There is an unmet need for novel anticancer therapeutics that work differently to current standard-of-care therapies. BOLD-100 is a unique clinical-stage anticancer compound that is based on the rare metal, ruthenium. Understanding the bioactivity of BOLD-100 can accelerate its development towards approval and into clinical practice. The aim of this study was to use a large panel of cancer cell lines to formulate a sensitivity profile of BOLD-100 across various cancer types. BOLD-100 demonstrated increased activity in cell lines from esophageal cancer, blood cancers, and bladder cancer. These indications are in addition to the gastrointestinal cancers currently in clinical development, thus opening new opportunities. Using the sensitivity profile for downstream bioinformatics and pathway analysis revealed associations between cancer cell lines’ sensitivity to BOLD-100 and ribosomal gene expression, including several genes coding for large- and small-ribosomal subunits. These findings provide evidence that ribosomal processes may be a critical component of BOLD-100’s mechanism.BOLD-100 (sodium trans-[tetrachlorobis(1H indazole)ruthenate(III)]) is a ruthenium-based anticancer compound currently in clinical development. The identification of cancer types that show increased sensitivity towards BOLD-100 can lead to improved developmental strategies. Sensitivity profiling can also identify mechanisms of action that are pertinent for the bioactivity of complex therapeutics. Sensitivity to BOLD-100 was measured in a 319-cancer-cell line panel spanning 24 tissues. BOLD-100’s sensitivity profile showed variation across the tissue lineages, including increased response in esophageal, bladder, and hematologic cancers. Multiple cancers, including esophageal, bile duct and colon cancer, had higher relative response to BOLD-100 than to cisplatin. Response to BOLD-100 showed only moderate correlation to anticancer compounds in the Genomics of Drug Sensitivity in Cancer (GDSC) database, as well as no clear theme in bioactivity of correlated hits, suggesting that BOLD-100 may have a differentiated therapeutic profile. The genomic modalities of cancer cell lines were modeled against the BOLD-100 sensitivity profile, which revealed that genes related to ribosomal processes were associated with sensitivity to BOLD-100. Machine learning modeling of the sensitivity profile to BOLD-100 and gene expression data provided moderative predictive value. These findings provide further mechanistic understanding around BOLD-100 and support its development for additional cancer types. Metal-based compounds have the potential to be highly efficacious anticancer therapies with novel mechanisms of action ,2. The pPreclinical screening of novel therapeutics against panels of cancer cell lines remains a pertinent protocol in drug development . Since tThe ruthenium complex BOLD-100 is a clinical-stage anticancer compound that is composed of sodium trans-[tetrachlorobis(1H indazole) ruthenate(III)] with cesium as an intermediate salt form. Predecessor molecules include IT-139, NKP-1339, and KP1339. BOLD-100 has successfully completed a Phase 1 monotherapy trial and is currently in a Phase 1b/2 clinical trial in combination with chemotherapy in advanced gastrointestinal cancers (NCT04421820) . Beyond The depth of knowledge and data yielded by in vitro cell line screening assays presents an opportunity to assess the efficacy of BOLD-100 across a large panel of cancer types. In this study, a cell viability screen across 319 cancer cell lines spanning 24 tissues of origin using BOLD-100 monotherapy was utilized to determine cancer types with increased sensitivity. In order to build the knowledge base of BOLD-100’s potential bioactivity, the sensitivity profile of BOLD-100 across the cancer cell lines was compared to anticancer compounds in the GDSC database. Utilizing the depth of cancer cell line genomic data from CCLE, a statistical modeling analysis was performed to identify candidate genes which associated with BOLD-100 susceptibility, identifying the importance of ribosomal gene expression for BOLD-100 response. Finally, a machine learning model was trained using the sensitivity profile of BOLD-100 and cell line genomic data in order to assess the ability to predict BOLD-100 sensitivity in cancer cell lines. This incorporation of cell line screening, pharmacogenomics, drug response modeling, and multivariate machine learning has formulated a more cohesive understanding of BOLD-100’s therapeutic potential and provides opportunities for future research for clinical applications.The effect of BOLD-100  and cisplatin  on the growth inhibition in 319 cancer cell lines  across 2PharmacoGx (version 3.0.2) was used to fit a four-parameter log-logistic curve on the cell line viability data and compute the half maximal inhibitory concentration (IC50) [max) was calculated using the online web tool GRCalculator  [50 and GRmax were calculated in order to compare the two drugs’ sensitivity profiles. The within-tissue distributions of IC50 and GRmax were visualized in boxplots, where the length of the boxes represent the interquartile range (IQR)  and 75th (Q3) percentiles) and the bottom and top whiskers represent Q1 − 1.5xIQR and Q3 + 1.5xIQR, respectively, which denote the threshold for outliers. ANOVA was used to test whether BOLD-100 and cisplatin sensitivity profiles showed significant variability across the tissues of origin. Cell lines in the following tissues of origin were further classified into cancer subtypes according to data available in Cell Model Passports : blood, bone, brain, kidney, head and neck, thyroid, esophagus, lung, ovary, prostate, soft tissue, cervix, and uterus [50 measurements for the following cell lines could not be obtained due to the values being out of the range of the concentrations tested: HT-1376 (bladder), SK-N-AS (brain and nerves), BT-483 (breast), HeLa, MS751 (cervix), and 22RV1 (prostate). Therefore, these cell lines were excluded in the analysis of BOLD-100 sensitivity. All statistics and data visualizations were performed using the R programming language (version 4.0.5). The centrality and dispersion of the drug sensitivity values were reported using the median and the IQR and comparison of IC50 distributions were carried out using non-parametric tests such as the Wilcoxon signed-rank test and the Kruskal-Wallis ANOVA.R package n (IC50) . The noray 2021) . Within-d uterus . The fold uterus ,46, bread uterus , colon [d uterus , and livd uterus . The BOLhttps://www.cancerrxgene.org/; accessed on 10 May 2021). Data from two separate drug screen cohorts termed GDSC1 and GDSC2 were collected. In cases where a drug entry appeared in both datasets, data from the GDSC2 cohort was used, as per the database documentation [50 data were log transformed and separated based on whether cell lines originated from solid or liquid tumors. Spearman rank correlation analysis was performed between BOLD-100 IC50 profile and the individual sensitivity profiles within the GDSC dataset in order to compare the BOLD-100 response signature to the 412 other drugs (50 profile. The associated pathway annotations for each drug were obtained from the GDSC database (accessed on 10 May 2021).Drug sensitivity profiles of anticancer compounds across cancer cell lines were obtained from the GDSC database . The growth property and MSI status data were obtained from the study by Iorio et al. [The sensitivity profile of BOLD-100 and cisplatin was tested for possible confounding variables in the experimental design. Variables included age, gender, doubling time, mutation rate, microsatellite instability (MSI) status, growth property , and cell culture media. The age, gender, doubling time, and mutation rate data for the cell lines were obtained from the CCLE database  were treated as continuous variables. For mutation data, X1 were binarized based on the absence or the presence of a given mutation prior to regression. A generalized additive model method was used to fit the regression model using the R package gam (version 1.20.2). The Benjamini-Hochberg FDR correction was performed on the regression p-values , which included gene expression, mutations, metabolomics, and reverse-phase protein array data . For thep-values .clusterProfiler (version 3.10.1) [REVIGO  and the R package treemap (version 2.4.3) [Significant gene expression associations (Benjamini-Hochberg FDR < 0.1) were used as input for functional enrichment analysis using the R package  3.10.1) . The Genn 2.4.3) .50. For this, a random forest regression model was used with the BOLD-100 IC50 obtained from the cell screen as the continuous target variable using the R package caret (version 6.0.92) [2 and root-mean-squared-error (RMSE). Using the Gini impurity method, genes with high influence on the model performance were identified. The model training and evaluation process was repeated using 500 and 600 features instead in order to investigate any potential biases of feature selection on model fitting and performance.To investigate whether cancer cell lines’ gene expression showed predictive ability in estimating BOLD-100 sensitivity, a nonlinear machine learning model was trained to predict BOLD-100 IC 6.0.92) . For fea 6.0.92) . The fit50 showed significant variability across the different tissues   showed a significantly lower median IC50  compared to cell lines derived from solid cancers .To identify cancer types with greater susceptibility to BOLD-100, sensitivity profiles were obtained from 319 cancer cell lines and assessed based on tissues of origin. Non-cancerous cell lines were not assessed in this study. The distribution of BOLD-100 IC × 10−6) A, with vn = 8; ANOVA, p = 0.02) and breast  showed a statistically significant variation in response to BOLD-100 across the defined subtypes  , which