ERCC2 inactivation associated NER deficiency and also substantial levels of PTGR1 expression. These patients may be responsive to irofulven, a previously abandoned anticancer agent that has minimal activity in NER-proficient cells.Due to a demonstrated lack of DNA repair deficiencies, clear cell renal cell carcinoma (ccRCC) has not benefitted from targeted synthetic lethality-based therapies. We investigated whether nucleotide excision repair (NER) deficiency is present in an identifiable subset of ccRCC cases that would render those tumors sensitive to therapy targeting this specific DNA repair pathway aberration. We used functional assays that detect UV-induced 6–4 pyrimidine-pyrimidone photoproducts to quantify NER deficiency in ccRCC cell lines. We also measured sensitivity to irofulven, an experimental cancer therapeutic agent that specifically targets cells with inactivated transcription-coupled nucleotide excision repair (TC-NER). In order to detect NER deficiency in clinical biopsies, we assessed whole exome sequencing data for the presence of an NER deficiency associated mutational signature previously identified in ERCC2 mutant bladder cancer. Functional assays showed NER deficiency in ccRCC cells. Some cell lines showed irofulven sensitivity at a concentration that is well tolerated by patients. Prostaglandin reductase 1 (PTGR1), which activates irofulven, was also associated with this sensitivity. Next generation sequencing data of the cell lines showed NER deficiency-associated mutational signatures. A significant subset of ccRCC patients had the same signature and high PTGR1 expression. ccRCC cell line-based analysis showed that NER deficiency is likely present in this cancer type. Approximately 10% of ccRCC patients in the TCGA cohort showed mutational signatures consistent with  BRCA1, BRCA2, RAD51 etc.). Furthermore, ccRCC cases rarely display DNA scarring signatures associated with HR deficiency1. Therefore, it is likely that patients with ccRCC will not show sensitivity to PARP inhibitors via the mechanisms that confer sensitivity in ovarian or breast cancer. It was proposed recently that other genetic events, such as the inactivation of PBRM1, often observed in ccRCC, may confer PARP inhibitor sensitivity in this disease2. However, the clinical relevance of this observation remains to be determined.Synthetic lethality driven therapy has become a successful treatment approach in the context of PARP inhibitor-based therapy for homologous recombination (HR) deficient ovarian, breast, prostate and pancreatic cancer. However, patients with clear cell renal cell carcinoma (ccRCC) have not benefitted from this treatment strategy thus far due to the absence of identifiable HR deficient cases. ccRCC cases almost never harbor inactivating mutations coupled with loss of heterozygosity (LOH) in the key HR genes (3. NER is initiated through two separate mechanisms of lesion recognition: transcription-coupled repair (TC-NER) is activated by RNA polymerase stalling at lesions, while global genome repair (GG-NER) is able to recognize distorted DNA structures throughout the genome. TC-NER and GG-NER converge on a common NER pathway that excises and replaces the damaged DNA strand in an error-free manner.Nucleotide excision repair (NER) deficiency is another therapeutically targetable DNA repair deficiency in cancer. It is a highly conserved DNA repair pathway that recognizes and repairs bulky intrastrand DNA adductsERCC2 detected in urothelial carcinoma of the bladder cause NER deficiency in cell line models and that ERCC2 mutations are associated with platinum sensitivity in some bladder cancer clinical cohorts5. Thus, preliminary evidence for NER deficiency and associated platinum sensitivity was established in at least one solid tumor type. Indirect evidence for the presence of NER deficiency is also presented in other solid tumor types as well, such as breast cancer6. We previously reported increased risk for breast cancer due to recurrent ERCC3 variant and demonstrated lower cell survival in mutant mammary epithelial cell line (HMLE), when exposed to Illudin S, a DNA damaging sesquiterpene7.It has been known for decades that inactivation of NER activity in experimental models leads to increased cisplatin sensitivity. This is believed to be primarily driven by the ability of the NER pathway to remove platinum-induced DNA crosslinks. However, establishing a causative link between NER deficiency and platinum sensitivity in the clinic proved to be difficult due to the lack of diagnostic tools that detect NER deficiency in clinical biopsies. Recently, it was shown that mutations in the NER helicase gene ERCC2 inactivation in bladder cancer are also present in a subset of ccRCC cases. Finally, we show that the mutational signature of NER deficiency detected in ccRCC cell lines is associated with increased sensitivity to irofulven, a semisynthetic DNA alkylating agent with synthetic lethal activity in NER deficient cells.Here we provide experimental evidence for the presence of NER deficiency in ccRCC cell lines. We also demonstrate that the specific mutational signatures associated with Cell lines 786O, 769P, A498 were purchased from ATCC. SLR26, CAKI1, ACHN and RXF393 were kindly supplied by the Kaelin laboratory (Dana Farber Cancer Institute). Cell lines were grown in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), incubated at 37 °C in 5% CO2, and regularly tested for Mycoplasma spp. contamination.ERCC4. Cas9 nuclease was purchased from Horizon Discovery. The crRNA was annealed with ATTO™ 550-tracrRNA, and ribonucleoparticles (RNPs) were then assembled by adding Cas9. RNPs were delivered into cells using electroporation-based nucleofection (Lonza system). Flow cytometry was utilized to sort ATTO-550 positive single cells 24 h following nucleofection. Next, single cells were expanded and clonal populations were screened by immunoblot to identify clones with complete loss of expression of the ERCC4 protein.The NCI-H460 cell line was purchased from ATCC. The Alt-R™ CRISPR-Cas9 System (IDT Technologies) was used to delete P-values were calculated using student’s t-test. P-values < 0.05 were considered statistically significant.Exponentially growing cell lines were seeded in 96-well plates (3000 cells/well) and incubated for 24 h to facilitate cell attachment. Identical cell numbers of seeded parallel isogenic lines were verified by the Celigo Imaging Cytometer after attachment. Cells were exposed to Irofulven  for 72 h, and cell growth was determined by the addition of PrestoBlue (Invitrogen) and incubated for 2.5 h. Cell viability was determined by using the BioTek plate reader system. Fluorescence was recorded at 560 nm/590 nm, and values were calculated based on the fluorescence intensity. IC50 values were determined by using the AAT Bioquest IC50 calculator tool. An siRNA against PTGR1 , shown to induce > 90% reduction of PTGR1 transcript levels over 48–72 h, or an Alexa Flour non-targeting control siRNA were transfected at 25 nM into the HMLE cell line using Lipofectamine RNAiMAX (Thermo Scientific). Cells were seeded at 3000 cells per well into a 96-well plate during reverse transfection. Following 24 h, the cells were treated with either vehicle (0.01% EtOH) or irofulven at 300 nM and 600 nM doses. Cell viability was measured after 72 h using the CellTiterGlo reagent (Promega).Freshly harvested cells were lysed in RIPA buffer. Protein concentrations were determined by Pierce BCA™ Protein Assay Kit (Pierce). Proteins were separated via Mini Protean TGX stain free gel 4–15% (BioRad) and transferred to polyvinylidene difluoride membrane by using iBlot 2 PVDF Regular Stacks (Invitrogen) and iBlot transfer system (LifeTechnologies).Membranes were blocked in 5% BSA solution (Sigma). Primary antibodies were diluted following the manufacturer’s instructions: anti-beta Actin, [AC15] (HRP-conjugated) ab 49,900, Abcam  and antiPTGR1 [EPR13451-10], ab181131, Abcam (1:1000). Signals were developed using Clarity Western ECL Substrate (BioRad) and Image Quant LAS4000 System .Removal of 6–4 pyrimidine-pyrimidone photoproducts (6–4PP) as a function of NER was quantified using an immunofluorescent assay. Cells were seeded on coverslips in a 12 well plate. After overnight growing, cells were irradiated with 40 J/m2 under UV lamp directly with 254 nm wavelength. Cells were fixed in cold methanol for 10 min right after UV damage or after 7 h recovery. Then cells were triton extracted (0.5% Triton X-100 in PBS) for 4 min at room temperature followed by 2 M HCL/PBS incubation at 37 °C for 15 min. After washing twice with PBS, once with 1% BSA/PBS, once with PBS, cells were incubated with 6-4PP primary antibody  for 45 min at 37 °C followed by incubation with secondary antibody for 30 min at 37 °C. Coverslips were then washed twice with PBS and mounted using DAPI.https://portal.gdc.cancer.gov/) for the analysis. From the TCGA data portal the vcf files for the somatic mutations from the MuTect2 pipeline were used.This study evaluated 389 whole exome sequenced (WES) pretreatment samples of RCC patients from the TCGA-KIRC cohort. The normal, tumor bam and vcf files were retrieved from the TCGA data portal  for the cancer cell line samples (DepMap version 22Q2).Variants were collected from the DepMap portal (The application of the MuTect2 default filters (FILTER =  = "PASS") for filtering the called mutations ensured the high accuracy of germline and somatic changes reported. Utilizing additional stringent filters on somatic samples provided the high accuracy of reported variants: TLOD ≥ 6 and NLOD ≥ 3, NORMAL.DEPTH ≥ 15 and TUMOR.DEPTH ≥ 20, TUMOR.ALT ≥ 5 and NORMAL.ALT = 0 and TUMOR.AF ≥ 0.05. Additionally, samples with less than a total of 50 variants were removed, since mutational signature extraction is less reliable when the number of mutations is fewer than 50.8 289 samples were further analyzed.After applying these filters and keeping only one sample per patient (by removing the samples with whole genome amplification) and removing the FFPE samples and samples indicated having MSI (Microsatellite Instability) using the MANTIS toolIntervar (version 2.0.2) was utilized to classify the variants as "Benign," "Likely Benign," "Uncertain Significance," "Likely Pathogenic," and "Pathogenic." Deleterious mutations were defined for exonic SNVs with "Pathogenic" or "Likely Pathogenic" labels, nonsense SNV-s and indels with "Pathogenic" or "Likely Pathogenic" labels. All the ERCC gene family mutants represented in the figures are deleterious mutations.For genotyping of the cell line samples, variants were defined as deleterious if the column “isDeleterious” was indicated as “True” in the CCLE.mutations.csv data file.9 described single base substitutions (SBS) signatures, doublet base substitution (DBS) signatures and small insertion and deletion (ID) signatures. In this study we calculated the number of ID8 signatures since we previously found this signature most significantly associated with NER deficiency10. The identified matrix of ID signatures was downloaded from https://www.synapse.org/#!Synapse:syn12025148. ID mutations in each sample were classified into 83-dimensional indel catalog using the ICAMS R package11. The resulting matrices were used in a non-negative least-squares problem to estimate the matrix of exposures to mutational processes.Using techniques based on non-negative matrix factorization, Alexandrov et al.The ID8 signature extraction was performed the same way on the patient and cancer cell line samples.https://portal.gdc.cancer.gov/) for the patient samples, and The Fragments Per Kilobase of Transcript per Million Mapped Reads (FPKM) technique was used to normalize the data, and the data were log2-transformed using a pseudo-count thereafter.RNA expression data were downloaded from the TCGA data portal  and the TPM-normalized data were log2-transformed using a pseudo-count. For comparison with the TCGA-KIRC PTGR1 FPKM values, cell-line expression data in FPKM were downloaded from the CellMiner website (https://discover.nci.nih.gov/cellminer/).For the cancer cell line samples, the RNA expression data were obtained from the DepMap portal  has not benefitted from this therapeutic approach due to a lack of evidence for the presence of specific DNA repair pathway deficiencies. Here, we demonstrate that ccRCC harbors a therapeutically targetable DNA repair pathway aberration, nucleotide excision repair (NER) deficiency. ccRCC cell lines displayed robust signs of NER deficiency as determined by functional assays and some of these cell lines also displayed NER deficiency induced mutational signatures. These cell lines are also sensitive to irofulven, an abandoned anticancer agent that creates DNA lesions which can only be repaired by the NER pathway. We estimate that up to 10% of ccRCC cases may respond to NER-directed therapy with irofulven based on NER deficiency associated mutational signatures and PTGR1 expression levels, which is an enzyme required to activate irofulven.12. Drug sensitivity experiments from the NCI60 drug screening program reported that RXF393, a kidney cancer cell line, showed high sensitivity to irofulven (https://dtp.cancer.gov/services/nci60data/colordoseresponse/jpg/683863). Recently it was also reported that the ccRCC cell lines A498 and RXF393, also show significant sensitivity (IC50 ~ 20 nM) to a recently developed analog of irofulven13. We expanded these experiments to include a panel of seven kidney cancer cell lines  and low sensitivity (786O and 769P) to irofulven, in the non-malignant immortalized HK-2 kidney epithelial cell line. As mentioned above, this assay monitors the cells’ ability to remove UV-induced 6–4 pyrimidine-pyrimidone photoproducts (6–4PP). 6–4PPs can be removed by both GGR  and TCR (transcription coupled repair) pathways of NER and their removal is closely correlated with NER efficiency15. Using this assay, we found that surprisingly all five kidney epithelial cell lines, including the non-malignant HK2 cells, showed NER deficiency to varying degrees. In contrast, the control cell line (the NER proficient H460 cell line) was NER proficient and efficiently removed the 6–4PP photoproducts by 7 h post UV irradiation  increases its efficacy by inducing an unstable cyclohexadiene intermediate by reduction at the C8 position16. Here we provide direct functional evidence that the presence of PTGR1 is a key determinant of drug response by demonstrating that suppression of PTGR1 expression in an otherwise irofulven sensitive, NER deficient cell line renders those cells irofulven resistant. A heterozygous truncating mutation in ERCC3 (p.R109X) was previously introduced by CRISPR editing into the HMLE cell line17. The mutation rendered these cells sensitive to irofulven. We depleted PTGR1 in these cells with siRNA and found that depletion of PTGR1 rendered those cells resistant to irofulven , all other kidney cancer cell lines expressed PTGR1 and a trend could be observed between PTGR1 expression levels and irofulven sensitivity, although the limited number of cell lines did not allow establishing a statistically significant correlation  that showed NER deficiency by the functional assay also had a high level of ID8 deletions . We identified four cases with predicted deleterious mutations in ERCC6, three cases with predicted deleterious mutations in ERCC2, one case with a predicted deleterious in ERCC3, and one case with multiple NER gene mutations . These cases with NER gene mutations showed a statistically significant association with higher ID8 events . We previously established that more than five ID8 deletions detected in WES analysis indicates the likely presence of NER deficiency in bladder cancer10. We used the same threshold in kidney cancer and found that 43 out of 289 cases (~ 15%) had ID8 deletion numbers consistent with NER deficiency.We have shown previously that NER deficiency associated mutational events are enriched in actively transcribed genomics regions, therefore whole exome sequencing data can be used to detect likely NER deficient casesPTGR1 using TCGA RNA-seq data and compared those to the PTGR1 expression levels detected in the ccRCC cell lines. 36 of the 43 cases with > 5 ID8 deletions had the same or higher level PTGR1 expression as the A498 cell line that had the highest level of PTGR1 expression at the protein level and also had a high level of irofulven sensitivity  indicated the presence of both NER deficiency and significant apy Fig. B.Tumor DNA repair deficiency can be therapeutically targeted by synthetic lethal–based strategies. The success of the synthetic lethal approach is dependent on the identification of the relevant DNA repair pathway deficiency in clinical tumor specimens and the availability of a therapeutic agent that can specifically target such DNA repair–deficient cells. ccRCC in general has not benefitted from this therapeutic strategy because the presence of specific DNA repair pathway deficiencies has not been demonstrated in this tumor type. Here we show that NER deficiency can be detected in several ccRCC cell line models by functional assays and that a subset of clinical ccRCC cases have mutational features consistent with NER deficiency.ERCC2 helicase inactivating mutations10. We were particularly encouraged by the fact that ccRCC is one of the solid tumor types where the highest proportion of cases harbor this mutational signature and also that the frequency of ID8 deletions is also among the highest across the various solid tumor types9. In our analysis, ID8 was present both in some of the ccRCC cell lines and patient biopsies at levels detected in ERCC2 mutant bladder cancer cases. Furthermore, this mutational signature was also associated with either the presence of functional NER deficiency (cell lines) or inactivating mutations in NER genes (TCGA biopsies). This suggests that the ERCC2 mutation associated mutational signature we previously described may also indicate the presence of NER deficiency in ccRCC. The ID8 signature may, however, be caused by other mechanisms as well. A rare somatic mutation of topoisomerase II alpha was previously described to be associated with this signature before18. This may lead to an overestimation of truly NER deficient ccRCC cases.Since currently there are no functional or IHC assays available to reliably identify NER deficiency from clinical specimens, we used a specific mutational signature (ID8) associated with 19. Cells with inactivated transcription couple repair (TCR) or NER show an approximately 100-fold increased cytotoxic activity relative to normal cells with active DNA repair12. This suggests an exploitable therapeutic index for NER deficient cases. However, although well-tolerated, irofulven showed only modest clinical benefit as a single agent in phase I/II clinical trials across a variety of tumor types22 including a phase II trial for advanced renal cell carcinoma23. The failure of irofulven to show clinical benefit in this limited set of thirteen renal cell carcinoma patients may be due to the fact that patients were not selected according to the two criteria for irofulven activity: NER deficiency and the expression of PTGR1. According to these criteria, we estimate that approximately one in ten ccRCC patients may respond to irofulven. Therefore, in the case of thirteen unselected patients, it is not surprising that no NER-deficient cases were included. In a basket trial of irofulven/cisplatin combination therapy, four ccRCC patients were included and one experienced a significant partial response24.Our interest in the diagnostic detection of NER deficiency in ccRCC was inspired by the remarkable sensitivity of some of the commonly used ccRCC cell lines to irofulven, which is a semisynthetic, DNA alkylating agent that is a derivative of the fungal sesquiterpene, illudin S4. Early disappointing phase II clinical trials with single agent platinum or platinum combination therapies suggested limited use for this form of treatment in ccRCC26. In these trials a 5% objective response rate was observed with limited cohort sizes (~ 20 patients each). The low objective response precluded the development of larger, biomarker directed trials for platinum. Therefore, currently we do not know whether the likely NER deficient subset of patients would benefit from platinum-based therapy. It is notable, however, that one of the NER deficient, irofulven sensitive cell line in our analysis, RXF393, has been reported to be as sensitive to platinum treatment as the NER deficient breast cancer cell line, MDA-MB4686 