Ionizing radiation is known to be DNA damaging and mutagenic, however less is known about which mutational footprints result from exposures of human cells to different types of radiation. We were interested in the mutagenic effects of particle radiation exposures on genomes of various human cell types, in order to gauge the genotoxic risks of galactic cosmic radiation, and of certain types of tumor radiotherapy. To this end, we exposed cultured cell lines from the human blood, breast and lung to fractionated proton and alpha particle (helium nuclei) beams at doses sufficient to considerably affect cell viability. Whole-genome sequencing revealed that mutation rates were not overall markedly increased upon proton and alpha exposures. However, there were modest changes in mutation spectra and distributions, such as the increases in clustered mutations and of certain types of indels and structural variants. The spectrum of mutagenic effects of particle beams may be cell-type and/or genetic background specific. Overall, the mutational effects of repeated exposures to proton and alpha radiation on human cells in culture appear subtle, however further work is warranted to understand effects of long-term exposures on various human tissues. Spaceflight is usually defined as crossing the Kármán separation line i.e. 80–100 km altitude above the sea level4. Above this boundary, the environment dramatically changes, notably in terms of microgravity, temperature, and radiation5. Various pathological consequences on different human organ systems arise from travelling in space, including cardiovascular, musculoskeletal, and immune changes6. However, a better understanding of space travel impact at the cellular and sub-cellular levels is needed, for instance how the genome integrity is affected.Humans have a keen interest in travelling in space, from deep space exploration to extraterrestrial colonization. In addition, the space environment was also considered for biomedical research purposes, for example the “tumors in space” experiment aims to study the effects of microgravity and exposure to space radiation on tumor organoids8. A study of the mutagenic mechanisms of different cosmic radiation types upon the human genome would be helpful to understand the basis of the increased risk of cancer or of reproductive harm resulting from space travel.Important effects of space travel are exerted by the various types of radiation to which tissues are exposed outside the Earth’s atmosphere and magnetosphere. Those include the solar radiation, the radioactive environment of the planet, and galactic cosmic radiation , which originates mainly from the Sun or from outside of the Solar system but within the Milky Way galaxy . The GCR encompasses photon-based radiation as well as particle radiations, including neutrons and charged particles; among the latter, protons and helium nuclei are the most abundant. Cosmic radiation has emerged as an issue of concern, since exposure to radiation may increase cancer risk in astronauts9. However, there was a radiation dose-dependent increase of fusion driver events, as well as more generally of certain types of structural variants (rearrangements)10. Consistent with this mutational impact that can generate somatic driver mutations, ionizing radiation (IR)-generating incidents  also increased cancer risk proportionally to the dose of exposure12.Photons are the better-studied component of the GCR, since they consist of gamma rays and X-rays, whose biological impact was studied in other contexts. For instance, gamma rays were released during the Chernobyl nuclear power plant disaster. The genomic profiles of thyroid carcinomas in irradiated children post-Chernobyl contained similar driver mutations and gene fusions as in non-radiation-associated thyroid tumors14. For instance, this entails a particular signature of indels (the PCAWG Signature ID8) characterized by larger deletions (≥ 5-bp) without flanking micro-homology, previously linked to double-strand break repair15. Consistently with their mutagenic effect, medical use of X-rays has been reported to increase cancer risk16.In addition to nuclear incidents, radiation exposures can result from medical uses. Commonly they involve X-rays, used for both diagnostic purposes, and tumor radiotherapy. The mutational footprint of therapeutic X-ray exposure was examined in the genomes of radiation-associated second malignancies and of post-treatment metastatic tumors, reporting an increase of small and large deletions, and also certain mutational signatures17. This suggests mutagenic impacts of various radiation types, however research on the specific mutational footprints of charged particles on human genome stability has been limited.Regardless of the source of the radiation—either the cosmos, nuclear accidents or medical uses—it is not clear if IR consisting of charged particles, such as protons or alpha beams, would have similar mutagenic effects to more commonly studied photons (X-rays and gamma-rays). Generally, all types of IR, including gamma-rays, X-rays, energetic charged particles [protons, alpha particles (helium nuclei) and heavier ions} and neutrons have been classified as human carcinogens by the World Health Organization (WHO)18, and appear promising with respect to toxicity profiles22. Proton therapy may reduce the health risks (compared to X-rays) of secondary malignancies23, and of severe lymphopenia25. The above provides motivation for studying the mutagenic effects of protons, and of helium nuclei  radiation, which have the potential to result in long term health effects in exposed individuals.Mutagenesis induced by particulate radiation is of interest because during transit beyond low Earth orbit, every cell nucleus within an astronaut’s body is traversed, on average, by an energetic proton every few days, and by a helium nucleus once every few weeks. The mutational effect of proton radiation is of interest also for medical reasons, as protons are increasingly adopted in the clinic to treat cancer26, mathematical constructs that describe the differential frequencies of mutation types across many genomes. Such signatures are commonly based on trinucleotide spectra of single nucleotide variants (SNVs), however they can also be based on small insertions and deletions (indels), structural variants (rearrangements), or mutation clusters28. SNV and indel mutational signatures in human cancer are well characterized and organized in comprehensive catalogues29, including signatures in tumors pre-treated with photon IR30.The activity of mutational processes in DNA—which may arise from both endogenous factors such as DNA repair deficiencies and exogenous factors such as tobacco smoking—can be captured by mutational signatures31, and also in cultured cells from healthy human tissues33. Additionally, signatures of IR were studied in exposed mice35 and in the worm C. elegans36; both reported SNV signatures enriched in C>T transitions15.In addition, some genomic studies have reported mutational signatures on experimental models, including experimentally-induced mutational signatures of (non-ionizing) UV radiation in human cell linesTo increase our understanding of the potential impact of particle radiation encountered during space travel on human genome integrity, it is important to systematically examine the DNA mutations arising from the exposure in cells originating from different human tissues. To this end, we analyzed the whole genome sequences of three human cancer cell lines, A549 , HAP1  and MCF7 . Those cell lines were irradiated repeatedly with two types of particle radiations—proton or helium fluxes—in order to identify the mutational footprints induced and their potential cell type-specificity.The dosage of radiation was determined experimentally in order to achieve between 40 and 50% lethality , independently across the two types of particle beams Fig. a,b.FigurThis led to a dose of 0.5 Gy for helium ions and a dose of 1 Gy for protons. After an expansion of the three cell lines collected , the cells were irradiated using the 5.5 MV Singletron accelerator at the Radiological Research Accelerator Facility , indels, and structural variants (SVs) in all three cell lines assayed, HAP1, A549 and MCF7, and regardless of the radiation type, protons or helium ions. As a control, we considered clones generated from sham-irradiated  cells for all three cell lines; these clones also accumulated certain numbers of SNVs, small indels and SVs, consistent with them undergoing cell divisions and also potentially being exposed to various stresses during handling. The global number of observed mutation events (SNVs and indels) does not markedly differ between conditions Fig. a, sugges37. The second pattern is one peak at 0.5 which suggests that one cell was collected and that mutations were found within a diploid segment; this was common for the A549 cells. Finally, a third pattern, a peak at 1, was observed for the HAP1 cell line, as expected due to the unusual genome-wide haploid state of that cell line , or the proportion of sequencing reads that contain a particular variant, is a proxy for the proportion of cells in the sequenced population that harbor the mutation. In our experiments, we aimed to select a single treated cell and expand it into a clone, thus the VAFs observed should be centered at 0.5 for heterozygous diploid genome segments. In practice, three different types of VAF patterns were observed  than in the two other cell lines after exposure to either particle as well as in the MCF7 untreated cells Fig. a. This m14 which were treated with photon radiation, typically X-rays. The globally most-mutated clone is a single proton-irradiated sample of the HAP1 cell line, which contains 4695 SNVs . Moreover, an abundance of C>A mutations was observed in recent mutation-accumulation experiments on human cell lines similar to ours39, plausibly due to exposure to atmospheric oxygen during cell culture conditions. Control (unirradiated) clones also had high C>A exposures.Upon classifying SNVs into 6 different categories, tallying them DNA strand-symmetrically, i.e., C>A, C>G, C>T, T>A, T>C, T>G, the