Dear Editor,1. Therefore, genes essential in cancer but not normal cells are good therapeutic targets. However, while genetic dependencies for many cancer cell lines have been defined through genetic screens, much less was known for normal cells. To overcome this problem, the DepMap project has developed a method to identify genes selectively essential in a subset of cancer cell lines, making them less likely to be in core pathways critical for normal cells. Based on genome-wide CRISPR-based fitness screen data from ~1000 human cancer cell lines4, DepMap calculates CERES scores to measure the effect of gene knockout on cell fitness . Next, DepMap calculates a Normality Likelihood Ratio Test (NormLRT) score for each gene. A higher NormLRT score indicates that the distribution of this gene’s CERES scores across cancer cell lines is more deviated from a normal distribution, and the gene may be more selectively essential. Identification of selectively essential genes will not only deepen our understanding of cancer biology but also guide cancer drug development. However, manually sorting out which of them is understudied and warrants investigations is time-consuming and probably impractical.An ideal cancer therapy drug should kill cancer cells while displaying limited toxicities toward normal cells4, we collected 347 potential selectively essential genes and ranked them by PubMed publication count5. In our ranking system, the top nine most understudied genes are C11orf53, C3orf38, TMEM164, ZNF511, KCNK13, BEST3, CYB561A3, C12orf49, and COLCA2  reported previouslyCA2 Fig. . In fact7. However, the mechanistic details of how POU2F3 drives the expression of the tuft cell-specific program remain unclear. Based on our analysis, we hypothesized that C11orf53/COLCA2 and POU2F3 may work in the same pathway to regulate the growth of tuft cell-like SCLC.We subsequently focused on C11orf53 and COLCA2 because of the significant CERES scores in their dependent cell lines where they are highly expressed, suggesting strong growth phenotypes Fig. . Remarka9. Since copy number increase was observed at C11orf53 locus in NCI-H526 cells  as a co-activator for POU2F3 and demonstrate that their physical interaction is critical for the growth of SCLC.Our co-activator hypothesis predicts that C11orf53 and COLCA2 physically interact with POU2F3. Indeed, co-immunoprecipitation detected physical interactions between COLCA2 and POU2F3 Fig. . FurtherP-value < 0.05) showed similar changes upon POU2F3 knockout  upon COLCA2/POU2F3 knockout 15 and both C11orf53 (renamed as POU2AF2/OCA-T1) and COLCA2 (renamed as POU2AF3/OCA-T2)14 in SCLC, and our conclusion is essentially the same as those reached by these investigators. We noticed that in addition to SCLC, COLCA2 is also essential in a multiple myeloma cell line, JJN-3, where POU2F1 instead of POU2F3, is highly expressed and essential  functions as a co-activator for POU2F3 to drive the transcriptional program important for tuft cell-like SCLC. The highly selective nature of these co-activators in cancer coupled with the reports that ial Fig. , suggestial Fig. . How theial Fig. , suggestSupplementary information


Tumor regression was induced at doses less than 5 mg/kg, while maximal activity with complete cures were observed starting at 10 mg/kg. In MCL Mino and Jeko-1 xenografts, STRO-001 starting at 3 mg/kg significantly prolonged survival or induced tumor regression, respectively, leading to tumor eradication in both models. In summary, high CD74 expression levels in tumors, nanomolar cellular potency, and significant anti-tumor in DLBCL and MCL xenograft models support the ongoing clinical study of STRO-001 in patients with B-cell NHL.Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. Here we report results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL. STRO-001 displayed nanomolar and sub-nanomolar cytotoxicity in 88% (15/17) of cancer cell lines tested. STRO-001 showed potent cytotoxicity on proliferating B cells while limited cytotoxicity was observed on naïve human B cells. A linear dose-response relationship was demonstrated  The addition of rituximab to regimens used to treat B-cell non-Hodgkin lymphomas (NHLs) marked a milestone in the treatment of hematologic malignancies , 2. SincCD74 is a type II transmembrane glycoprotein primarily involved in MHC class II antigen presentation, with additional roles in B-cell maturation, T-cell responses, and macrophage migration inhibitory factor-induced signaling . CD74 exWe previously reported the cell-free synthesis of STRO-001, a site-specific, predominantly single-species anti-CD74 ADC incorporating a non-cleavable linker-maytansine warhead with a drug-antibody ratio (DAR) of 2 . STRO-00D = 1.75 nM) and cynomolgus monkey (KD = 2.70 nM) CD74 ECD (extra-cellular domains), but not to mouse CD74 ECD. SP7219 bound with high affinities to CHO cells transfected with human (KD = 0.93 nM) and cynomolgus monkey (KD = 1.1 nM) CD74. Conjugation to the non-cleavable linker-maytansine warhead did not affect the binding affinity of SP7219 since STRO-001 retained high binding affinity to CHO cells expressing human (KD = 1.1 nM) and monkey CD74 (KD = 1.0 nM) (In SPR (surface plasmon resonance) based Biacore binding assay, the anti-CD74 antibody SP7219 bound to recombinant human (K 1.0 nM) . Neither 1.0 nM) .CD74 surface expression was determined in 9 GCB-like DLBCL cell lines, 4 ABC-like DLBCL cell lines, and 4 MCL cell lines. Among the 13 DLBCL cell lines, CD74 copy number varied from high levels  to below the lower limit of detection . Among t2 = 0.41) toward increased STRO-001 potency with higher CD74 receptor number was observed but appeared to be weak. Notably, nanomolar potency was observed for STRO-001 in 2 of the 3 cell lines with low CD74 expression level below detection limit , anti-CD74 antibody SP7219 was conjugated to DBCO-Alexa 647 to generate SP9241, which was used to stain cells isolated from bone marrow aspirates for 5 healthy human and 5 healthy cynomolgus monkey donors. An anti-GFP antibody produced by cell free Xpress+ system was also conjugated to DBCO-Alexa 647 and used as an isotype control (SP9367). The B cells, T cells, NK cells, monocytes, myelocytes, neutrophils, granulocytes and stem cells from human and cynomolgus monkey BMA were labeled with species specific commercial antibodies, respectively (Supplementary Tables 2 and 3). Relative expression of CD74 was expressed as MFI ratio of SP9241 over SP9367 on different immune cell populations. CD74 expression on human B cells was 1.4–2.0-fold higher relative to background, while other cell types did not show CD74 expression above GFP background (Supplementary Table 4). Relative CD74 expression was found to be 2.0–7.3-fold higher on cynomolgus monkey B cells, while other cell types did not show CD74 expression above GFP background (Supplementary Table 5).in vitro was associated with upregulation of cell surface markers CD86 and HLA-DR, as well as increased CD74 expression, after 5 days of treatment . Expansion of B cell populations reatment . B cell r 5 days .The cytotoxic activity of STRO-001 toward naïve B cells and proliferating B cells was determined using a FACS based cell viability assay after 6 days of treatment. in vivo anti-tumor activity of STRO-001 was evaluated in NHL models including DLBCL and MCL subcutaneous and disseminated tumor xenografts. Two DLBCL cell lines, SU-DHL-6 and U2932, were chosen based on moderate to low CD74 expression levels (p = 0.053) with 87% TGI and 4 out of 6 complete responders. Maximal activity was observed at 20 mg/kg administered either once-weekly or twice-weekly, led to tumor regression  with all animals (7/7) in both groups remaining tumor free through the end of the study (The n levels  and reprn levels . Althougn levels . At 10 mhe study .p < 0.01)   and 5B.  < 0.01)  and 4D.3, mean tumor sizes of animals in all three STRO-001 dosing groups were significantly smaller than those in the vehicle control group (p < 0.0001)  . Signs o 0.0001) . At 10 m 0.0001)  and 5B.In the disseminated Mino model, STRO-001 was administered starting on day 14 post-tumor inoculation. The survival endpoint criteria included body weight change loss >20% and clinical signs of morbidity. Mean survival in the vehicle group was 81.3 days, while treatment with STRO-001 at all the doses tested significantly prolonged survival up to 54 days relative to the vehicle control . Almost in vitro expansion. Taken together, these findings provide a strong rationale for potently and selectively targeting of CD74 in patients with B-NHL.In considering novel cancer therapies, the selection of an appropriate target depends on its sufficient expression in or on targeted cells, with limited expression elsewhere. The present study investigated CD74 expression in B-NHL cell lines and patient tumor samples. Biotinylated SP7219, the antibody portion of the ADC STRO-001, which displayed high affinity for human CD74, was used to determine expression levels by immunohistochemistry. We found CD74 expression in 88% 15/17) of DLBCL and MCL cell lines, as well as near-ubiquitous expression in DBCLC, FL and MCL tissue microarrays. These results are consistent with a previous study using a limited number of patient biopsies /17 of DL. Other iWe previously reported a pharmacodynamics/safety study of STRO-001 in cynomolgus monkey that identified bone marrow toxicity as dose-limiting in this species . At an iSTRO-001 displayed nanomolar to sub-nanomolar potency in 88% (15/17) of cell lines, including both GCB-like and ABC-like DLBCL cell lines as well as MCL cell lines. Among these, CD74 surface expression varied considerably. Interestingly, although a weak correlation between CD74 expression and STRO-001 cytotoxicity was noted, the high internalization rate of CD74 may drive robust activity in spite of low target expression as observed in several cell lines. And as expected, the antibody-only portion of STRO-001, SP7219, did not show any cytotoxicity on any of the cell lines tested. Similarly, SP7219 has been shown to lack cytotoxicity in multiple myeloma cell lines  and no ein vitro expanded B cells. Also of note, the major and minor catabolites of STRO-001 containing the maytansinoid warhead, SC246 and SC401, were >15-fold less cytotoxic in MM cell lines, suggesting that the clinical safety profile of STRO-001 may benefit from the consequent minimization of post-catabolic non-specific toxicity (bystander effects) /Winitial) × 100 where Wintial is the weight on the first day of treatment (day 0 post-treatment). Significant toxicity was defined as a >20% decrease in animal weight, at which point affected animals were euthanized. All graphs are presented as mean or individual values ± standard error of measurement (SEM). All results were reproduced in at least 2 independent studies, and representative data from one experiment is shown.In all models, STRO-001 and vehicle control (PBS) were administered intravenously (IV) at the intervals and doses indicated. Body weight change (BWC) was calculated relative to weight on the first day of treatment using the formula BWC = ([Wp < 0.05) was considered statistically significant. Annotations for p-values are: *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001; ns: not significant.Statistical analyses of final tumor sizes were performed using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test using Prism software (version 6). When Brown-Forsythe and Bartlett’s tests showed significant data heterogeneity, non-parametric analysis was subsequently performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. A probability of <5% (


Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies. Triple negative breast cancer (TNBC) represents between 10–15% of all breast cancers and can be defined histologically as a subtype of breast cancer that lacks expression of the three key receptors: estrogen, progesterone, and HER2 . The resIn clinical practice, chemotherapies including platinum-based drugs continue to be the mainstay of standard treatments for TNBC. The use of chemotherapy in TNBC is supported by clinical evidence that demonstrates the benefit of treating breast cancer patients with cisplatin . HoweverIn this study, we aimed to reconcile these approaches by testing how cisplatin-based chemotherapy interacts with signal transduction pathways in order to identify targeted therapies that can overcome resistance to cisplatin. Most targeted therapies, such as kinase inhibitors, target components of signal transduction pathways. There is also evidence that DNA-damaging drugs synergize with tyrosine kinase inhibitors (TKIs) that inhibit EGFR to kill TNBC cells . Therefo2.TNBC cell lines BT20, HCC1143, MDA-MB-157, MDA-MB-231, MDA-MB-468, MFM-223, and the immortalized mammary epithelial cells MCF10A cells were obtained from the American Tissue Culture Collection . TNBC cell lines CAL120, CAL851, and HDQP1 were obtained from the German Tissue Repository DMSZ . All cell lines were tested for mycoplasma and authenticated by short tandem repeat (STR) typing. The HCC1143, MDA-MB-231, and MDA-MB-468 cells were cultured in RPMI  containing 10% fetal calf serum ; the HDQP1 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FCS; the CAL120 and CAL851 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FCS, 1 mM sodium pyruvate, and 2 mM glutamine (Life Technologies); the BT20 cells were cultured in DMEM-HAM F12 (Sigma-Aldrich) containing 10% FCS; the MDA-MB-157 cells were cultured in Leibovitz L15 (Sigma-Aldrich) containing 10% FCS. MCF10A cells were cultured in MEGM with 0.5 mL insulin, 0.5 mL hydrocortisone, 0.5 mL rhEGF and 2 mL BPE. Cells were incubated at 37 °C and 5% COIpatasertib (S2808), TAK-632 (S7291), PF-00562271 (S2672), Dorsomorphin (S7840), CHIR-98014 (S2745), Everolimus (S1120), PF-4708671 (S2163), and Stattic (S7024) were purchased from SelleckChem, whilst AICAR (Acadesine) (NA03552) and QNZ (EVP4593) (1237744-13-6) were purchased from Carbosynth, and U0126 (V1121) was purchased from Promega.QNZ (EVP4593), Ipatasertib, Stattic, CHIR-98014, PF-4708671, AICAR (Acadesine), PF-00562271, and U0126 were made up to 10 mM stocks, whilst Dorsomorphin (7.4 mM) and Everolimus (10μM) were also reconstituted in DMSO to the stock concentrations listed. Cisplatin (Selleckchem Cat #S1166 50 mg) was prepared in a stock solution in DMF at a concentration 12 mg/mL (39.99 mM).3 cells/well for MDA-MB-157 cells, 4 × 103 cells/well for CAL851 cells, and 3 × 103 cells/well for the other cell lines were seeded in 96-well plates. Following overnight incubation at 37 °C, drugs were added at the indicated concentrations and incubated for 5 days at 37 °C. Cell proliferation was determined using the acid phosphatase assay, as described previously [50 values) and combination index (CI) values were determined using the Chou–Talalay equation on CalcuSyn software. Loewe synergy was calculated using Combifit Analysis ™ [A total of 5 × 106 cells per well whilst CAL851, MCF10A, and HDQP1 cells were seeded at 0.3 × 106 cells per well, and MFM-223, MDA-MB-468, and MDAMB-157 cells were seeded at 0.5 × 106 cells per well in 6 well plates. Cell density was chosen to reflect 75–80% confluence prior to starting treatment. Afterward, cells had the attached media removed and replaced with serum-free medium at least 16 h prior to treatment with inhibitors.On day 0 of the experiment, MDA-MB-231 cells were seeded at 0.45 × 10On day 1, cell lines were treated with the relevant drug as per Protein quantification was performed on samples using the bicinchoninic acid assay (Pierce Biotechnology) and subsequently stored at −80 °C until required.Samples were prepared for RPPA analysis by adjusting protein concentrations to 1 μg/μL and by the addition of 4× SDS Sample buffer  +1/10 Bond breaker TCEP solution . Samples were heated to 96 °C for 5 min and the prepared sample was collected and stored at −80 °C until further analysis.Baseline and on-treatment determination of proteins/phosphorylated proteins in the panel of 6 TNBC cell lines and the MCF10A cell line were determined by RPPA as described previously . The anthttps://github.com/dirkfey/TNBC-RPPA accessed on 15 February 2022).Network inference was performed for each cell line using the corresponding perturbation data by applying the BMRA algorithm described previously, and available as MATLAB code . The priHierarchical clustering using Euclidean distance and average linkage was performed using the MATLAB R2019b clustergram function.p-value < 0.05 was considered significant.Spearman’s rank correlation was conducted using the PRISM software package, and a 50 to cisplatin in the immortalised normal breast epithelial cell line MCF10A (5.25 ± 0.55 µM). Taking their IC50 as a guide to avoid harming normal epithelial tissue, we categorised our panel of TNBC cell lines as cisplatin-sensitive if they had an IC50 of < 5 µM, or cisplatin-resistant if they had an IC50 to