThus, this RNA-Seq analysis revealed that the canine lymphoma and leukemia cells shared expression of a majority of DDR genes, but that the expression of certain key components differed substantially between the tumor types.4.2.The first screening for the basal expression of DDR proteins  indicate4.2.1.ATR was detected in all the cell lines at the mRNA level . The obtATR gene is widely expressed in the canine lymphoma/leukemia cell lines. However, as ATR has a high molecular weight of 220 kDa, and high-molecular-weight proteins are difficulted to transfer . Indeed, Claspin protein expression was detected in all the cell lines of our panel as a single band with a molecular mass of 180 kDa as seen in human cell lines . Claspinell line . This coell line .Claspin has been described to be highly expressed in prostate cancer cells in comparison with non-cancerous prostate cells . Many ca4.2.3.Chk1 showed a 96.7% identity between the human and canine protein sequences in the BLAST alignment . ConsistUpregulation of Chk1 has been proposed as a target for anticancer therapies. Different studies have confirmed Chk1 inhibitors acting as apoptosis inducers in various human and canine tumor cells, indicating, for example, proliferation decrease in human neoplastic B-cells and mast tumor cell (MTC) canine cell lines , 69. Cur4.2.4.in vivo mice models , highlighting the similarity of both species in the way naturally occurring cancer and lymphoma respond to therapies , 99. Cel5.To conclude, we propose the use of canine lymphoma/leukemia cells as a model to study DDR in cancer, with ATR, Claspin, Chk1, and Rad51 as promising targets for further analysis. Our results will facilitate further investigation on DDR in canine cancer by identifying validated antibodies for ATR, Claspin, Chk1, p-Chk1, and Rad51, and primers for ATR and Chk1, and bringing numerous opportunities to develop new targeted-anticancer therapies which later may be also implemented in human medicine.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/AP: conceptualization and project administration. BH-S and AP: methodology. BH-S: software, validation, formal analysis, data curation, writing–original draft preparation, visualization, and funding acquisition. BH-S, AP, PK, and ED: investigation. BO-M: resources. AP and DG: writing–review and editing. AP, DG, and BO-M: supervision. All authors have read and agreed to the published version of the manuscript.The publication was financed by the project “UPWR 2.0:international and interdisciplinary program of development of Wrocław University of Environmental and Life Sciences,” co-financed by the European Social Fund under the Operational Program Knowledge Education Development, under contract No. POWR.03.05.00-00-Z062/18 of June 4, 2019 and by the Polish National Agency for Academic Exchange under Grant No. PPI/APM/2019/1/00044/U/00001. DG was an Agustín de Betancourt Investigador of the Universidad de La Laguna.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metabolomics analysis showed clear separation between the parental MDA-MB-231 cell line and the drug-resistant cell lines. Pathway analysis revealed that arginine and proline metabolism, glutathione metabolism, and beta-alanine metabolism were significantly perturbed in the drug-resistant cell lines compared to the parental cell line. After matching signals to an in-house library of reference standards, significant decreases in short- and medium-chain acylcarnitines and significant increases in long-chain acylcarnitines, 5-oxoproline, and 7-ketodeoxycholic acid were observed in the resistant cell lines as compared to the parental MDA-MB-231 cell line. In addition to baseline metabolic differences, we also investigated differences in metabolic responses in resistant cell lines upon a second exposure at multiple concentrations. Results indicate that whereas the parental MDA-MB-231 cell line had many metabolites that responded to doxorubicin in a dose-dependent manner, the two resistant cell lines lost a dose-dependent response for the majority of these metabolites. The study’s findings provide insight into how metabolism is altered during the acquisition of resistance in TNBC cells and how the metabolic response to doxorubicin changes upon repeated treatment. This information can potentially identify novel targets to prevent or reverse multi-drug resistance in TNBC, and also demonstrate the usefulness of metabolomics technology in identifying new mechanisms of drug resistance in cancer and potential drug targets.This study aimed to investigate metabolic changes following