most prevalent class of SNVs in all cell lines/clones was C>A Fig. b. One im15. These transition mutations were particularly abundant in the MCF7 genomes, suggesting a mutational process specific to that cell line.Additionally, we observed considerable numbers of the C>T and T>C transition mutation types Fig. b, which To further investigate mutational processes, the SNV mutations were classified into 96 categories, considering both the observed class of mutation and the trinucleotide context , in order to infer the 96-component mutation spectrum of SNVs for each clone  155 cell line genomes treated with various mutagens, largely chemicals, in which a specific SNV signature was able to be identified as well as gamma-irradiated genomes (with no identifiable signature)31, and (ii) an additional set of 38 cell line genomes generated from CRISPR-Cas9 gene knockouts of 9 DNA repair genes reported to exhibit mutagenesis upon knockout39. By a joint analysis of this mutation accumulation data, we identified 10 independent mutational signatures ; the signature activities were high in the experimental exposure to certain PAHs (polycyclic aromatic hydrocarbons)31  and gamma radiation . This signature dominated by T>C and C>T transitions, had high exposures in genomes of cell lines previously treated with methylating agentsMLH1, MSH2 and MSH6 gene knockouts39, resembling a signature of a defective DNA mismatch repair. SBS96G was assigned to the known mutational signatures associated with ultraviolet light exposure , and had high exposure in the simulated solar  radiation treatment of a cell line31 . SBS96H is a mix of SBS4 (tobacco smoking-associated) and SBS24 (aflatoxin-associated) and was seen in cells exposed to BPDE and PhIP chemicals in previous data31, thus reflecting mutagenesis due to bulky adducts in DNA. These signatures, SBS96E–H, tend to generate few mutations in our samples; however, the latter, signature H, is somewhat more abundant in both treated and untreated samples , C (possible DNA repair defect and/or artifacts), and I . These were found at various levels in the three cell lines and in the majority of the samples of each line and were also seen in the non-irradiated control samples Fig. b.versus helium treatments  in our data Fig. a,b. WhilIn addition to the common C>A signature of oxidative stress SBS96B mentioned above, we noted some evidence for radiation treatment association in our cell line experiments with two other signatures. Firstly, the T>A transversion signature SBS96E Fig. a,b, assoThe mechanisms that underlie signatures SBS96B, E and H have a connecting thread: these mutational signatures are thought to result from large, replication-blocking DNA lesions. Thus, it appears that proton/helium radiation effect on DNA can mimic the mutational footprint of mutagens generating bulky adducts. Speculatively, such a DNA lesion might be generated by the radiation causing intrastrand nucleotide cross-links, or by cross-linking DNA with proteins. Since these SNV mutational signatures were found in both protons and alpha radiation treatment groups , they do appear broadly relevant to various particle radiation types present in GCR. Moreover, they would be expected to result from other sources of particle radiation  in the susceptible cell types and/or genetic backgrounds.39 in our signature extraction dataset, as described above, does not have undue influence on the NMF point mutation signatures identified here . In brief, the indels are separated into 1-bp or longer indels, insertions or deletions, at DNA repeats or not, and finally deletions with micro-homology are counted separately. These classes can further be divided into subclasses based on the gained/lost nucleotide or based on the alteration length. Each observed indel is assigned to one category, thus providing the mutational spectrum of indels for each cell line and each condition   so ID83A and ID83C do not appear to be a general signature of particle radiation exposure. The activities of the indel signatures correlated with activities of some SNV signatures across our cohort . The spectrum of ID83B consists of various lengths (1–5 bp with similar frequency) insertions and deletions outside of DNA repeats, as well as a notable component of deletions with short flanking microhomology . The activities of ID83B in prior cell line WGS data correspond to exposures to UV radiation and to propylene oxide and to furan , this suggests a universal indel mutational footprint of small deletions caused by particle-based radiation in human cells. However, due to a small number of cell lines studied herein, we do not rule out that different cell types and/or genetic background might be differentially affected by different radiation types generating indel changes.Consistent with trends toward association with radiation treatments in our study, certain features of ID83B and ID83D spectra are similar to the indel mutational signature induced by ionizing photon radiation in clonal organoids from mouse and human cellsLarge structural variants  were grouped into four broad categories: large deletion (DEL), large insertions (INS), tandem duplications (DUP) and inversions (INV). No particular SV category has been found to be the most prevalent across the cell lines and across the cell lines/conditions Fig. .Figure 5The rarest event type—insertions—is similarly observed across the cell lines and clones; their low frequency may however stem from technical difficulties in calling insertion variants from short-read WGS, resulting in many undetected variants. More generally, in our experiments, we observed globally a low number of SVs of any type, suggesting that the impact of the proton and alpha particle treatment on rearrangement rates in human cells may not be remarkable, at least in the treatment regimen applied here. The helium and proton-treated clones from all cell lines have higher numbers of large deletion SVs compared to the untreated clones . As reported for IR-associated second malignanciesnes Fig. . Overall48. We were interested to know if there is an enrichment of radiation-associated mutation rates in regions with a specific RT.DNA replication timing (RT) is correlated with regional variability in somatic mutation rates in human tissues. There is an increased mutation rate in late-replicating heterochromatic regions of the genome, because DNA repair mechanisms preferentially protect the early-replicating euchromatic, gene-rich regionsOverall SNV mutation rates were, as expected, associated with RT in all 3 cell lines  mutation clusters. In the A549 cell line, 102 mutations were clustered in control clones, 171 in helium-irradiated clones and 143 in proton-irradiated samples. In the HAP1 cell lines, a total of 88 mutations were clustered in control clones, 178 in helium-irradiated clones and 255 in proton-irradiated samples. Finally, in the MCF7 cell line, we detected 130 mutations in the control clone, 325 clustered mutations in the helium-irradiated clones and 370 in the proton-irradiated clones  in all cell lines, indicating that alpha and proton radiation generates mutation clusters in various human cell types Fig. a. This pnes Fig. a.We next asked if the clustered mutations, depending on the cell line and the radiation type, were found at closer or longer intermutational distances. In HAP1 cells, clustered mutations were spaced significantly further apart for both helium and proton radiations compared to the control clones, with the helium group displaying the largest inter-mutational distance Fig. b. The trkataegis-like phenomenon49 and the former to an omikli-like phenomenon51. We applied a graph-theory approach to quantify this  clusters, the latter case corresponding to a his see “”. Considhis see “c. Of notons Fig. c.Finally, we were interested in the mutational spectrum of the clustered SNV mutations, which can identify or rule out a mechanism underlying the clusters. Here, because of the relatively modest number of SNVs, we considered the 6 mutation type-spectrum of clusters  also in unirradiated cells, and that radiation may occasionally generate multi event, In this study, we analyzed the genomes of three human cancer cell lines irradiated with two different components of GCR—proton and helium exposure—in order to characterize the spectra of DNA alterations that GCR would produce in different human tissues. Overall burden of SNV mutations, indels and SV events was not markedly different between treated and untreated cells. This suggests that particle radiation exposure—at least in the regime applied in our experiments—is not grossly mutagenic (see below for further considerations).14, for indels we observed an enrichment of deletions compared to insertions, and this was even more the case for large (SV) events 55. Mechanisms would need to be clarified with additional experiments in future work.We found that particle radiation generates point mutations that are more often clustered. This is in line with recent work suggesting clustered mutations to be more specific indicators of certain types of mutagenic processes in human50, suggesting, speculatively, that particle radiation may generate lesions resembling bulky adducts.We were also interested in the SNV trinucleotide mutational signatures that helium and proton exposures can generate, and whether known biological mechanisms can be linked to those footprints. While most observed SNV signatures were consistent across all treated and untreated samples, e.g., reactive oxygen species mutagenesis was noted universally, some samples exhibited signatures consistent with MMR deficiency. This MMR deficiency was however unlikely to have resulted from, or have been selected by irradiation, because we detected it also in a control sample. Interestingly, we observed a modest enrichment of certain SNV signatures in both irradiation conditions: a C>A signature linked with oxidative stress, and an T>A rich signature, resembling previous signatures of aristolochic acid and some chemotherapy treatments45; additional data appears required to gain statistical power to establish to what extent the indel signatures