or the homologous recombination deficient breast cancer cell line, MDA-MB436 .NER deficient cells are particularly sensitive to platinum treatmentPTGR1, the enzymatic activator of irofulven, is a NADPH-dependent alkenal/one oxidoreductase with high expression levels in the kidney, the tissue from where it was originally cloned27. The significant expression of this enzyme in the majority of kidney cancer cases is perhaps a result of the retention of a key pathway for kidney metabolism of leukotrienes. Since PTGR1 is not expressed in every cell type, , several normal tissues are not affected by the toxicities of irofulven treatment due to lack of enzymatic activation of the drug. This significantly contributes to the good tolerability, including its lack of hematological toxicity20, while potentially retaining the majority of NER deficient kidney cancer cases as a potential therapeutic target. Downregulation of PTGR1 will likely render tumor cells resistant to irofulven, therefore the expression of this enzyme should be verified in the diagnostic setting before administration.28. Therefore, it is possible that the NER deficiency we detected in several kidney epithelial cell lines may in fact be the result of culturing those cells under conditions, in this case normoxia, that would lead to the inactivation of NER. This would also suggest that under hypoxic conditions, when the risk of the various forms of DNA damage is increased, NER would be reactivated. In theory, if the underlying molecular mechanisms can be identified, then inactivating NER by such an oxygen sensing mechanism could also sensitize a wider range of ccRCC cases to NER deficiency targeted therapy. Oxygen concentrations can be increased in the cancer tissue in the clinical setting by carbogen inhalation as it was clinically evaluated in the context of improving radiotherapy29. In principle a similar strategy can be used to induce NER deficiency in ccRCC.One of the unexpected results of our experiments was the fact that all kidney epithelium cell lines, including a non-malignant cell line, showed signs of NER deficiency. It was shown before that hypoxia-inducible factor-1α regulates the expression of nucleotide excision repair proteins in keratinocytesEven without further induction of NER deficiency, we estimate that about 10% of ccRCC cases may be responsive to irofulven therapy due to persistent NER deficiency and high PTGR1 expression. A biomarker directed clinical trial could identify this population and evaluate the efficacy of irofulven or its more recent analog LP184, which has just received its investigational new drug approval from FDA, in such patients.Supplementary Information.


Upper Palaeolithic sites in southwestern France attributed to the Upper Gravettian and the Solutrean yielded sub spherical gravels with a highly shiny appearance that have intrigued researchers since the 1930s. In this work, we analyze specimens from five sites, including the recently excavated Solutrean site of Landry, to establish whether their presence in archaeological layers and peculiar aspect are due to natural processes or human agency. We study the spatial distribution of gravels at Landry and submit archaeological gravels from the five sites, natural formations, Landry sediment sieving, and polishing experiments with a rotary tumbling machine to morphometric, colorimetric, microscopic, and textural analyses. Our results indicate the lustrous gravels found at the five sites result from deliberate selection and suggest their shiny appearance is the consequence of human agency, possibly resulting from prolonged contact with a soft material such as animal skin. Ethnographic accounts indicate that these gravels may have been used for magico-religious ritual purposes , in games, as elements of musical instruments, and as items serving other social and personal purposes. We argue that these objects reflect a cultural innovation emerged during the Gravettian and continued into the Solutrean. Upper Palaeolithic sites in southwestern France attributed to the Upper Gravettian and the Solutrean have yielded sub spherical gravels with a highly shiny appearance that have attracted the attention of Palaeolithic archaeologists since the 1930s . PeyronyAnswering these questions has been made difficult by the fact that these gravels were uncovered during excavations for which we lack relevant contextual data. We do not know, for example, whether gravels were systematically collected nor do we have precise information on their spatial distribution and eventual association with other archaeological remains. The discovery of about a hundred lustrous gravels on the recently excavated Solutrean site of Landry  , and the application of new analytical methods make it possible to approach the question of the origin and eventual anthropogenic modification of these objects in new ways.In order to address this topic we combine in our study four approaches: 1) spatial analysis of lustrous gravels and their relationship with other archaeological remains unearthed at Landry, 2) morphometric and colorimetric analysis of gravels discovered at this site, four other Upper Palaeolithic sites from Dordogne, and gravels from geological deposits accumulated by known sedimentary processes, 3) surface texture analysis of lustrous and non-lustrous gravels from archaeological sites and natural accumulations as well as gravels experimentally polished to mimic natural and anthropogenic actions, 4) microscopic analysis of the natural, archaeological and experimental gravels, and 5) a survey of ethnographically-attested uses of gravels and small pebbles. The objective of the spatial analysis was to establish whether the gravels’ distribution was random or not, and connected with that of other archaeological remains. The morphometry and colorimetry of the lustrous and non-lustrous gravels aimed at verifying whether the former represents a selection of the gravels naturally available on the site. The textural analysis of archaeological, natural and experimentally polished gravel was intended to document and quantitatively compare their wear patterns to establish whether shine on archaeological objects could be found in nature, and which experimental polishes generated by a tumbler and a metallographic polisher better mimic archaeological wear. The microscopic analysis, conducted at different magnifications, was aimed at a more finely tuned characterization of natural, archaeological and experimental gravel’s surface conditions and intra-site comparisons.The last decade has seen a substantial development of studies applying confocal microscopy and roughness parameters to archaeological artifacts with the objective of understanding the function of tools, the intensity of use, and taphonomic processes affecting the artefact after its production and use –17. The Lustrous gravels are reported from four sites of the South-West of France: Fourneau du Diable, Casserole, Pech de la Boissière and Laugerie Haute . The recMB excavated in 2011, Landry is an open-air site located close to Boulazac, Dordogne, France . The sitSpatial analysis identified five distinct concentrations of artefacts  spread oThis site is located on the right bank of the Dronne River, near Brantôme, Dordogne, France . It consLocated near Carsac-Aillac, Dordogne, France , the Pec14C AMS on burned bones to 20.5–23.3 cal BP  was performed . The search was restricted to the 186 Standard Cross Cultural Sample (SCCS) cultures, a subset of eHRAF with the most detailed and robust ethnographic descriptions. This yielded 23022 paragraphs across 175 cultures. The eHRAF Outline of Cultural Materials (OCM) has subject codes tagged to each paragraph. The top five for this search were Sacred Objects and Places, Prayers and Sacrifices; Mythology; Literary Texts; Spirits and Gods. Utensils, Tools, Weapons, and Burial Practices were in the top 20. Because each paragraph is usually tagged with multiple OCM codes, we developed a bespoke coding scheme for specificity.To explore historical and contemporary uses of small stones similar to the lustrous gravels examined, FMJ used the electronic Human Relations Area Files (eHRAF), a collection of full-text, subject-coded global ethnographic materials  was read and coded for how pebbles were used. FMJ noted if pebbles were present in multiples of four or more at one time , and any information about bags or other carrying items. We did not code keyword mentions that were irrelevant to our study, especially where stones were described as larger than a fist  or when the keywords were associated with technologies not relevant for our study . Other rare mentions, such as being described in fantastical folklore, or stones referring to the seeds of fruit, were also not assigned to a code.Lustrous gravels are concentrated in squares R11, R12, and Q11, sector 3, and to a lesser extent in adjacent squares . A few gThe spatial distribution of flints and metamorphic rocks appears strongly correlated with that of the lustrous gravels . As indiMorphometric analysis identifies significant differences (p < 0.05) see  between The archaeological lustrous gravels are characterized by homogeneous surfaces on which depressions are either absent, or rare and of dimensions not exceeding 10μm. Curved micro-cracks corresponding to incipient fracture cones are also discernable . The edgAt higher magnification, the surface of all natural gravels and those recovered in the sieving at Landry retain a rough appearance associated with micro concavities ranging from 20 to 100 μm in length . Rare luRoughness parameters identify significant differences (p < 0.05) see  between The principal component analysis of all recorded parameters  highlighAs expected, all experimental treatments significantly reduced the roughness of the gravels. The tumbling process, entailing impact of the gravels on each other and their contact with the skin, causes a gradual flattening of the surface resulting in a shiny appearance . AbrasioUses were first documented at a low level (e.g. “as luck charm”), and then on completion of the sample, our coding system aggregated initial categories into a higher level , as described in In the full sample, ritual uses  were the most commonly described (n = 44), followed by games (n = 25) and