Colorectal cancer is a global issue with millions of patients in need for new treatment options. Surgical resection is often the first clinical intervention and can be, especially for primary tumours, curative. However, advanced tumours, recurrences, and metastases require drug treatment. Our aim was to compare material of primary tumours and corresponding metastases to draw conclusions from the first one to the latter. The feasibility of therapy prediction using the primary tumour material for the metastatic situation creates a time window for functional testing, which can realistically be integrated into the strict timeframe of clinical procedures. A reliable treatment recommendation, based on the tumour drug response, would improve treatment success in patients with metachronous metastases and spare them from unnecessary side effects of unsuccessful therapies.The prognosis of metastatic colorectal cancer (CRC) remains poor. Patients and physicians are in need of individual therapies and precise response predictions. We investigated the predictive capacity of primary tumour material for treatment response of metastases. Mutational landscapes of primary tumours and corresponding metastases of 10 CRC patients were compared. Cell line characteristics and chemosensitivity were investigated pairwise for primary and metastatic tumours of four patients. PDX models of one patient were treated in vivo for proof of concept. Driver mutations did not differ between primaries and metastases, while the latter accumulated additional mutations. In vitro chemosensitivity testing revealed no differences for responses to 5-FU and oxaliplatin between primary and metastatic cell lines. However, irinotecan response differed significantly: the majority of metastases-derived cell lines was less sensitive to irinotecan than their matching primary counterpart. Therapy recommendations based on these findings were compared to clinical treatment response and mostly in line with the predicted outcome. Therefore, primary tumour cell models seem to be a good tool for drug response testing and conclusion drawing for later metastases. With further data from tumour-derived cell models, such predictions could improve clinical treatment decisions, both recommending likely effective therapeutic options while excluding ineffective treatments. Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, with 1.4 million new cases annually and almost 700,000 deaths worldwide . TreatmeThese agents can be applied in a neoadjuvant or adjuvant setting and even in cases of advanced tumour progression or in the metastatic situation. A common first-line chemotherapeutic agent for CRC is fluorouracil 5-FU) which is often applied in combination with oxaliplatin and/or irinotecan to increase response rates . Even th-FU whichTo further this individualised therapeutic approach, we established patient-derived cancer models from primary tumours as well as matched metastases from CRC patients with different tumour locations, molecular subtypes and mutational profiles. Either patient tumour material or patient-derived xenograft tissues were used to start the cell line establishment process .We could successfully establish cell line pairs of primary carcinomas and their corresponding metastases from seven different CRC patients. In one of those cases, we were even able to generate a cell line out of the primary tumour, a synchronous as well as a metachronous metastasis. Four of these cell line pairs were analysed in detail with regard to growth behaviour, molecular pattern and chemosensitivity. We studied the response of these matching cell lines side-by-side to the first-line chemotherapeutics 5-FU, oxaliplatin and irinotecan in vitro. For the set consisting of primary, synchronous and metachronous model, we additionally analysed drug responses in PDX models in vivo.The aim of the study was to unveil commonalities and differences between primaries and metastases. And more importantly, we explored the feasibility of drawing conclusions from primary tissue characteristics  to metastasis. This option would pave the way for predicting treatment success for metastases using the primary material and thus save precious time in the clinical context.Cell lines were established as described in detail before . In shorNames of the cell lines consist of the following information: Place of material collection (HRO = Hanse City of Rostock), tumour entity (C = Colon), patient number. In the case of a collected metastasis, “Met” is added after the patients’ ID, and in the case of xenograft-derived cell lines number of tumour transfer in mice  and mouse identifier  are included in the cell line name.Foxn1nu mice  weighing 18–22 g. Mice received standard pellet food and water ad libitum. Tumour pieces from previously xenografted patients’ tumours with less than 5 mouse transfers were used and implanted subcutaneously into the animals’ right flank under anaesthesia . Tumour growth was regularly monitored, and therapy was initiated upon tumour establishment (ca. 65 mm3). Tumour size was measured using a slide scale, and tumour volume was calculated with the following formula:PDX were established as described in detail before . Under s3) were randomised into four treatment groups: (1) 5-FU (30 mg/kg bw) (2) irinotecan (20 mg/kg bw) (3) FIRI (5-FU 30 mg/kg bw and irinotecan 20 mg/kg bw) (4) control group receiving equal amount of 0.9% NaCl solution. All therapeutic regimens were applied i.p. thrice weekly for a total of 6 injections. Tumour growth was monitored thrice weekly, within therapy as well as during one week of follow up time. Termination criteria were weight loss >20% vs. start of therapy, impaired socio-physiological behaviour, or tumour sizes >1500 mm3. For detailed examinations, tumour tissues were resected, dissected, and cryopreserved as well as embedded in paraffin. H&E sections (4 µm) were prepared for histopathological examinations, according to standard protocols for surgical pathology reports. In a blinded study, morphology of tumour architecture, cytological features, and signs of therapy-induced necrosis/apoptosis of all obtained PDX material was examined and scored  by an expert pathologist (F.P.).Mice with solid tumours  were seeded in triplicates in 150 µL/well standard medium in a flat 96-well plate and allowed to attach overnight. Then, chemotherapeutics were added in 50 µL medium yielding the following final concentration ranges: (1) 1 nM–5000 µM 5-FU; (2) 0.1 nM–1000 µM irinotecan; (3) 1.3 nM–20 µM oxaliplatin and (4) FIRI. Medium exchange was performed after 72 h with the addition of fresh chemotherapeutics. After a second 72 h treatment period, viability of the cells was measured.Wells were washed with PBS to remove dead cells and fragments before the staining. For 96-well plates, 50 µL/well crystal violet staining solution was used. After 20 min incubation at room temperature, wells were washed three times with PBS and dried. To solubilize the colorant, 100 µL of 1% sodium dodecyl sulfate solution was added and incubated for 10 min on a plate shaker. Measurement of absorbance at 590 nm was performed using a Tecan Infinite 200 Pro plate reader and percentage of living cells was determined as follows:IC50 values were calculated using GraphPad Prism 5 and heatmaps were created using TBtool .After the preparation of a single cell suspension 100 cells/well were seeded in quadruplets in a 96-well plate in 200 µL standard medium. Outgrowing colonies were stained after 14–20 days with crystal violet as described above and counted manually.After the preparation of a single cell suspension, 100,000 cells/well were seeded in a 6-well plate for suspension cell culture in 5 mL defined serum free medium. Spheroid formation was evaluated qualitatively after 7–14 days and photographically documented.https://www.ncbi.nlm.nih.gov/clinvar/, accessed on 28 August 2021) followed by a manual evaluation.Tumour DNA was isolated from PDX samples snap frozen in liquid nitrogen by using the Precellys Tissue DNA Kit . Quality criteria for DNA included a concentration ≥25 ng/µL and a DNA integrity number ≥5. Next Generation Sequencing was than performed by Centogene  applying the augmented Solid Tumour