indeed differ between photons and particle radiations, providing more insight into mechanisms.Similar to SNVs, some indel signatures were radiation-associated, being consistently enriched (with modest effects) exposed samples, thus suggesting a likely general effect over tissues for indel-generating mechanisms. The indel signatures bore certain similarities with indel signatures previously reported for photon IR31 (presumably generated by the UV radiation) were observed at low intensities in our irradiated samples, likely stemming from imprecisions during statistical analysis. This is consistent with prior knowledge that ionizing cosmic radiation affects the DNA differently than the non-ionizing UV radiation56.Regarding non-ionizing radiation, the solar radiation-generated SNV signatures identified previouslyOur study also has several limitations. For instance, some components of GCR were not considered in this study, in particular the heavy atom nuclei (often referred to as “HZE ions”), highly energetic particles likely originating from supernova explosions. Despite their small proportion in cosmic radiation, their biological impact might be large, and their mutagenic effects on genome stability remain to be studied.Next, we considered only a single dose of radiation that resulted in a ~ 40% to 50% reduction of cell viability assessed by the colony formation assay, and a fractionation regime of four total fractions (with each fraction given every 4 days) exposures of this dose. The dose studied here had rather strong biological effects on cell viability and does not correspond to what would be encountered in space travel (where any vessel would need to be shielded to prevent such exposures that would have very deleterious acute effects on the organism). These exposures were chosen as an experimental setup, with the rationale that doses with notable effects on cell viability would probably be well sufficient to observe the mutagenic effects of particle radiation, if any. Since a gross increase in mutation rates was not observed, we infer that particle beam exposures are not highly mutagenic. However, we cannot rule out the (less parsimonious) scenario where lower doses of particle radiation than those employed here might be more mutagenic e.g., by failing to trigger cell cycle checkpoints, thus ‘slipping under the radar’ of the mechanisms protecting genome integrity and introducing mutations.Moreover, given that for various mutation patterns we observed responses that appear specific to one cell line, this suggests there may be tissue-specific and/or genetic background specific responses (these two scenarios cannot be distinguished from our data). Additional experiments on different cell lines or other experimental models would be required to ascertain tissue-specific responses to various radiation types. Furthermore, this study was performed on cancer cell lines, and mutational responses might be different in healthy, noncancerous cells, which remains to be investigated in future work.In conclusion, our study suggests that particle radiation components of the galactic cosmic radiation are not overly mutagenic to human cells, with very modest effects on point mutation spectra, and with some effects on SV distributions, indels and on clustered mutation spectra. However, the repeated exposure regime that we employed is different from longer-term, chronic exposures to GCR expected e.g. during space flight. Even modest increases in mutation rates under chronic GCR exposures might have detrimental effects on cancer risk, and possibly neurodegeneration and reproductive health and on genetic disease incidence in the progeny. Therefore, we highlight the necessity of further experimental work on cell and animal models, using longer-term exposures to various components of galactic cosmic radiation to characterize their effects on the stability of the genome.We used three human cell lines: A549, HAP1 and MCF7. The lung adenocarcinoma cell line A549 (CCL-185) and the breast adenocarcinoma cell line MCF7 (HTB-22) were purchased from ATCC . The near-haploid cell line derived from the KBM-7 cell line HAP1 (C859) was purchased from Horizon . All cell lines were cultured and maintained according to recommended protocols. All the cell lines were authenticated by STR profiling by the corresponding repositories.2 humidified incubator; the A549 cells were grown in Eagle’s Minimum Essential Medium (MEM), MCF7 in Dulbecco modified MEM, and HAP-1 cells in Iscove’s modified Dulbecco's medium (IMDM). All the media were supplemented with 10% heat-inactivated  fetal bovine serum (FBS), and 100 U/ml penicillin and 100 mg/ml streptomycin .All cells were grown at 37 °C in a 5% CO57 pilot experiment was conducted to assess the dose that resulted in 40–50% lethality  to 11 MeV helium ions (LET = 65 keV/µm) or 5.4 MeV protons (LET = 10 keV/µm), which was 0.5 and 1 Gy, respectively  and, finally, to calibrate the online beam monitor in terms of the absorbed dose delivered to the sample. In the second step, the dosimetry wheel was replaced with the sample-carrying wheel that can accommodate up to 20 custom made dishes. The dishes were manufactured by gluing 6 µm thick mylar foil over metal rings having a diameter of 5 cm. The mylar foil serves as the bottom of the dish on which the cells are attached, allowing thus the ions to penetrate through and reach the cells without losing much of their energy. The in-house developed software drives the stepper motor and controls the movement of sample dishes over the beam. A desired dose was delivered to the sample by exposing it to the beam until the appropriate number of monitor counts was reached according to dosimetry measurements performed during the first part of the irradiation protocol.The 5.5 MV Singletron accelerator at the Radiological Research Accelerator Facility (RARAF) served as a source of energetic protons and alpha particles. The accelerator was operated at the maximum terminal voltage generating beams with nominal energies of 5.5 MeV and 11 MeV for protons and alphas, respectively. Cells were irradiated at the so called “track segment” irradiation platform whose name indicates the traversal of thin samples  by a short segment of the ion’s trajectory resulting in a small variation of the linear energy transfer (LET) throughout the sample. It is valid therefore to assume that radiation doses at the track segment platform are delivered by mono-LET beams. The LET values of the applied beams were 10 keV/µm for protons and 65 keV/µm for alphas. Detailed description of the track segment irradiation platform and its operational principles can be found elsewhereThe obtained survival curves were fitted with the linear quadratic (LQ) model. Doses of 1 Gy and 0.5 Gy were selected for investigating the mutational signatures of protons and alpha particles, respectively. According to the LQ fits, the selected doses resulted in preservation of between 50 and 60% clonogenic capacity for all cell lines. Four fractions of the same dose were delivered to the cells every 96 h to amplify the number of mutations in surviving cells. Between the fractions, the cells were not removed from the mylar dishes. To prevent the cells from overgrowing the dish size before the end of the irradiations, the initial number of plated cells was < 1000.58. GATK Base Quality Score Recalibration was applied to adjust quality scores assigned during the sequencing process59. The average coverage of the various samples was 18.5X to 44.8X.Total genomic DNA was extracted from pelleted cells using PureLink™ Genomic DNA Mini Kit . DNA was sequenced on NovaSeq 6000, 150 nt paired-end mode. Reads were aligned to the reference human genome Hg38 (GRCh38.d1.vd1) using BWA v0.7.1760. Strelka2 was launched in the joint genotyping mode: briefly, mutations were called for each clone individually (in one batch), but genotyped jointly across all clones belonging to a given cell line. Mutations were then annotated using annovar, function table_annovar.pl61 with the gnomAD genome database v2.1.1 allele frequencies62. This allowed us to identify mutations shared by all clones (which were acquired prior to treatment) as well as discrete mutations acquired by only one clone during treatment. To arrive at the final set of mutations used in our analyses, a number of filters were applied: (1) variants passed both sample and call filters from Strelka2, (2) variants were found in uniquely mapping region of the genome (based on the Umap k50 mappability tracks), (3) variants were either not present in the gnomAD database or found at frequencies lower than 0.1%, (4) variants were genotyped in only one of the cell line clones, (5) variants were not multiallelic and (6) there were 2 or more variant supporting reads in the genotyped clone. For small insertions and deletions, we also imposed a genotype quality equal or higher than 10 and removed variants where more than two other clones have one or more variant supporting reads and variants where more than one other clone has two or more supporting reads, in order to remove putatively germline mutations only genotyped in one clone by Strelka2.Strelka2 variant calling algorithm v2.9.10 was used to detect point mutations and small insertions and deletions63, in the same manner we used Strelka2. For inversions, Manta returns a list of breakpoints that describe the inversions it detected. To transform those breakpoints into inversions, we applied the convertInversion.py python script provided by the Manta developers (https://github.com/Illumina/manta). Filters 1–5 described above were also used to filter the structural variant calls; further, we required variants to have 2 or more supporting split-reads in the genotyped clone, with no requirement for the number of supporting spanning reads.To call structural variants, i.e., large insertions and deletions, duplications and inversions, we used the Manta v1.6.0 tool in the joint calling modeomikli (“mutation fog”)51. To consider large, multi-mutation clusters resembling a kataegis (mutation shower) event64, we used an approach based on graph theory. Roughly, we consider each mutation to be