music (n = 18). A range of other uses accounted for the remainder. Hunter-gatherer societies differed in the most common use  but followed the trend for music (n = 11), ritual (n = 10) and games (n = 9) as most common . Eating/Polished pebbles have been found repeatedly at archaeological, including ancient, sites , 68. FewThe geoarchaeological study of the Landry site indicates that the spatial distribution of archaeological remains has not been marginally affected by post-depositional disturbances . Landry At all the analyzed sites, what makes the gravels peculiar is also their shiny appearance. Since the mode of formation of the sedimentary layers is not different at Landry between the concentrations and the areas outside them, there is no reason for thinking that the site formation processes could have affected the gravels inside and outside the concentrations in a different way and create a gloss only on the former. No evidence of chemical alteration or a similar gloss is observed on the remainder of the archaeological remains found inside the concentrations of artifacts , 22. TheIn summary, the roughness analysis of natural, experimental and archaeological gravels supports the hypothesis that after being selected by their size and shape, the archaeological gravels were subjected to a human action that has strongly polished them.The differences in roughness observed between the lustrous gravels from Landry and other archaeological sites may be due to the fact that 1) only the most lustrous gravels were collected in early excavations; 2) at Landry non- or low-gloss gravels were naturally present in archaeological concentrations, which created a sample that included both lustered and non-lustered gravels; 3) the activity that lustered the gravels at Landry was not as extensive as at other sites; 4) their mineralogical composition is different, the Landry gravels could not reach the degree of lustering observed at other sites, where the gravels are composed of other rocks, while being subjected to the same modifications. Selective collection of gravels from older excavations is possible, but two of the collections examined were from recent excavations in which the sediment was systematically sieved with water. The degree of luster of the gravels from these sites is not substantially different from that observed in the ancient collections, which contradicts the hypothesis of selective gathering. The possibility that the rock of which they are composed may determine the lower luster of the Landry gravels seems partly contradicted by the fact that the experimentally tumbled gravels, composed of quartz, attained a degree of luster similar to that observed on the lustered gravels from other archaeological sites. Although it is difficult to rule out the possibility that some of the discarded factors may be a contributing factor to the differences in luster between Landry and the other sites, the most likely hypothesis appears to be that the luster on the Landry gravels is either the result of the same action to which the gravels from the other sites were subjected, but for a shorter period of time, or the result of a slightly different action.Microscopic analysis of archaeological, natural and experimental gravels provides essential additional elements for documenting and interpreting the differences between these groups. On the one hand, it confirms that the wear pattern present on archaeological gravels is absent on natural gravels, which further supports its anthropogenic origin. On the other hand, it reveals that the shine observed on the archaeological gravels, similar in terms of roughness values, include in reality at least two major categories of wear, one characterized by the presence of striated areas and the other by their absence, incipient micro fractures and large homogeneously lustered areas with rare crisscrossing striations. The first category results from directional rubbing of the gravels against a surface rich in abrasive particles of variable size, the second from repeated contact with objects of same hardness than the gravels in an environment with very few abrasive elements. This last wear pattern is comparable to that observed on tumbled gravels, which show, however, as documented by texture analysis, a slightly less developed shine. These two wear patterns are consistent with 1) intentional rubbing of the gravels against a variety of slightly abrasive materials with or without the adding or abrasive particles, and 2) prolonged tumbling of numerous gravels in leather containers to polish or transport the gravels. The differences observed in the first wear pattern can be attributed to the materials against which the gravels were rubbed at different archaeological sites, the motions involved and the state of preservation of the wear pattern. The local disappearance of striations on some gravels may result from transport of previously intentionally polished gravels or taphonomic processes. The variability observed in the second wear pattern may have resulted from the type and treatment of the soft materials used for the containers in which the gravels were tumbled or transported.The long duration of the tumbling experiments necessary to produce a luster similar to the one observed on the archaeological specimens seems to contradict the hypothesis that sporadic contact with soft material or human skin, for example when handling the gravels, may have produced such a developed luster. In summary, results from textural and microscopic analyses concur to identify the lustrous appearance of the archaeological gravels as resulting from intentional polishing, producing oriented striations, probably aimed at making the gravels shinier, and their transport. This hypothesis is consistent with results of texture analysis indicating that although not significantly different from that recorded on the experimentally tumbled gravels, the luster on most archaeological collections reach substantially higher degrees.These results do not allow us to identify with certainty the function for which the gravels were intended. The luster may, for example, be the result of polishing by abrasion to increase the gravels’ lustrous appearance tumbling in leather containers to obtain a similar effect or transporting the gravels from one site to another. Intentional polishing may have been instrumental, for example, to create visually catching ornaments, as suggested by Peyrony or to use the gravel to play. It is less probable that gravels were intentionally polished with the aim of putting them in a container to produce a rhythmic sound. The actual function may have produced little or no luster.It is worthwhile for this reason to turn to the ethnographic and archaeological record. The former may inform us on the uses of gravels in different traditional human cultures. The latter may identify cases in which contextual information would allow us to understand the function that gravels had in past populations. Both records open the possibility of analyzing in the future, with the same methodologies applied in our study, ethnographic and archaeological gravels and compare the results of these analyses with ours.Our ethnographic survey shows that the most common functions for small pebbles are magico-religious ritual uses, and as game pieces. Worldwide, pebbles are used as talismans, amulets, and charms –72 see .While many of the ethnographically attested functions imply a choice of gravels on the basis of size, shape, weight and raw material criteria, exact remarks on size were uncommon in the sources. Anthropologists occasionally noted qualitative features such as color or shape e.g. “marble-like stones” , “preferThe results of our study indicate that there are means to distinguish objects modified by natural processes from artifacts minimally modified by humans. Use-wear analysis has been widely applied to prehistoric artefacts to identify their function. However, this approach has been implemented on artefacts recognized as the outcome of human agency. In our case, instead, we have faced the challenge of identifying such agency exclusively from surface modification. The chosen means to pursue this endeavor involved evaluation of contextual data, creation of natural and experimental frames of reference, and characterization of natural, experimental and archaeological material using multiple, adapted analytical tools. These analyses leave no doubt: the lustrous gravels discovered in several Gravettian and Solutrean sites in southwestern France were lost or abandoned in these sites after having been selected on the basis of size and shape criteria and having been involved in specific activities that produced their lustrous appearance. These gravels were not discovered in older Gravettian layers during the numerous excavations conducted in the region despite the fact that many of these excavations were conducted in recent times, with modern excavation techniques and by archaeologists aware of the possibility of discovering glossy gravels in Upper Palaeolithic archaeological layers. This leads us to conclude that the activity in which the gravels were involved, which is impossible to identify precisely but could involve a long-time use or curation in a leather bag, reflect a cultural innovation that developed during the Gravettian period and continued into the Solutrean period at the least.S1 TableRegion, Culture, Outline of World Cultures (OWC) code, Subsistence Type*, Source, Page, Focus Date, Outline of Cultural Materials (OCM) Subject, and Paragraph are taken direct from eHRAF. Focus Date is the earliest given in the source, or when not given, the publication date. Use denotes a low-level classification of the use. Coded Use is the final code given in the text. Multiples refers to if pebbles were present in multiples of four or more at one time . Notes carries any pertinent information about bags or other carrying items, and qualities of the stones . Subsistence Type: AP Agro-Pastoralists, HG Hunter-Gatherers, HC Horticulturalists, IA Intensive Agriculturists, OSC Other Subsistence Combinations, PA Pastoralists, PHG Primarily Hunter-Gatherers.(XLSX)Click here for additional data file.S2 Tablehttps://osf.io/9wpn7/.R script and data to produce Figs (XLSX)Click here for additional data file.