Panel including 105 fully sequenced genes plus mutational hot spots from additional 146 genes see . A coverConcerning the NGS data, for this study, we focused on mutations classified as either pathogenic or likely pathogenic as well as mutations of uncertain significance. Excluded were all synonymous and benign as well as likely benign mutations. Quality criteria included a variant allele frequency of at least 25.We collected tumour and corresponding metastatic material of 10 patients  aged 44–82 years . Detailed patient characteristics and clinical information can be depicted from TNM classification of the primary tumours revealed medium and high-grade adenocarcinomas  classified to UICC III (3 patients) or IV (7 patients). The majority of metastases were found in the liver (9), but three patients developed metastases either in the peritoneum (2) or in the brain (1) see . Six patMost tumours and metastases were untreated when resected. Only one patient (HROC348) received neo-adjuvant irradiation and only HROC277Met2 was drug-treated before metastatic resection see . FollowiThe ten analysed patient tumours’ molecular subtype could be classified as sporadic standard CRC (6/10), CpG island methylator phenotype high and microsatellite stable CRC (3/10) as well as sporadic microsatellite instable CRC (1/10) .The analysis of genetic aberrations in tumours and metastases revealed a relatively low number of mutations detected in the selected panel. This included 251 tumour relevant genes. After application of quality and exclusion criteria we detected 30 genetic aberrations  including well-known driver mutations like G12D in the oncogene KRAS and truncating mutations of the tumour suppressor gene APC. Furthermore, there were two genes  mutated exclusively in tumours and eight  solely in metastases. Especially in the metastases, there was an increase of mutations classified as ‘uncertain significance’, which represent more than 40% of all detected alterations.In four of the ten cases, the number of mutations did not vary between tumour and metastasis, whereas six pairs presented with more mutations in the metastasis. The tumour and Met2 of HROC103 showed one mutation while the Met1 had an additional mutation. There were commonalities as well as differences between tumours and metastases regarding the mutated genes. Comparing their mutational profile, three sets of tumour and metastases were similar, whereas the detected mutations of seven patients differed between tumour and metastasis see . We founAPC, KRAS and TP53. Mutations in the APC gene were detected in the samples of four patients, which showed all individual mutations  pathogenic mutations were detected in BRAF, HNF1A, SMAD4, MUTYH, FBXW7 and TSC2. Pathogenic and likely pathogenic mutations were mainly consistent in tumours and metastases. Interestingly, HROC103 showed only mutations of uncertain significance and no (likely) pathogenic mutation was detected.Focusing on (likely) pathogenic mutations, we identified 33 mutations in nine genes (0–3 mutations per sample). The most frequently mutated genes were ions see . NotablyFor in vitro experiments, cell line pairs of the following patients were selected based on feasibility criteria  and further characterised: HROC147, HROC277, HROC278, and HROC348. For an overview, see HROC147 T0 M1 was established from a xenograft, whereas the cell line of the corresponding metastasis HROC147Met1 originates directly from patient material. Cells of both cell lines grew in dense, clearly outlined round colonies limited in size and did not form a confluent monolayer. Doubling times of HROC147 T0 M1 and HROC147Met1 were 63 h and 58 h, respectively.The cell lines of patient HROC277 differed in their morphology as well as their proliferation rate. HROC277 T0 M1 and HROC277Met2 appeared to grow in colonies without contact inhibition, leading to the establishment of a confluent monolayer. On the other hand, HROC277Met1 T0 M2 favoured growth in single colonies not spreading over the culture vessel. The lowest proliferation rate in this set of cell lines was observed for HROC277Met1 T0 M2 with a doubling time of 66 h. The doubling times of HROC277 T0 M1 and HROC277Met1 T0 M2 were similar with 62 h.The xenograft-derived matching cell lines of patient HROC278 were dissimilar in their cultivation appearance. The cell line of the tumour, HROC278 T0 M1, formed small and dense colonies with miniature adjacent colonies. HROC278Met1 T2 M2 grew in bigger and less dense colonies. According to their morphology, their rate of proliferation differed, too. The cell line of the metastasis, HROC278Met1 T2 M2, grew slightly faster than the tumour-derived cell line (54 h versus 61 h).HROC348 and the corresponding metastasis HROC348Met1 were directly patient-derived cell lines. They both favoured growth on collagen-coated cell culture vessels and built similar irregularly shaped colonies that were characterised by small cells. Doubling times of HROC348 and HROC348Met1 were 74 h and 66 h, respectively.p < 0.001). The opposite was observed in the cell line pair of HROC348. Here, the metastasis-derived cell line formed significantly more colonies than the tumour (p < 0.001).All cell lines used were tested positive in the colony formation assay, thereby proving their capacity to form colonies out of single cells see . A signiIn the spheroid formation assay, most tested cell line pairs grew into spheroids with no morphological differences between tumour-derived and metastasis-derived cell lines. Only the spheroids of HROC278 T0 M2 and HROC278Met1 T2 M2 did not resemble each other. HROC278 T0 M2 formed spheroids of irregular shape with dark inclusions, whereas spheroids of HROC278Met1 T2 M2 were round and of light colour.50 values ranged from 0.5 to 9.2 µM . The results for the cell lines from case HROC277 were even more diverse. HROC277Met2 was least responsive , followed by HROC277Met1 T0 M2  and HROC277 T0 M1, which was relatively sensitive (IC50[HROC277 T0 M1] = 0.1 µM).A remarkable difference was observed for the responses to irinotecan see B. HROC1450[HROC348] = 2.1 µM; IC50 = 16.8 µM), the IC50 exceeded the normal plasma level of irinotecan (up to 2 µM [Our in vitro chemosensitivity testing showed the most diverse results for irinotecan. All tested CRC cell lines reacted to the treatment and no intrinsic resistance was observed (acquired resistance under long term treatment was not tested). But the metastasis-derived cell lines of three patients were less sensitive towards irinotecan than the tumour cell lines. This is in accordance with results from Takebayashi et al. who also observed lower sensitivity, especially in liver metastatic cells compared to paired CRC primary tumour cells when treating with irinotecan, but not with 5-FU or oxaliplatin . In the  to 2 µM ) by far;We further tested if combinations of the chemotherapeutics 5-FU and irinotecan act synergistically in lower doses. Contrarily to what was expected, viability decreasing effects of irinotecan were abrogated by 5-FU.Exemplarily, we selected an in vivo PDX model set of one CRC patient from whom we had PDX models of the primary tumour (HROC277), the synchronous (HROC277Met1) and the metachronous (HROC277Met2) metastases for validation of these in vitro results. All PDX models were treated with 5-FU, irinotecan and their combination FIRI. The results confirmed that a combinational approach with these chemotherapeutic substances is not superior to the best-acting single chemotherapy, i.e., irinotecan; and thus corroborating the in vitro results of HROC277. In contrast, the different responsive behaviours of the metastasis-derived cell clones compared to the primary-derived ones under irinotecan treatment obtained in vitro could not be reproduced. One explanation for this phenomenon could be the comparatively low activity of irinotecan compared to its hydrolysed compound SN-38 . MetabolBased on these in vitro and in vivo results, we retrospectively stated therapy recommendations and compared them with the actual clinical treatment success of the patients. For patient HROC147, we would have proposed chemotherapy including 5-FU and oxaliplatin. Indeed, FOLFOX  therapy led to a complete remission of the