a node of the graph, and edges connect two nodes if the distance between corresponding mutations is lower than 1 kb. Then, we computed the components of the graph, that corresponds to a connected sub-graph, and subsequently the size of the components, i.e., how many nodes or mutations are present in the component.Point mutations were considered to be clustered if they appeared at positions with a genomic distance lower than 1 kb; such mutations would be classified as . study39, which analysed 38 clones generated by CRISPR-Cas9 knockouts of 9 DNA repair/replicative pathway genes  which generated mutational signatures in human induced pluripotent stem cells . We then pooled the data with the mutations reported in the Kucab et al. study31, which reported 153 clones  yielding a mutational signature , as well as 2 clones treated with gamma radiation (with no identified signature).This pooled dataset of 214 individual clones was used for SNV and indel signature extraction. In order to extract SNV signatures generated by our radiation treatments, we applied SigProfiler ExtractorR v1.1.16 algorithm15 with default settings on the set of detected mutations, classified into the 96 SNV categories. The suggested solution contained 10 extracted signatures with high stability and low reconstruction error. Extracted signature spectra were compared and assigned to a known PCAWG “SBS” signature set15 (COSMIC_v3.3.1_SBS_GRCh38 from https://cancer.sanger.ac.uk/signatures/downloads/) if the cosine similarity between the two mutational profiles was at least 0.85. Further, all signatures were decomposed into the same set of PCAWG signatures using the python SigProfiler Assignment v0.0.24 tool (https://github.com/AlexandrovLab/SigProfilerAssignment), function decompose_fit.We pooled our samples (21 clones) with two already published datasets in order to help NMF converge and in order to compare our datasets to signatures of DNA damage and of various mutagens. We first pooled our data with the mutations reported in the Zou et al. and Kucab et al. samples influences the extracted SNV signature spectra and their respective exposures in our dataset, we implemented a subsampling approach whereby half of each external dataset is randomly removed thrice from the original dataset and the NMF algorithm is run on the remaining 117 samples , once for every subsampled set. Then, mutational signature spectra and exposures in our samples are compared to the original set.In order to determine the extent to which the addition of Zou et al.15. Then, the SigProfiler ExtractorR v1.1.16 tool was launched on the same set of 214 samples from Kucab et al., Zou et al., and our experiments, using the generated mutation matrices. We considered the optimal solution given by the tool, resulting in a total set of 4 extracted indel signatures with high stability and low reconstruction error. As for the SNV signatures, we compared and decomposed our extracted spectra into the known PCAWG “ID” signature set15 (COSMIC_v3.3_ID_GRCh37.txt from https://cancer.sanger.ac.uk/signatures/downloads/).For indel signatures, we used the SigProfiler Matrix Generator for R v1.2.4 tool to correctly classify the observed indels into one of the 83 categories defined in Alexandrov et al28. To detect significant association of mutations in specific genomic regions, we fitted a negative binomial regression using the glm.nb function from the MASS R package, on the counts of mutations per bins, controlling for the 3-nucleotide context and type of mutation in the case of SNV, as in Supek and Lehner28. The regional enrichment analysis source code is implemented using Nextflow65 and is available on the GitHub platform .We performed a regional enrichment analysis in order to estimate if the mutations detected in the irradiated clones were located in regions with a specific epigenetic pattern compared to the mutations detected in the non-treated clones. We tested three genomic features: the replication timing, the DNA repair histone mark H3K36me3 and the gene expression levels based on RNA-seq data. Replication timing bins, genomic bins extracted from chromatin mark H3K36me3, and regions based on variable gene expression were computed as in Supek and Lehner66 R package v1.32 was used to read the VCF files. The R packages BSgenome.Hsapiens.UCSC.hg38 v1.4.1 and BSgenome.Hsapiens.UCSC.hg19 v1.4.0 were used to load the human genome references inside R. MutationalPatterns67 R package v1.12 was used to generate the trinucleotide contexts. Ggraph v2.0.5 and Igraph v1.2.6 R packages were used to identify the cluster components. The statistical tests used to derive the p-values within some figures are described in the figure legends.All the statistical analyses were performed using R software version 3.6.0. VariantAnnotation68, where p = (r + 1)/(n + 1), r is the number of randomised differences > = or < = than the real mean difference and n is the total number of iterations . Then, all p-values from all comparisons across SNV signatures, ID signatures and mutation burdens were corrected (separately for each category) to account for the multiple comparisons problem using the p.adjust function from R core package stats v3.6.0, method “BH”/”fdr”69. Comparisons with an original p-value of 0.1 or lower were reported with their corrected values.In order to systematically compare the mutation burdens and mutation signature exposures between all cell lines and treatments, we implemented a randomization test. Taking every mutation type (in the case of mutation burden comparison) and every signature (in the case of exposure comparisons), we randomly shuffled the clone labels (n = 21) 100,000 times. Then, we calculated the real mean mutation burden or exposure for each cell line (stratified by treatment and pooled) and for each treatment (stratified by cell line and pooled) and the mean from each shuffling iteration, resulting in 1 real mean and 100,000 randomised means for each comparison. Then, we calculated the pairwise difference between the mean of all cell lines and treatments (stratified or pooled) and compared the real mean difference to the distribution of randomised mean differences. For each such comparison, we calculated two empirical p-valuesSupplementary Figures.Supplementary Figure 11.Supplementary Figure 12.Supplementary Figure 13.Supplementary Table 1.Supplementary Table 2.


Endometriosis is an invasive condition that affects 10% of women  worldwide. The purpose of this study was to characterize the relative invasiveness of three available endometriotic cell lines  to cancer cell lines  and assess whether the relative invasiveness was consistent across different invasion assays. All cell lines were subjected to transwell, spheroid drop, and spheroid-gel invasion assays, and stained for vimentin, cytokeratin, E-Cadherin and N-Cadherin to assess changes in expression. In all assays, endometriotic cell lines showed comparable invasiveness to the cancer cell lines used in this study, with no significant differences in invasiveness identified. EEC12Z cells that had invaded within the assay periods showed declines in E-Cadherin expression compared to cells that had not invaded within the assay period, without significant changes in N-Cadherin expression, which may support the hypothesis that an epithelial-to-mesenchymal transition is an influence on the invasiveness shown by this peritoneal endometriosis cell line. Endometriosis is a chronic condition  where ceEndometriosis is a non-neoplastic invasive condition , with thEndometriosis is believed to be able to spread via both lymphatic and hematogenous means to extra-pelvic locations as diverse as the umbilicus, thorax, vulva , brain, While classified as a benign condition, endometriosis exhibits many of the hallmarks of cancer . The halA 1995 paper by Gaetje et al. (The Lancet) found that in collagen assays, cells from non-endometriotic endometria were non-invasive, while cells from peritoneal endometriotic lesions had similar invasive indices to the metastatic bladder carcinoma cell line EJ28 . In anotOne process thought to permit the invasion of endometriotic cells is the epithelial-to-mesenchymal transition (EMT). EMT involves reprogramming the epithelial cells which decreases adhesion, enhances invasion, and can be a contributing factor to the malignancy of epithelial tumours ,50. In eThe purpose of this investigation is to characterize the invasiveness of peritoneal and ovarian endometriosis patient-derived cell lines and endometrial stromal cells from endometriosis patients, relative to the invasiveness shown by the triple-negative breast cancer cell line (MDA-MB-231), a hormone-driven endometrial cancer cell line (EM-E6/E7/TERT), and a bone chondrosarcoma cell line (SW1353). The intent of this avenue of analysis is to leverage the greater wealth of knowledge surrounding the invasion of cancers and to uncover more detail about the degree to which endometriosis is an invasive non-lethal metastatic disease.2.EEC12Z, an SV40-transformed epithelial endometriotic cell line derived from red peritoneal lesions , and iEcMDA-MB-231 is a triple-negative cancer cell line that is one of the most studied breast cancer cell lines and it is known to be highly invasive ,61,62. TEEC12Z was immortalized by Starzinski-Powitz et al. in Germany in 2001 . PrimarytHESCs were immortalized by Holdsworth-Carson et al. in Australia in 2019 . As withiEc-ESCs were immortalized by Fazleabas et al. in Michigan, USA in 2020 . This ce5 cells (in DMEM/F12 medium with 0.1% FBS) were seeded onto 24-well plate inserts with 8 μm pores  coated with 30 μL of Geltrex . The inserts were placed into wells of a 24-well plate containing 600 μL of 10% FBS DMEM/F12 medium. After 48 h of incubation, the medium was removed from the lower and upper chambers. An amount of 600 μL of methanol from the fridge was added to the lower chamber and the transwells were placed into the wells for ten minutes to fix the invading cells. The non-invading cells were removed from the Geltrex by adding 80 μL of refrigerated Corning cell recovery solution  to the upper chamber. After 15 min of incubation at room temperature and triturating