was within the expected range for cisplatin [50 in response to cisplatin  compared to solid cancers . In within-tissue subtypes, the response to cisplatin showed a statistically significant variation within breast cancers  (n = 9)  . The ranisplatin ,50. Simi 0.0026) , but, di (n = 9) . Cisplat50 values were used to directly compare the sensitivity profiles of BOLD-100 and cisplatin (p = 0.002), suggesting that the majority of tissues shared similar susceptibility to BOLD-100 and cisplatin. However, esophagus, bile duct, liver, breast, and colon cancer cell lines showed a relatively greater degree of susceptibility to BOLD-100 compared to cisplatin. In breast cancer, the luminal A (LA) subtype showed the highest median IC50 in response to cisplatin  and the efficacy  thus provides additional dimensions in assessing the differential drug effect across disease types. Therefore, to infer the efficacy of BOLD-100, the growth rate metric GRmax was calculated using the cell screen viability data. GRmax is defined as the primary growth metric for drug efficacy and is bound by values −1 and +1, where positive values indicate partial growth inhibition, negative values indicate cytotoxic effects, and a value of 0 indicates a completely cytostatic effect [50 values, the distribution of GRmax values in cell lines treated with BOLD-100 also showed statistically significant variability across the different tissues   A. The mamax values between BOLD-100 and cisplatin showed that cell lines derived from liquid cancers tended to show the greatest degree of cytotoxicity in both treatments, which suggests that liquid cancers may be more predisposed to showing cytotoxic effects, or there may be assay biases related to cell growth conditions and drug exposure  with cisplatin showing a higher degree of cytotoxic activity  compared to BOLD-100   C. Both d50 and culture media in both solid and liquid cancer derived cell lines (α < 0.05). In both stratifications, cells grown in RPMI + FBS media showed greater susceptibility to BOLD-100 based on the median IC50 (median of 139 μM in solid cancers and 112 μM in liquid cancers) compared to cells grown in DMEM+FBS media (median of 178 μM in solid cancers and 138 μM in liquid cancers). In contrast, comparing the IC50 of cisplatin across the two culture conditions showed no significant differences, which suggests this bias is related to BOLD-100 sensitivity. No other associations with BOLD-100 or cisplatin response were found across the confounding variables , a total of 12 drugs’ sensitivity profiles showed a significant correlation with that of BOLD-100 (n = 58), a total of 39 drugs’ sensitivity profiles showed significant correlations with BOLD-100 , while 35 drugs were significantly correlated with cisplatin.A comparative analysis of drug sensitivity profiles allows for the identification of underlying similarities in the drugs’ MOA and has previously been used to infer the bioactivity of novel compounds ,63,64. TCisplatin had a high degree of similarity to itself  and the top associated drugs were exclusively from pathways related to DNA replication and genome integrity, which corresponds to the known mechanism of cisplatin’s cytotoxicity  7,65,6666,67. InMultiple regression analyses were performed to identify potential associations between BOLD-100 sensitivity and various genomic modalities. For gene expression data analysis, a total of 294 cell lines were used. At a significance cut-off of α < 0.1, a total of 124 genes’ expression values showed an association with BOLD-100 response . Using cn = 124 genes) were used as input for functional enrichment analysis. Over-representation analysis using the GO annotation database returned a total of 51 enriched terms after FDR correction (α < 0.05) (REVIGO for summarization based on representative pathways [REVIGO treemap based on the GO: BP over-representation result (n = 38) showed that the GO terms were grouped into the following families: processes related to protein targeting, RNA metabolic processes and translation, viral gene expression, and ribosomal biogenesis and assembly  . The top < 0.05) . To conspathways . The REVassembly .50 from the cell panel. This finding suggests that the expression levels of genes associated with ribosome biogenesis are inversely related to the susceptibility to BOLD-100 treatment in the cell panel. Though the interaction between BOLD-100 and RPs have been elucidated in the past [To elucidate the directionality of associations between BOLD-100 response and individual ribosomal gene expressions, correlation analyses were carried out . There wthe past , the pos50 in cancer cell lines. To select the number of features in the model , n = 300 genes sorted by statistical significance from the multiple regression model output were used for model training. Higher numbers of features  were also tested and no significant differences in model performance was found  identify possible cancer indications with greater susceptibility to BOLD-100 treatment and (2) generate a sensitivity profile of BOLD-100 to investigate its potential MOA. Specific cancer types, including esophageal, bladder and liquid cancers, were identified as having strong potential for further investigations. Using an integrated bioinformatics approach, this study also expanded the understanding of BOLD-100’s potential mechanism, with the identification of the importance of ribosomal proteins, thus providing avenues for future validation studies.By screening broad categories of cancer types, this study supports the current clinical strategies with BOLD-100. BOLD-100 is currently being tested in a multiple-arm Phase 1b/2 clinical study (NCT04421820) in combination with fluorouracil, oxaliplatin and leucovorin (FOLFOX) for patients with advanced metastatic colorectal, bile duct, gastric, and pancreatic cancer. Encouragingly, this cell screen identified colorectal, bile duct, and pancreatic cancer as potential indications where BOLD-100 might have increased efficacy. Colorectal cancer has previously been identified as an indication with strong potential for BOLD-100 efficacy. In a previous Phase 1 monotherapy study of KP1339/IT-139 (a precursor version of BOLD-100 with the same active ingredient), the two patients with the largest decrease in target lesion size were both colorectal cancer patients . This maCancer types with development potential were identified. Esophageal cancer is of particular interest due to it being the most responsive cancer type by median to BOLD-100 in the screen, while having high resistance to cisplatin. Within the esophageal cell lines, four out of five cell lines profiled were squamous cell carcinomas (SCCs), a cancer type that has low progression-free survival and overall survival for advanced patients, even with immunomodulating agents ,77,78. Lmax values with BOLD-100 compared to cisplatin was observed, suggesting that the pathways altered by BOLD-100 may be cell-line dependent. Similarly, comparison of the drug sensitivity profile against that of other compounds, a method that has previously been used to elucidate drug mechanisms [The mechanism of action of BOLD-100 is likely to be multimodal and complex, with contributions from the regulation of multiple cellular stress pathways. Previous work on BOLD-100, and earlier generation molecules KP1339  and KP1019, have shown that ER stress and the downregulation of the stress regulator GRP78 are critical for BOLD-100’s efficacy ,35,37. Ichanisms ,63, provchanisms ,84,85,86Ribosomal biogenesis was identified to be associated with the response to BOLD-100, with the expression levels of the RPs showing an inverse relationship with BOLD-100 sensitivity. In target identification studies, BOLD-100 was shown to bind RPLs and BOLD-100 increased expression of RPLs in treated HCT116 cells . These r2 of 0.399, which suggests the model explained the variability in the dataset to a reasonable degree, in line with the previous literature on drug sensitivity prediction tasks using random forests [Drug sensitivity prediction modeling is an opportunity to develop unique response profiles for therapeutics. This study employed a simple generalized linear model as a means of feature selection and a classical machine learning model in the random forest regressor, which is a standardly utilized approach in the field of pharmacogenomics ,91,92. T forests . However forests . Indeed, forests  with no The cell screening approach and subsequent bioinformatics analysis using gene expression provided a wealth of new information around BOLD-100. However, no significant associations in the other genomic datasets were identified, including oncogene or tumor suppressor gene mutations or protein expression patterns. Gene expression data has been shown to be the most predictive modality in drug sensitivity prediction tasks ,23,24. FThe current study employed a cancer cell line panel to infer the differential sensitivity of BOLD-100 across different tissues and build a sensitivity profile that can be used for downstream pathway analysis. This enabled the finding of potential routes for clinical development of BOLD-100 in cancer types which were not identified previously, including esophageal, bladder, and liquid cancers. BOLD-100 generally exhibited cytotoxic effects across the cell panel and shared moderate correlations to a heterogeneous group of anticancer drugs found in the literature, with no clear overarching theme in bioactivity. Furthermore, potential molecular determinants of BOLD-100 sensitivity were identified, namely the association with genes coding for RPs and related pathways. This study thus provides a platform for translational research in BOLD-100 to expand the current findings to in vivo models and additional avenues towards next-generation machine learning tasks in oncology and drug prediction.