Jarosch et al. Jarosch et al. have deeply characterized immune cell infiltrates in gastrointestinal (GI) biopsies from individuals with GI graft-versus-host disease (GI-GvHD) using single-cell RNA sequencing and ChipCytometry. Individuals with severe GI-GvHD demonstrated increased clonally expanded cytotoxic CD8 T cells in GI biopsies. Graft-versus-host disease (GvHD) is an alloreactive T cell-mediated systemic inflammatory disease and remains a major obstacle in allogeneic hematopoietic stem cell transplantation (aHSCT). The gastrointestinal (GI) tract is often targeted by GvHD, and severe GI-GvHD tends to have a poor prognosis.Here, Jarosch and colleaguesEvaluating infiltrating immune cell populations using scRNA-seq, the authors observed in individuals with aHSCT compared to control cases increases in monocytes, CD4 memory cells, and CD8-activated T cells, as well as reductions in B cells, plasma cells, and mast cells. Among aHSCT individuals, CD8-activated T cells and regulatory CD4 T cells (Tregs) were increased in samples from individuals with severe GvHD compared to those with mild GvHD. To complement these findings, the authors employed ChipCytometry, a multi-parameter tissue section imaging method, and found that severe GvHD samples indeed showed significantly more infiltration of CD8 T cells compared to mild GvHD samples although there was no clear increase in Treg infiltration. T cell receptor (TCR) repertoire analysis showed that this expansion of CD8 T cells was clonal in aHSCT individuals compared to control cases. The degree of increased clonality and consequently decreased TCR diversity were significantly associated with GvHD severity. Importantly, these findings were observed at multiple time points after aHSCT.Immune cell phenotypes of GI biopsies in aHSCT individuals are heterogeneous because of complex clinical backgrounds. It is necessary to carefully consider the potential for confounding effects of the state of immune reconstitution when analyzing biopsies from different time points. Another potential modulator of immune cell phenotypes from GI biopsies includes the use of antibiotics, which leads to dynamic alterations of the microbiota composition. To investigate these factors, the authors grouped affected individuals into antibiotic-exposed and non-antibiotic-exposed groups. In the non-antibiotic-exposed group, severe GvHD cases showed a trend toward higher Treg infiltration compared to mild GvHD cases. Interestingly, this association was not observed in the antibiotic-exposed group, suggesting that Treg infiltration was affected by exposure to antibiotics or subsequent alterations to the microbiota composition.FOXP3, a hallmark of Tregs. One of them showed significantly higher gene signatures associated with Treg functions, whereas others showed an effector T cell-like signature, indicating reduced Treg functionality. The authors next investigated whether specific bacterial taxa were linked to differentiation of functional Tregs using 16S rRNA-seq. They found that the classes Clostridia and Bacteroidia were associated with functional Treg differentiation. Intestinal Clostridia and Bacteroidia are the primary producers of short-chain fatty acids, including butyrate and propionate, which are known to introduce Tregs in the GI tract,,Importantly, the authors identified three distinct Treg clusters by examining expression of Finally, to investigate how therapeutic interventions targeting the microbiome composition could act on the Treg/CD8 T cell balance, the authors evaluated GI biopsies collected from one individual with severe GvHD before and after undergoing fecal microbiota transplantation (FMT). This individual experienced a significant increase in microbial diversity and complete response of GI-GvHD at 9 months of follow-up. Interestingly, treatment with FMT led to a trend of decreased CD8 T cell infiltration along with increased Treg infiltration. While results of a single case study are not definitive, these findings suggest that the immunosuppressive potential of Tregs can be tuned by microbiota composition and altered abundances of Tregs as well Treg functionality and suppression of CD8 T cells.+ GM-CSF+ T cell subset that contributes to pathologic damage in the GI tract.scRNA-seq is a next-generation sequencing method that enables unbiased and high-resolution genome-wide assessment of individual cells. This technique is becoming the gold standard for investigating cell heterogeneity. However, scRNA-seq for analysis of biopies from individuals with GI-GvHD has been understudied thus far. One previous study employing scRNA-seq in a murine model of GvHD identified an intestinal CD4In conclusion, this study has uncovered new insights regarding GI tissue infiltrating immune cell populations in individuals with GvHD utilizing a combination of cutting-edge technologies. Clonally expanded CD8-activated T cells in the GI tract appear to be important mediators of severe GI-GvHD. However, how clonally expanded CD8 T cells induce severe GI-GvHD and how Tregs are linked to CD8 T cells are still unclear. In addition, the impact of the microbiota or microbe-derived metabolites on GvHD severity has not yet been completely explored. To elucidate these points, further studies will be necessary, including large prospective cohort studies and well-designed animal models.