cisplatin of >5 µM. We also classified cisplatin response in our panel of TNBC cell lines based on their molecular subtype, as determined by Lehmann et al. [We tested the antiproliferative effects of cisplatin in a panel of eight TNBC cell lines representing the main subtypes of TNBC . To defiWe utilised a systems biology approach with reverse protein array data to determine inferred networks that predict inhibitor sensitivity. We performed RPPA analysis on a panel of seven TNBC cell lines that included three cisplatin-resistant and three cisplatin-sensitive cell lines and the normal MCF10A cell line. We tested a panel of 11 inhibitors that target nodes of pathways found altered in breast cancer, including the AMPK, STAT, NFkB, PI3K/AKT, and MAPK/ERK pathways in each cell line. The RPPA contained a panel of 72 proteins, which was enriched for members of the PI3K/AKT and MAPK pathway to determine differences in protein expression and phosphorylation between untreated and inhibitor-treated cell lines.First, we wanted to understand if there were any drug-specific response patterns across the cell lines. Thus, we clustered the treatment-induced fold changes for each drug across the seven cell lines . A few oNext, we identified the cell-line specific interaction networks from these data using Bayesian molecular response analysis (BMRA) . BMRA usp-value < 0.05, two-sample students t-test) in the resistant versus sensitive network were (1) markedly increased inhibition of p38 MAPK by AKT, (2) change from activation to inhibition of CHK1 by GSK3B, (3) change from activation to inhibition of PTEN by AMPK, (4) a loss of negative feedback from AKT to PI3K, and (5) a change from inhibition to activation of PDK1 by FAK  should be active in the basal state. Thus, we assessed the networks’ basal activity states in the unperturbed condition . Severaln = 299), AKT1 mRNA expression was downregulated compared to both HER2 positive n = 127) and hormone receptor positive patients , but not PIK3CA (p = 0.032 > 0.01) and PDPK1 (p = 0.14), significantly associated with poor relapse-free survival in triple negative patients  is upregulated in resistant cell lines, (ii) features several crosstalk and feedbacks that are more active in resistant cell lines, (iii) overrides DNA damage checkpoints by inhibiting CHK1, (iv) enhances survival by suppressing p38 activity, and (v) promotes cell proliferation by inactivating the cell-cycle inhibitor p27kip .n = 237) showed that TP53 mutations occur in 81.9% of TNBC patients whilst PIK3CA mutations occur in 14.8% TNBC patients. Further, we determined that PIK3CA and TP53 mutations are neither mutually exclusive nor do they significantly co-occur in TNBC patients [The results of this computational analysis reverberate with biological findings. PTEN and PI3KCA  are often mutated in TNBC resulting in the constitutive activation of the PI3K-AKT pathway . Howeverpatients ,18, sugg50s ranging from 0.11 ± 0.02 μM in MFM-223 cells to 7.34 ± 3.40 μM in HDQ-P1 cells. We correlated sensitivity of the S6-kinase inhibitor with cisplatin and found no significant correlation . We further assessed the GSK3B inhibitor CHIR-98014 in our panel of TNBC cell lines, but found limited antiproliferative response with only one cell line achieving an IC50 at 1 μM (MDAMB-468). Lastly, we tested the PI3K (αδ specific) inhibitor taselisib and the AKT inhibitor ipatasertib in a panel of TNBC cell lines. As expected, cell lines with PIK3CA mutations were most sensitive to the PI3K/AKT inhibitors.Therefore, we focused our investigation on possible links between TP53 and PIK3CA mutational status and cisplatin response in TNBC cell lines, which were treated with cisplatin and a PI3K/AKT pathway inhibitor. For this, we tested inhibitors for multiple points along the PI3K/AKT/S6-kinase axis that were identified from our systems biology analysis . The S6-In our TNBC cell-line panel, we demonstrated that the proliferation of cisplatin responsive TNBC cell lines with or without TP53 mutations could be decreased by inhibiting key nodes of the PI3K/AKT S6 kinase pathway. We therefore determined the antiproliferative effects of combining a PI3K inhibitor (taselisib) or the AKT inhibitor ipatasertib with cisplatin in a panel of cisplatin-sensitive/resistant TNBC cell lines . The resWhen we analysed the synergism to PI3K inhibitor and cisplatin in the remaining cisplatin-resistant cell lines, we found that the combination showed limited synergy in cell lines that harboured TP53 mutation but were PIK3CA wildtype (WT) . We also found that the CAL-51 cell line which is cisplatin sensitive and PIK3CA WT but harbours a TP53 mutation showed reduced synergy to the combination of cisplatin and a PI3K inhibitor. Finally, the BT20 cell line which is cisplatin sensitive but has mutations in both PIK3CA and TP53 exhibited strong synergism to the combination of cisplatin and PI3K inhibition. However, the strongest synergism between cisplatin and PI3K was observed in MFM223 cells. To test whether this synergy extends down the pathway, we combined an AKT inhibitor with cisplatin, also observing a strong synergy B.Overall, our analysis suggests that cisplatin-resistant TNBC cell lines can be resensitised to cisplatin by combination therapies with either a PI3K inhibitor or AKT inhibitor, if they also harbour mutations in PIKC3A and TP53.Chemotherapies that target DNA including anthracycline- and platinum-based drugs contribute towards the standard treatments for many cancers . In TNBCDespite these advances, cisplatin’s efficacy to treat cancer can be limited due to either acquired or intrinsic resistance—a clinical observation also observed in TNBC . ResistaWe utilised RPPA data and applied BMRA to reconstruct signalling pathways and their rewiring in cisplatin-sensitive and cisplatin-resistant cell lines. It is important to acknowledge that the TKIs used in this study—similar to all kinase inhibitors—may have off-target effects. However, it is not currently possible to determine every single off-target effect of specific TKIs . TherefoThe BMRA analysis reconstructed two consensus networks, one from cisplatin-sensitive and the other from cisplatin-resistant cell lines showing that a large number of connections were rewired either qualitatively or quantitatively. Looking at how the drug-resistant network could be converted into a sensitive configuration initially suggested that cisplatin-resistant TNBC cell lines should have greater sensitivity to inhibition of either p70S6-kinase or GSK3B. However, in our panel of TNBC cell-line sensitivity to either p70S6-kinase (M2698) or GSK3B (CHIR-98104) did not correlate with cisplatin resistance. We showed that the MFM223 cell line which is cisplatin resistant is the most sensitive to M2698, and previous studies also displayed M2698 effectively inhibited in vivo TNBC tumour growth. However this antitumour effect could be associated with cell lines that harboured PIK3CA mutations , such as50s < 1 μM. Again, the PIK3CA mutant MFM223 cells had the greatest sensitivity to both taselisib (PI3Kα/δ) and ipaterisib (AKTi).It is known that PTEN and PI3KCA  are often mutated in TNBC, resulting in the constitutive activation of the PI3K-AKT pathway . The frep-AKT in cancer cells also increased [Several studies have targeted the PIK3/AKT pathway in TNBC ,30 with ncreased . HoweverTNBC features frequent TP53 mutations, and previous studies have linked TP53 mutation status with increased activity of the PIK3CA gene and downstream PI3K-AKT signalling . AstanehTherefore, to confirm that targeting TNBC cell lines that harbour either or both TP53 and PIK3CA mutations is an effective treatment for cisplatin-resistant TNBC, we tested combinations of either PI3Ki or AKTi with cisplatin. We found that TNBC cell lines responded synergistically to the combination; however, the MFM223 cell lines which harbour a dual PIK3CA TP53 mutation and were cisplatin resistant responded best to both combinations.Further assessing why this combination of drugs has robust anticancer effects in cisplatin-resistant TNBC cell lines; studies have associated that cell lines with PIK3CA mutations are associated with chemotherapy resistance . SpecifiIn conclusion, our results demonstrate that TP53- and PIK3CA-mutant TNBC cell lines with innate resistance to cisplatin are sensitive to drug combinations of cisplatin and PI3K/AKT pathway inhibitors, representing a potential therapeutic approach that warrants further investigation.


ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs ( High-grade osteosarcoma (HGOS), the most common malignant tumor of bone, is treated by surgery and systemic neo-adjuvant multidrug chemotherapy ,2. CisplPharmacogenetic studies have revealed several single nucleotide polymorphisms (SNPs) of genes belonging to either DNA repair, drug transport, folate metabolism, and detoxification pathways to be associated with therapy-related parameters in HGOS, as survival and drug response, or development of drug-associated toxicity . The genIn the last decade, pharmacogenomic approaches have been increasingly applied to the study of HGOS providing a series of interesting insights related to genetic polymorphisms, which may be causally related to drug resistance or susceptibility to develop treatment-related adverse toxicities ,6. HowevThis analysis was performed by using an innovative multimodal targeted next generation sequencing (mmNGS) approach that allowed for the contemporary study of the selected SNPs on both DNA- and RNA-derived libraries. Data obtained by mmNGS on DNA-derived libraries were validated by TaqMan genotyping. RNA expression level of the 14 genes in CDDP-resistant variants compared to their parental cell lines was also determined. Heatmap analysis was performed, including all CDDP-resistant and drug-sensitive cell lines.Data obtained for the 28 SNPs on DNA-derived libraries by the custom mmNGS approach were validated by TaqMan genotyping in 24/28 SNPs. Variants with an allele frequency greater than 3% were considered reliable. Those SNPs that were homozygous wild-type for the reference allele were not reported in By comparing the data obtained from sequencing and genotyping, we found that for 11/18 (61%) cell lines data obtained by both techniques matched in 100% of the SNPs, whereas in 4/18 (22%) cell lines the match ranged from 90 to 93%, and was below 90% in 3/18 (17%) cell lines. The fact that 39% of the cell lines did not show a complete match could be explained by the presence of different subpopulations within the same cell line.ABCC2 rs17222723, ACYP2 rs1872328, TPMT rs12201199, rs1142345, and rs1800460 the homozygous wild-type genotype was identified in all 18 cell lines.Interestingly, for five SNPs, ERCC2 gene, (rs13181 and rs1799793), which exhibited a genotype change in relation to the acquisition of CDDP resistance , while in the variant with the highest resistance level (U-2OS/CDDP4µg) the genotype of the polymorphism shifted to homozygous wild-type (TT).In the CDDP-sensitive, parental cell line and in the two variants with the lower level of CDDP resistance (U-2OS/CDDP300 and U-2OS/CDDP1µg), the genotype of ERCC2 rs13181 in U-2OS/CDDP1µg variant for which mmNGS reported GT but TaqMan genotyping a TT genotype. This apparent discordance may be due to the different sensitivity of the techniques and the presence of subpopulations with TT and GT genotypes. However, these data indicate that in these cells the transition toward a TT genotype is associated with development of CDDP resistance.The data obtained for these SNPs by TaqMan genotyping and mmNGS were concordant for all cell lines except for ERCC2 rs1799793, the sensitive cell line and the two U-2OS/CDDP300 and U-2OS/CDDP1µg resistant cell lines showed a heterozygous variant genotype CT, which became homozygous (CC) in the variant with the highest resistance level  GAT, was detected.All genotype variations identified at the DNA level and described above were also identified on the RNA level, indicating that these changes had been selected and maintained during development of CDDP resistance. Differently, the ibraries . The genGSTP1 rs1695 variant allele was identified by the CLC GWB in all three CDDP-resistant U-2OS variants , which estimates the fold-change in RNA expression, for each CDDP-resistant variant compared to its drug-sensitive parental cell line is graphically shown in ABCC2, ABCC3, ACYP2, COMT, ERCC2, and XRCC3 emerged to be increased more than 2-fold compared to the parental U-2OS cell line, whereas four genes, ATM, ATR, TP53, and XPA were downregulated in CDDP-resistant variants. Considering all three CDDP-resistant variants together, the differential gene expression tool of the CLC GWB identified the downregulation of ATM, ATR, and TP53 as significant with a Bonferroni corrected p-value < 0.05.In U2OS-derived CDDP-resistant variants, six genes, ABCB1, ABCC2 and XRCC3 in all three variants, whereas ACYP2, COMT and ERCC2 only in Saos-2/CDDP300, the variant with the lowest resistance level. CDDP-resistant variants also presented downregulation of ATM, ATR, GSTP1, TPMT, and XPA genes. Evaluating all three CDDP-resistant variants together, a significant difference after Bonferroni correction with a p-value < 0.05 was identified for upregulation of ABCB1 and downregulation of TPMT and XPA.In Saos-2-derived CDDP-resistant variants, six genes were increased more than 2-fold: Similarities between CDDP-resistant and CDDP-sensitive cell lines were assessed by using the heatmap tool of the CLC GWB, including all 14 genes . Two maiThe 10 drug-sensitive cell lines formed the second main cluster, which was mostly separated from that of CDDP-resistant variants.As also shown in In this study, a custom mmNGS approach has been used to study 28 SNPs of 14 genes, contemporarily on the DNA and RNA level, in 6 CDDP-resistant and 12 CDDP-sensitive human HGOS cell lines. To our knowledge, this innovative approach has not been used so far for pharmacogenomic studies.The successful validation of the DNA variant calling by TaqMan genotyping confirmed that this approach is an appropriate method to study even rare SNPs. Compared to genotyping by single TaqMan assays, the custom mmNGS approach is faster and also offers the possibility to identify additional SNPs mapping to the target region. Moreover, small targeted panels allow the pooling of higher sample numbers compared to whole-genome NGS. Another advantage of the mmNGS approach is the low amount of starting material that is required for library preparation, which facilitates the application of this method to tumor tissue samples. In addition, the simultaneous analysis of SNPs on the DNA and RNA level, as well as the possibility to estimate the level of RNA expression associated with the polymorphic gene status allow for a direct correlation between the genotype status with the biological function of each SNP.The frequencies of variant alleles per cell line ranged from 9 (found in IOR/OS15 and MG-63) to 22 (detected in IOR/SARG), confirming the heterogeneity and high genetic instability of HGOS.ACYP2 rs1872328 [ABCC2 rs17222723 [TPMT rs12201199, rs1142345, and rs1800460 [ABCC2 rs717620 and GSTP1 rs1695, were found as homozygous wild-type or heterozygous but not as homozygous variant. These findings suggest that the variant allele of these seven SNPs could be of biological disadvantage in HGOS tumor cells. Differently, the two SNPs of TP53, rs1042522 and rs1642785, were identified either in a homozygous wild-type or variant but not heterozygous status.Particular genotype distributions were found for 9 SNPs. Five SNPs that had been reported in association with CDDP-related ototoxicity, s1800460 ,11, wereGSTP1 rs1695, ERCC2 rs13181, ERCC2 rs1799793, and ERCC1 rs11615.The most relevant SNPs that emerged in this study to be associated with the development of CDDP resistance were GSTP1 rs1695 was the only SNP for which the genotype changes found in the RNA-derived libraries differed from those revealed in DNA-derived libraries. Interestingly, in all five drug-sensitive cell lines with the heterozygous genotype in the DNA , the genotype status in the RNA was homozygous wild-type. The presence of the variant also in the RNA of all six CDDP-resistant cell lines, with the consequent amino acid change Ile105Val, strongly suggests that the AG genotype is associated with reduced CDDP response. These findings further support the previously demonstrated relevance of GSTP1 enhanced enzymatic activity in these CDDP-resistant HGOS cell lines [The ll lines . The phaThis observation is concordant