The structure of trans complex 2 was additionally confirmed by X-ray diffraction. The cytotoxicity of the investigated compounds was examined in vitro on three human cancer cell lines  in both normoxia and hypoxia conditions. LogPs of complexes were measured using the shake-flask method. The trans complex 2 showed much better cytotoxic activity than cisplatin for all the tested cancer cell lines. Cis complex 1 was inferior to its trans isomer against all the cancer lines tested in normoxia conditions but proved superior to the reference cisplatin against the MCF-7 and A549 lines, and showed similar activity to cisplatin against the ES-2 line. To gain additional information that may facilitate the explanation of the pharmacological activity of the tested compounds, cellular platinum uptake and stability in L-glutathione solution were determined for both compounds 1 and 2.Two novel platinum(II) complexes ( Cytotoxicity evaluation on a panel of three tumor cell lines revealed a significant potential of trans-[PtCl2 (1-methyl-4-nitropyrazole)2], which exhibited cytostatic activity in the micromolar concentration range, with the highest cytotoxicity against ES-2 cells, surpassing that of cisplatinum, while the appropriate cis congeners showed only moderate activity. An attempt was also made to elucidate the molecular mechanisms underlying the in vitro biological activity of trans [PtCl2 (1-methyl-4-nitropyrazole)] by examining its effect on the cell cycle, and we evaluated the mechanism of cytotoxic action as a result of the ability of the complex to bind intracellular DNA and proteins. We also conducted a study of the reactivity of the obtained compounds with glutathione in order to assess the rate of their deactivation in living organisms. This work is a continuation of this topic, but now we have directed our interests to the area of 4-nitroisoxazole derivatives as potential ligands. This choice was related to our previous studies, which strongly suggested that the cytotoxicity of nitroazole complexes with Pt(II) requires the presence of a heteroatom–heteroatom bond in the ligand molecule used. The anticancer properties of Pt(II) complexes with nitroisoxazoles have never been investigated so far. However, a number of methods for the synthesis of these types of nitroisoxazole ligands are known, including the direct nitration of isoxazoles in the HNO3-H2SO4 system  2.88 , 3.01 . Far-IR (nujol) ν[cm−1]: 355 , 347 . Visualizations of far-IR and 1HNMR spectra of compound 1 are in the Combustion and spectral analyses for the trans complex 2: Elemental analysis for formula C10H12N4O6Cl2Pt of the trans complex 2 ): C 21.83/22.12, H 2.20/2.56, N 10.18/10.50, Cl 12.89/12.31. 1H NMR : δ[ppm] 3.00 , 3.06 . Far-IR (nujol) ν[cm−1]: 344 . Visualizations of far-IR and 1HNMR spectra of compound 2 are in the Combustion and spectral analyses for the 1 and 2: The calculated value of the parent peak mass for the formula C10H12N4O6Cl2Pt is 548.97817 u. The ESI-MS (negative ionization in methanol) of 1 and 2 revealed, amongst others, the following peaks given as a ratio m/z [u/e] (relative intensity to base peak [%]): 548.965197 (100%) [PtL2Cl2+1-H]− —isotope peak of quasi-molecular ion (base peak), 547.967118 (73.7%) [PtL2Cl2-H]−—quasi-molecular ion (parent) peak, 405.929436 (31.8%) [PtLCl2-H]−—fragmentation ion peak, where L means ligand, i.e., 3,5-dimethyl-4-nitroisoxazole. Visualizations of ESI-MS spectra are in the Supplementary Materials Analysis of mass spectra (ESI-MS) of both complexes trans complex 2 suitable for XRD measurement was obtained by slowly evaporating the solvent from its acetone solution. Crystallographic measurement for compound 2 was performed with Κ-geometry diffractometers: Xcalibur Ruby, Gemini ultra with graphite monochromatized Mo-Kα radiation (λ = 0.71073 Å) at 100(2) K, using an Oxford Cryosystems cooler. Data collection, cell refinement, data reduction and analysis were conducted with CrysAlisPro . The data were subjected to analytical absorption correction using CrysAlisPro. The crystal structure was solved using SHELXT . CCDC reference number for compound 2: CCDC2215593  and partition coefficients were calculated. For analysis, all samples were dissolved 1:1000 in 2.5% HCl. Each measurement was repeated four times, and the final logP was calculated as the arithmetic mean of the four values. Directly after the experiment, the organic and aqueous phases were additionally tested (TLC) for homogeneity, but no signs of decomposition or isomerization of the tested Pt complexes were found.The partition coefficient was measured using the methods described earlier ,30. An a1 or 2 in dioxane (30 μL) were added to 3 mL of a 2 mM solution of GSH (reduced L-glutathione) pre-saturated with argon in a medium containing 10 mmol/L NaCl and 10 mmol/L Tris xHCl buffer, pH 7.4, reaching a final concentration of 6 µM of the tested complex, and warmed to 37 °C. The progress of the reaction was monitored by UV absorption spectrometry at 260 nm. In this experiment, dioxane was used to dissolve the tested Pt complexes due to its lack of UV absorption at the wavelength used. Argon saturation of GSH was performed to remove oxygen from the solution and thus prevent the formation of S-S bonds that would distort the measurement results.A total of 60 mM solutions of compounds The following cancer lines were used for the study: MCF-7 (breast adenocarcinoma), ES-2 (ovarian adenocarcinoma) and A549 (lung adenocarcinoma). Normal cell line BALB/3T3 (murine embryonic fibroblast) was also used for comparison purposes. All these lines were purchased from the American Type Culture Collection . The preparation of these lines for testing is described in detail in the following paper .1 and 2 were prepared ex tempore by dissolving the complexes in acetone, and the solutions obtained were added to the culture medium (1:9 (v/v), respectively) to obtain a stock solution of 1 mg/mL.The acetone solutions of complexes The test was prepared and performed in the same manner as detailed in the following cited works ,35,36. TThe survey was conducted exactly according to the method described in the following paper . The res50 values of all of the compounds within a single cell line and in the mean IC50 values of each individual compound for cancerous cell lines and healthy reference cells was tested with conservative post hoc tests for multiple comparisons, such as Tukey’s HSD and Bonferroni tests. Moreover, the statistical significance of the differences in the mean IC50 values of the tested compounds in relation to the mean IC50 values of the reference drug was also tested with Dunnett’s post hoc test. The confidence level (limit) was set as 95% and the threshold for significance level was set at α = 0.05; hence, the differences between the means with a p-value  <0.05  were considered statistically significant.Statistical analysis was carried out using following software: Statistica , version 13.3  and PTC Mathcad Express Prime 6.0.0.0. . The normal distribution of the data within the groups was verified with the Shapiro–Wilk test and the Kolmogorov–Smirnov (K-S) test with Lilliefors correction. The homogeneity of variances was checked with Levene’s and Brown–Forsythe tests. To avoid a type I error, the statistical significance of differences in the mean ICStudies on the complexation of platinum(II) ions by 4-nitroisoxazoles indicate that this process occurs much more difficultly than in the case of 1-methylnitropyrazoles, where only 1-methyl-3-nitropyrazole did not give the Pt complexes, probably because of the steric hindrance of both methyl and nitro groups that constrain access to lone pairs of pyrazole nitrogen electrons. Furthermore, the electrostatic repelling of the approaching tetrachloroplatinate anion by electron-dense oxygen atoms of the nitro group may also be an important factor that can hinder and/or prevent the formation of an appropriate Pt–nitropyrazole complex . Of the 1 and 2 showed high cytotoxicity in biological tests, especially compound 2 of trans configuration was surprisingly extremely active, clearly exceeding (more than 15 times) the reference cisplatin against all tested cancer cell lines. Cis complex 1 appeared to be more active than cisplatin against MCF7 and A549 cancer cell lines, and comparable to cisplatin against the ES2 cancer cell line. The Pt uptake study showed that the cytotoxicity of compounds 1 and 2 was not due to excessive platination of the tumor cells. Moreover, it was revealed that the platination of cells was significantly lower for the tested compounds than those of cisplatinum, which suggests a molecular mechanism of their biological action. It has been previously shown that the sensitivity of neoplastic tissues to platinum drugs strongly depends on their ability to metabolize and excrete them from the cell [1 and 2 to GSH, finding that trans complex 2, which is more cytotoxic than cisplatin, undergoes this reaction about three times faster, while the compound 1 of cis configuration reacts at a similar rate. This fact, coupled with its high cytotoxicity to normal cells, discouraged us from testing compound 2 for its in vivo antineoplastic activity in a murine tumor model.Both complexes the cell . In turnThe experimental data described in this paper, combined with the conclusions from our previous investigations concerning Pt(II) nitropyrazole complexes, suggest two ways to continue further research in our field of interest. The first is the synthesis of appropriate Pt(II) complexes with various nitroisothiazole ligands  in the hCis-and trans-platinum(II) complex compounds with 3,5-dimethyl-4-nitroisoxazole having antitumor activity, process for their preparation and separation of, Polish patent PL231434B1, 29 February 2019.The results of the research have been patented: Regiec, A.; Mastalarz, H.; Mastalarz, A.; Milczarek, M.; Ryng, St.; Wietrzyk, J.;


Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth  Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma of childhood that includes two major histological subtypes: embryonal and alveolar . About 8Intracellular Domain (ICD). Then, from the cytoplasm, NOTCH (N)ICD migrates into the nucleus where it binds the DNA-binding transcription factor CSL/RBP-Jk (CBF1/RBPj/Su (H)/Lag-1) and the mastermind-like proteins (MAML1-3) displacing the co-repressor complex and allowing the transcription of canonical NOTCH target genes of the basic helix-loop-helix (bHLH) Hes (HES1-7) and Hey  families of transcriptional repressors . SoCip1 increase in both RMS cell subtypes.As already reported for other types of cancer cells , ARQ treHowever, although significant activation of caspase 3/7 was induced by NOTCHi in all four cell lines, it was able to significantly increase the percentage of Annexin V positive cells only in RD and RH4 cell lines. Similarly, a cell line-specific response was seen for ARQ, which activated caspase 3/7 and enhanced Annexin V positivity in three cell lines with the exception of RH30 cells. Furthermore, only METi promoted PARP1 cleavage in all the tested cell lines, even if the increase in cleavage is mild in RH4 cells. These results suggest that NOTCHi induces PARP1-independent while METi PARP1-dependent apoptosis in RMS cells. In RH30 and JR1 cells, where neither Annexin V positivity nor PARP-1 cleavage increase was seen in response to NOTCHi, cell death could be due to a transient cell activation of caspase 3/7  or, altevs each single drug. These findings suggest that this combinatorial targeting triggers apoptotic cell death, which is dependent by PARP1 in RMS cells.All these effects were significantly enhanced combining the two inhibitors. Indeed, in all the RMS cell lines treated with the combination, the levels of MET and its phosphorylated form were similar or even decreased compared to those in vehicle-treated control cells demonstrating that ARQ in the combination counteracted the PF-0308-induced increase of MET protein levels. In addition, the RMS cell lines proliferated significantly less than control cells under drug combination, with the stronger effect on FP-RMS cells. Furthermore, NOTCH-MET co-inhibition was capable of triggeringsustained apoptosis in RMS cells as testified by the marked enhancement of the activity of caspase 3/7 and the concomitant augmentation of Annexin V positivity and PARP1 cleavage in vitro studies are needed to clarify whether the pharmacological co-inhibition of NOTCH and MET pathways have synergistic or additive effects or both on the different malignant features in our tumor context.Moreover, while single treatments mildly affected the capacity of RMS cells to migrate, form colonies, and grow in an anchorage-independent manner, the PF-0308 and ARQ combination had a remarkable inhibitory effect on these tumorigenic properties. NOTCH-MET co-inhibition also strongly affected the survival of RMS cells grown as spheroids increasing cell death as compared to individual drug treatments. The drug combination showed a different impact on the proliferation ability of the two RMS subtypes resulting in a larger inhibitory effect than the sum of the effects of each individual drug in FP-RMS but not in FN-RMS cells. Further, the marked activation of caspase 3/7 and the induction of Annexin V positivity in three cell lines were greater than that due to additive effects, with the exception of RH4 cells. However, further in vitro studies showed that ARQ affects cell proliferation in cells not expressing MET and does not inhibit MET phosphorylation in lung cancer cells with low basal levels of MET expression  #15312 to RR and #24696 to FM; Italian Ministry of Health (Ricerca Corrente) to BDA, CQ, and RR; AIRC 5xmille #9962 to FL; Italian Ministry of Health (Fondi 5xmille 2021-2022) to RR; Alleanza Contro il Cancro (ACC) Italian Network-Working Group Sarcomas to BDA, RM, and RR; Fondi Ateneo 2019 to FM; MIUR-Italy: Grant to Department of Science, Roma Tre University  to MCe and PM.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.


Stenocereus queretaroensis (F.A.C. Weber ex Mathes.) Buxb is a cactus that has long been used as a source food in central and northern México. Its fruits, commonly called pitayas, biosynthesize high amounts of betalains. These molecules are water-soluble nitrogenous compounds; that compared to other pigments, such as anthocyanins or carotenoids, stand out for their physicochemical stability in industrial processes. Due to genetic and environmental factors involved in the biosynthesis and accumulation of secondary metabolites in plants, we tested different stress-inducing agents  to induce betalains accumulation in cell culture from fruits of Stenocereus queretaroensis. This work aimed to understand stress conditions that induce the metabolic pathways required for the accumulation of betalains. The results show how betacyanin concentration increases under high sugar conditions, thus affecting the expression of L-DOPA 4, 5 dioxygenase resulting in a strong dark red coloration. This suggests this enzyme is part of a rate-limiting step in betalain production. In addition, we found that betalains accumulation occurs under particular stress conditions. Cells that have a high level of betacyanins show better resistance to stress in the cell culture, as well as an overall different behavior including cell aggregation and alterations in nuclear size. Together the results shown here may provide new strategies to manipulate and mass produce the pigments from Stenocereus queretaroensis in cell culture. Stenocereus queretaroensis (F.A.C. Weber ex Mathes.) Buxb. was recognized as a natural source of betalains [In general, plant pigments are secondary metabolites except for the chlorophylls which are photosynthetic pigments in all autotrophic organisms . Accordietalains ,17,18,19etalains . Betalaietalains . They aretalains ,22. Betaetalains ,4,5,6,7,etalains . Betalaietalains .in vitro. Additionally, we focused on exploring the pathways of betalains bioproduction. These pathways can lead to improved high-throughput models to produce pigments that can satisfy different industries and reduce the cost of additional sugars needed as a signal for the production of betalains in vitro.This work focused on the use of an elicitor , osmostressors (PEG 6000 and sucrose), salts (NaCl), and temperature (45 °C) with the aim to test how various stress signals modulate or affect the biosynthesis of betalains and cell behavior −1 FW). In the case of betacyanins, they present a small increase throught the culture time with an accumulation after 24 days from reseeding the culture (4.69 µg g−1 FW) (−1 FW), whereas betaxanthins reached their maximum on day 16 (78.76 µg g−1 FW)  a. In the g−1 FW) b.ID: OP609705), and 4,5 dioxygenase (DODA ID: OP609705), as key genes in the betalains biosynthetic pathway. The sequences were obtained from Stenocereus queretaroensis transcriptomic analysis (accession number: GSE173230). TyrAa gene transcripts showed a constant expression  after 48 h of exposure to salt stress a, whereaThe addition of 100 mM NaCl in the sqY1 line promoted orange-red cell aggregates in contrast to the normal media as a control. e: A,B, wTo induce osmotic stress to the cells, different concentrations of polyethylene glycol  were used in sqY1 presenting a light brown color a and in The formation of callus from cells suspension of the sqY1 line resulted in callus with brown coloration e: A–D; oExposure to 45 °C for 8 and 16 h in sqY1 and sqR1 cell lines did not induce pigmentation or cell aggregation a,b. HoweS. queretaroensis in an open field.The coordinates in CIELab space show that the color variation in the sqY1 line has a significant difference concerning the control line in the presence of all stress signals, among which the distribution of the points for each treatment in the chromatic circle of CIELab space showed distance from each other a; whereaIn recent years, betalains have received a renewed interest due to their