patient.50 values for irinotecan. The promising results from the in vivo experiments probably trace back to the much higher plasma level in the mouse [50 value of all tested cell lines for irinotecan. With this knowledge, the patient could have been spared from irinotecan-containing treatments including their side effects and other promising therapy options should have been used in first-line treatment. Even the primary tumour presented a rather high IC50 for irinotecan in the range of reachable plasma levels, indicating potential treatment difficulties.Patient HROC277 underwent surgical resection of the tumour and its synchronous metastasis before chemotherapy started with FOLFOX, followed by FOLFIRI. The metachronous metastasis HROC277Met2, detected under FOLFIRI treatment, was resected subsequently. But the following FOLFIRI cycles did not result in tumour control and the disease progressed with further metastases. Our in vitro studies match these potential treatment failures with relatively high IChe mouse  comparedThe cell lines of HROC278 were highly sensitive for 5-FU and irinotecan and intermediate for oxaliplatin proposing an excellent treatment success. Nevertheless, FOLFIRI plus bevacizumab therapy after resection failed to prevent the development of a further metastasis, and the patient died 26 months later. As a likely explanation, the relatively high tumour burden due to the incomplete resection may have caused rapid disease progression by proliferation of chemo-resistant subclones.The initial question of this study was whether it is possible to predict biological behaviour of metastases by concluding results obtained from CRC cells of the primary tumour. As an ideal tool to answer this question, the cell line pairwise comparison confirmed that both phenotype and growth behaviour of CRC cells did not differ significantly between tumour- and metastasis-derived cells. Also, the chemosensitivity tests revealed no significant differences between tumour and metastasis for the majority of cell line pairs and tested substances. Consequently, we would conclude that analysing and testing cells from primary CRC allows for identifying promising treatments for metastases likely to develop in the future. Besides approaching chemo response with 2D cell cultures, patient-derived organoids (PDOs) delivered also encouraging results. Studies investigating PDOs from either tumour or metastatic material could confirm a correlation between PDO reactivity and patient’s response to chemotherapy ,46. Howe


Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general.mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology.To investigate if the miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.The online version contains supplementary material available at 10.1186/s12864-022-08346-6. High-grade osteosarcoma is the most prevalent primary malignant tumour of bone, affecting both children, adolescents and, more rarely, elderly people. Following the introduction of multi-agent chemotherapy in the 1970s, the 5-year survival rate increased considerable reaching 60–70% among patients with conventional high-grade osteosarcoma , and alsOsteosarcoma is characterized by considerable phenotypic and genomic heterogeneity, and few recurrent targetable genetic changes have been reported. Osteosarcoma exhibits a complex karyotype with high genetic and chromosomal instability seen as multiple rearrangements across the genome, kataegis and chromothripsis –6. The gEpigenetic mechanisms are fundamental drivers of tumour initiation, development and progression . SeveralMicroRNAs (miRNAs) are a key class of epigenetic regulators as they act to post-transcriptionally silence large numbers of genes without modifying the DNA . miRNAs mir-486 locus in detail. Finally, the in vitro effect of miR-486-5p overexpression on cell morphology and proliferation was investigated.In an effort to advance the understanding of osteosarcoma biology, we aimed to investigate if the expression of miR-486-5p was epigenetically regulated through methylation of promoter regions. We analysed miR-486-5p expression and DNA methylation levels in a cohort of osteosarcoma cancer cell lines and patient samples. Qualitative and quantitative methylation analyses were performed, allowing us to characterize the n = 9), osteosarcoma cell lines (n = 17) and normal bone samples (n = 6) by quantitative real-time reverse-transcription PCR (qRT-PCR). The mean expression of miR-486-5p was reduced 3-fold in patient samples (p < 0.01) and 300-fold in cell lines (p < 0.0001) compared to normal bone  gene. Several transcript variants of human ANK1 exist, with multiple transcriptional start sites (TSS), promoter regions and CGIs. The ANK1 variants 1–4 represents medium-sized transcripts, ANK1 variant 9 is a longer transcript while sANK1 represents the short variants 5, 7 and 10  and normal bone samples (n = 4) using Illumina HumanMethylation27 BeadChips. This microarray contains one CpG site in the CGI CpG79  at the TSS (position chr8:41655140) of ANK1 (variant 1–4). The average difference of methylation in cell lines versus normal bone samples was 0.36 (DeltaBeta) for CpG79, providing a first evidence for methylation of the mir-486 locus.p < 0.0001) and the methylation was higher in cell lines than in bone (p < 0.01). Taken together, this indicated an association between low expression of miR-486-5p and CpG methylation of ANK1  were treated with the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza). After 72 h, the relative expression levels of miR-486-5p in untreated and treated cell lines were quantified by qRT-PCR. A significant difference between the cells before and after treatment was observed (p = 0.02). miR-486-5p showed at least a 1.3-fold (30%) induction in 8/12 tested cell lines  before 5-Aza treatment and r = 0.92 (p < 0.0001) after treatment of the cell lines  was quantified by qRT-PCR. pri-mirR486 showed at least 1.3 fold (30%) induction in 9/12 cell lines after 5-Aza treatment, confirming the epigenetic effects seen on the mature miRNA  could be observed across the cell line panel if one of the cell lines, MG-63, was omitted . The cell line MG-63 showed very high expression levels of ANK1 variants 1–4, however as the sample seems to be a biological and not a technical outlier, it was not removed from the dataset.In osteosarcoma cell lines, ANK1 variant 1–4 expression was induced in 8/12 cell lines upon 5-Aza treatment  could be seen between the fold change observed for ANK1 and the fold change observed for miR-486-5p upon Aza treatment across all 12 cell lines  analysis was done for CGI CpG79 and CpG171, both located upstream of nes Fig. . InteresDirect bisulfite sequencing was performed on osteosarcoma cell lines. The analysis was done on an extended region of the CGI CpG79, covering a total of 34 CpGs including the region for the MSP on CpG79 Fig. . The resmir-486 was analysed quantitatively by quantitative methylation-specific polymerase chain reaction (qMSP) in a larger cohort of samples, including osteosarcoma cell lines (n = 20), xenografts (n = 41), patient samples (n = 14) and bone (n = 5). Methylated percent of methylated reference (PMR) was set to > 5.2. High qMSP methylation pattern was only observed in cancer samples, comprising both cell lines, xenografts and patient samples . For the cell lines, 17/20 (85%) showed increased methylation (>2x) compared to the bone average. However, increased methylation was also observed for 8/14 (60%) of the patient’s samples and 17/41 (40%) of the xenografts. Together, these observations suggest that the CGI CpG79 is highly methylated in osteosarcoma.The methylation of a region of CpG79 of p = 0.2). However, a clear change in cell morphology was observed in all three cell lines where cells became flatter, rounder and smaller  repressed both ANK1 and miR-486-5p [It has been shown that alternative promoters give individualized regulation of transcripts with distinct first exons, providing an expression pattern with a strong tissue preference . The regid cells , and hasid cells , 34. Theiant 1–4 . An earl variant . The sANR-486-5p , and wasThe induction of miR-486-5p expression in osteosarcoma cells resulted in a change in cell