the acquisition of resistance to doxorubicin in the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Two drug-resistant cell lines, DOX-RES-50 and DOX-RES-100, were generated by treating MDA-MB-231 cells with doxorubicin for 24 h and allowing them to recover for six weeks. Both drug-resistant cell lines demonstrated an increase in doxorubicin IC In the United States, breast cancer is the most diagnosed cancer for women (excluding skin cancers) and is the second leading cause of cancer deaths in women . One subDoxorubicin is a commonly used chemotherapeutic drug for the treatment of TNBC . The mecAn understudied factor in the development of doxorubicin resistance is cancer cell metabolism. Metabolic reprogramming is a hallmark of cancer and has been shown to play a critical role in drug resistance . Cancer Metabolomics technologies can be used to identify new mechanisms of drug resistance in cancer by analyzing the metabolic profile of cancer cells before and after treatment with drugs . By anal50 values, demonstrating the acquisition of drug resistance  in DOX-RES-50 and DOX-RES-100 cells, respectively, compared to the parental MDA-MB-231 cell line.After the preprocessing of metabolomics data, 4063 features remained for analysis. Principal component analysis (PCA) showed clear separation between the parental MDA-MB-231 cell line and the drug-resistant cell lines. Additionally, the DOX-RES-50 and DOX-RES-100 cell lines showed noticeable overlap in the PCA plot, indicating similarities in metabolic profiles B. Pairwimparison . Signifip-values for the MDA-MB-231 versus DOX-RES-50 and the MDA-MB-231 versus DOX-RES-100 comparisons. Results revealed that arginine and proline metabolism, glutathione metabolism, and beta-alanine metabolism were significantly perturbed between the MDA-MB-231 and DOX-RES-50 cells  in DOX-RES-50 and DOX-RES-100, respectively, compared to the parental MDA-MB-231 cell line  significantly increased metabolites in both resistant cell lines  in baseline secreted metabolite profiles of doxorubicin-resistant cell lines compared to the parental MDA-MB-231 cell line . Statistell line . Volcanoll lines A,B. Metall lines . Also, ill lines . Methylt50 values in both resistant cell lines. While the fold increase in IC50 values is more modest compared to other studies, this degree of resistance is more clinically relevant—indeed, other methods of generating drug resistance can lead to fold changes of 100–1000 in IC50 levels, but cause cellular changes that are not reflective of this in cancer patients [50 levels allows for more relevant biological changes to be observed, and may even reflect more accurately the early stages of resistance development. We used an untargeted metabolomics approach to gain insights into metabolic reprogramming during this stage by assessing metabolic differences between drug-resistant and parental MDA-MB-231 cells. Our results showed clear metabolic differences in baseline intracellular and secreted metabolomes of parental and drug-resistant cells. Moreover, we demonstrated that while parental MDA-MB-231 cells had metabolite sets with clear dose-response activity following a dose range of doxorubicin treatment, drug-resistant cells lost dose-response relationships with the majority of these metabolites. Interestingly, a small number of metabolites were significantly correlated with doxorubicin treatment in the resistant cell lines but not the parental MDA-MB-231 cells, suggesting that these metabolites may play a role in suppressing the metabolic changes associated with doxorubicin exposure in resistant TNBC cells.This study created doxorubicin-resistant cell lines by exposing MDA-MB-231 cells to a single dose of doxorubicin followed by a six-week recovery period. The acquisition of drug resistance was confirmed through an increase in doxorubicin ICpatients . Our genPathway analysis (using KEGG maps) of all peaks revealed arginine and proline metabolism, glutathione metabolism, and beta-alanine metabolism as significantly altered in both resistant cell lines. The arginine and proline metabolism pathway connects arginine, proline, and glutamate, as well as their intermediates. This metabolic pathway connects key processes of cancer cell metabolism, including biosynthesis of other amino acids, nucleotide biosynthesis, TCA cycle activity, and polyamine biosynthesis. Interestingly, this pathway is being investigated as a therapeutic target of several cancers through starvation of specific amino acids in this pathway, including arginine, proline, and glutamine . This paUpon investigating