with the data reported in almost all germline studies. A significant association between AG+GG genotypes and poor histological response as well as decreased event-free and overall survival was observed in five studies ,14,15,16GSTP1 rs1695 was excluded from further analyses in the study by Goricar and co-workers because the genotype frequencies of rs1695 were not in Hardy Weinberg Equilibrium [Interestingly, ilibrium . Since tERCC2 rs1799793 and rs13181 and the synonymous ERCC1 rs11615 at the DNA and RNA level. All three SNPs have been reported to be associated with survival and toxicity, but the data are quite discordant [Genotype changes in relation to CDDP resistance were found for the two non-synonymous SNPs scordant ,27,28,29ERCC2 rs1799793 GG genotype was reported in association with poor event-free survival compared to the GA +AA genotypes and ERCC2 rs13181 AA with poor response to chemotherapy compared to AC+CC [However, the ERCC2 rs13181 and ERCC2 rs1799793 SNPs were associated with survival [In one germline study performed on 130 patients with osteosarcoma treated with neoadjuvant cisplatin-based therapy in combination with doxorubicin, methotrexate, and ifosfamide the survival . The autERCC2 rs1799793 and rs13181 on the protein structure and stability have recently been elucidated [The functional consequences of ucidated ,31. MoleERCC2 rs13181 variant caused the loss of C-terminal alpha-helix and beta-sheet [In addition, the ta-sheet . AlthougERCC1 rs11615, which changed to the homozygous wild-type genotype status in the Saos-2 CDDP-resistant variant with the highest level of resistance, two germline studies reported similar evidence being better survival associated with the TT compared to the CC genotype [For genotype ,25. Howegenotype ,21,22,23ATM and ATR. It has been shown that cancer cells that are deficient in one DNA repair pathway can activate other functional repair pathways, which underlines the importance to study not only one of them for treatment optimization [Differential gene expression analysis identified dysregulations in CDDP-resistant variants compared to their parental cell lines suggesting that development of CDDP resistance influences not only genes of the NER pathway, which is known to be mainly responsible for the removal of CDDP-associated DNA adducts, but also genes belonging to other DNA repair mechanisms, such as mization .ATM and ATR observed in Saos-2/CDDP-resistant variants is a relevant finding that needs to be further explored, since inhibitors against ATR have already been used in clinical trials in other cancers [ACYP2 and COMT, although not significant, warrants attention because SNPs of these two genes had been described to be associated with ototoxicity after CDDP treatment [The biological consequence of the significant downregulation of  cancers . On the reatment ,9,10,11.In conclusion, the mmNGS approach emerged to be an innovative, reliable tool to detect genetic polymorphisms at both DNA and RNA level, allowing for the identification of genetic changes causally related to CDDP resistance in HGOS cells. Once further validated in tumor samples series, these SNPs could be useful to identify patients with reduced sensitivity to CDDP-based therapy and/or increased susceptibility to CDDP-related adverse toxicities.The study was performed on a panel of 12 drug-sensitive human HGOS cell lines: U-2OS, Saos-2, MG-63, and HOS  and IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS18, IOR/OS20, IOR/MOS, IOR/SARG, which were established from tumor specimens at the Laboratory of Experimental Oncology of the Orthopaedic Rizzoli Institute .The panel of 6 CDDP-resistant variants derived either from U-2OS  or Saos-2  CDDP-sensitive cell lines, as previously reported . ResistaCDDP sensitivity of each cell line was expressed as IC50 (drug concentration resulting in 50% inhibition of cell growth after 96 h of in vitro treatment). The fold-increase in CDDP resistance of each variant was determined by comparing its IC50 value with that of its corresponding parental cell line and, as previously described, ranged from 4.0- to 62.5-fold for U-2OS variants and from 7.4- to 112.1-fold for Saos-2 variants [2 at 37 °C. Drug resistant variants were continuously cultured in the presence of the CDDP concentrations used for their selection. Cell pellets were prepared according to standard procedures when cells were confluent, snap-frozen, and stored at −80 °C.All cell lines were cultured in Iscove’s modified Dulbecco’s medium (IMDM) added with 10% fetal bovine serum  and maintained in a humified atmosphere with 5% CODNA fingerprint analyses were performed for all cell lines using 17 polymorphic short tandem repeat sequences confirming their identity.DNA and RNA were simultaneously isolated and purified from the same pellet obtained from each cell line by using the AllPrep DNA/RNA mini kit  according to the manufacturers’ instructions. During this process, the DNA and RNA were isolated from the entire sample by passing the lysate first to the AllPrep DNA spin column to isolate high molecular weight total genomic DNA and through the AllPrep RNA spin column to isolate total RNA.A DNA and RNA quality check was performed for all samples by spectrophotometry . All RNA samples were run on a 2100 Bioanalyzer system  using the RNA 6000 kit .Library preparation was performed according to the QIAseq Multimodal Panel handbook v06/2020  for small panels. The primers for the libraries derived from DNA were designed for 28 SNPs of 14 genes related to DNA repair, CDDP transport and detoxification, and TP53 . For theDNA- and RNA-derived libraries were prepared for all 18 cell lines. Prior to library preparation, the nucleic acid concentrations were determined fluorometrically by Qubit high-sensitivity assays on a Qubit reader version 4.0 .For library preparation, the input amount was 40 ng of DNA and 100 ng of RNA. All libraries were run on a 2100 Bioanalyzer system  using the High Sensitivity DNA kit  to check the profile of the samples. The fragment lengths of all libraries ranged between 400 and 600 base pairs, as expected according to the protocol.In order to provide an accurate quantification of the amplifiable libraries, the QIAseq Library Quant Assay kit  was performed on a real-time PCR system  for all of them.For sequencing, libraries were diluted to 1.2 pM, pooled together and analyzed by paired-end sequencing on a NextSeq 500 instrument  using a mid-output reagent kit v2.5 (300 cycles) with a custom sequencing primer provided with the library preparation kit.All bioinformatic analyses were performed using the CLC GWB software  v22.04. FastQ files were downloaded from the BaseSpace cloud  and imported in the CLC GWB .For the detection of DNA variants and gene expression, the FastQ files were analyzed using the Biomedical Genomics Analysis plugin running the Qiaseq Multimodal Analysis workflow.The DNA and RNA reads were aligned to the human genome hg38 reference sequence and filtered using a coverage of 100× and a variant allele frequency (VAF) higher than 3%.For RNA variant calling a custom workflow was provided by the Qiagen bioinformatics support. This workflow worked with UMIs, mapped the reads on the human genome hg38 and filtered with specific parameters for rare RNA variant calling.p-value < 0.05 were considered significant.For differential gene expression analysis between the groups of drug-resistant variants and their respective parental cell line, the differential expression tools of the CLC GWB were used and changes with a Bonferroni corrected For hierarchical clustering analysis the tool for creating heatmaps of the CLC GWB was used with Euclidean distance and complete linkage.In total, 24 of the 28 selected polymorphisms were validated by real-time genotyping PCR . TaqMan The genotyping experiments were performed according to standard protocols using 10 ng DNA as input material using the VIIA 7 DX realtime PCR system  and the results were analyzed with the TaqMan Genotyper software , which generated allelic discrimination cluster plots to determine the genotype of each SNP.