to break up the Geltrex, the solution from each well was transferred to 1.5 mL Eppendorf tubes and centrifuged for five minutes at 1000× g. The supernatant was removed, and the cells were plated on a 96-well plate . The cells were incubated overnight to attach, then fixed with methanol from the fridge for ten minutes. To conduct a cell count to assess the degree of invasion [Transwell invasion assays allow the mimicking of the invasion of metastasizing cells through the basement membrane and are the gold standard of characterizing invasive cell behaviour . A totalinvasion ,66,67, t5 cells were centrifuged at 1000× g in a 1.5 mL Eppendorf tube. The supernatant was removed, and the cell pellet resuspended in 30 μL of Geltrex . An amount of 10 μL of the suspended Geltrex cell solution was seeded onto the well of a 48-well plate . The well plate was placed upside down into the incubator to incubate for 20 min at 37 °C and 5% CO2. Once removed from the incubator, 2 mL of DMEM/F12 with 10% FBS medium was added to each well. The medium was changed every 48 h. The spheroids were imaged daily and the area of the Geltrex spheroid and invading cells were measured in ImageJ.The spheroid drop invasion assay was conducted in accordance with previously published methods . A total4 cells from mono-layer culture to three wells of ultra-low attachment round-bottom 96-well plates  with 100 μL of medium and cultured for seven days [2. On top of the Geltrex gel, 200 μL of DMEM/F12 medium was added. The spheroids were imaged on day zero to determine the initial spheroid area. The invasion was characterized by measuring the change in the spheroid area and the sprouting area in ImageJ [Scaffold-less spheroids were constructed by adding 6.0 × 10ven days  with haln ImageJ .Imaging was conducted using the EVOS M5000 Imaging System  with five images taken of each well at 400× total magnification. For each antibody, the definition of the positive threshold was selected in ImageJ based on a cell that was identified to be strongly positive. The same threshold was used consistently to identify the percentage of positive cells identified in each figure. To quantify the Cadherin staining, bright spots were treated as non-specific. To set the threshold, an image was chosen that had staining clearly associated with the cytoplasm of the cell. The threshold was set to identify this cell as positive, and this threshold was applied to all images. Cells were only counted as positive for Cadherin if the stain could be associated with the cytoplasm of the cell.Between each staining step, the cells were washed with PBS by filling the well with PBS, leaving for five minutes, then removing and discarding the PBS. The cells were washed three times in each wash step. Cells were washed in PBS, fixed with cold methanol for 10 min, and blocked with 5% goat serum  in PBS for 30 min at room temperature (RT). Invasive assays were characterized with vimentin , cytokeratin , N-Cadherin  and E-Cadherin . Cells were incubated with primary antibodies at 1:200 dilution in PBS for 90 min at RT. Secondary goat anti-Ms 594  and secondary goat anti-Rb 488  were added at 1:200 dilution in PBS, along with Hoechst  at 1:1000 dilution, to the cells for 45 min. The cells that had invaded in the transwell assay were fixed to the membrane of the Boyden chamber. The membrane was gently removed with a scalpel and placed into a 48-well plate for imaging.TM Gold Antifade Mountant  was added to the cells before imaging.Cells were washed in PBS, and fixed with cold 1:1 acetone/methanol solution from the fridge for 10 min at −20 °C, and blocked with 5% goat serum in PBS overnight at 4 °C. Cells were incubated with primary antibodies overnight at 1:200 dilution in PBS at 4 °C. Secondary goat anti-ms 594 and secondary goat anti-rb 488 at 1:200 dilution in PBS were added to cells for 2 h at RT. Hoechst at a 1:1000 dilution with 20% glycerol and 20% ProlongSpheroid drop assays, scaffold-less spheroids and spheroid gel invasion were imaged using the EVOS M5000 Imaging System. The entire spheroid was imaged by taking multiple images at 100× total magnification. The images were stitched together using Hugin (macOS Version 10.16). The lens type was set to rectilinear, and the horizontal field of view was set to 1°. The images were saved without compression and the freehand tool was used in ImageJ to draw around the spheroid and the area taken up by the cells. To compare the areas over time, the percentage increase in spheroid areas relative to day zero was calculated for each spheroid.p < 0.05, (**) indicates p < 0.01, and (***) indicates p < 0.001. The post hoc tests were carried out by Bonferroni correction (multiple comparisons) and the Mann–Whitney test (pairwise comparison).All experiments were repeated in triplicate, with the cells cultured separately for at least one passage for all cell lines. Data are presented as mean ± standard error of the mean . All tesThe comparison of the level of invasion in the transwell assay is shown in All cancer and endometriosis-related cell lines exhibited increased invasive areas in the six-day period of cell culture , while MDA-MB-231 and EEC12Z formed larger spheroids , and the spheroids formed by iEc-ESCs and tHESCs were intermediary in size   . The sph0.16 mm2 B.Most cell lines showed a steady increase in invaded area , indicatSimilarly to the spheroid droplet assay, two groups can be seen forming when examining the percentage increase over time D. The grThe cell lines that showed the greatest invasion, as determined by the number of Hoechst-stained nuclei present on the bottom of the membrane of the transwells, were the peritoneal endometriosis cell line EEC12Z with 90% ± 2% of cells invaded and by the breast cancer cell line MDA-MB-231 with 89% ± 4% of cells invaded within the 48 h of culture. Prior assessments of invasion of EEC12Z through transwells has shown an invasion of over 80%, comparable to the invasiveness shown by the bladder carcinoma cell line EJ28 . MDA-MB-With a one-way ANOVA analysis, there were no statistically significant differences between the percentage of cells that invaded within 48 h of culture between the breast cancer cell line MDA-MB-231, the endometrial cancer cell line EM-E6/E7/TERT, the peritoneal endometriosis cell line EEC12Z, the ovarian endometriosis cell line iEc-ESCs, or the endometrium cell line derived from endometriosis patient samples tHESCs . Cancer When the percentage increase in spheroid drop and cell invasion area was determined on the final day of cell culture was compared, the cell line showing the highest invasiveness was again the peritoneal endometriosis cell line EEC12Z, with 206.7% ± 24.6% percentage area increase, followed by EM-E6/E7/TERT with 204.0% ± 21.5% percentage area increase. As with the transwell assays, there are no statistically significant differences in invasiveness between cancer cell lines MDA-MB-231, EM-E6/E7/TERT, and endometriosis-related cell lines EEC12Z, iEc-ESCs, and tHESCs. Unlike the transwell assays, there were no significant differences in the invasiveness of SW1353, and the other cancer cell lines or endometriosis-related cell lines.The percentage increase in spheroid area through the formation of invasive edges was determined on the final day of cell culture and compared. The cell line showing the highest invasiveness was EM-E6/E7/TERT and tHESCs E after tWhen comparing the pattern of invasiveness across all three assays , it is iA previous study compared primary endometriosis cells to a metastatic bladder carcinoma cell line (EJ28) and assessed their invasive potential  by charaIt has been postulated that the success of endometriosis cell invasion is related to the epithelial-to-mesenchymal transition ,50. ThisThe spheroid drop assay showed EEC12Z was behaving with similar invasiveness to two of the cancer cell lines (MDA-MB-231 and EM-E6/E7/TERT), and more invasively than the bone chondrosarcoma cell line SW1353. The extra-cellular matrix used here was Geltrex which is made up of laminin, collagen IV, entactin, and heparin sulfate proteoglycans . This exRecent studies have focused on organ-derived ECM using various decellularization methods to create materials to make scaffolds for recellularization . These sThe self-assembling spheroid assay successfully showed the ability not only for the invasion of the cells, but this is also the first time we are aware that two of the three cell lines (iEc-ESCs and tHESCs) have been shown to self-assemble into spheroids. A study in 2011 used the hanging drop method to create EEC12Z collagen I and Matrigel spheroids . In addiFurther work could be carried out to examine how the size of the cell influences the size of the spheroid formed. EEC12Z cells are one of the smallest cell lines in this experiment; however, they formed the largest spheroids. This could result in the EEC12Z cells requiring to migrate further through the Geltrex before they are able to proliferate outside of the spheroid. A similar pattern is seen with the MDA-MB-231 cells which could indicate their invasion rate was underestimated because the initial area is larger. Future work could examine tagging the cells, along with fluorescent cell cycle indicators, to track individual cell behaviour to better understand the mobility associated with each cell line.The future direction for this work includes the development of primary endometriosis spheroids and a comparison of the invasive behaviour of primary cells isolated from the different sub-types of endometriosis, as well as at different stages of disease. The stage of disease of endometriosis is classified in the same way as cancer (Stage I–IV) and it would be beneficial to identify how these factors impact the invasive behaviour of endometriosis cells. If endometriosis invasion can be compared more directly to cancer and similar invasive mechanisms can be identified, it is likely this will result in increased research funding for endometriosis and better awareness for this chronic condition that affects 6–18% of women . In addition, it could result in the translation of targeted cancer treatments to develop a cure for this disease.