physicochemical characteristics (high stability at wide ranges of temperature and pH) that can be of utility in many industries, as well as bioactive compounds for the prevention of some diseases ,26,27,28S. queretaroensis fruits represent an important source of betalains, which accumulate in large quantities in their pulp. However, the fruits can be collected in limited numbers in only specific periods of time. Therefore, the uses of cell lines derived from fruits represent a viable alternative to study the regulatory mechanisms that control their synthesis [S. queretaroensis cell suspensions seemed to favor the production of polyphenols over betalains accumulation. The use of sequencing tools, particularly RNA sequence analysis for de novo assemblies, have proven to be very useful in determining genes of importance for the synthesis of a large number of plant metabolites, including secondary metabolites derived from tyrosine such as alkaloids or other pigments such as anthocyanins [S. queretaroensis, whose fruits have the ability to produce a large number of betalains, giving them different colors, make it very attractive to deepen one’s knowledge of the synthesis of these pigments and metabolic engineering.ynthesis ,30,31. Tynthesis . The useocyanins ,33,34 inArabidopsis thaliana during cell growth [The TyrAa down-regulation in sqY1 cell line has been shown in l growth ; howeverl growth . Howeverl growth ,38. Abiol growth . Generall growth , and alsl growth .A change in pigmentation (orange-reddish hue) could be observed in sqY1 cells in the presence of 100 mM NaCl, while this same treatment did not affect the sqR1 cells. When yellow cells were subjected to an increase in the concentration of sucrose from 3% to 8% sucrose, a reddish coloration appeared, which may be related to an increase in betacyanin, perhaps by redirecting the synthesis pathways to form the red pigments. The nuclear condensation on sqR1 cells reflects the sensitivity to salt stress in this cell line in comparison with the osmotic stress induced by the addition of PEG 6000 in the yellow cells. The changes by this particular stress in sqY1 resulted in remarkable changes in pigmentation and cell aggregation at all tested concentrations. sqR1 cells, which are cultivated in 8% sucrose, did show any significant change, suggesting that an additional osmotic disturbance do not further alter their betacyanin accumulation. Temperature stress did not induce any disturbance in pigmentation or cell development; therefore, it is suggested that betalains have a protective function at high temperatures, which is linked to the origin of the cell’s lines. The color change analysis using the CIELab system objectively demonstrated that the greatest color variation was present in the sqY1 cell line in all stress conditions compared to the control. The sqR1 line presented minimal variations in color change but without affecting the phenotype and cell proliferation in all stress conditions compared to the control as compared to the sqY1 line that is sensitive to all types of stress.Stenocereus queretaroensis (Weber) Buxbaum cell lines (sqY1 and sqR1) previously established from an immature pitaya fruit brought from Querétaro [w/v), depending on the cell line, as well as 1-naphthaleneacetic acid  (5.3 µM), 2,4-dichlorophenoxyacetic acid (Sigma-Aldrich) (4.5 µM) and kinetin (Sigma-Aldrich) (4.6 µM). Media pH was adjusted to 5.8 ± 0.02 and it was distributed at a rate of 40 mL in 250 mL Erlenmeyer flasks. Subsequently, these were sterilized in an autoclave at a pressure of 1 kg/cm2 and a temperature of 121 °C for 20 min. Each line produces mainly a particular class of betalains: the sqR1 line produces red-violet pigments (betacyanins) and the sqY1 line produces yellow pigments (betaxanthins).Two uerétaro  were useCell suspensions were propagated by subculturing every 15 days and maintained under the following conditions: temperature 25 ± 2 °C, light (4700 lumens) and continuous agitation (120 rpm). Each subculture was carried out by transferring 15–20 mL of the cell suspension to Erlenmeyer flasks with 40 mL of fresh culture medium.In order to determine the growth cycle of the cell lines, an initial stock was prepared by pouring 15 flasks of suspensions into a one-liter Erlenmeyer flask (under sterile conditions). Once the cell stock was homogenized, 10 mL of the suspension, equivalent to 1.8 ± 0.2 g of fresh weight, were transferred to each of the 250 mL Erlenmeyer flasks with 40 mL of modified Murashige and Skoog culture medium for cacti. After the inoculation, the flasks were kept in the culture room under continuous light to 4700 lumens, a temperature of 25 ± 2 °C and 120 rpm, throughout the culture cycle.®). The application was exogenous, adding 50 µM, 100 µM and 200 µM from the stock solution described above; at the same time, a blank was made by adding distilled water to the cell suspensions in the same volume as the elicitor, the treatments were performed in triplicate for each dose. Flasks with each treatment were placed in continuous light with orbital shaking at 120 rpm at a temperature of 25 ± 2 °C until harvesting after 24, 48 and 72 h of exposure to SA. Harvesting was performed using a vacuum pump (Model AR-1500 dry pump from Arsa®) and the filtered cells were stored in aluminum envelopes labeled with the corresponding treatment and stored at −80 °C until use.Cells from the sqY1 and sqR1 lines were collected on day 18 of their cell cycle, taking into account cell density. A 20 mM salicylic acid (SA) stock solution (Sigma-Aldrich) was prepared, dissolved in distilled water and sterilized by filtration at pH 5.7  with 3% sucrose for the sqY1 line and 8% sucrose for the sqY1 line. The sqR1 line, then it was sterilized in an autoclave  at a pressure of 1 kg/cm2 and a temperature of 121 °C for 15 min; subsequently, using pipettes, 10 mL of cells from 14-day-old sqY1 and sqR1 lines were added due to their abundant cell development. The flasks were kept under continuous light and at a temperature of 25 ± 2 °C with agitation at 120 rpm. After 9 days of exposure to treatment, cells were harvested by vacuum filtration, stored in aluminum envelopes and kept at −80 °C until use. Treatments were performed in triplicate.Osmotic stress was simulated using polyethylene glycol 6000 ,4,5,6  and heated to 45 °C. During the induction of heat stress, the cells were maintained under normal growth conditions, agitation at 120 rpm and continuous light 4700 lumens Flasks were maintained there for 8 h at 120 rpm. Images were recorded before and after temperature exposure.w/v) ratio. With the help of the polytron, the sample was homogenized for 2 min; subsequently, it was centrifuged at 13,000 rpm for 20 min. The supernatant was transferred to a test tube, which was used to carry out all the determinations.For pigment extraction, two flasks were used for the sqY1 line and two for sqR1, to remove the culture medium a vacuum filter was used, 1g of fresh weight was used for each cell line, samples were placed in a cold mortar, liquid nitrogen was added, and they were then ground to a fine, uniform powder. The powdered sample was transferred to a test tube and ethanol was added in a 1:5  for 24 h at 4 °C. Cells were subsequently mounted in fluoroshield-DAPI (Sigma Aldrich) on glass slides and incubated for 24 h. Cell images were taken on a Axioplan Carl Zeiss microscope Axio Scope. A1 and confocal microscope  using 388 nm for DAPI detection in blue auto fluorescence was detected at an excitation wavelength of 488 nm.w/v), depending on the cell line, as well as 1-naphthaleneacetic acid (Sigma-Aldrich) (5.3 µM), 2,4-dichlorophenoxyacetic acid (Sigma-Aldrich) (4.5 µM) and kinetin (Sigma-Aldrich) (4.6 µM). The pH of the medium was adjusted to 5.8 ± 0.02 and sterilized in an autoclave at a pressure of 1 kg/cm2 and a temperature of 121 °C for 20 min. It was distributed at a rate of 25 mL in plastic Petri dishes. Sterile 10 mL tips were used to collect cells in suspension from flasks of the sqY1 and sqR1 lines by placing a cluster of cells in the center of the Petri dish. The Petri dishes were then cellared and placed in continuous light at a temperature of 25 °C. Each line mainly produces a particular class of betalains: the sqR1 line produces red-violet pigments (betacyanins) and the sqY1 line produces yellow pigments (betaxanthins).Both lines were maintained in Murashige and Skoog medium adapted for cacti, based on inorganic MS salts , with a total calcium concentration of 4.4 µM and without nicotinic acid and glycine. It contained 3% or 8% sucrose  to obtain SD with three independent replicates for each treatment and analyzed as a completely randomized factorial design.Group means were calculated and compared using one-way independent ANOVA analyses performed with Graphpad Prism 9.0.2 software.p ≤ 0.05) was performed. The error bars are shown in charts with the represented error bars in the histogram. All figures were prepared with PowerPoint , and ImageJ  for bans quantification and size of the nucleus.For comparisons between treatments, Tukey’s honestly significant difference (HSD) tests differences in the means were considered statistically significant  and finally stored at −80 °C until use. For total RNA extraction, the Chomczynski and Sacchi (1987) protocol was used, with slight modifications. Initially, 0.1 g per sample of cells stored at −80 °C from each line and day were used, individually homogenized in 1.5 mL Eppendorf tubes, in 750 μL of Trizol reagent  with glass beads and vortexed. The tissue was manually homogenized with 300 μL of chloroform and incubated on ice for 3 min. Subsequently, the phases were separated by centrifuging at 12,000× g for 15 min at 4 °C, the aqueous phase was recovered in a new tube to which 500 μL of isopropanol was added, it was shaken and incubated at room temperature for 10 min, the mixture was centrifuged at 12,000× g for 15 min at 4 °C, carefully discarding the supernatant. The RNA pellet was washed in 1000 μL of 70% ethanol, vortexed, and centrifuged at 7500× g for 5 min. Three consecutive washes of the pellet were performed, adding 100 μL of 70% ethanol and centrifuging at 8000× g for 5 min at 4 °C each time. Finally, the tablet was resuspended in 30 μL of water with diethylpyrocarbonate (DEPC) and stored at −22 °C until use. The concentration and purity of the RNA obtained in each extraction method was confirmed by spectrophotometry  in a NanoDrop-1000 ND-1000 Spectrophotometer UV/VIS . The integrity and quality of the total RNA was verified by electrophoresis on a 1% agarose gel stained with SYBR® Gold nucleic acid gel stain .Cells from each line were collected at specific stages of the growth cycle, based on betalains quantification. The sample collection was performed by vacuum filtration, where 0.1 g of cells were deposited in 20 μL cryotubes  and 4, 5-extradiol dioxygenase , in addition to the reference gene elongation factor alpha 1 , were used.The analysis of the expression of the ® III RT (200 U/μL) and initially incubated at 50 °C for 50 min and finished by heating at 85 °C for 5 min. Finally, each reaction was treated with 1 μL of RNase H per tube and incubated at 37 °C for 20 min.cDNA synthesis, the SuperScript ™ III First kit was used. Strand synthesis System for RT-PCR (Invitrogen). 1 µg of the total RNA previously extracted and treated with DNAses was taken and deposited in a mixture containing 1 µL of oligo (dT), 1 µL of dNTP mix (10 mM) brought to a volume of 10 μL with DEPC water and incubated at 65 °C for 5 min. For complementary strand synthesis, the cDNA synthesis mix was added, consisting of 2 μL of RT buffer (10X), 4 μL of MgCl2 (25 mM), 2 μL of DTT (0.1 M), 1 μL of RNAseOUTTM and 1 μL of SuperScriptA total of 1 μg of cDNA was taken and incubated in a PCR mix containing 25 μL of DreamTaq Green PCR Master Mix (2X) and 10 μM of specific primers, brought to a total volume of 50 μL with nuclease-free water. The program consisted of 26 cycles that provided quantitative differences to be detected by PCR. The PCR products were resolved on 2% agarose gels and visualized in a transillumination system.S. queretaroensis as an experimental model. The cell line sqY1 obtained from fruits of S. queretaroensis show a bright yellow color that under high sugar content changes to a dark red coloration due to increase betalain concentration resulting in the sqR1 cell line. The metabolic pathway for betalains production requires conversion of Tyrosine-by-Tyrosine hydroxylase to obtain L-DOPA followed by L-DOPA 4,5 dioxygenase to form betalamic acid. These two genes were explored in this analysis and show an increase in L-DOPA 4, 5 dioxygenase when sugar is added suggesting that this is a rate limiting step that may trigger betalains production. Obviously, other controlling mechanisms may be involved, including posttranscriptional modifications. Nevertheless, our results clearly point out that different conditions result in different behaviors from these two cell lines. With a lower sensitivity to salicylic acid, Sodium Chloride, temperature, and Osmotic stress in betalains containing cells. A deeper understanding of betalains production pathways should allow us to genetically engineer high-throughput models to produce pigments in sufficient amounts that can satisfy different industries. Here, the conditions presented not only show some possible mechanism that can be used to induce and affect betalains production, but also the stability of the culture under different conditions. The results may suggest that betalains accumulation under unfavorable conditions may represent not only an advantage for the plant in the field, but also provide us with strategies to manipulate their synthesis in more suitable experimental models, and hence, obtaining valuable basic knowledge of that in the future could substitute the use of some synthetic dyes.Results obtained from this work had been aimed to attain a better understanding of the regulatory expression of betalains synthesis, using a cell line of


The outcome of allogeneic hematopoietic cell transplantation  largely depends on the development and management of graft-versus-host disease (GvHD), infections, and the occurrence of relapse of malignancies. Recent studies showed a lower incidence of chronic GvHD and severe acute GvHD in patients receiving naive T cell depleted grafts compared to patients receiving complete T cell depleted grafts. On the other hand, the incidence of acute GvHD in patients receiving cord blood grafts containing only naive T cells is rather low, while potent graft-versus-leukemia (GvL) responses have been observed. These data suggest the significance of naive T cells as both drivers and regulators of allogeneic reactions. The naive T cell pool was previously thought to be a quiescent, homogenous pool of antigen-inexperienced cells. However, recent studies showed important differences in phenotype, differentiation status, location, and function within the naive T cell population. Therefore, the adequate recovery of these seemingly innocent T cells might be relevant in the imminent allogeneic reactions after allo-HCT. Here, an extensive review on naive T cells and their contribution to the development of GvHD and GvL responses after allo-HCT is provided. In addition, strategies specifically directed to stimulate adequate reconstitution of naive T cells while reducing the risk of GvHD are discussed. A better understanding of the relation between naive T cells and alloreactivity after allo-HCT could provide opportunities to improve GvHD prevention, while maintaining GvL effects to lower relapse risk. Allogeneic hematopoietic cell transplantation  is an intensive, but potentially curative treatment for refractory hematologic malignant disorders, and life-threatening, non-malignant diseases, such as primary immune deficiencies and inborn metabolic disease. Complications associated with this therapy, such as graft rejection, acute or chronic graft-versus-host-disease (aGvHD and cGvHD respectively), infections, and disease relapse are life-threatening and limit allo-HCT success. T cell reconstitution after allo-HCT plays an important role, as T cell numbers associate with the occurrence of relapse in malignant disease and the outcome of GvHD responses –7. The eTransplantation with T cell depleted (TCD) grafts results in lower GvHD incidence and graft rejection, but also leads to delayed immune reconstitution (IR) and thereby increases risk of disease relapse and viral infections , 3, 8–11In this review, we will provide an overview from recent literature on the heterogeneity and composition of the naive T cell population in different graft types and discuss their role in allogeneic reactions associated with GvHD and GvL after allo-HCT. Furthermore, possible naive T cell-targeted strategies to enhance allo-HCT outcome will be suggested. More knowledge about the underlying mechanisms of naive T cells in alloreactivity after allo-HCT will allow us to gather insights in how to prevent adverse reactions such as GvHD, while maintaining GvL responses to prevent leukemia relapse.via the lymphatic system. Although the exact compartmentalization is tissue- and age-dependent, naive T cells are also widely distributed in non-lymphoid tissues, and some cells are suggested to hardly even recirculate through the blood  of naive T cells are responsible for the TCR repertoire diversity and size of the naive T cell pool after allo-HCT , 30. DurEarly naive T cell reconstitution after allo-HCT highly depends on the number of naive T cells transferred with the graft , 37–39. via thymopoiesis might also be different when starting with fetal- compared to adult stem cell sources. It is known that fetal and adult HSCs give rise to thymocytes with distinct gene signatures  in the setting of an inflammatory environment and typically occurs in the first few months after transplantation , 59. Howin vitro  were used to predict donor