Chemokines exert crucial roles in inducing immune responses through ligation to their canonical receptors. Besides these receptors, there are other atypical chemokine receptors (ACKR1–4) that can bind to a wide range of chemokines and carry out various functions in the body. ACKR2, due to its ability to bind various CC chemokines, has attracted much attention during the past few years. ACKR2 has been shown to be expressed in different cells, including trophoblasts, myeloid cells, and especially lymphoid endothelial cells. In terms of molecular functions, ACKR2 scavenges various inflammatory chemokines and affects inflammatory microenvironments. In the period of pregnancy and fetal development, ACKR2 plays a pivotal role in maintaining the fetus from inflammatory reactions and inhibiting subsequent abortion. In adults, ACKR2 is thought to be a resolving agent in the body because it scavenges chemokines. This leads to the alleviation of inflammation in different situations, including cardiovascular diseases, autoimmune diseases, neurological disorders, and infections. In cancer, ACKR2 exerts conflicting roles, either tumor-promoting or tumor-suppressing. On the one hand, ACKR2 inhibits the recruitment of tumor-promoting cells and suppresses tumor-promoting inflammation to blockade inflammatory responses that are favorable for tumor growth. In contrast, scavenging chemokines in the tumor microenvironment might lead to disruption in NK cell recruitment to the tumor microenvironment. Other than its involvement in diseases, analyzing the expression of ACKR2 in body fluids and tissues can be used as a biomarker for diseases. In conclusion, this review study has tried to shed more light on the various effects of ACKR2 on different inflammatory conditions. Chemokines are small chemotactic cytokines produced by many cell types  that are primarily involved in regulating the movement of immune and non-immune cells during normal immune surveillance, inflammatory situations, and development . These cChemokine receptors, which are considered members of a family of seven transmembrane G-protein-coupled receptors (GPRs), mediate the particular actions of chemokines on their target cells . These rApart from the conventional chemokine receptors, a subfamily of atypical chemokine receptors (ACKRs) has been discovered . These rACKR2 cDNA showed 86% similarity and 71% identity to the murine Ackr2 gene, and regarding protein, both differ from other chemokine receptors as they replace the highly conserved DRYLAIVHA motif with DKYLEIVHA in human ACKR2 and mouse Ackr2, bringing an extra charged residue into the region (https://www.ncbi.nlm.nih.gov/gene/1238), the ACKR2 gene comprises three exons mapped to chromosome 3p22.1, a region of the genome that contains a cluster of genes encoding chemokine receptors. Moreover, according to the UniProt database (https://www.uniprot.org/uniprot/P21741), ACKR2 protein contains 384 amino acids, of which amino acid 19 is N-glycosylated, which is considered dispensable for ligand binding and high expression , in which a single amino acid change at position 41 in its binding site, i.e., the first three extracellular loops, sets V41A apart from wild-type ACKR2. It has been shown that this allele is structurally similar to the wild-type ACKR2, and there was no noticeable variation in the predicted folding patterns of proteins, and both have similar expression levels in transfected CHO-k1 cells. When compared to wild type, ACKR2-V41A is estimated to have minor hydrophilicity. In terms of binding affinity, the binding affinity for CCL2 and CCL4 is decreased in V41A, leading to a decreased scavenging efficiency of this allele. Regarding receptor recycling, it was also demonstrated that ACKR2-V41A has decreased receptor recycling and expression levels on the cell surface for a long period of time. Furthermore, Ca2+-dependent signaling is considerably lower in CCL2-activated cells that express the ACKR2-V41A allele than in the wild type, resulting in decreased signaling following CCL2 activation in this allele. These variations in binding, recycling, and signaling have shown different efficiency in various conditions, possibly leading to different responses in diseases , was first cloned by Nibbs et al. in 1997. The human e region . Accordipression , and thediseases . In sepadiseases . In addidiseases . Anotherdiseases . A possivia activating a β-arrestin-dependent pathway , some lymphocytes , myeloid progenitor-derived cells (such as mast cells and dendritic cells), blood endothelial cells, and lymphatic endothelial cells (LECs)—the major ACKR2-expressing cells in adults—have been shown to express ACKR2 –22. Acco pathway , 24. It  pathway . For theThis receptor is a highly versatile ACKR capable of binding to the majority of inflammatory CC chemokines, including CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, and CCL22 , 26. AltGATA1 gene induced ACKR2 expression in these cells, suggesting that the ACKR2 promoter possibly contains a binding site for GATA1 , cytokines (IL-6), leukocytes, the coagulation cascade, and the complement system. Regarding ACKR2, although it has been shown that placental ACKR2 is decreased at transcriptional and protein levels in preeclampsia in Cho et al.’s study, this downregulation has not been observed in Tersigni et al.’s investigation. Cho et al. have hypothesized that ACKR2 downregulation leads to higher inflammatory responses, resulting in excessive recruitment of macrophages to the maternal–fetal interface and an inappropriate response to systemic inflammation . In contnterface . Additionterface .+ macrophages into the mammary glands and subsequently controls the process of mammary gland branching morphogenesis during development. Ackr2-deficient mice have displayed premature development of mammary glands. This was linked to elevated infiltration of macrophages into the developing gland, resulting in an increased density of the ductal epithelial network. These findings show that ACKR2 is a key regulator of branching morphogenesis in a variety of biological situations and support the involvement of chemokines, playing critical roles in postnatal development processes , are developed at the epithelial ducts and promote network formation. A stromal component composed of fibroblasts, extracellular matrix (ECM), adipocytes, and immune cells leads to the achievement of this process. Mammary glands and surrounding TEBs contain macrophages, and their phagocytic and cytokine-producing characteristics are essential for several tissue remodeling events during developmental processes. In macrophage-deficient animals, mammary gland development is substantially disrupted, accompanied by abnormal TEB formation, ductal elongation throughout puberty, and lobuloalveolar development during pregnancy. In summary, these investigations demonstrate that macrophages play a critical role in the control of ductal branching in the developing mammary gland , 47. Macrocesses , 49. Furrocesses .The lymphatic vessels (lymphatics) and LECs play a vital role in the flow of fluid and leukocytes such as APCs from the peripheral tissues to the secondary lymphoid organs, especially lymph nodes. Human LECs but not vascular endothelial cells have been shown to express ACKR2 within non-inflamed tissues. Since this receptor has been found in a subset of lymphatic vessels, proposing that the heterogeneity in the expression of ACKR2 within the lymphatic vasculature might be a sign of function in this microenvironment. For example, only afferent lymphoid vessels in regions near the epidermis and deeper in the dermis express ACKR2 in the human skin sections. ACKR2 is also expressed in small and large intestines (in the villi), colon (lamina propria mucosa and muscular layer), appendix (in lamina muscularis externa), and other secondary lymphoid organs, including spleen (in red pulp) and tonsils (on sinuses like channels and parafollicular areas) . AccordiWhen the body’s tissues are exposed to harmful stimuli such as infections, damaged cells, or irritants, inflammation occurs. It is a defensive reaction involving immune cells, blood vessels, and molecular mediators, and it is a complicated biological response involving several factors. An inflammatory response’s primary goal is to remove the source of cell damage, which helps remove necrotic cells and tissues that have been destroyed by the initial harmful stimuli as well as the inflammatory response, leading to tissue healing . During Numerous investigations employing human samples or ACKR2-deficient animals have shown that ACKR2 expressed by lymphatic vessels plays a critical role in inflammatory diseases and malignancy. While the increasing consensus is that ACKR2 works as a negative regulator of inflammation, conflicting findings have been reported about its involvement in the modulation of adaptive immune responses and the development of autoimmune disease.The tumor microenvironment is the main interaction site between cancer cells and the host immune system. Through interactions between chemokines and chemokine receptors, diverse immune cell subsets are attracted to the tumor microenvironment, and these populations have unique impacts on tumor growth and treatment outcomes .de novo tumor development. CCL3 is highly elevated in tetradecanoyl phorbol acetate/dimethylbenz(a) anthracene -induced inflammation in B6/129 ACKR2-deficient mice, leading to recruitment and infiltration of polymorphonuclear cells (PMNs) and CD3+ T cells into papilloma. This hyperactive inflammatory response enhanced papilloma formation and made ACKR2-deficient mice more susceptible to the higher proliferation of keratinocytes  and elevated leukocyte infiltration  in the inflamed mucosa of the colon (The two most common types of inflammatory bowel illness are Crohn’s disease and ulcerative colitis (IBD). The production of pro-inflammatory chemokines is common in people with IBD. Additionally, the persistent mucosal inflammation seen in IBD has been linked to a higher incidence of colon cancer, also called colitis-associated cancer. Inflammatory cytokines have also been shown to play a role in the development of colonic inflammation into colon cancer. If pro-inflammatory chemokines are not properly controlled, it may lead to persistent intestinal inflammation and even cancer . In an ehe colon . These hhe colon . These dhe colon .