Bladder cancer (BLCA) is considered as a highly prevalent disease that is strongly associated with elevated morbidity, mortality, and cost. Strategies designed to be targeting critical components and processes orchestrating BLCA cells’ evolutionary trajectories, towards metastasis and chemoresistance, are necessitated to be promptly developed, and successfully pass the proof-of-principle tests in pre-clinical models and clinical trials. To this direction, DepMap and PRISM project-derived findings, combined with transcriptomics and epigenetic data analysis, have herein unveiled the cardinal roles of microtubule dynamics in the survival and growth of BLCA cells. Most importantly, they can foresee the therapeutic promise of pathway’s targeted perturbation, with paclitaxel single scheme and paclitaxel-containing drug-cocktails opening new therapeutic windows for the disease.Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy. FGFR3, H-Ras, and mTOR pathway member genes. In contrast, the progression to high-grade invasive urothelial carcinoma is correlated with loss of function of p53 and Rb tumor-suppressor network genes  primers . Accordihttps://depmap.org/portal, accessed on 23 March 2023). Gene effect difference was calculated as a T-statistic from the DepMap portal, as described in [https://depmap.org/repurposing, accessed on 27 March 2023) includes data from 4518 compounds screened against 578 cell lines [Genome-scale, CRISPR-Cas9-mediated screening has been previously described ,13. The ll lines . In contll lines . The celll lines .Normalized bigWig files containing ATAC-seq. reads from bladder, breast, lung, uterine, cervical, and kidney cancers were downloaded from the NCI portal, as described in . Normalip-value < 0.05 was considered statistically significant.Statistical analyses for DepMap and PRISM data were generated and obtained from the DepMap portal. Using GraphPad Prism v.9, the statistical significance of differences in sqPCR was determined using a one-way ANOVA with Tukey’s test for multiple hypothesis correction. A Given the poor prognoses of patients suffering from BLCA, we sought to identify gene dependencies and small molecules to which BLCA cells are preferentially sensitive by interrogating the DepMap project databases ,19,20,21We analyzed the results from the PRISM repurposing drug screens to further identify therapeutic agents that could selectively target BLCA cells ,14. We fKATNB1 as a readout, the DepMap data suggest that BLCA cells are presented as the most sensitive to KATNB1 perturbation (sgRNA targeting) compared to all other cell lines analyzed , we further investigated the dependency of BLCA cells on microtubule assembly-associated proteins compared to all other cell lines . Using Kanalyzed A.KATNB1 will rely on a specific chromatin state that will facilitate abundant transcription of KATNB1 compared to other cancer cells. We systematically investigated the open chromatin at the KATNB1 locus to test our hypothesis using normalized ATAC-seq. data from a large collection of primary human cancers [KATNB1 gene locus compared to the other cases examined .Our analysis of the DepMap data and chromatin accessibility suggests that BLCA cells are sensitive to microtubule dynamics perturbations. To further confirm our findings, we used a collection of BLCA cell lines derived from different disease stages and treated them with a dose range of paclitaxel for 24 and 48 h. RT4 cell line originates from a grade I carcinoma, RT112 from grade II, T24 from grade III, and TCCSUP from grade IV. RT4 and RT112 have wild-type Paclitaxel inhibits microtubule depolymerization, which is critical for mitotic spindle assembly. To test the effects of paclitaxel on the cell cycle, we treated RT4, RT112, T24, and TCCSUP cell lines with a dose range of the drug for 24 h. We also analyzed the effects using Fluorescent Activated Cell Sorting (FACS)-based cell cycle analysis. We found that a low concentration of paclitaxel-induced a robust cell cycle arrest at the G2/M phase in all cell lines analyzed, independently of their malignancy grade or p53 status .To further investigate the effects of paclitaxel on survival and growth, we treated our panel of BLCA cell lines with a dose range of the drug for either 24 or 48 h and monitored their survival and growth using an MTT assay. Consistent with the effects of paclitaxel on the cell cycle, drug treatment also induced growth inhibition. Although the results varied among the four cell lines tested, all cells exhibited significant growth inhibition 48 h after treatment, especially with the higher drug concentrations C,F,I,L.Therefore, our in vitro study is in accordance with the in silico analysis regarding BLCA cells’ sensitivity to microtubule dynamics inhibition.To investigate the induction of caspase-mediated apoptosis in BLCA cells following exposure to paclitaxel, we treated the panel of RT4, RT112, T24, and TCCSUP cell lines with varying doses of the drug for 24 h. We analyzed the cleavage profiles of Caspase-8, -9, -3, and PARP proteins using Western immunoblotting analysis. Our results clearly demonstrate that paclitaxel treatment induces cleavage of the caspase repertoire and the typical PARP substrate A. AlthouFASL, FAS, CIAP-1, CIAP-2, XIAP, and SURVIVIN  RT-PCR as an orthogonal assay. We analyzed the expression of several genes encoding proteins involved in apoptotic cell death . RT-sqPCSURVIVIN . Most imtrations B–D. TherOverall, our immunoblot and RT-sqPCR results so far demonstrate that paclitaxel treatment induces a robust apoptotic program in all BLCA cell lines tested, further confirming the sensitivity of BLCA cells to microtubule dynamics perturbation.BCL2 locus and sustain its expression [BCL2 locus in BLCA cells that could suggest an oncogenic addiction to BCL2 expression. To answer our question, we investigated the open chromatin status of BLCA compared to breast, uterine, cervical, lung, and kidney cancers. We found that BLCA cells have preferentially open chromatin at the BCL2 locus by plotting the normalized ATAC-seq. reads for each type of cancer  nor lung cancer demonstrated such a signal, with kidney cancer serving as a negative control  even at the lower concentration(s) of paclitaxel used in all cell lines tested  or total protein levels observed upon paclitaxel treatment  and TCCSUP (IV), we did not detect any phospho-specific, or total, p53 protein forms before and after paclitaxel administration, which confirms the lack of protein due to damaging mutations A. To ourreatment A. This iPUMA and NOXA. Our analysis showed no transcriptional upregulation of the PUMA or NOXA genes, further confirming the absence of a p53 activation program following paclitaxel treatment of BLCA cells . We found no association between the expression of TP53 and the sensitivity of the cells to paclitaxel (TP53 gene in the BLCA cell lines used in the PRISM screen based on data from the Cancer Cell Line Encyclopedia (CCLE) [TP53 and the response of BLCA cells to paclitaxel  . We cateclitaxel B, in linATM, ATR, CHEK1, and CHEK2 genes (mRNA levels). Specifically, we found a strong and significant association between the expression of ATM and CHEK2 kinases and the sensitivity of BLCA cell lines (n = 24) to paclitaxel  scored as the top dependency  CRISPR screens has been validated in numerous publications . At the We first focused on the genetic dependencies specific to BLCA cells and the biological processes they control. Many of the genes scored as dependencies are involved in microtubule dynamics, revealing the importance of this biological process for the survival of BLCA cells. Microtubules are dynamic structures that play crucial roles in cell homeostasis and, most importantly, cell division, as they form the mitotic spindle that facilitates chromosomal segregation during mitosis and meiosis ,37. DuriPaclitaxel binds to beta-tubulin and blocks microtubule depolymerization. This stabilizes microtubules and prevents the proper execution of anaphase and telophase, leading to errors in chromosomal segregation and, ultimately, cell death . Thus, wTP53 gene. Since we focused our analysis on the early stages of cell response to paclitaxel (24 h), it is possible that p53 is activated at a later time point and its status affects the later stage(s) of the response. However, since we detected activation of apoptosis, as determined by cleaved Caspase-8, -9, and -3, without transcriptional upregulation of the p53 targets PUMA and NOXA genes, it is evident that paclitaxel can induce apoptosis in BLCA cells without activating the p53 pathway. Moreover, when looking at the response to paclitaxel in the large collection of BLCA cell lines used in the PRISM project, we found no association between the response and the status of the TP53 gene. This further confirms the p53-independent nature of the response.Paclitaxel treatment can cause DNA damage and activate repair mechanisms to fix the damage and maintain genomic integrity. However, if the DDR is impaired or the DNA damage is too severe, cells can undergo apoptosis. Given the extremely high sensitivity of BLCA cells to paclitaxel, we tested markers of DDR in our system. We found a robust upregulation of the DNA DSB marker phospho-H2AX, but to our surprise, we did not observe an activation of p53, at least in the two wild-type cell lines for the BCL2 locus   E. This sThe strong correlation between sensitivity to paclitaxel and expression of DNA DSB repair enzymes, such as the ATM/ATR and CHEK1/2 kinases, underscores the need to further investigate the combination of paclitaxel with ATM/ATR inhibitors as novel therapeutic regimens in BLCA. Similarly, the combined administration of paclitaxel with PPARG inhibitors may also benefit BLCA clinical treatment. Most importantly, our major finding that the growth inhibitory effect of paclitaxel on BLCA cells is independent of p53 activity strongly suggests that paclitaxel can treat a wide range of BLCA types independently of their p53 genetic status.Our study combined a variety of in silico and in vitro approaches in a collection of human BLCA cell lines representing different stages of the disease. It demonstrated the importance of microtubule dynamics for BLCA cell survival and growth. It also unveiled the therapeutic potential of disrupting the pathway’s integrity and functionality independently of p53 oncogenic activity .