morphology, as well as a pattern of reduced cell proliferation in 2 out of 3 cell lines tested. A morphological change in MG-63 cells following overexpression of miR-486-5p has been described previously, possibly related to an EMT-like phenotype . Throughmir-486 in various tissue types would be highly interesting.There were a couple limitations to our study. The sample size of the normal samples is smaller than the included osteosarcoma samples. Furthermore, the manuscripts only showed that the hypermethylation in the differentially methylated region correlated with downregulation of miR-486-5p expression. Although a direct effect is not proven, an inverse pattern between methylation and gene expression is shown and the other results performed support the observation that the miRNA is epigenetic regulated. Further investigations regarding the epigenetic regulation of ANK1 variant 1–4. This implies a tumor suppressive role of miR-486-5p in osteosarcoma, and the findings may lead to clarify the tissue/disease-specific regulation of the miR-486-5p expression. The main effect of miR-486-5p overexpression was morphological changes in osteosarcoma cells. The epigenetic regulation should be further correlated with clinical characteristics, and the functional role needs to be further validated in extended model systems.In conclusion, the present study of miR-486-5p shows that the low expression observed in osteosarcoma is caused by cancer-specific methylation of the upstream promoter region of n = 21) composed of 143B, CAL-72, G-292, HAL, HOS, IOR/MOS, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/SARG, KPD, MG-63, MHM, MNNG/HOS, OHS, OSA, Saos-2, U-2 OS and ZK-58 were obtained from ATCC (www.lgcstandards-atcc.org), DSMZ (http://www.dsmz.de/) or research partners in the EU funded EuroBoNeT project [A panel of human osteosarcoma cell lines ( project . These c project  and authn = 19) and at the University of Valencia (n = 22) [The xenograft cohort comprised osteosarcoma xenografts established at the Norwegian Radium Hospital ((n = 22) , 53. In (n = 22) .The osteosarcoma patient sample panel comprised fresh-frozen tissue from 14 high-grade osteosarcoma samples collected at the Norwegian Radium Hospital. The tumours were diagnosed by an osteosarcoma pathologist, according to the current World Health Organization classification .n = 2) or obtained from amputations of cancer patients at the Norwegian Radium Hospital (n = 4), where bone samples were collected distant from the tumour margin. DNA copy number analysis of the latter four samples (Bone 1–4) showed normal diploid karyotype [Six bone samples were used as normal controls. Normal bones were purchased from Capital Biosciences   compliant data can be downloaded from the GEO repository (www.ncbi.nlm.nih.gov/geo/), accession number GSE28425.Total RNA from osteosarcoma cell lines and bones was extracted and quality controlled as previously described . miRNA eDNA was isolated using the Wizard Genomic DNA Purification Kit . For the initial analyses, DNA methylation profiling of 19 cell lines and four bone samples was performed using the Illumina HumanMethylation27 BeadChip , covering 27,000 CpG sites across the genome . For an The association between CpG methylation and miRNA expression data in osteosarcomas and normal samples was calculated using Pearson’s Correlation between miR-486-5p expression and methylation level (Beta) using the Methylation module v1.9.0 of GenomeStudio. DeltaBeta values for the groups of samples were calculated .ANK1 expression quantification, the Fast Cells-to-Ct reagents (Life Technologies) were used to generate cDNA. Transcript-specific TaqMan Gene Expression Assays (Life Technologies) were used for quantitative PCR. Gene expression was normalized towards RNU44 for miRNA quantification and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for pri-miRNA hsa-mir-486-1 and ALK1 quantification. Overview of TaqMan assays are found in Additional file T method.qRT-PCR was performed using the ABI PRISM 7500 DNA Sequence Detection System . TaqMan MicroRNA Reverse Transcription Kit and TaqMan MiRNA Assays (Life Technologies) were used to generate cDNA and to quantitatively detect the expression of mature miR-486-5p. For pri-miRNA hsa-mir-486-1 and Osteosarcoma cells were cultured in the presence of 1 μM 5-Aza , as previously described . The celGenomic DNA from tumour and normal samples was isolated by standard phenol chloroform extraction or using the Wizard Genomic DNA Purification Kit (Promega). DNA (1.3 μg) was bisulfite treated using the EpiTect Bisulfite Kit (Qiagen) and purified using the QIAcube (Qiagen).Primers for MSP were designed using Methyl Primer Express 1.0 (Life Technologies)  and flanked the MSP amplicons  were transiently transfected into osteosarcoma cells at a final concentration of 15 nM using the INTERFERin siRNA transfection reagent . The cellular growth after transfection was measured every 3 h, by a live-cell imaging system, IncuCyte ZOOM  and the percent of cells confluence was calculated with the corresponding software application (version 2013BRev1).p-values were derived from two-sided tests using a significance level of 0.05. Similarities between samples were calculated using Pearson’s correlation (r). Statistical differences between the group of cell lines before and after 5-Aza treatment were calculated using a paired t-test.Analyses were performed with the GraphPad Prism 9 software. Non-parametric Mann-Whitney U test was applied to compare groups of samples. Additional file 1: Supplementary Table S1. Overview of TaqMan assays for miRNA and mRNA qRT-PCR experiments. Supplementary Table S2. Primers used for qualitative Methylation-Specific Polymerase Chain Reaction (MSP), bisulfite sequencing (BS) and quantitative Methylation-Specific Polymerase Chain Reaction (qMSP). Supplementary Figure S1. Genomic overview of the mir-486/ANK1 locus. Supplementary Figure S2. Expression of ANK1 transcript variants in untreated and 5-Aza treated osteosarcoma cell lines. Supplementary Figure S3. Methylation level of miR-486 as assessed by methylation specific PCR (MSP).


The epidermal growth factor receptor (EGFR) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). Resistance to EGFR-targeted therapies, such as cetuximab, poses a challenging problem. This study aims to characterize acquired cetuximab resistance mechanisms in HNSCC cell lines by protein phosphorylation profiling. Through this, promising combination treatments can be identified to possibly overcome acquired cetuximab resistance in HNSCC. Protein phosphorylation profiling showed increased phosphorylation of Akt1/2/3 after cetuximab treatment in acquired cetuximab resistant cells compared to cetuximab sensitive cells, which was confirmed by western blotting. Based on this protein phosphorylation profile, a novel combination treatment with cetuximab and the Akt1/2/3 inhibitor MK2206 was designed. Synergy between cetuximab and MK2206 was observed in two cetuximab sensitive HNSCC cell lines and one acquired cetuximab resistant variant in simultaneous treatment schedules. In conclusion, this study demonstrates that increased Akt1/2/3 phosphorylation seems to be characteristic for acquired cetuximab resistance in HNSCC cell lines. Our results also show an additive to synergistic interaction between cetuximab and MK2206 in simultaneous treatment schedules. These data support the hypothesis that the combination of cetuximab with PI3K/Akt pathway inhibition might be a promising novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC patients. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide and remains one of the most challenging malignancies to treat , 2. ThroOver the past decades, research has revealed that the epidermal growth factor receptor  plays an integral role in the tumorigenesis of HNSCC. Increased or sustained activation of the EGFR signaling pathway can induce malignant transformation through sustained signaling for cell proliferation, anti-apoptotic signaling, angiogenesis and metastasis . FurtherIn 2006, the anti-EGFR monoclonal antibody (mAb) cetuximab, was approved by the Food and Drug Administration (FDA) in combination with radiotherapy for locoregionally advanced HNSCC  of 4.1 years versus 2.4 years with radiotherapy alone) and in combination with platinum-based therapy and infusional 5-fluorouracil (EXTREME regimen) for the first-line treatment of patients with recurrent/metastatic (R/M) HNSCC  . In partThe reason for resistance to pembrolizumab treatment in HNSCC remains largely unknown. In this regard, several biomarkers, such as PD-L1 expression, immune infiltration, tumor mutational burden and immune-gene expression profiling, have been explored for their predictive potential. However, none of them could be validated in HNSCC so far . ConcernSens) HNSCC cell lines and their acquired cetuximab resistant (AcqRes) variants. Based on this protein phosphorylation profile, a novel combination treatment was designed to overcome acquired therapy resistance.Therefore, the present study aims to investigate a novel combination strategy to overcome acquired cetuximab resistance. We hypothesized that acquired cetuximab resistance in HNSCC may arise from the activation of compensatory signaling pathways following cetuximab treatment, which are able to reverse the inhibitory effects of cetuximab through phosphorylation of key proteins, thereby promoting cell survival. Pharmacological inhibition of these phosphorylated proteins might be essential to overcome acquired cetuximab resistance in HNSCC. Therefore, we first characterized the protein phosphorylation profile of two cetuximab sensitive (CetSens following cetuximab treatment for 168h using the colorimetric sulforhodamine B (SRB) assay. Isogenic AcqRes HNSCC cell lines (SC263-R and SCC22b-R) were previously generated by chronically exposing these initially CetSens cell lines  to cetuximab, starting with the IC50 concentration of cetuximab . The identity of each cell line was validated through short tandem repeat profiling .HPV-negative human HNSCC cell lines SC263 and SCC22b were kindly provided by Prof. Dr. Sandra Nuyts  and Prof. Dr. Olivier De Wever . These HNSCC cell lines were previously identified as Cetetuximab –14. In petuximab –14. All Sens and AcqRes cell lines after cetuximab treatment. The human phospho-kinase antibody array was performed according to the manufacturer’s protocol. In short, cells were lysed on 6-well plates after treatment with cetuximab . Twenty minutes before lysis, epidermal growth factor  was added to the medium. Protein concentrations were determined using the Pierce BCA protein kit . Next, samples were prepared and 450 μg of proteins were incubated overnight with nitrocellulose membranes which are spotted with 46 capture antibodies in duplicate. The specific target proteins  was used to determine the relative levels of protein phosphorylation in the Cetproteins . Only prIn order to validate findings of the human phospho-kinase antibody array with western blot, cell lysates were three times independently prepared as described above. Twenty micrograms of proteins were separated by SDS-page  and western blot was performed . Blocking of non-specific binding sites was done by incubation of the membrane with Odyssey blocking buffer TBS  for 1h at room temperature while shaking. Next, primary and secondary antibody incubation and washing was performed using the SNAP id 2.0 protein detection system (Merck Millipore) according to the manufacturer’s instructions. Membranes were incubated with the following antibodies: phospho-Akt (S473) rabbit mAb  and total Akt (pan) rabbit mAb . Anti-β-actin was used as an internal standard . Goat-anti-rabbit  or goat-anti-mouse  fluorescently labeled secondary antibodies were used and fluorescent detection was performed using the Odyssey imaging system (Li-Cor). Protein levels were quantified using Image Studio™ Lite, a software program available on the Odyssey imaging system. Phospho-Akt and Akt expression levels were corrected for loading differences based on beta-actin expression. Fold changes were calculated by dividing the mean fluorescent signal of the treatment and control groups.The baseline mRNA expression of Akt1/2/3 in HNSCC patients was examined using the RNA sequencing data from The Cancer Genome Atlas (TCGA) dataset . RNASeqV2 from TCGA was processed and normalized using the software package RNA-Seq by Expectation Maximization (RSEM) in order to generate transcripts per million. This dataset was downloaded from cBioportal.Cell survival was assessed using the colorimetric SRB assay, as previously described , 17. ThiCetuximab plus MK2206 with total treatment duration of 72hCetuximab for 168h with MK2206 added during the last 72h of treatment50 values (i.e. drug concentration causing 50% growth inhibition) were calculated using Graphpad Prism 9 software. Possible synergism between cetuximab and MK2206 was determined by calculation of the combination index (CI) using the Additive Model as described by others  and further dilutions were made in cell culture medium. ICy others –20. CI <We performed all experiments at least three times independently, unless otherwise stated. In cytotoxicity experiments, each condition was tested in triplicate in each of the three experiments. Differences in total and phosphorylated Akt1/2/3, determined with western blot, were statistically analyzed using the non-parametric Kruskal-Wallis test. Significant values were adjusted by the Bonferroni correction for multiple testing. One-way ANOVA with Tukey-Kramer HSD posthoc analysis was used to assess significant differences in Akt1/2/3 mRNA expression of HNSCC patients (RNASeqV2 TCGA data). The influence of cetuximab treatment on the effect of MK2206 alone was evaluated for each cell line using linear mixed models in case of non-independent observations. More specifically, combination treatment was set as fixed effect. A random intercept for experimental replicates was added in order to account for the dependence of observations between experiments. The influence of cetuximab resistance status on the effect of MK2206 alone was also evaluated using linear mixed models. Resistance status was set as fixed effect and a random intercept for cell line and experimental replicates was added to account for the dependence of observations between cell lines and experiments. GraphPad Prism 9 was used for data comparison and artwork. All statistical analyses were performed in JMP Pro 15 and SPSS v25. P-values below 0.050 were considered significant.Sens HNSCC cell lines and their isogenic AcqRes variants. Data analysis of the protein phosphorylation profile revealed a differential response of CetSens HNSCC cell lines and AcqRes variants to cetuximab treatment. As this change was most pronounced in substrates involved in the Akt signaling pathway, we focused further on the interpretation of these results in the next paragraph.In order to characterize acquired cetuximab resistance, a protein phosphorylation profile was established using the human phospho-kinase antibody array. Hereby, the effect of cetuximab treatment on the phosphorylation of various proteins (Sens HNSCC cell lines and AcqRes variants (Sens HNSCC cell lines (i.e. SC263-S and SCC22b-S) demonstrated decreased phosphorylation of Akt1/2/3 at T308 after cetuximab treatment. T308 is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) in the phosphoinositide 3-kinase (PI3K)/Akt pathway, leading to Akt activation  , proline-rich Akt substrate 40kDa (PRAS40) and WNK lysine deficient protein kinase-1 (WNK1)] in AcqRes variants, especially in SCC22b-R. This means that the Akt pathway is still activated in AcqRes cells following cetuximab treatment, leading to anti-apoptotic and pro-proliferative effects.The effects of cetuximab treatment on the phosphorylation of EGFR, Akt and other substrates involved in the Akt pathway were quantified in Cettivation . As a re(mTORC2) , was alsSens HNSCC cell lines and AcqRes variants . There was no statistical difference in the change of expression of total Akt1/2/3 following cetuximab treatment across cell lines , but not between SC263-S and SC263-R . This data demonstrated that there is a significant difference in mRNA expression between Akt1, Akt2 and Akt3 . As mentioned above, we previously identified CetSens cell lines based on the outcomes of experiments with a total treatment time of 168h  in CetSens HNSCC cell