T cell mediated recognition of mismatched-HLA derived peptides following allo-HCT, with peptides presented on HLA-I as PIRCHE-I score, and peptides presented on HLA-II as PIRCHE-II score. Those scores were correlated with patient survival, disease relapse and complication development . In BMT,Some centers are successfully using T cell depleted grafts to reduce GvHD risk. Despite initial success, complications, such as increased disease relapse and viral reactivation have emerged, caused by delayed or absent early T cell reconstitution after allo-HCT . Recent + and CD8+ T cell numbers recovered much earlier, and no significant increase in disease relapse was observed after naive TCD grafts relative to patients that received complete TCD grafts (Since naive T cells show higher alloreactive capacity and are more potent inducers of GvHD compared to effector memory T cells , 100–102D grafts , 14. FurD grafts , 106. ThD grafts . Two othD grafts , 73. Howde novo production of naive T cells will contribute to a high TCR repertoire diversity and thus high antigen specificity, thereby support a long-lasting functional immune recovery (Thymic regeneration, thereby recovering TCR repertoire diversity , may takrecovery .Current insights show that the naive T cell compartment consists of a heterogenic population of T cells highly involved in GvHD and GvL responses after allo-HCT. The naive T cell pool contains the most diverse collection of TCRs, which is of great importance for the formation of adequate immune responses against newly encountered pathogens. Together with the capacity of naive T cells to promptly switch to effector phenotype and proliferate upon antigen encounter, this high TCR repertoire diversity potentiates the naive T cell pool for GvL. On the other hand, TCR diversity might be responsible for the recognition of allo-antigens and contribute to GvHD risk. Naive T cells derived from distinct graft types, such as CB versus BM and PB, show a different behavior regarding GvHD risk and GvL effect, and might thus have an intrinsic difference in the balance between GvHD and GvL alloreactivity. The differences between CB-derived and BM/PB-derived naive T cells need to be further explored and their role in alloreactivity needs to be elucidated. For this, the recent advances in tracking naïve T cells for single cell sequencing using natural barcoding, would be of major interest to better understand their role in GvHD and GvL effects . MoreoveLD and ES wrote the manuscript; ES designed the figure; CK and SN conceptualized and revised the manuscript; CL revised the manuscript. All authors have read and agreed to the published version of the manuscript.This work was supported by the Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.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.


Malignant mesothelioma is a rare, fatal cancer with limited treatment options. Photodynamic therapy is an emerging treatment option which uses a drug (known as a photosensitizer) and a specific type of light to selectively destroy cancerous cells. We have synthesized new photosensitizing drugs by linking organic chemical structures (variable length fatty acid chains) to existing photosensitisers. These new photosensitizing drugs were characterized, and their cancer-killing capabilities were also assessed in malignant mesothelioma cells in the laboratory. We have found that one of the newly synthesized photosensitisers (C5SHU) appeared to cause a significant amount of cell death in the malignant mesothelioma cells in combination with light. C5SHU-induced cell death did not occur in the absence of light. Therefore, we propose that this photosensitizer—C5SHU—is a suitable anti-tumour drug candidate in this hard-to-treat cancer.Clinical trials evaluating intrapleural photodynamic therapy (PDT) are ongoing for mesothelioma. Several issues still hinder the development of PDT, such as those related to the inherent properties of photosensitizers. Herein, we report the synthesis, photophysical, and photobiological properties of three porphyrin-based photosensitizers conjugated to truncated fatty acids (C5SHU to C7SHU). Our photosensitizers exhibited excellent water solubility and high PDT efficiency in mesothelioma. As expected, absorption spectroscopy confirmed an increased aggregation as a consequence of extending the fatty acid chain length. In vitro PDT activity was studied using human mesothelioma cell lines (biphasic MSTO-211H cells and epithelioid NCI-H28 cells) alongside a non-malignant mesothelial cell line (MET-5A). The PDT effect of these photosensitizers was initially assessed using the colorimetric WST-8 cell viability assay and the mode of cell death was determined via flow cytometry of Annexin V-FITC/PI-stained cells. Photosensitizers appeared to selectively localize within the non-nuclear compartments of cells before exhibiting high phototoxicity. Both apoptosis and necrosis were induced at 24 and 48 h. As our pentanoic acid-derivatized porphyrin (C5SHU) induced the largest anti-tumor effect in this study, we put this forward as an anti-tumor drug candidate in PDT and photo-imaging diagnosis in mesothelioma. Photodynamic therapy (PDT) consists of the use of a tumor-specific photosensitizer (PS) and laser irradiation to induce the production of reactive oxygen species in cancer cells ,2,3. MolThe photoactivated PS generates reactive oxygen species (ROS) either through sensitizing biomolecules to radicals/ions (type I reaction) or via the generation of singlet oxygen (type II reaction) . The disMalignant pleural mesothelioma (MPM) is an incurable cancer of the lungs primarily associated with occupational asbestos exposure. MPM has a limited median survival time <12 months) and long latency period, which can reach up to 70 years in some cases . Treatme months aIn the clinic several photosensitizers have been used in the treatment of mesothelioma, based around the tetrapyrrole scaffold: hematoporphyrin derivatives, Photofrin for example, Foscan, and HPPH ,15,16. TOver the last 30 years, the most common PS used to treat mesothelioma in the clinic is Photofrin. Photofrin is typically given at a dose of 2 mg/kg, 24 h preoperatively and the PS is excited intraoperatively using 630 nm laser light. With the development of modern surgical treatments for MPM the addition of photodynamic therapy as multi modal treatment became an interesting option, both Photofrin and Foscan have been used to develop safe intraoperative PDT treatments ,20. Thes2 groups 5 to 6, 6 to 7 providing an additional; 75 Å2 of SASA, and 68 Å3 of volume per one carbon extension. These changes in carbon chain length give SlogP values of 13.9, 15.4, and 17 for molecules C5SHU, C6SHU, and C7SHU, respectively, highlighting the increase in lipophilicity as the truncated fatty acids get longer. Considering previous experimental measurements relating SASA to hydrophobic interaction the increase of SASA of 75 Å2 per one carbon extension of the hydrocarbon chain increases the potential for hydrophobic interactions by 20 kJ mol−1 [The porphyrin-based PS in this work were designed with truncated fatty acid chains conjugated to the phenyl rings in the 5, 10, 15 and 20 carbon positions around the porphyrin core. The fatty acid truncations are connected to the porphyrin’s phenyl rings via an ether linkage four, five, and six carbons from the carbonyl carbon of a straight chain fatty acid giving porphyrin C5SHU, C6SHU, and C7SHU, respectively. The fatty acid-type conjugation is designed to allow cell permeability through diffusion, low-density lipoprotein (LDL) transport or fatty acid specific transport protein activity ,25. SelekJ mol−1 ,27,28,29kJ mol−1 ,31,32.The aim of this initial report was to assess the PDT effect of a panel of three porphyrin derivatives in the human MSTO-211H and NCI-H28 mesothelioma cell lines. These cell lines were chosen as models due to their varying characteristics. MSTO-211H is of biphasic origin, consisting of epithelioid and sarcomatoid cell types. NCI-H28 is from a stage 4 mesothelioma consisting of purely epithelioid cells ,34,35. TCommercial reagents. Cell death staining reagents were all acquired in the FITC Annexin V Apoptosis Detection Kit with PI . WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium) reagent was part of a pre-made solution in the Cell Counting Kit-8 .