metabolites that were matched to our in-house library, we identified several acylcarnitines as significant differentiators of parental MDA-MB-231 cells and the doxorubicin-resistant cell lines. In general, we observed a decrease in short- and medium-chain acylcarnitines and an increase in long-chain acylcarnitines in the doxorubicin-resistant cell lines. Acylcarnitines are intermediates in fatty acid metabolism and are generated during the process of mitochondrial and peroxisomal β-oxidation of fatty acids, making them markers of energy metabolism. Additionally, they have been identified as indicators of metabolic diseases including cardiovascular disease, diabetes, and certain cancers . More reOne of the unique contributions of this study is the analysis of metabolites that reacted in a dose-responsive manner to a range of doxorubicin doses in the drug-resistant cell lines and the parental MDA-MB-231 cell line. The loss of dose response in the metabolome, particularly at lower doxorubicin levels, suggests that the resistant cells rewire cellular metabolism to survive doxorubicin exposure. Our analysis revealed novel metabolic features that are perturbed during resistance acquisition and may represent novel targets that could be targeted to reverse or mitigate doxorubicin resistance in TNBC. Additionally, these metabolites may have potential to be used as markers to be monitored during therapy to guide treatment dose, treatment length, and/or number of treatments. The correlation data also provide additional insights into the mechanism of action of doxorubicin in breast cancer cells, which, despite its widespread clinical use, still remains unclear . MethyltIn conclusion, the findings of this study contribute to our understanding of metabolic reprogramming in drug-resistant breast cancer cells and suggest potential mechanisms and treatment strategies for this challenging clinical problem. Our findings point to a significant role of acylcarnitines/fatty acid oxidation, as well as specific metabolites such as MTA, 5-oxoproline, and 7-ketodeoxycholic acid in the process of acquired drug resistance. Surprisingly, metabolic investigations regarding the acquisition of drug resistance are sparse in the literature, particularly for TNBC. Because of this, our study provides many novel insights into the metabolic changes that occur during TNBC drug resistance, forming the basis for future research to further investigate this process and generate novel findings, such as metabolic targets that could be manipulated to potentially increase drug sensitivity. Further studies are needed to validate the findings of this study and to explore the functional significance of the metabolic changes observed in the drug-resistant cell lines. Additionally, in vivo model systems should also be investigated to determine if these metabolic changes are observed in this setting as well.Optima grade solvents (water with 0.1% formic acid and methanol with 0.1% formic acid) and fetal bovine serum (FBS) were purchased from Fisher Scientific . Dubelcco’s Modified Eagle Medium (DMEM) with high glucose and phosphate-buffered saline (PBS) was purchased from Gibco . Doxorubicin was purchased from SelleckChem . The MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC) .4 cells per well and were allowed to attach overnight. Cells were then treated with an 11-point dose curve of doxorubicin with a top concentration of 10 µM and a dilution series of 1:1 (n = 3–6 per group). Cell viability was assessed using the alamarBlue assay according to manufacturer instructions (Thermo Scientific). GraphPad was used to generate viability dose curves and calculate doxorubicin IC50 values. DMSO was used as a vehicle, with a final concentration of 0.1% for all treatments.MDA-MB-231 cells were cultured according to manufacturer guidelines. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. To generate doxorubicin-resistant cells, MDA-MB-231 cells were treated with 50 or 100 nM doxorubicin for 24 h and were allowed to recover in supplemented DMEM for six weeks. To verify resistance, treatment-naïve and treatment-experienced cells were plated into 96-well plates at 2 × 10g at 4 °C for 10 min, and supernatants were transferred to autosampler vials for analysis by ultra-high-pressure liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS). Quality control study pools (QCSP) were created by combining 10 µL of each sample into a single mixture. Method blanks were created by adding 500 µL of 80% methanol to empty tubes and were processed in an identical manner as the study samples.Cells