Comparing curcumin (4) and its pyrazole derivative (5), curcumin showed a better cytotoxic effect on SCC-25 cells, while curcumin pyrazole was more potent on FaDu cells. The highest sonotherapeutic activity, compared to its individual components, was demonstrated by a structural linker mode hybrid containing both curcumin pyrazole-oleanoyl hydrogen succinate units within one complex molecule (7). This study can be beneficial in the context of new perspectives in the search for effective sonosensitizers among derivatives of natural organic compounds.Sonodynamic therapy (SDT) is a non-invasive therapeutic modality in cancer treatment that combines low-intensity ultrasound (US) and sonosensitizers. Tumor cells are destroyed through the synergistic effects of ultrasound and a chemical sonosensitizer. This study focused on the synthesis and in vitro evaluation of the sonodynamic effect of natural curcumin, triterpene oleanolic acid, and their semi-synthetic derivatives on tongue cancer SCC-25 and hypopharyngeal FaDu cell lines. The combination of the tested compounds with sonication showed a synergistic increase in cytotoxicity. In the group of oleanolic acid derivatives, oleanoyl hydrogen succinate ( The progress of the reaction was checked by thin layer chromatography (TLC). After complete conversion, the mixture was cooled to room temperature, and the precipitate formed was filtered under vacuum and air-dried. Then, the crude product was subjected to column chromatography on silica gel using a chloroform/methanol  mixture as eluent. The pure product was obtained as a red solid. Yield: 79%; m.p. and spectral data were in good agreement with the reference [A mixture of curcumin (eference .4-{[(3β)-28-hydroksy-28-oxoolean-12-en-3-yl]oxy}-4- oxobutanoic acid, Oleanoyl hydrogen succinate (6)1, 0.94 g, 0.002 mol) and 4-(dimethylamino)pyridine  were added to a solution of succinic anhydride  in dry pyridine (10 mL). Then, the mixture was refluxed for 8 h. The reaction mixture was left overnight and poured into 100 mL of water. The precipitate formed was filtered under reduced pressure, washed with 5% HCl, air-dried, and finally crystallized from ethanol. Yield: 90%; m.p. and spectral data were in good agreement with the reference . Furtheand head ,62,63. Hand head ,61,64 anand head ,64,65,66MTT assay was used to study the cytotoxicity of compounds tested and their combinations with US in SCC-25 and FaDu cells. Ultrasound parameters were selected based on available literature data ,64,65,661) alone was decreased by 27% as compared to the control  was lower than that of 2 combined with US. On the contrary, we observed only slightly reduced viability of the SCC-25 cell line treated with Oleanolic acid oxime methyl ester (3) alone as well as 3 used in combination with US (~24–30%). The cytotoxic activity of the curcumin (4) was similar to that of 3. However, the number of SCC-25 cells exposed to 4 and the US was reduced significantly by ~50%, as compared to the control. The viability of the SCC-25 cell line treated with both curcumin pyrazole (5) alone, as well as the combination of 5 and US, was slightly inhibited (~25%) compared to untreated cells. However, the latter one was not statistically significant. Oleanoyl hydrogen succinate (6) was shown to decrease the cell viability of SCC-25 cells by ~10% as compared to the control. On the contrary, a hybrid of curcumin pyrazole and oleanoyl hydrogen succinate (7) alone has no statistically significant effect on cell proliferation. However, the exposure to 6 combined with US, as well as the combination of 7 and US, reduced the number of viable cells by ~50% compared to the untreated control.The number of SCC-25 cells treated with oleanolic . The US exposure in the presence of 1 was shown to decrease the number of viable cells by ~46% as compared to the control. The derivatives of OA, compounds 2 and 3, were shown to similarly inhibit the viability of the FaDu cell line (~20%). The combination of US with 2 and 3 reduced the number of viable cells by ~34% and ~29%, respectively, as compared to the control. The cytotoxic activity of the curcumin was only slightly lower than that of 4 and UV. However, statistical significance was observed as compared to untreated cells. The curcumin pyrazole (5) alone reduced the viability of FaDu cells by ~34%, while the exposure to compound 5 combined with US inhibited the number of viable cells by 44% as compared to control. The succinyl derivative of OA (6) and the hybrid pyrazole curcumin and oleanoyl hydrogen succinate (7) were shown to slightly decrease cell viability (~10%) compared to the control; however, the combination of US with 6 and 7 inhibited the cell viability by ~60% and ~80%, respectively.As shown in 1-7 in monotherapy and the action of their combinations with ultrasounds might bring different effects in epithelial cell lines derived from different tumors. Comparing the results obtained in the SCC-25 tongue cancer cells and the FaDu throat ones, we cannot clearly state which cell line is more sensitive to the SDT since we showed a marked decrease in cell viability after treatment with compounds 1-7 alone and their combinations with US in both cell lines.The observed difference between cell survival after the action of the tested compounds in monotherapy and cell viability after treatment in combination with ultrasounds suggests that separate mechanisms might be responsible for the cytotoxic effect. The results of the MTT test suggest that both the action of compounds 4) showed a better cytotoxic effect on SCC-25 cells than oleanolic acid (1), both in monotherapy and in combination with US. In the FaDu cell line, the effect of both compounds was comparable in monotherapy, while oleanolic acid (1) was found to exert significantly stronger cytotoxicity after exposure to US. In the group of oleanolic acid derivatives, oleanolic acid (1) showed the most potent monotherapy effect in FaDu cancer cells, while oleanoyl hydrogen succinate (6) indicated the higher cytotoxic effect in SDT therapy. However, when comparing curcumin and its pyrazole derivative, the latter revealed a stronger cytotoxic effect, both individually and in combination with US. In the group of triterpene compounds in the SCC-25 cell line, the highest cytotoxic activity in monotherapy was shown by oleanolic acid oxime (2), while after exposure to ultrasounds, the strongest effect was exerted by compound 6. Comparing compounds 4 and 5, curcumin (4) showed higher cytotoxicity against SCC-25 cells, both in monotherapy and in SDT. Compound 7, a hybrid of curcumin pyrazole and oleanoyl hydrogen succinate, was found to be the most effective sonosensitizer in both tested cell lines , its oxime (2), its methyl ester oxime (3), and oleanoyl hydrogen succinate (6). In parallel, curcumin (4) and its heterocyclic pyrazole (5) were evaluated. A complex hybrid derivative (7) combining the above-mentioned structures of triterpene and curcumin in one molecule was also obtained and investigated.Among the triterpene derivatives, the following were tested: oleanolic acid (7), exhibit a high sonosensitizing potential and have a chance to be used in SDT therapy supporting the treatment of head and neck cancers.Based on the available literature data and above all our results, we suggest that oleanolic acid, curcumin, and their derivatives, especially the triterpene–curcumin type hybrid (


More insight into the biological diversity of colorectal cancer (CRC) is needed to improve therapeutic outcomes. We aimed at establishing a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC with regards to the molecular subtypes, allowing bioluminescence imaging-assisted analyses. Comparative characterization of stable luciferase expressing derivatives of well-established CRC cell lines, derived spheroids and subcutaneous xenograft tumors showed that regarding primary tumor characteristics, the 3D-spheroid cultures resembled xenografts more closely than 2D-cultured cells do. Xenograft tumor growth resulted in metastatic spread to the lungs. Furthermore, a bioluminescence-based spheroid cytotoxicity assay was set up in order to be able to perform dose–response relationship studies in analogy to typical monolayer assays. Thus, the model systems can be used in preclinical research applications to study new therapy approaches and represents the biological heterogeneity of CRC.Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose–response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications. Cancer cell lines are valuable in vitro model systems that are widely used in basic cancer research and drug discovery . Here, tColorectal cancer (CRC) is among the most common cancers and a major cause of cancer mortality ,8. CRC iGene expression profiles of tumor tissue samples represent the sum of signals derived from the cancer cells and their surrounding tumor microenvironment, the latter of which can impede gene expression analysis, depending on the model used. Moreover, the stromal component in a tumor has been suggested to be crucial for the determination of CMS4 ,19. In cIn the present work, we generated stable, luciferase expressing cell clones from established CRC cell lines, in order to analyze growth, therapy response and metastasis in derived spheroid and nude mouse xenograft models utilizing the endogenous signal. Tumor spheroids are useful in vitro models as they can represent several important aspects of real tumor tissues, e.g., (1) three-dimensional growth with structural organization and physiologically relevant cell–cell and cell–matrix interactions; (2) establishment of tumor microenvironmental characteristics such as nutrient gradients, hypoxia and acidosis; (3) localization-dependent heterogeneous cell growth and differentiation; (4) drug resistance mechanisms ,27. Mous2 in a humid atmosphere. The colorectal cancer cell lines HT29 (HTB-38), DLD1 (CCL-221), LOVO (CCL-229), SW48 (CCL-231), LS1034 (CRL-2158), SW1463 (CCL-234), COLO205 (CCL-222), LS174T (CL-188), HCT116 (CCL-247) and SW480 (CCL-228) were originally obtained from the ATCC and were authenticated in 2010 at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH . All cell lines were cultivated in RPMI medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin (Sigma-Aldrich) at 37 °C/5% COThe cDNA of the red-shifted firefly luciferase PLR1 , kindly ® GTG™ Agarose, Lonza, Basel, Switzerland) before. The cell lines HT29-Luc, DLD1-Luc, LS174T-Luc, LS1034-Luc and SW1463-Luc were able to form compact spheroids within 2 days. For the cell lines LOVO-Luc, SW48-Luc, COLO205-Luc, HCT116-Luc and SW480-Luc, the used culture medium was supplemented with 10 µg/mL collagen I  in order to support spheroid formation. Compact spheroids were formed within 7 days. In order to generate single tumor spheroids, tumor cells resuspended in culture medium were seeded onto 96-well plates, which were coated with 0.7% agarose  were seeded onto agarose-coated 96-well plates. The starting point of the assay (d0) was chosen after the formation of spheroids within two or seven days. On d0, 20 µL D-luciferin solution was added per well and luciferase activity was measured after 15 min incubation time on the Spark microplate reader. Further measurements were performed on d2, d5 and d7. Growth kinetic curves were established by plotting