Similar substantial variability was seen in the other parameters, including baseline DNA damage, plating efficiency, and ploidy. In the CRISPR-modified cell lines, residual DSBs were good predictors of cell survival , as were induced levels of DSBs . However, amongst the normal and cancerous cells, none of the measured parameters correlated strongly with MID (R2 < 0.45), and the only metrics with statistically significant associations are plating efficiency  and percentage of cell in S phase . While these data provide a valuable dataset for the modelling of radiobiological responses, the differences in the predictive power of residual DSBs between CRISPR-modified and other subgroups suggest that genetic alterations in other pathways, such as proliferation and metabolism, may have a greater impact on cellular radiation response. These pathways are often neglected in response modelling and should be considered in the future.Intrinsic radiosensitivity is a major determinant of radiation response. Despite the extensive amount of radiobiological data available, variability among different studies makes it very difficult to produce high-quality radiosensitivity biomarkers or predictive models. Here, we characterize a panel of 27 human cell lines, including those derived from lung cancer, prostate cancer, and normal tissues. In addition, we used CRISPR-Cas9 to generate a panel of lines with known DNA repair defects. These cells were characterised by measuring a range of biological features, including the induction and repair of DNA double-strand breaks (DSBs), cell cycle distribution, ploidy, and clonogenic survival following X-ray irradiation. These results offer a robust dataset without inter-experimental variabilities for model development. In addition, we used these results to explore correlations between potential determinants of radiosensitivity. There was a wide variation in the intrinsic radiosensitivity of cell lines, with cell line Mean Inactivation Doses (MID) ranging from 1.3 to 3.4 Gy for cell lines, and as low as 0.65 Gy in Lig4 The intrinsic radiosensitivity of normal and tumour tissues is a major determinant of the outcome of any radiation-based treatment. A significant correlation between the intrinsic radiosensitivity of tumour cells, measured by in vitro clonogenic assays, and patient response to radiotherapy has previously been demonstrated ,2. The aCurrently, radiotherapy, for a given cancer type, is typically given with a standard dose determined from clinical experience and population-level clinical trials. However, cancer is a highly heterogeneous disease, driven by numerous genetic alterations which significantly affect radiosensitivity. This genetic variability is reflected in estimates which suggest that cancers at the same site which receive the same treatment may vary in radiosensitivity by 25% or more ,4. As a The clonogenic assay has been the gold standard method for measuring cellular radiosensitivity, providing a large amount of useful data. However, most of these studies were completed in different conditions and by different investigators, giving substantial variability in reported survival values, as elegantly demonstrated by Nuryadi et al. in the A549 cell model . This enFurthermore, the clonogenic assay has limitations, including the extended time required for colony growth , and the low efficiency of clone formation in certain cell lines, which can make this assay unsuitable for such cells. This highlights the need for the application of new rapid predictive assays and models of radiation response if intrinsic sensitivity is to be translated as a clinical tool.The other obvious candidate as a radiosensitivity marker is DNA double-strand breaks (DSBs). Their relationship to clonogenic survival has been previously studied, and some investigators found correlation between the two assays ,9,10,11,The aim of this study was to characterize radiosensitivity across a diverse panel of human cell lines. This combined clonogenic assays with measuring different biological features as surrogate markers of radiation response, including induction and repair of radiation-induced DSBs, cell cycle distribution, and ploidy. This was first performed in a controlled panel of cell lines with known DNA repair defects introduced using CRISPR-Cas9. This was then expanded into a more diverse panel of human cells with different genetic backgrounds. This enabled the evaluation of the predictive power of these baseline features, while at the same time beginning to build a robust dataset with reduced inter-experimental variabilities to support future modelling.Radiation survival curves for CRISPR-modified cell lines with known DNA repair defects are shown in Additionally, cellular DSB repair characteristics were examined by immunofluorescence staining of 53BP1 for various times after 2 Gy exposure. The DSB repair curves are shown in A summary of CRISPR-modified cell features and radiation response parameters, such as SF2 (fraction of cells surviving exposure to 2 Gy), D10% (radiation dose required to inactivate 90% of cell population), MID (mean inactivation dose), and plating efficiency, as well as distribution of cells in each phase of cell cycle, is given in 2 = 0.61, p = 0.013), residual DSBs , and percentage of cell in S-phase of cell cycle . Plating efficiency is also statistically significantly correlated with MID , but this is dominated by the single outlying Lig4 KO cell line, and no significant trend is observed amongst other cell lines; however, and perhaps surprisingly, no significant correlation was seen between baseline DSBs and MID .To identify if any cellular features provided an effective surrogate for radiosensitivity, we evaluated the correlation between MID and baseline DSBs (indirectly measured by the number of 53BP1 foci in control samples), radiation-induced DSBs (indirectly measured by the number of 53BP1 foci 30 min after irradiation), residual DSBs (indirectly measured by the number of 53BP1 foci 24 h after irradiation), cell cycle distribution and plating efficiency. The resulting correlation for each feature is plotted in 2 = 0.78, p = 0.001)  H 15], s, s2 = 0.We then performed the same evaluation of radiation response in a diverse panel of human cell lines, including normal lines, and cancerous lines originating from the prostate and lung. Significant variation was seen in sensitivity across all cell lines, with MID ranges of 1.5–3.4, 1.9–3.1, and 1.3–3.4 Gy, for lung, prostate, and normal, respectively. Notably, the range of sensitivity in supposedly ‘normal’ cells is the greatest among all the groups of cell lines considered here.Radiation-induced DSB repair curves are shown in p = 0.87 and p = 0.83, respectively, 1-way ANOVA), although this is almost significantly different (p = 0.053 1-way ANOVA) in the prostate cancer panel. Additionally, considering repair rates, there is a significant difference (p = 0.014) in the repair rates of cell lines in the lung cancer group. In contrast, prostate and normal cell lines have statistically similar repair rates within their respective groups . The best fit repair rate for each of these two groups of cells are 0.21 ± 0.04 and 0.26 ± 0.04 h−1 for prostate and normal cells, respectively. Notably, unlike the CRISPR-modified cell lines, these lines all show a high degree of repair (95 ± 3%).It is interesting to note that there is no statistically significant difference between the initial number of foci for the cell lines in the lung cancer and normal cell panels (Yields of ell line .We measured the cell cycle distribution of each cell line by flow cytometry. The percentage of cells in each phase of the cell cycle ranged from 47.7% (PNT2) to 66.3% (LNCAP) for G1 phase, from 9.4% (AGO1522) to 15.1% (WPMY-1) for S phase, and from 19.1% (RWPE) to 39.9% (PNT2) for G2/M phase . p < 0.0001). As expected, all normal cell lines have a ploidy value around 2, with exception of WPMY-1 that was immortalized with a SV40-large-T antigen gene and which underwent significant genome duplication. A wide range of values is observed in the cancer cell lines, from 2.12 for 22RV1 to 3.95 for PC-3. Interestingly, our data showed no statistically significant correlation between cell ploidy and the initial number of DSBs induced by 2 Gy (p = 0.31)  , despiteTo identify if any radiobiological parameters provided an effective surrogate for survival in this more complex and diverse panel of cells, we evaluated the correlation between MID and ploidy, baseline DSBs, radiation-induced DSBs, residual DSBs, and cell cycle distribution, as well as plating efficiency. The resulting correlation heatmap is plotted in 2 = 0.72, p = 0.031), and plating efficiency  , no significant correlation was observed between any DNA damage endpoint and MID. Indeed, normal and prostate cancer cell lines showed particularly poor correlations between cellular features and survival, with no metric showing significant predictive power. Some significant correlations were observed in the lung cancer subgroup with some other features, including the percentage of cells in G2 phase of the cell cycle (R= 0.018) .2 = 0.31, p = 0.013) and percentage of cells in S phase  showed statistically significant predictive power, although correlation coefficients were relatively low. Together, these observations suggest that while DSBs do play a significant role in radiation response, in a diverse panel of genetically heterogeneous cell lines they are not individually strong predictors of response.Unfortunately, most of these metrics perform poorly in the population as a whole. Only plating efficiency . This is in agreement with previous reports of radiosensitivity across a wide variety of human tumours by Amundson et al. (SF2 range from 0.038 to 0.95) . The lowsitivity .