–/– mice develop high levels of IL-1β, IL-6, IL-12, IL-17, TNF-α, CCL2, CCL3, CCL4, CCL5, CCL12, CCR1, CCR2, CCR5, FGF, and VEGF in oral SCC lesions, leading to higher infiltration of Ly6G+ neutrophils, mast cells, and F4/80+ macrophages in tumor tissues and proposing an unleashed inflammation in the tumor site. This inflammatory milieu results in the creation of highly proliferative tumor cells -treated ACKRor cells . The prior cells . Therefong VEGFA .+ NK cells, leading to enhanced tumor-homing efficiency of NK cells. ACKR2–/– mice have been demonstrated to have increased killing activity of NK cells through enhanced CCR2 expression and NK infiltration into the CCL2-expressing melanoma tumor microenvironment. As a result, ACKR2 expression in the tumor microenvironment decoys CCL2 and impairs the infiltration of NK cells  during the early stages of the metastasis process, losing cell-to-cell connections and epithelial properties in favor of mesenchymal ones that enable them to invade and metastasize to the surrounding or distant tissues. In this process, several transcription factors, including Twist, Slug, Snail, Gsc, and Zeb, are involved, leading to downregulation of epithelial markers (E-cadherin and cytokeratin) and upregulation of mesenchymal markers  . Cancer-As discussed, LECs are the main expressors of ACKR2 in the adult human body. Therefore, analyzing vascular tumors in terms of ACKR2 expression to find the origin of the tumor might be promising in detecting the type of cancer. In this regard, Nibbs et al. showed that ACKR2-immunoreactive tumor epithelial cells might be exclusively derived from LECs, suggesting that ACKR2 can be useful in detecting lymphatic tumor cells .Cervical cancer is the female reproductive system’s second most prevalent malignancy. Regional lymph node metastases are a critical prognostic factor in individuals with cervical cancer. Around half of the cervical cancer patients with pelvic lymph node metastases will have a disease recurrence, with the majority suffering from uncontrolled tumor growth. The reported 5-year survival rate for individuals with a recurrence is between 3% and 13%, and prognostic indicators for lymph node metastases in patients with cervical cancer are lacking . AnalyziAlthough there is no defined correlation of ACKR2 expression with other carcinogenic factors in cervical cancer, ACKR2 downregulation has been accompanied by dysregulation of metastatic factors such as MMP9 and VEGF in breast cancer. In this cancer, expression of ACKR2 was accompanied by ACKR1 and ACKR4 which were negatively correlated to axillary lymph node metastasis of breast tumor cells, suggesting favorable outcomes for co-expression of ACKR2, DARC, and CCX-CKR in breast cancer , 79. FurSince ACKR2 downregulation has been shown to contribute to the progression of colon adenocarcinoma, the correlation of ACKR2 expression in tumor tissues was analyzed to introduce its capability of being a biomarker in colon adenocarcinoma. Results showed that ACKR2 mRNA expression in colon adenocarcinoma tissues was decreased compared to unaffected mucosa. ACKR2 was also highly expressed in endothelial cells of the blood and lymphatic vessels of unaffected tissues compared with tumor tissues. Moreover, ACKR2 expression in tumor tissues was negatively correlated to tumor invasiveness, as its expression was lower in T3 and T4 grades than in T1 and T2 ones. All of the correlation was sex- and age-independent. In addition, ACKR2 expression was correlated to the expression of its counterpart ligand, i.e., CCL2, but not CCL5 and CCL8, suggesting that ACKR2 downregulation increases CCL2 in the tumor microenvironment to recruit more inflammatory cells and promote inflammation. CCL22, a Treg-recruiting chemokine, was another chemokine that was increased in the ACKR2-decreased tumor tissues, showing that ACKR2 downregulation also promotes tumor development by creating an immunosuppressive tumor microenvironment . TherefoIn another study, ACKR2 expression was tracked to analyze the trend of expression through the conversion of an adenoma into an adenocarcinoma sequence. Results have demonstrated that the expression ratio of ACKR2 was decreased through normal to tumor and increasing depth of tumor invasion. Moreover, the expression ratio of ACKR2 was decreased along with elevating tumor grade (adenoma to adenocarcinoma), whereas its expression was elevated through normal to pathological in adenoma tissues. Collectively, during the adenoma procedure, ACKR2 expression was insignificantly increased, and then its expression declined insignificantly in the process of malignancy. Moreover, the highest downregulation of ACKR2 was at the point that adenoma cells are capable of being malignant . Thus, tToday, it is well known that chemokines, their receptors, and other mediators are part of an interactive network. Therefore, analysis of a set or group of mediators would be helpful in predicting disease progression and prognosis. In a study to analyze the profile of expression of chemokine ligands and receptors in salivary adenoid cystic carcinoma (SACC), it has been shown that the expression of ACKR2 alongside CXR1, CXCR2, CMKLR1, and ACKR4 was higher in patients without recurrence, while inflammatory chemokines such as CXCL11, CCL5, and CCL20 were higher in metastatic cell lines compared with non-recurrent and less-invasive cells . AlthougACKR2 gene on cancer, it has been shown that rs2228468 is correlated with breast cancer metastasis. In SNP rs2228468, serine substitutes with tyrosine at location 373, and it has been found that this SNP shows limited differential effects on chemokine degradation ability. Analysis of this SNP in breast cancer has concluded that SNP rs2228468 has been associated with TNM and that rs2228468 serves as an indication of a changed metastatic propensity in Han Chinese patients  and Ly6Clow (after acute phase) subsets of monocytes in ischemic zones, and excessive infiltration of innate immune cells has been demonstrated to induce unfavorable remodeling of the left ventricle and heart rupture. The recruitment of inflammatory cells to the ischemic heart is mainly dependent on the CC chemokine members, namely, CCL2, CCL5, and CCL3 and their receptors, CCR2. ACKR2 was shown to be expressed in infarcted myocardium in humans and mice  include a wide range of conditions that affect the blood vessels and heart and may present themselves in life-threatening events such as myocardial infarction (MI), aneurysm, and stroke. Myocardial infarction (MI) is histologically characterized by the infiltration of leukocytes, namely, neutrophils (within the first 24 h) and Ly6Cand mice , 86. ACKfraction .Other than MI, recent research has found that pro-inflammatory molecules are overexpressed in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHR) when compared to normotensive rats (Wistar–Kyoto rats: WKY), implying that the NTS of SHR may be inflamed abnormally. Therefore, the expression of inflammatory chemokines and cytokines might be involved in the formation of hypertensive phenotype in rats . RegardiAD is the most frequent neurodegenerative condition in the aged population and is the leading cause of dementia in this generation. AD is defined pathologically by the accumulation of amyloid-beta (Aβ) peptides in the gray matter, resulting in insoluble aggregated plaques that are considered to cause neurodegeneration and cognitive impairment. CCL2 is involved in the regulation of monocyte and dendritic cell migration across the blood–brain barrier, as well as the differentiation and migration of macrophages. CCL2 is mostly released by astrocytes and microglia in the brain, with only trace amounts secreted by endothelial cells. CCL2 is produced in the central nervous system (CNS) due to an immunological response to infection, damage, or inflammatory reaction in the CNS .In the animal model of AD, CCL2 is essential for causing chronic inflammation and activation of immune cells, as well as the release of other chemokines. The overexpression of CCL2 causes activated microglia cells to remain in close proximity to the inflamed location. This leads to enhanced activated microglia and elevation of key features of AD, such as amyloid plaques, plaque aggregation, and cognitive loss . In AD mCells of the microglia and astrocytes predominate in the CNS when it comes to ACKR2 expression , 94. Folvia the production of reactive oxygen species, nitric oxide, and glutamate from infiltrating immune cells and brain-resident microglia . Inflammation is a natural reaction to trauma and has neuroprotective and regenerative effects because it enables the clearance of cellular debris and the release of neurotrophic substances , 96. Theicroglia , 97. Furicroglia –100. Furicroglia , 100. Siicroglia , 94. Folfter TBI .