lines and the AcqRes variants  due to prolonged treatment duration. These fixed concentrations were based on the outcome of previous monotherapy experiments with a total treatment duration of 168h  was added during the last 72h of treatment. The goal of this prolonged treatment schedule was to decrease the used drug concentrations. In contrast to the first treatment schedule, Cet of 168h .Sens HNSCC cell lines and their AcqRes variants. The addition of MK2206 during the last 72h of treatment still resulted in a concentration-dependent cytotoxic effect of this compound. The IC50 values for MK2206 ranged from 1.073 ± 0.038 µM to 4.372 ± 1.182 µM . Furthermore, in this treatment regimen, the CI ranged from 0.578 to 0.867 , Akt1/2/3 (T308 and S473) and downstream substrates after cetuximab treatment. Although EGFR phosphorylation was decreased, Akt1/2/3 was still phosphorylated and activated following cetuximab treatment in AcqRes HNSCC cell lines. This was supported by the observed increase in phosphorylation of several downstream substrates of Akt, such as mTOR, p70S6 kinase, GSK-3α/β, PRAS40 and WNK1. Thus, in AcqRes HNSCC cell lines, the Akt pathway was still able to exerts its anti-apoptotic and pro-proliferative effects under cetuximab treatment, possibly leading to therapy resistance. Importantly, it is worth mentioning that the number of cell lines used in this study is a limiting factor. Therefore, our data needs to be validated in additional isogenic HNSCC cell lines  in order to strengthen the results. Although further confirmation in HNSCC cell lines and patient samples is still needed, based on our results, Akt represents a potential target to improve outcome of cetuximab-based treatment in HNSCC patients.Protein phosphorylation profiling showed a differential response of Cet2) to generate phosphatidylinositol 3,4,5-triphosphate (PIP3). The binding of PIP3 to Akt locates Akt to the plasma membrane and allows its phosphorylation and activation by PDK1. Akt can also be phosphorylated by other substrates and in response to cellular stress, such as ischemia, hypoxia and oxidative stress. The tumor suppressor phosphatase and tensin homolog (PTEN) catalyzes the dephosphorylation of PIP3 and is the major negative regulator of Akt signaling. Activated Akt exerts its effects by phosphorylating various downstream substrates, all resulting in anti-apoptotic or pro-proliferative effects  is characteristic for acquired cetuximab resistance in HNSCC cell lines. Importantly, this observation needs to be validated in a cohort of HNSCC patient samples in order to elucidate its clinical value. Nevertheless, previous research has already suggested that enhanced Akt activation can play a role in resistance to cetuximab, not only in HNSCC but also in colorectal cancer and non-small cell lung cancer –39. Incr3CA gene . For instivation . Accordification , 42. In  pathway , 44. Accectively , 42. Basand mTOR , 46. As Res HNSCC cell lines and Akt1, Akt2 and Akt3 are expressed in HNSCC patients, we hypothesized that the combination of cetuximab with an Akt inhibitor might be a potential novel therapeutic strategy to overcome acquired cetuximab resistance. To date, Akt inhibitors are not yet included in clinical practice , the Flemish cancer society (OZ7410). IDP and HZ are supported by research grants of Kom op tegen Kanker (Stand up to Cancer), the Flemish cancer society (OZ7410). We would like to thank Mr. Willy Floren for funding some of the equipment used in this study.JBV has had in the last three years consulting/advisory relationships with Immunomedics, Innate Pharma, Merck-Serono, Merck Sharp & Dome Corp, PCI Biotech, Synthon Biopharmaceuticals, Debiopharm, Cue Biopharma, and WntResearch and has received honoraria from Merck-Serono, MSD, and BMS.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mixed Lineage Leukemia (MLL/KMT2A) gene have a poor prognosis, especially when the disease strikes in infants. The poor clinical outcome linked to this aggressive disease and the detrimental treatment side-effects, particularly in children, warrant the urgent development of more effective and cancer-selective therapeutics. The aim of this study was to identify novel candidate compounds that selectively target KMT2A-rearranged (KMT2A-r) leukemia cells. A library containing 3707 approved drugs and pharmacologically active compounds was screened for differential activity against KMT2A-r leukemia cell lines versus KMT2A-wild type (KMT2A-wt) leukemia cell lines, solid tumor cells and non-malignant cells by cell-based viability assays. The screen yielded SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), that limited the viability of 7 out of 11 KMT2A-r leukemia cell lines including 5 out of 7 lines derived from infants, without affecting KMT2A-wt leukemia cells, solid cancer lines, non-malignant cell lines, or peripheral blood mononuclear cells from healthy controls. The compound also significantly inhibited growth of leukemia cell lines with a CALM-AF10 translocation, which defines a highly aggressive leukemia subtype that shares common underlying leukemogenic mechanisms with KMT2A-r leukemia. SID7969543 decreased KMT2A-r leukemia cell viability by inducing caspase-dependent apoptosis within hours of treatment and demonstrated synergy with established chemotherapeutics used in the treatment of high-risk leukemia. Thus, SID7969543 represents a novel candidate agent with selective activity against CALM-AF10 translocated and KMT2A-r leukemias that warrants further investigation.Patients whose leukemias harbor a rearrangement of the  The conventional drug discovery path involves several drug development stages, from compound discovery, through to extensive preclinical drug optimization and characterization, which includes Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) and efficacy studies in animal models, culminating in rigorous testing in humans through clinical trials. Due to this time-consuming process, it has typically taken more than a decade for new agents to advance from bench to bedside, with the vast majority of compounds failing to reach the final stages of clinical trial testing. As a result, an alternative and potentially more effective discovery approach based on drug repurposing has recently gained traction. Drug repurposing entails the use of drugs for indications other than those for which they were originally intended and therefore bypasses many development and optimization steps, shortening the timeline and decreasing the overall costs for drug approval.MLL gene, recently renamed KMT2A, present in approximately ten percent of patients with acute leukemia -approved drugs and pharmacologically active compounds with known targets against a KMT2A-r leukemia cell line with counter screening against a KMT2A-wildtype (KMT2A-wt) leukemia cell line. We identified SID7969543, an inhibitor of transcription factor Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), as a novel selective candidate inhibitor against a subset of KMT2A-r and NR5A1 siRNA (M-003429-00-0010) targeting NR5A1 and On-TARGETplus control pool non-targeting scrambled siRNA (D-001810-10-20) were purchased from Millennium Science Pty Ltd.SID7969543 was purchased from Tocris Bioscience . 2-Cl-ATP was procured from Santa-Cruz Biotechnology . 2-CADO and cladribine were purchased from Sigma Aldrich . For the generation of lipid-based nanoparticles used to transfect leukemia cells with siRNA, cholesterol and the lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B sulfonyl] (DOPE-Rho) were obtained through Merck & Co., Inc. D-Lin-MC3-DMA was purchased from Assay Matrix Pty Ltd. siGENOME Human All cell lines used in this study were mycoplasma-free and have been authenticated using STR profiling. Cells were cultured as previously described , 8. Char®1280 library , Tocriscreen Plus library  and Selleck Inhibitor Library , together combining 3707 compounds. PER-485 (KMT2A-r) and CCRF-CEM  leukemia cells were seeded into 384-well culture plates. Test compounds were added to the assay plates for a final concentration of 5 μM (in DMSO stock). Plates were incubated for 72 hours at 37°C, 5% CO2. Resazurin was added and plates were incubated for 7 hours. The difference in relative fluorescence units at time zero and 7 hours of resazurin incubation was calculated for each well. The percentage cell viability for each test compound was calculated relative to the cells treated with vehicle (100% viability).The following chemical libraries were used in the screening as previously described : Prestwi50) was calculated as previously described . Quantitative PCR was run and analyzed on Applied Biosystem’s QuantStudio5 Real-Time PCR Detection System, using Taqman gene expression assays for genes NR5A1 (Hs00610436_m1), GUSB (Hs99999908_m1) and HPRT (Hs99999909_m1) Relative expressions were determined as previously described .Compound half-life was estimated by mouse liver microsomal stability assays as described .