−/− cells, despite employing different methods to assess the impact of DNA repair deficiencies on radiosensitivity. Specifically, while we investigated clonogenic survival, the screen study quantified raw numbers of cells with the loss of particular genes [2 = 0.98, p = 0.002). This discrepancy in BRCA1 data could potentially be attributed to the limitations of CRISPR dropout screens in genes that are essential for cell proliferation, as well as genes with multiple cellular functions, as has been previously reported in BRCA1 [Indeed, our findings show remarkable concordance with previously published results from a CRISPR screen studying radiation response in RPE-1 p53ar genes . This noin BRCA1 . NonetheThe determination of radiosensitivity by the clonogenic assay is a lengthy process, so there is a need to understand cellular mechanisms and parameters that might enable the prediction of intrinsic radiosensitivity. The development of such rapid techniques would enable them to inform clinical decision-making and allow personalized treatment planning.Here, we evaluated the relationship of cellular radiosensitivity with different cellular characteristics in a two-step approach; first in a controlled environment where the only difference between cell models is targeted mutations in specific DNA damage repair genes, then in diverse panel of human cell lines where cells have a more complex genetic profile. A wide variation was observed in cellular features, such as ploidy, plating efficiency, and DNA damage repair rates, among the cells used in this study.p = 0.009). This excellent performance in the CRISPR-modified cell lines is likely due to the very controlled environment where the only difference between cell models is one mutation in a specific DNA damage repair gene.Radiation-induced DSBs and their repair is attractive as a clinical and pre-clinical test, as results can be available in a few hours. Moreover, DSB repair is an obvious candidate marker for radiation sensitivity because DSBs are considered the most critical type of DNA damage caused by radiation. The relationship between residual levels of DSBs or repair rates to cell survival in cancer has been a topic of numerous investigations ,10,11,12p = 0.542), as do other metrics associated with DSBs, such as baseline levels of damage and levels of induced damage. This is despite many of the cell lines we investigated having mutations in DSB repair pathways  and pooled cell lines (p = 0.005).Cellular sensitivity is known to vary during different phases of the cell cycle. Typically, cells are most sensitive to irradiation during mitosis and in late G2, less sensitive in G1, and least sensitive during the latter part of S phase . Thus, cp = 0.032), as this metric performed poorly for all the other subgroups (p > 0.12).Interestingly, although S-phase is typically suggested to be a more resistant phase of the cell cycle, in the CRISPR-modified cell lines this correlation is negative E. This sp = 0.013) across the pooled cell lines. This metric performed well in the subgroup of lung cancer cell lines (p = 0.018), but poorly for the prostate cancer cells (p = 0.77). The lack of correlation between plating efficiency and survival has also been previously reported [p = 0.048), although this is dominated by the LIG4 knockout line. Taken together, we can conclude that plating efficiency may give some indications of sensitivity but is not a generally reliable predictive feature alone.Similar behaviour was observed with plating efficiency, where it had some predictive power for survival  and other damaging molecules, overwhelming the cellular antioxidant defence mechanisms. Subsequently, this can cause oxidative stress, further DNA damage and cell death. Antioxidant pathways, such as the glutathione system, superoxide dismutase, and catalase, help to neutralize ROS and prevent oxidative damage . ModulatIn summary a better understanding of the interplay between cellular metabolism, proliferation, antioxidant pathways, and radiation response is essential for elucidating the mechanisms of radiation response and may lead to the development of strategies to increase radiation efficacy in cancer treatments.In this study a total of 27 cell lines were used, divided in four groups: (1) 6 Human lung cancer cell lines acquired from ATCC ; (2) 5 Human prostate cancer cell lines acquired from ATCC ; (3) 8 Human normal cell lines acquired from ATCC or ECACC ; and (4) 8 CRISPR-modified cell lines, derived from the RPE-1 cell line with stable knock-out of p53, ATM, DCLRE1C (Artemis), BRCA1, BAX, LIG4, FANCD2, and PRKDC (DNA-PKcs).2.All cell lines were maintained in supplier recommended complete medium . All culThe RPE-1 cells were selected for CRISPR-Cas9 studies due to their stable epithelial cell characteristics, with minimal genetic modifications, which closely mimic normal tissue biology. Additionally, its widespread usage in previous CRISPR studies provides a robust foundation of data for comparison, confirming its suitability as a reliable and well-established cellular model.The process of selecting tumoral and normal human cell lines in this study was carried out based on factors such as cell availability, previously described levels of radiosensitivity and their widespread use in radiation studies. Lung and prostate cell lines were selected in this initial comparison as radiotherapy is frequently used as a primary treatment in these cancers.CRISPR-Cas9 modified cell lines were generated using a transient transfection of CRISPR-Cas9 in RPE-1 cells, by delivering a ribonucleoprotein (RNP) complex to recombinant Cas9 coupled to a specific guide RNA following manufacturer’s instructions. In brief, an RNP complex was assembled by mixing the tracrRNA (100 µM) and crRNA (100 µM)  in equimolar concentrations and Cas9 Nuclease V3 (61 µM) . A list of crRNA used in this study can be found in 5 cells suspended in 20 µL of Nucleofector Solution  supplemented with 5 µL of previously assembled RNP complex and 1 µL of IDT electroporation enhancer (100 µM)  were used for each nucleofection. After nucleofection cells were expanded for clonal selection.Delivery of the RNP complex to cells was performed via Lonza nucleofection. For that, 3.5 × 10All cell lines were generated as single knockouts, apart from BRCA1 which was combined with P53 knockout, as cells with only BRCA1 knockout were not viable.The RPE-1 cells were selected for CRISPR-Cas9 studies due to their stable epithelial cell characteristics, with minimal genetic modifications, which closely mimic normal tissue biology. Additionally, its widespread usage in previous CRISPR studies provides a robust foundation of data for comparison, confirming its suitability as a reliable and well-established cellular model.v/v NP-40, 0.5% v/v sodium deoxycholate (10%) and 0.1% v/v SDS (10%)) supplemented with protease inhibitor .Stable knock-out was validated by Western Blot of the protein of interest in populations derived from single clones . Cells wA 40 µg total protein sample was loaded onto an SDS-PAGE gel, and, after electrophoresis, the proteins were blotted on a nitrocellulose membrane . The membranes were blocked with 5% non-fat milk in PBS-Tween (0.1%) and incubated overnight at 4 °C with primary antibody diluted in milk . After wThe colony formation assay was carried out according to published methods . Cells wAfter irradiation, cells were incubated for 7 to 11 days depending on the cell line. The colonies were stained with 4% crystal violet solution in ethanol and were manually counted, with a colony defined as consisting of at least 50 cells. From these counts, plating efficiency (PE) and survival fraction (SF) were calculated. Survival fraction was defined as the number of colonies formed after irradiation divided by the number of cells seeded, corrected for the PE of unirradiated cells. Data were fit to the linear quadratic equation  at predetermined time points before being blocked in blocking buffer (5% FBS an 0.1% Triton X-100 in PBS) and stained with 53BP1 primary antibody (1:5000)  and https://cellmodelpassports.sanger.ac.uk (accessed on 8 November 2021)) where available, showing an Exponentially growing cells were harvested before being fixed in 100% ice-cold ethanol and left at 4 °C overnight. Samples were then centrifuged, the excess ethanol was removed, the cell pellets were then resuspended in PBS and centrifuged again before being resuspended in 500  of 0.93 .t-test and one-way ANOVA were used for statistical evaluation as appropriate. All statistics and graph plotting used GraphPad Prism 9.0 .All experiments were performed in triplicate, data are presented as mean values and respective standard deviation. Unpaired Student’s Our results indicate that this diverse panel of human cell lines have a wide variation in radiosensitivity and cellular characteristics. Residual radiation-induced DSBs were clearly related to intrinsic radiosensitivity in the subgroup of CRISPR-modified cells with well-defined DNA repair mutations, but they failed to predict survival in a group of cells with a more complex genetic profile. These data suggest that purely DNA damage-based analyses have a limited predictive power in diverse cell lines and supports further investigation into different metabolic and proliferative cellular pathways and their relationship with radiation response. Additionally, these data may assist the development of new predictive models of radiation response.