−2–1 × 10−1 mM). For each porphyrin aliquots of working solutions were added to a quartz cuvette containing DMSO (2 mL), the absorption spectra was recorded from 400-800 nm after each addition. Absorbance values at the λmax of the Soret band and four Q-bands were plotted as a function of PS concentration for use in the calculation of molar absorption coefficient.UV/vis absorbance coefficient measurement in DMSO. Volumetric solutions of porphyrins 2–7 (0.5–2 mM) in DMSO were diluted to give working solutions  fetal bovine serum (Gibco) and 1% (v/v) Penicillin and Streptomycin . The cell culture flasks were incubated at 37 °C and maintained in a 5% CO2:95% air humidified atmosphere. All cell lines passed routine mycoplasma testing using the MycoAlertTM Mycoplasma Detection Kit .The human mesothelioma cell lines, MSTO-211H and NCI-H28, and the non-cancerous mesothelial cell line, MET-5a  were grown and routinely maintained in RPMI 1640 media . The complete growing media was supplemented with 2 mM L-glutamine (Gibco), 10% (v/v) exclusion was used to count viable cells before seeded into 96-well plates (Nunc) at a density of 5 × 103 cells per well in 200 μL of complete RPMI. Cell culture plates were seeded approximately 24 h before PDT. Media was aspirated and replaced with equal volumes of the respective PS treatment. For the purpose of background subtraction, PS treatments were also added to wells without cells. Following 3 h of incubation , plates were irradiated for 15 min on a 15 W light box . Experiments performed parallel to dark-treated control plates.Cells were detached using a trypsin/EDTA solution (Lonza). Trypan blue (0.4% 2:95% air), 10 μL of WST-8 was added to each cell-containing well of the 96-well plates. The plates were incubated for 3 h  and then analyzed at 450 nm using a Clariostar Plate Reader . Corrected absorbance was calculated in each case by subtraction of the media only controls. Finally, percentage cell viability was expressed relative to untreated control values.Cell viability was assessed by the colorimetric WST-8 assay. Following 24 h of incubation  at a density of 2 × 10g)  and resuspended in Annexin V Binding Buffer. Following a second centrifugation, Annexin V Binding Buffer was discarded, and cell pellets were resuspended in the residual volume. Cells were stained with Annexin V-FITC (5 μL) and PI (10 μL) and incubated at room temperature for 15 min protected from light. 200 µL of Annexin V Binding Buffer was added prior to immediate analysis using a BD FACSCalibur  and data from 10,000 events was collected. Flow cytometry results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences). Quadrants consisted of Viable (Annexin V-FITC−/PI−), early stage apoptotic (Annexin V-FITC+/PI−) and late-stage apoptotic/necrotic (Annexin V-FITC+/PI+) populations.Annexin V-FITC/PI staining and cell death analysis was performed at both 24- and 48 h time points following PDT  of three independent experiments. Statistical analysis was performed using a two-way Analysis of Variance (ANOVA) with Bonferroni post hoc tests in GraphPad Prism . Data points where Cells were harvested in accordance with methods previously described and seeded into 24-well plates  at a density of 25,000 treated with porphyrin working solutions made up from the porphyrin PS stock solutions and incubated for 24 h. The cells were visualized using a Cytation 5  high content microscopy platform at 20× magnification. The SSIPTools suite of software, made available by the Hunter group, was used to describe the solvation properties of the fatty acid chain sections ,31. A raThree PS, porphyrins conjugated to truncated fatty acid chains were synthesized from 5,10,15,20-Tetrakis(3-hydroxyphenyl)-21H,23H-porphyrin, 1 . Bromo-eThe fluorescent behavior of the porphyrin PS arises from the 18-electron aromatic π system modulated by the additional carbon-carbon double bonds in the carbon 2 to 3 and carbon 12 to 13 positions around the ring. This fluorescence provides the opportunity to observe the uptake of the PS in the MSTO-211H, NCI-H28, and Met-5a cell lines being used in this study using a fluorescence microscopy approach. Treatment of a cell line followed by a 24 h incubation and visualization using a high-content microscopy platform allowed detection of the fluorescent PS in the chosen cell line. The C7SHU PS can be seen to localize in the cytosolic region of the MSTO-211H cell line in For these experiments, a high-content approach was taken for the initial assessment of photosensitizer activity, allowing the irradiation of multiple PS conditions and multiple cell lines simultaneously using a white light source. Cell viability was assessed using the WST-8 assay quantifying viable cells through the metabolism of a water-soluble tetrazolium salt to a formazan dye. This was performed to establish the initial PDT effect of each PS in differing cell lines. Individually the MSTO-211H, NCI-H28, and Met-5a cell lines were incubated with various PS concentrations  in light and dark treatment conditions at both 24- and 48 h intervals post treatment. This was performed in the presence of untreated and vehicle controls, with cell viability being presented as a percentage of the untreated cells.The MSTO-211H cell line demonstrated a sensitivity to all three PS C5SHU, C6SHU and C7SHU in both the 24 h and 48 h treatments . The C5SThe NCI-H28 cell line displayed some sensitivity to the C5SHU and C6SHU porphyrins, which also appeared to potentiate at the 48-h time point . C5SHU dThe non-cancerous cell line, Met-5a demonstrated a similar C5SHU sensitivity to NCI-H28. Cell viability in light treatments plateaued to approximately 28% 24 h after the 5 μg/mL treatment. This was potentiated at the 48 h time point (approximately 11% after the 5 μg/mL treatment). A similar PDT effect was observed with C7SHU treatment, but to a lower potency. A reduction to 66 and 30% viability was observed in 5 μg/mL light treatments at 24- and 48-h time points, respectively .50).In summary, the WST-8 cell viability permitted the observation of light toxicity in all three cell lines  with all three PS , but further confirmation is needed. Therefore, the mode of cell death was assessed by flow cytometry using the Annexin V-FITC/PI assay. Both the MSTO-211H and NCI-H28 cell lines were assessed 24- and 48 h following PDT. Viable, early stage apoptotic and late-stage apoptotic/necrotic cell populations were determined by FITC-labelled Annexin V and PI staining. Cell viability can provide an initial measure of dose response. 50 values of 7.8 and 7.7 μg/mL, respectively 24 h time point) (C5SHU demonstrated a potent PDT effect in both NCI-H28 and MSTO-211H cells (ECe point) . In lighe point) . In darke point) . The Mete point) . Negligie point) .50 value of >20 μg/mL) . At the 10 μg/mL treatment, NCI-H28 displayed a high proportion of viable cells (75% at the 48 h time point)  (The C6SHU PS did not induce a potent PDT effect in NCI-28 (IC0 μg/mL) . A subst14 μg/mL ; Table 114 μg/mL  was lesse point) . The MSTThe porphyrins C5SHU, C6SHU, and C7SHU were synthesized and then characterized using spectroscopic and mass-spec techniques to confirm their identity and purity , this was the most dose-sensitive cell line/PS combination in light treatments. The NCI-H28 cell line gave the most archetypal response to C5SHU, demonstrating a light-specific induction of cell death; in dark conditions, the proportion of viable cells (approximately 95%), was not impacted by C5SHU concentration.The ability of all three photosensitizers  to induce light-mediated cytotoxicity and mode of cell death induced was evaluated via a combination of methods. The WST-8 cell viability permitted the observation of light toxicity in all three cell lines  with all three PS . The Annexin V-FITC/PI assay confirmed and provided further evidence of specific cell death populations in the light conditions. The five-carbon porphyrin derivative (C5SHU) was shown to be the most effective PS as demonstrated by the Annexin V-FITC/PI assay. The MSTO-211H displayed significant dark toxicity at the 48 h 15 and 20 μg/mL treatments  could be produced for an expanded group of PS. The QSARs produced will give a high-quality understanding of which of the available PS is most efficacious and the direction in which the development of PS and use in treatment can take.The C5SHU PS demonstrated the highest efficacy in all the cell lines investigated, in the stacking studies this PS demonstrated the least evidence of stacking in aqueous solutions. If we combine this observation with our initial EC50 values, it is possible to differentiate between the PS activity of all the PS C5SHU, C6SHU, and C7SHU in an individual cell line. The same EC50 values provide a difference in activity of an individual PS in the three cell lines each with different origins and phenotypic characteristics. This tells us that the C5SHU molecule with the shortest chain length and lowest hydrophobic surface area is the most efficacious PS, and it targets the MSTO-211H cells most effectively. This provides a range of parameters that we can correlate with activity in future work to better synthesize PS and target these PS to MPM tumor cells as part of a treatment.An initial comparison of the molecular description of the three PS molecules has been made in terms of molecular volume, fatty acid chain length, surface area, and hydrophobicity. The physical behavior of the PS has been initially probed using a measure of aggregation induced by stacking of the PS molecules. Considering the measure of PS activity in the cell lines used, with the initial EC