® instrument (Precision Nanosystems) as previously described (Lipid-based nanoparticles encapsulating siRNA targeting NR5A1 and scrambled siRNA (LNP-siRNA) were prepared using a NanoAssemblr™ Sparkescribed . Prior t6 cells/mL in 12 wells culture plates and incubated at 37°C, 5% CO2, with LNPs encapsulating anti-NR5A1 siRNA or scrambled siRNA at 250 nM siRNA concentration for up to 72 hours.Cells were seeded at 0.5 x 10KMT2A translocation found in infants and pediatric patients [t], and a KMT2A-wt leukemia cell line CCRF-CEM (in vitro models for high-risk disease. The viability of cells treated with a fixed concentration (5 µM) of the library compounds was assessed in resazurin-based cytotoxicity assays after a 72-hour incubation to allow selection of drugs acting rapidly on cell viability as previously described  in PER-485 cells, confirming our results from the original screen, it was less potent against the other KMT2A-r leukemia cell lines tested  that included the PER-485 and CEM cells used in the original screen, as well as four additional KMT2A-r cell lines (both ALL and acute myeloid leukemia (AML)) with either t (n=2) or t (n=2) CALM-AF10 translocated leukemias, which are KMT2A-wt but represent an aggressive leukemia subtype that shares underlying molecular etiological pathways with KMT2A-r leukemias, such as their dependency on DOT1L histone-lysine methyltransferase and an upregulation of HOXA cluster genes  KMT2A-r leukemia cells with IC50 values ranging from 2.5 to 5.9 µM . Corroborating our observations, previous studies have reported sensitivity of KMT2A-r ALL cells to nucleoside analogs , 18. To 50 < 10 µM) were leukemias with either a KMT2A or CALM-AF10 translocation , four out of six t and one t translocated KMT2A-r leukemia cell were sensitive to SID7969543, whereas the remaining two t KMT2A-r leukemia cells did not display sensitivity  and pyruvate kinase (PKM)  remained unchanged, demonstrating that SID7969543 hit its reported target   in sensise cells . Taken tWe next determined whether SID7969543 synergized with conventional chemotherapeutics used in the treatment of high-risk leukemia, namely, cytarabine, daunorubicin, etoposide, mitoxantrone, and topotecan Figure 4via the induction of apoptosis. In addition, the compound potentiates several standard-of-care chemotherapies used for treatment of high-risk leukemia. However, microsomal stability assays showed that SID7969543 has a short half-life and thus limited in vivo stability , a future larger scale screening in an expanded panel of KMT2A-r leukemia cell lines is warranted.SID7969543 preferentially inhibited the growth of a subset of KMT2A-r leukemia cells, which included various subtypes with different  subtype . While Kpathways –27. Thispathways –16 and fSID7969543 is an isoquinolinone derivative first identified in an ultra-high-throughput screen of approximately 65,000 compounds from the National Institute of Health’s Molecular Libraries Small Molecule Repository in a cell-based transactivation assay to search for potent and selective inhibitors of NR5A1 . NR5A1 iNR5A1 as a crucial gene for leukemic cell survival in one out of six primary AML samples  analysis with SID7969543 derivatives will be needed to identify the targeted pathway in KMT2A-r leukemia cells and develop clinically viable inhibitors. SAR analysis will pinpoint the functional groups essential for the KMT2A-r cell killing effect of the drug, guiding the development of analogs suitable for tagging and affinity chromatography to allow the identification of the molecular targets of SID7969543 and its derivatives in KMT2A-r leukemia cells. Silencing of candidate targets followed by downstream phenotypic analysis will subsequently provide insight into the identity of the true molecular target or targeted pathway for this class of compounds that mediates the observed KMT2A-r cell killing selectivity. These analyses will also direct the development of pharmacologically viable SID7969543 derivatives that have better drug-like properties and are suitable for evaluation in animal models of KMT2A-r leukemia.in vitro findings of our study demonstrating the rapid killing of a subset of KMT2A-r leukemia cells, particularly those arising in infants, support further study of SID7969543 to identify its molecular target, develop drug-like derivatives and use as a tool compound in vitro.While SID7969543 cannot be moved directly into the clinic, the strong KMT2A and CALM-AF10 translocated leukemia, warranting further investigation.Overall, our results show that SID7969543 represents a novel candidate agent with selective activity against The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MKar conducted the majority of the experiments with technical support from AB and ER. MKar, LX, and KS analyzed the data. AM, TF, and GA executed the drug screening at the Drug Discovery Centre, Children’s Cancer Institute Australia. EM generated lipid nanoparticles for silencing experiments. LC and RK provided guidance and access to cell lines used in the study. MKav, MH, and MN provided guidance and support with study design. MKar, MJH, LX, and KS designed the experiments. MJH and KS conceived the project. MJH and KS supervised the project. KS, MKar, and LX wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by a grant from Cancer Council NSW (Program Grant PG-1601) and philanthropic funds from the Tenix Foundation. For this work, MKar was supported by a PhD topup scholarship by CRC Cancer Therapeutics. KS received funding from the Kids Cancer Alliance. LX was supported by early-career researcher fellowships from CINSW and Tour de Cure.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few cell lines have been investigated in vitro, and previous results are inconsistent. Assuming that oxygen depletion accounts for the FLASH sparing effect, no sparing should appear for cells irradiated with low doses in normoxia.Irradiation with ultra-high dose rate (FLASH) has been shown to spare normal tissue without hampering tumor control in several Seven cancer cell lines  and normal lung fibroblasts (MRC-5) were irradiated with doses ranging from 0 to 12 Gy using FLASH (≥800 Gy/s) or conventional dose rates , with a 10 MeV electron beam from a clinical linear accelerator. Surviving fraction (SF) was determined with clonogenic assays. Three cell lines were further studied for radiation-induced DNA-damage foci using a 53BP1-marker and for cell cycle synchronization after irradiation.subclone. Similar cell cycle distributions and 53BP1-foci numbers were found comparing FLASH to CONV.A tendency of increased survival following FLASH compared with CONV was suggested for all cell lines, with significant differences for 4/7 cell lines. The magnitude of the FLASH-sparing expressed as a dose-modifying factor at SF=0.1 was around 1.1 for 6/7 cell lines and around 1.3 for the HeLain vitro. The magnitude of the FLASH effect differed between the cell lines, suggesting inherited biological susceptibilities for FLASH irradiation.We have found a FLASH effect appearing at low doses under normoxic conditions for several cell lines  Experimental evidence for the FLASH effect has been demonstrated 00 Gy/s, settings , as wellsettings .in vivo data available suggest that more sparing may occur in physoxic normal tissues than in severely hypoxic or nearly anoxic tumors  were acquired from American Type Culture Collection (ATCC). The human colon cancer cell line WiDr was acquired from LGC Promochem . The