mean values (8 spheroids) over time using GraphPad Prism8. Based on these analyses, a cell line-specific cell amount to be used in our cytotoxicity assays was determined: HT29, DLD1, LS174T, LS1034, SW1463–1500; HCT116, LOVO–300; SW48, SW480–500; COLO205–150.50 values including standard deviations were carried out using GraphPad Prism8. The IC50 values of the groups were then compared using a Welch test.For the cytotoxicity assay, cells were seeded onto agarose-coated 96-well plates and were treated with serial dilutions of oxaliplatin  for seven days after compact spheroids formed. Measurements of luciferase activity were performed as described above. Dose–response curves and calculation of IC3. Tumors were sliced in half and, afterwards, one half was fixed in formalin for histological analyses, whereas the other half was immediately processed, performing cell separation using the MACS cell separation technology from Miltenyi . Preparation of the tumor mass was performed on a gentleMACS Octo by using the Tumor Dissociation Kit (130-095-929), followed by separation using the Mouse Cell Depletion Kit (130-104-694) and the Death Cell Removal Kit (130-090-101) according to the protocols of the manufacturer. For histological analyses, formalin-fixed samples were embedded in paraffin and then cut to perform hematoxylin/eosin (HE) staining according to standard protocols, as described previously [Cell lines were harvested 48 h after seeding and different samples per cell line were pooled. Spheroids were harvested on day 7  or day 12  and were pooled. Xenograft tumors see  were resion Kit 1-095-929,RNA was extracted using the TRIzol™ Reagent  according to the manufacturer’s protocol. For transcriptomic analyses, the Clariom™ D Assay from Applied Biosystems™ (Thermo Fisher Scientific) was used. The processing of the microarrays was performed in the core facility “analyses” of the Center for Medical Basis Research (ZMG) of the Medical Faculty  according to the instructions of the manufacturer. Microarray data will be available from the Gene Expression Omnibus (GEO) database. In addition to our preclinical samples, ten tumor samples and ten normal colon tissue samples from the GEO database (accession number GSE115261 ), which Microarrays were analyzed using the robust multi-array average (RMA) algorithm with the Transcriptome Analysis Console . Gene filtering and cluster analysis was performed with TAC4.0. For the genes included in the cluster analysis focusing on cell line-specific genes and differences between tumor and normal samples were filtered for a false discovery rate (FDR) F-test < 0.0001 and a tumor-versus-normal log2 fold change of >5/<−5 (749 probe sets passed these filters). Normal samples were set as the baseline. Genes included in cluster analysis focusing on the different preclinical model systems were filtered for a FDR F-test < 0.01 and 2D cultures were used as baseline. Genes were further filtered for spheroid-versus-2D and xenograft-versus-2D log2 fold changes of >1.5/<−1.5 (152 probe sets) or >2/<−2 (51 probe sets).https://github.com/peterawe/CMScaller (lastly accessed on 10 August 2023). In addition, the datasets were classified using the CRCassigner [https://github.com/syspremed/CRCAssigner (lastly accessed on 10 August 2023).The gene expression data were used to classify the samples according to the CMS classification system applying the CMScaller . The claassigner  accordinFor classification of the intrinsic epithelial tumor cell type, the specific iCMS2 and iCMS3 gene sets provided in the original study  were useAdditional datasets from public databases were used for iCMS classification of additional cell lines. From ArrayExpress, the cel files from dataset E-MTAB-2971 were downloaded. In addition, the following the GEO datasets were used: GSM1374426, GSM1374451, GSM1374452, GSM1374456, GSM1374463, GSM1374516, GSM1374517, GSM1374518, GSM1374561, GSM1374562, GSM1374563, GSM1374564, GSM1374627, GSM1374628, GSM1374629, GSM1374630, GSM1374632, GSM1374633, GSM1374759, GSM1374919, GSM1374920, GSM1374925, GSM1374926, GSM1374927, GSM1374928, GSM1374929, GSM1374930, GSM1374933, GSM1374934, GSM1374935, GSM1374937, GSM206450, GSM206455, GSM206459, GSM206463, GSM206467, GSM206501, GSM206517, GSM206519, GSM206524, GSM206547, GSM206548, GSM206552, GSM206553, GSM206554, GSM843481, GSM843482, GSM844580, GSM844713, GSM887141, GSM887274, GSM887277, GSM887278, GSM887303, GSM887479, GSM887632, GSM887644, GSM887667, GSM887668, GSM887674, GSM887675, GSM887677, GSM887679 .Generation of subcutaneous xenograft tumors was performed by inoculation of 5 million tumor cells into the right flank of male athymic nude mice . Monitoring of tumor growth was performed by caliper measurement and volume calculation using the formula ayses see , three tn = 3) with similar mean tumor volumes of about 0.150 cm3 and equal volume distribution at the start of treatment. Treatments comprised weekly intraperitoneal applications of oxaliplatin (8 mg/kg BW) and normal saline (control). Mouse weight and behavior were controlled daily during the course of treatment. The impact of treatment was calculated as the increase in tumor volume after treatment relative to the tumor volume at the start of the treatment on day 0 . Selected models were used to analyze the anti-tumor activity of oxaliplatin. For this purpose, mice were divided into two groups , CMS3  and CMS4  . Both ceIn a very recent study, two intrinsic epithelial tumor cell states, iCMS2 and iCMS3, were identified in colorectal tumors, which can be used to refine the CMS classification system . In thisWe noticed that neither of our initially selected models represented the second main group of CMS4 tumors CMS4/iCMS3-MSS . In ordeThus, including the three cell lines in preparation, the completed model comprises thirteen CRC cell lines, with four cell lines clearly representing the iCMS2 epithelial subtype , whereas seven cell lines  clearly belong to the iCMS3 subtype. The cell lines COLO205 and HT29 seem to represent intermediate types with a similarity to iCMS3. Furthermore, Joanito et al. demonstrated that one of the defining features of iCMS2 was the enrichment in copy number variations (CNV), whereas iCMS3 tumors were diploid or showed infrequent and inconsistent CNV . Data reThe combined data show that tumor spheroids are more suited to be compared to xenograft tumors in regards to characteristics of patient tumors in comparison with the respective cell lines, making them useful models for preclinical drug research. Taking advantage of the endogenous luciferase expression in these models, we set up a bioluminescence-based cytotoxicity assay in order to be able to perform dose–response relationship studies in analogy to typical monolayer assays. In order to determine the optimal assay conditions, we first studied the growth kinetics of each spheroid model in order to prove the expected correlation between cell amount and signal intensity as an important prerequisite. A direct correlation between spheroid mass and signal intensity was confirmed in freshly formed spheroids. However, a near linear growth kinetic in growing and compacted spheroids is highly dependent on the amount of cells seeded at the start of the experiment. The relative decrease in signal intensity in growing spheroids can be explained by their typical characteristics such as the induction of oxygen and nutrient gradients leading to hypoxia, acidosis and heterogeneous cell growth, with proliferating cells at the rim area, and less proliferating/differentiated or even apoptotic/necrotic cells in the core area ,36,37. TApplying the established assay, we studied the efficacy of oxaliplatin in the spheroid models of the whole panel. Oxaliplatin treatment induced a dose-dependent inhibition of spheroid growth and resulted in a typical dose–response pattern. Next, IC50 values were calculated and compared with existing data obtained from monolayer assays. This revealed clear differences throughout the whole panel . In geneWe next questioned to what extent the different in vitro sensitivity of spheroids, represented by their specific IC50 value, can predict a specific response towards oxaliplatin treatment in vivo. We therefore analyzed the impact of an oxaliplatin therapy in selected xenograft models . Clear rBased on the proven oxaliplatin resistance, we performed analyses of differentially expressed genes comparing the xenograft tumors of SW1463 and LS174T with those of LOVO, SW48 and HCT116 in order to find targets associated with the differential oxaliplatin sensitivity. CAV1 (caveolin 1) was identified as the top-ranked gene with a 400-fold increased expression in resistant vs. sensitive xenograft tumors, . FurtherCancer is among the world’s greatest health problems and one of the leading causes of death worldwide . Since cAs CRC is both a leading cause of cancer mortality as well as a very diverse and poorly understood disease, establishing an aforementioned model is of utmost importance, in order to improve preclinical research with the aim of improving therapeutic options. Previous research has already suggested that CRC cell lines have the potential to be used as representative models . The CMSOur panel of luciferase-labeled, cloned CRC cell lines was able to robustly recapitulate the main subtypes of CRC based on the CMS classification in the majority of cases, both in the spheroid as well as in the mouse xenograft model and to lesser extent in the monolayer model. The results for the cell lines were in accordance to the results obtained for the wild-type cell lines in previous reports ,24. FurtThe combined data showed that 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do regarding primary tumor characteristics, which can be expected. While unrestrained proliferation is the main task for cancer cells in monolayer models, three-dimensional growth now requires cell–cell interactions and the establishment of a tumor microarchitecture . This noTumor spheroids are useful in vitro models and are also an easier, cheaper and faster way to create a three-dimensional preclinical model compared to mouse xenograft tumors ,27. ReceAnalysis of the differential oxaliplatin sensitivity with respect to the molecular subtypes represented by the models revealed some interesting clues. For example, a subset of patients with CMS2 featured tumors benefited from oxaliplatin containing chemotherapy regimen compared to other subtypes in the adjuvant setting . AccordiWith our work we aimed to establish a combined 2D/3D, in vitro/in vivo model system capable of representing the heterogeneity of CRC with regards to the molecular subtypes, and allowing bioluminescence imaging-assisted analyses. Our work has also shown that spheroid models do exhibit a higher similarity towards xenograft tumors compared to monolayer models both in terms of expression patterns as well as in terms of response to drug treatment due to the establishment of a three-dimensional structure and the associated mechanisms. Although spheroids do not completely resemble the heterogeneity found inside xenograft and human tumor samples, they are very solid models, especially considering their easy manageability and availability in comparison to xenograft models, with a stronger validity when, for example, performing drug screening studies.


MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype  NOTCH1 mutations are present in 60–70% of T-ALL cases [FBXW7, a negative regulator of Notch signaling, present in an additional 15% of patients [CDKN2A locus encoding ARF in 80% of patients [There is an unmet need for improved therapies to treat T-cell acute lymphoblastic leukemia (T-ALL), given the high rate of relapse and poor outcomes, especially in adults , 2. ActiLL cases , with inpatients –8. Althopatients . ARF antpatients . Inhibitpatients . Importapatients , 13. BasBAX and BBC3 (PUMA) but only modest apoptosis. Based on these observations, we hypothesized that inhibition of anti-apoptotic proteins would be synergistic with MDM2 inhibition in T-ALL. Navitoclax is an orally available investigational small molecule inhibitor of Bcl-2 family proteins [In this study, we show that idasanutlin, a second generation small molecule inhibitor of MDM2, induces expression of pro-death BH3 domain family members proteins . Navitocproteins . As T-ALproteins , 17, we 2 in a humidified incubator. The mouse T-ALL PDX lines DFCI12, DFCI15, DFAT28537, DFAT27681, and CBAT27299 were previously established and kindly provided by the cBioPortal  [TM SFEM II , penicillin-streptomycin (100 U/ml), human IL-2 (0.2 IU/ml), human IL-7 (10 ng/ml), and human stem cell factor (50 ng/ml).The human T-ALL MOLT-3 cell line was previously kindly provided by the lab of Dr. Grant Challen  . MOLT-3 MA, USA) , 20. TheTP53 or the AAVS1 safe-harbor locus were purchased (Integrated DNA Technologies) and mixed with recombinant Cas9 protein (IDT), incubated at room temperature to generate ribonucleoprotein complexes, and transferred into MOLT-3 cells using the Neon transfection system at 1350 V, 35 ms, 1 pulse, following manufacturer recommendations. Isogenic clones were generated through outgrowth following single cell sorting. Insertion/deletion status was assessed by next-generation sequencing of the target locus on the Illumina MiSeq platform and analyzed using CRISPResso2 [For generation of knockout cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, single-guide RNA (sgRNAs) targeting SPResso2 , and knoThe methods followed for real-time PCR and immunoblotting were as previously described in  and are T-ALL cell lines and PDX cells were subjected to CellTiter-Glo assay  and Caspase-Glo 3/7 assay  as detailed in the Supplementary Methods.T-ALL cell lines and PDX cells were subjected to flow cytometry assessment of cell cycle, apoptosis, and viability as previously described in  and are n = 10 per treatment, per PDX line. Minimum cohort size determined based on estimated effect size following pilot experiment. Based on the law of diminishing returns [All animal studies were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis under protocol number 20-0495. DFCI12, DFCI15, DFAT28537, and CBAT27299 PDX cells were engrafted into NSG mice and treated with vehicle alone, idasanutlin, and/or navitoclax with hCD45 flow cytometry as described in the Supplementary Methods. Upon evidence of engraftment, mice were stratified by degree of tumor burden, and mice from each tier were randomized equally into treatment cohorts. These random groups were treated with a proportional mixture of both vehicles, idasanutlin  , navitoclax   score [n < 5. All data are presented as mean ± standard error of the mean. A p value of <0.05 was considered significant.Statistical testing was performed using Prism . The between-group differences in tumor burden were compared using unpaired Student’s y) score , 28. SynTP53-sufficient T-ALL is sensitive to MDM2 inhibition. To test this hypothesis, we initially studied MOLT-3 cells, an immortalized T-ALL cell line with wildtype TP53, and generated isogenic cell lines edited at TP53 or the AAVS1 safe-harbor locus (control). After identifying frameshift indels in each allele by sequencing, we validated p53 loss by assessing protein level following x-ray irradiation. We observed strong induction of p53 protein in the AAVS1-edited control lines, but completely absent p53 in both TP53 knockout (KO) lines tested, each of which carried different indel mutation combinations , and CDKN1A (p21) at the RNA  or navitoclax (targeting Bcl-2 and Bcl-xL). Since a prior study showed increased activity of navitoclax compared to venetoclax in T-ALL [The induction of expression of pro-death BH3 domain family members in T-ALL , we nextin T-ALL . GeneralNOTCH1 and/or FBXW7 mutations are present in three lines; CDKN2A biallelic loss is present in three lines; and all lines are wildtype for TP53. Two lines are ETP-ALL with wildtype NOTCH1, FBXW7, and TP53. For all five PDX lines tested, in vitro dual treatment led to robust cell death within 48 h. For two of the cell lines tested, in vitro treatment with 1 μM navitoclax alone was also highly efficacious  alone, idasanutlin alone, or the combination. At clinically relevant doses [There is evidence for increased JAK/STAT signaling in a subset of T-ALL , 36, witnt doses , 40, ruxINKA2, BAX, BBC3, and CDKN1A  of cells were viable and non-apoptotic as measured by Annexin-V staining Fig. S. PrincipN1A Fig. . FinallyFκB Fig. . Indeed,45B Fig. .Fig. 4TrHaving observed strong in vitro activity against a panel of T-ALL PDX lines, we next assessed the in vivo response of four different T-ALL xenografts to dual idasanutlin and navitoclax treatment. For each line, human T-ALL cells were injected into immunodeficient mice, and following engraftment, mice were randomized into treatment groups. Mice were treated for 14 days before withdrawal of therapy, and tumor burden as well as survival were analyzed Fig. . CombinaTreatment with navitoclax induced a significant decrease in T-ALL burden in all four xenotransplants Fig. . TreatmeT-ALL is an aggressive hematologic malignancy that comprises 15% of pediatric ALL and 25% of adult ALL , 41. CurMYC is among the numerous target genes activated by Notch1, and has been proposed to be one of the most critical for T-ALL growth and maintenance [MYC has been reported in the majority of T-ALL cases [FBXW7 mutations are present in ~15% of T-ALL patients [PTEN [Notch pathway overactivation is key to T-ALL pathogenesis. ntenance –50. IncrLL cases . FBXW7 mpatients . In addipatients . Activatts [PTEN , 54. A rts [PTEN .TP53 is rarely mutated in T-ALL, even in relapsed patients [Myc is a master transcription factor that regulates up to 15% of genes, including many involved in proliferation and metabolism . Howeverpatients , 13. HerIncreased Myc expression can induce oncogenic stress though multiple mechanisms, including induction of DNA replicative stress and ribosome biogenesis stress , 58. WheκB pathway gene expression. Whether NF-κB signaling directly contributes to induction of apoptosis in this setting will require further study.Given the evidence of upregulation of pro-apoptotic proteins in response to idasanutlin exposure, we explored a rational combination of idasanutlin and navitoclax, a Bcl-2/Bcl-xL inhibitor. We observed strong p53-dependent synergistic activity, and induction of apoptosis by idasanutlin and navitoclax in vitro against a panel of T-ALL PDX lines. RNA sequencing of treated PDX cells was consistent with a convergence on apoptosis, with idasanutlin inducing gene expression level upregulation of the p53 pathway and navitoclax leading to minimal additional RNA level changes. The addition of navitoclax to idasanutlin in combination therapy was associated with a significant increase in NF-We demonstrated that idasanutlin and navitoclax have strong activity in vivo in human T-ALL xenotransplantation models. Combination therapy demonstrated synergistic activity in suppression of tumor burden and increased survival in a panel of four T-ALL PDX. Hence, the present work provides strong support for therapeutic efficacy of simultaneous MDM2 inhibition and Bcl-2 family protein inhibition against T-ALL. There is concern that the dose-limiting thrombocytopenia observed with navitoclax treatment due to on-target Bcl-xL inhibition in circulating platelets may limit its use in the clinic , 59, 60.NOTCH1 and/or FBXW7 mutations, biallelic CDKN2A loss, and wildtype TP53; all T-ALL genetic hallmarks. These lines are representative and generalizable to T-ALL genomics across a majority of cases. Acknowledging this limitation, the strong and robust response across these lines, which carry commonly recurring T-ALL mutations, suggests that human trials for idasanutlin and navitoclax combination therapy in wildtype TP53 cases are merited.A limitation of this study remains that in vitro only five PDX lines were assessed, and in vivo only PDX lines from four different patients were investigated. Although our findings were robust across these lines, they cannot represent the complete diversity of all T-ALL mutational profiles. However, the PDX lines selected are broadly representative, harboring a combination of frequent Supplemental Materials and MethodsSupplemental FiguresTable S1