were plated at approximately 80% confluency in 6-well culture plates and were treated with 500, 200, 100, 50, or 0 nM doxorubicin with a final DMSO amount of 0.1% (n = 4 per group). After 24 h of treatment, metabolites were extracted from cell samples as described previously ,32,33,34m/z. QCSP and blank injections were placed at a rate of 10% throughout the study samples. An injection volume of 5 µL was used for analysis of each sample. Raw UHPLC-HRMS data were imported into Progenesis QI  for alignment, peak picking, and deconvolution. Background signals were removed by filtering out peaks with a higher average abundance in the blank injections as compared to the QCSP injections. Data were normalized using a QCSP reference sample using the “normalize to all” function in progenesis [Metabolomics data were acquired via previously published UHPLC-HRMS methods ,35,36,37ogenesis .Peaks were matched to an in-house library of reference standards or public mass spectral databases from the National Institute of Standards and Technology (NIST) and METLIN. Peaks were matched to metabolites by retention time , exact mass , and fragmentation pattern . An ontology system was given to denote the evidence basis for each metabolite assignment. OL1 refers to a match to the in-house library for RT, MS, and MS/MS; OL2a refers to an in-house match to the in-house library for RT and MS; OL2b refers to a match to the in-house library for MS and MS/MS; PDa refers to a match to public databases for MS and MS/MS; PDb refers to a public database match for MS and theoretical MS/MS (HMDB); PDc refers to a public database match for MS and isotopic similarity; PDd refers to a public database match for MS only.p-values were calculated for each peak for each treatment as compared to the vehicle control. p-values were calculated using Student’s t-test. Correlation analyses were performed by using the Statistical Analysis (metadata table) module in MetaboAnalyst 5.0 using Pearson r as the correlation measure. p-values were not adjusted for multiple testing due to the small sample size of this study and the exploratory, rather than confirmatory, nature of this study [p-value cutoff of 0.05.Principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) was performed in SIMCA 16  using the normalized, filtered data. Unit Variance (UV) scaling was used for all multivariate plots. PCA plots were used to assess data quality by verifying the clustering and centering of QCSP samples by PCA, and OPLS-DA plots were used to assess the separation of metabolomes between vehicle and treated cells, as well as to calculate variable importance to projection (VIP) scores for each peak. Heatmaps were generated using MetaboAnalyst 5.0 . Fold chis study . Pathway


Acquired chemoresistance during chemotherapy, often accompanied by cross- and multi-resistance, limits therapeutic outcomes and leads to recurrence. In order to create in vitro model systems to understand acquired doxorubicin-resistance, we generated doxorubicin-resistant sublines of canine prostate adenocarcinoma and urothelial cell carcinoma cell lines. Chemoresistance to doxorubicin, cross-resistance to carboplatin, and the reversibility of the acquired resistance by the specific MDR1-inhibitor tariquidar were quantified in metabolic assays. Resistance mechanisms were characterized by expression of the efflux transporters MDR1 and RALBP1, as well as the molecular target of doxorubicin, TOP2A, with qPCR and Western blotting. Six out of nine cell lines established stable resistance to 2 µM doxorubicin. Drug efflux via massive MDR1 overexpression was identified as common, driving resistance mechanism in all sublines. MDR1 inhibition with tariquidar extensively reduced or reversed the acquired, and also partly the parental resistance. Three cell lines developed additional, non-MDR1-dependent resistance. RALBP1 was upregulated in one resistant subline at the protein level, while TOP2A expression was not altered. Combination therapies aiming to inhibit MDR1 activity can now be screened for synergistic effects using our resistant sublines. Nevertheless, detailed resistance mechanisms and maintained molecular target expression in the resistant sublines are still to be examined.  