squamous cell carcinoma LU-HNSCC4 was established in our laboratory from a patient with a squamous cell carcinoma in the floor of the mouth . Cells wsubclone). Irradiation was performed under normoxic conditions at room temperature with the flasks lying flat and irradiated from beneath (beam angle 180 degrees). After irradiation, the flasks were returned to the incubator for 9-14 days. All flasks, including the non-irradiated controls, were terminated at the same occasion. Cells were fixed and stained with methylene-blue in 70% ethanol. Flasks were scanned using a flatbed scanner in 1,200 dpi resolution. Colony counts were performed with a standardized ImageJ-code  and manually checked. Surviving fraction (SF) was determined as the number of colonies with at least 50 cells divided by the number of plated cells (corrected for plating efficiency).Exponentially growing cells were trypsinized and plated in appropriate cell densities in 2.50 ml medium per Falcon T12.5 flask  and allowed to adhere overnight before irradiation. Control flasks for determination of the plating efficiency and the FLASH- and CONV-flasks were prepared identically on the same occasion, for each repetition. FLASH and CONV-flasks were irradiated minutes apart with doses from 0-9 Gy  and allowed to adhere overnight before irradiation with 3 Gy. At specific time points after irradiation, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After washing, cells were permeabilized with 0.5% Triton-X100 in PBS for 20 min, washed, blocked in blocking buffer  for 1 hour followed by 1 hour incubation with 53BP1 primary antibody (Invitrogen PA146147) and 1 hour of incubation with a secondary antibody (AlexaFluor anti-rabbit 488). Cell nuclei were counterstained with DAPI. Permeabilization, washing, blocking and staining steps were all performed at room temperature. Foci formation were assessed with a widefield fluorescence microscope, AxioOberver Z.1 , equipped with ×63/1.40 Plan-Apochromat oil-immersion objective lens and Colibri 7 solid state LED light source (Zeiss), and an ORCA-Flash4.0 V3 Digital CMOS camera . In each sample, at four different positions, fifteen Z-stack images were acquired, deconvoluted with a GPU-based deconvolution module and averaged using a Maximum Intensity Projection-algorithm . ImageJ was used for automated foci identification and quantification of DNA-double strand break (DSB) foci.subclone cells). At 24 h (6 h and 24 h for HeLasubclone cells), after irradiation cells were washed with PBS, harvested and fixated with ice-cold ethanol (70%). Cell nuclei were stained with propidium iodide  for 30 min at room temperature and DNA content was determined with an Accuri C6 Flow Cytometer . DNA-histograms were analyzed in ModFit LT 5.0 for Mac (BD Biosciences).150 000 - 500 000 cells were plated in 35 mm or 60 mm Petri Dishes  and allowed to adhere overnight before irradiation with 6 Gy (and 3 Gy for the HeLaIrradiation and dosimetry were performed as described previously . In summhttp://www.rstudio.com/) was used for statistical calculations. The parameters of the linear–quadratic model (2)) were fitted to the log (SF) using the nonlinear least-squares method (‘nls’ in RStudio). Two alternative models were fitted, one with separate α and β parameters for the CONV and FLASH data, and one with common α and β parameters for all data. The residuals were tested for normality using the Kolmogorov-Smirnov test, and the F-test was used to determine whether the fit was significantly improved by using separate parameters. Using the model fitted with separate α and β parameters for the CONV and FLASH data, dose-modifying factors (DMF) were determined as the ratio of DFLASH/DCONV at a survival fraction of 0.1 (SF=0.1) and 0.01 (SF=0.01). Boot-strapping was used to calculate the median and the interquartile range of the DMF. In addition, the difference in survival fraction at the individual dose levels were tested for statistical significance, without assuming normality, by using the Wilcoxon rank-sum test. All tests were two-sided with a chosen significance level of 5%. Experiments were repeated 2-4 times.RStudio v. 1.0.136 (RStudio Team (2015). RStudio, Inc., Boston, MA, URL ic model   show a distinct behavior compared to the other cell lines, with a larger DMF and also earlier cell cycle arrest. In comparison with HeLaearly passage, the HeLasubclone was considerably more radioresistant and lacked a shouldered survival curve for the CONV-irradiated samples. It has previously been reported that phenotype changes can occur with high passage numbers, affecting radiation responses  with two different doses (3 Gy and 6 Gy) indicated an earlier radiation induced G2/M-synchronization, suggesting a peak of the G2/M-arrest at a time point before 24 h , showed a lower number of foci for FLASH compared with CONV for normal lung fibroblasts, but no differences for the A549 lung cancer cell line . Cell cyFLASH effects are typically seen at doses ≥ 10 Gy. The current study showed a separation of the survival curves at doses below 10 Gy. Interestingly, also using a low dose of 4 Gy, Chabi et al. found FLASH irradiation to be more efficient than conventional dose rate exposures for two cases of T-cell lymphoblastic leukemia (T-ALL), but an opposite relation for a third case . The resin vivo experiments also suggest an iso-effective tumor control compared with CONV , the average dose rate varied between the different dose levels. However, the dose-per-pulse and the instantaneous/pulse dose rate were constant and the average dose rate exceeded 800 Gy/s at all dose levels. Further, we have chosen to perform a pure in vitro, and that the magnitude of the FLASH effect differed between the cell lines. The results indicate that the FLASH effect cannot be solely explained by TOD and that other mechanisms are involved. The nature of such possible biological susceptibilities and their dependence on oxygen concentrations will be subject to further investigations.To conclude, we have found a FLASH effect under normoxic conditions for several cell lines gabriel.adrian@med.lu.se.The datasets presented in this article are not readily available but the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Requests to access the datasets should be directed to GA, CC, SM, and KP designed the study. GA, SB, EK, KB, MG, and KP conducted the experiments. GA, CC, EK, SM, SB, KP, and AW analyzed and interpreted the data GA, KP, and CC drafted the manuscript. Critical revision of the manuscript for important intellectual content – all authors. GA and CC did the statistical analyses. All authors contributed to the article and approved the submitted version.This work was supported by Mrs Berta Kamprad Foundation (grant no 2020-19-301), Swedish Cancer Society (grant no 20 1298), John and Augusta Persson’s Foundation and governmental research funding (ST-ALF). Funding for AW was provided by the Swedish Society for Medical Research (SSMF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recently, we read a paper in Frontiers in Cell and Developmental Biology “Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction.” Oleic Acid (OA) may be a promising drug candidate for the treatment of hepatocellular carcinoma (HCC). However, some problems existing in this paper need to be commented.With great interest we read a paper “Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction” published in Frontiers in Cell and Developmental Biology  for 48 h . However, authors demonstrated that OA significantly suppressed cell proliferation and induced cell death of HCC cells after incubation with the same concentrations (300 μM) of OA for the same time (48 h) . These results strongly suggested that the cause responsible for inhibition of migration and invasion of cells could be due to apoptosis induction of OA. Authors could treat the cells with OA at a lower concentration or for a short time when studying the inherent effects of drugs on the migration and invasion ability of cells without apoptosis induction. This made the results more convincing.JL: study concept and design, data analysis, methodology, drafting manuscript, review and editing, and supervision.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.