Canine prostate cancer, regardless of whether adenocarcinoma (PAC) or the frequent urothelial or transitional cell carcinoma (TCC) of the prostate, is often diagnosed at a late stage and with a poor outcome . In partDoxorubicin is an effective and thus commonly used chemotherapeutic drug in canine oncology . IntercaABCB1, belongs to the ABC family and is a well-known multidrug-resistance protein with a broad spectrum of substrates in both dogs and humans  = ln(2)/ln(r).Growth kinetics were assessed as previously described . Cells w®, Corning inc., Corning, NY, USA), coated with basement membrane . The next day, the lower chambers were filled in duplicate with medium containing 10% serum as attractant. Serum-free medium served as negative control. Following 48 h incubation time, non-invasive cells were gently removed from the upper chamber by using moistened cotton swaps. The remaining invasive cells attached to the membranes were fixed in 10% formalin for 10 min, permeabilized in methanol for 20 min, and washed twice in PBS. Finally, the membranes with attached cells were stained for 2 min with 1% crystal violet, washed again three times in PBS, and allowed to dry. Three representative regions of each membrane were photographed with a DMI600 B microscope  at 100-fold magnification using the LAS AF 2.6.0 software. The proportion of area covered by invasive cells was determined with ImageJ 1.53c. [As described for the parental cell lines , cells iJ 1.53c. . ® NP80 . For cDNA synthesis, applying the QuantiTect Reverse Transcription Kit , 2 µg RNA per 10 µL reaction was used. Primer pairs  and purchased from Microsynth AG . Specific primer binding was confirmed by sequencing the amplicons . Reactions were run in triplicates on a LightCycler®96 real-time PCR System . Cycling conditions were as follows: 95 °C for 10 min, 45 cycles , followed by melting. Data analysis was carried out using Roche LightCycler®96 software (1.1.0.1320). A two-fold dilution series of pooled cDNA served for calculating primer efficiencies. GAPDH and ACTB gene expression was more stable compared to HPRT, which is why they were chosen as normalization genes. Gene expression values relative to Adcarc0846 were calculated according to an efficiency-corrected model, taking into account both normalization genes [In three independent experiments, cells were cultured for 96 h in presence of 2 µM doxorubicin (doxo lines only) or without doxorubicin . Afterwards, cells were washed with ice-cold PBS and harvested using cell scratchers. RNA was isolated with the SV Total RNA Isolation System (Promega GmbH) and quantified photometrically using a NanoPhotometerer pairs  were deson genes . ®TGX™ Gels, Bio-Rad Laboratories Inc., Hercules, CA, USA). After blotting onto a nitrocellulose membrane , unspecific binding was blocked by EveryBlot blocking buffer (Bio-Rad Laboratories Inc.). Primary antibodies  by scraping. Protein concentrations were assessed by Pierce™ BCA Protein Assay Kit . Without previous cooking, as required by the ABCB1-antibody, proteins (30 µg per lane) were separated by SDS-PAGE . For qPCR and Western blot analysis, Log2 transformed relative expression values were used. To compare fluorescence intensities as well as gene and protein expression of the parental cell lines with the resistant clones, cultured either with or without doxorubicin, a one-way ANOVA was performed followed by Tukey’s post-hoc test in case of normal distribution (Shapiro–Wilk test). If the data were not normally distributed, the Kruskal–Wallis test was used, followed by Dunn’s multiple comparisons test. Regarding cell numbers for doubling times and area covered by cells for invasion assay, parental and resistant cell lines were compared with Student’s t-test because of normally distributed data. Acquired chemoresistance limits the therapeutic outcome in veterinary and human oncology, yet no satisfying treatment strategy for canine prostate cancer exists. In order to create in vitro models to overcome acquired chemoresistance, we generated six doxorubicin-resistant sublines from nine well characterized canine prostate cancer cell lines. Drug efflux by massive MDR1 overexpression was identified as a common driving resistance mechanism in all sublines. Moreover, Adcarc0846doxo, Adcarc1508doxo, and TCC1506doxo developed additional, non-MDR1 dependent resistance mechanisms, which still need to be characterized. MDR1 inhibitors, such as the herein efficiently used tariquidar, reduce or reverse the acquired doxorubicin-resistance. Furthermore, tyrosine kinase inhibitors show potential in reversing MDR1-dependent drug-resistance, while exhibiting antiproliferative effects themselves. Potential synergistic effects can now be investigated with our six pairs of parental and resistant cell lines. However, in advance, the expression signature of molecular targets initially observed in the parental cell lines remains to be confirmed.