+ mice compared to ACKR2-knockout mice decrease inflammation and demyelination of the spinal cord and have a lower incidence and severity of EAE episodes  peptide-induced ACKR2episodes . The admammation . In contammation . It has ammation . Finallyammation . Given ARegarding the human counterpart of EAE, i.e., MS, it has been shown that ACKR2 mRNA expression in PBMC derived from relapsing-remitting MS subjects was decreased. However, no link was found between this receptor and other cytokines implicated in the pathogenesis of MS, such as TNF-α and IL-33 . TherefoACKR2–/– mice under identical settings, but what was evoked at primary sites (probably not only IFN-γ) that increases ACKR2 expression at distant locations is still unknown , indicating the inflammation-regulatory role of ACKR2 at distant sites . This re unknown –107.ACKR2 gene and decrease ACKR2 mRNA and protein levels in HEK293 cells. ACKR2 expression is further downregulated in psoriasis patients after cell damage, which is a key trigger for the creation of new plaques . When cells are subjected to tensile stress, ACKR2 is rapidly downregulated, and miR-146b is simultaneously upregulated  in both ACKR2–/– and WT animals. After bleomycin exposure, Ifnb–/– mice showed increased pro-fibrotic indices. There is evidence to indicate that ACKR2 is one of the key players in preventing chronic inflammation-induced skin scarring caused by promoting IFN-β production . Moreover, downregulation of ACKR2 increased the recruitment and influx of CCR2+/CCR5+ Tγδ cells (anti-fibrotic and homeostatic T cells) in the lungs, which resulted from upregulation of CCL5, CCL12, and IFNγ. In conclusion, ACKR2 is activated during bleomycin-induced pulmonary fibrosis, and its deletion reduces mortality, lung damage, inflammation, and fibrosis. ACKR2 is a determinant for managing the recruitment of CCR2+/CCR5+ IFN-γ–producing Tγδ cells, which generate IFN-γ and regulate the development of the Th17 response and fibrosis intensity (Idiopathic pulmonary fibrosis (IPF) is a lethal disorder resulting from aberrant fibroblast and immune system activation, which leads to irreversible fibrotic changes in the lungs. This disorder like fibrosis in other tissues is caused by excessive immune responses and production of chemokines such as CCL2, CCL3, CCL11, and CXCL1–3 in the lungs, leading to initiating the inflammatory reactions and inappropriate activation of fibroblast. Although ACKR2 has been implicated as a chemokine scavenging receptor to reduce inflammatory responses in several tissues, it has been shown that ACKR2 plays a promoting role in IPF. In bleomycin-induced IPF in ntensity .Obesity is the outcome of a persistent anabolic state defined by increased caloric intake, decreased energy expenditure, or both . ExperimRheumatic disorders, often known as rheumatism, are a group of diseases that affect the connective tissues, especially the joints, causing chronic and often intermittent pain. Rheumatism is a general term that refers to a wide range of diseases, including arthritis and non-articular rheumatism, including spondyloarthropathies, lupus, Sjogren’s syndrome, systemic sclerosis, and sarcoidosis.ACKR2 mRNA and a lack of increase in serum levels of CCL2, CCL3 (weakly), and CCL4 in patients with SSC compared with healthy control, indicating that ACKR2 might be a response to alleviate the SSC pathogenesis , is an autoimmune rheumatic condition characterized by excessive collagen production and deposition in the skin and internal organs, as well as injury to small arteries, which is collectively called fibrosis . Therefoogenesis . In contogenesis . A possi–/– mice rejected their syngrafts 1 week after surgery. This is a surprising finding, given that ACKR2–/–mice are completely histocompatible with WT littermate mice and form such syngeneic graft rejection. However, the impact was attributed to an enhanced innate immune response in ACKR2–/– mice, indicating the participation of damage-associated molecular patterns (DAMPs) under the sterile circumstances of transplant surgery. Specifically, it was claimed that DC-expressed ACKR2 contributes to DC behavior modification by increasing maturation and allosensitization. Despite the reduced allospecific T-cell response shown in ACKR2–/– mice, no difference in allograft survival was observed between WT and ACKR2–/– mice, suggesting that any such impact was limited to syngeneic grafts (+ cells (LEC cells) in ACKR2–/– mice at the early post-graft stage, although lymphatic density was comparable to that of WT grafted mice. It was proposed that deletion of ACKR2 increases the proximity of pro-lymphangiogenic macrophages to LECs. Therefore, it was proposed that ACKR2, through modulating lymphangiogenesis, might be involved in the suppression of corneal allograft rejection  process and present corneal alloantigens to naive host T cells. The induction of an allospecific Th1 response then facilitates corneal transplant rejection. Syngeneic corneal transplants done in naive hosts, in contrast to allografts, are accepted forever. Regardless of the result, both corneal syngeneic and allogeneic transplants stimulate corneal hem- and lymphangiogenesis, which is thought to have a substantial impact on the graft’s outcome or the effectiveness of a subsequent graft , 133. ACc grafts . The altc grafts . Syngeneejection .high/Gr1+ myeloid cells, which express CCR2, exert immunomodulatory and immunoregulatory functions and recruit into tissues whereby CCL2 is produced  is the most serious clinical issue that may occur following allogeneic BM cell transplantation. GVHD’s target tissues are invaded by T cells, and it has been shown that local chemokine synthesis is considered a mediator of this invasion . It is nproduced . Therefoe marrow . In concIn vitro studies have shown that ACKR2 reduces the amount of leukocyte infiltration, inflammation, and fibrotic tissue remodeling that occurs following renal IRI, hence avoiding the development of chronic kidney disease , as well as greater levels of inflammatory CCL2  activation in the lungs and might promote COPD progression  is the formation of granulomas. Mycobacteria-reactive T cells, especially IFN-γ-secreting ones, play a critical role in this process  were protected against IAV, with a reduced viral load and dysfunction of the lungs -induced inflammation is a critical process for viral clearance, inducing adaptive immune responses, and getting the lung to a hemostatic status. However, an increased immune response characterized by excessive chemokine production might result in severe lung damage that contributes to death. Certain chemokines are linked to an increase in immunopathology after IAV infection, whereas others are necessary for the virus to be effectively controlled , 157. CChe lungs . It is t failure . CCR5-de process .In vitro, in vivo, and clinical studies have indicated that ACKR2 is dysregulated in different conditions in the body. Therefore, down- or upregulation of ACKR2 in these conditions prompted investigations to find underlying mechanisms concerning ACKR2’s involvement in diseases. In cancer, ACKR2 has been shown to act as a double-edged sword and play a paradoxical role. In some studies, ACKR2, through inhibiting inflammation, mediates the resolution of inflammation in various conditions such as infections, autoimmune diseases, and cancer. Therefore, using mechanisms that lead to upregulation of ACKR2 might be promising in the treatment of those diseases. For example, it has been shown that some miRNAs, such as miR-146b, can suppress ACKR2 expression. In this regard, targeted modulation of miR-146b through siRNA might be promising in the upregulation of ACKR2 to induce scavenging inflammatory chemokines , that leads to the formation of enormous and densely linked networks is a fascinating area of research. Protein interaction networks effectively develop biological and biomedical applications by abstracting core scientific concepts . Proteinin vitro and in vivo studies, which are almost animal models. As a result, it is proposed that future investigations can be more humanized to be translated into clinical trials. Finally, it is hoped that using ACKR2-based approaches in the treatment and diagnosis of diseases might broaden our knowledge of pathogenesis to overcome complications.Most studies regarding ACKR2 function and its mechanism of action in physiological and pathological situations are based on observations extracted from AS: Conceptualization, Investigation, and Writing—Original Draft. FJ-N: Investigation and Writing—Original Draft. HM: Investigation and Writing—Original Draft. FE: Investigation and Writing—Review and Editing. MO: Investigation and Writing—Original Draft. AJ: Investigation and Writing—Original Draft. JN: Conceptualization, Writing—Original Draft, Methodology, and Supervision. All authors contributed to the article and approved the submitted version.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.


Calcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses  of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye. Uveal melanoma (UM) is a relatively rare disease with an incidence of approx. five cases per million in the USA and approx. 1–8 cases per million in Europe per year, however, constituting the most common primary intraocular tumor in adults . It arisFurthermore, the molecular pathogenesis of UM is characterized by chromosomal abnormalities and gene mutations. Most common are mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) and the guanine nucleotide-binding protein subunit alpha-11 (GNAQ/GNA11), which occur in a mutually exclusive manner in approx. 90% of posterior UMs . Other cTreatment options range from local therapy, including radiotherapy (brachytherapy or teletherapy), thermotherapy, and tumor resection  to enucleation . The proDespite advancements in the management of localized disease, adjuvant therapy of metastatic disease in the form of chemotherapy, adjuvant immunotherapy, and molecular targeted therapy may be necessary to prevent further progression, prolong life expectancy, and improve quality of life. The research and establishment of additional globe-preserving treatment strategies are, therefore, crucial for uveal melanoma patients .Electroporation-based techniques, such as irreversible electroporation, electrochemotherapy, and gene therapy, have already been established as effective alternative treatment modalities, especially for inoperable, recurring, and chemo-resistant malignancies in various tumor entities that do not adequately respond to the current treatment regimen. Electroporation (EP) is the induction of transient permeabilization of the cell membrane via short high voltage electric pulses, allowing passage of ions and molecules into and out of the cell. Electrochemotherapy is emerging (ECT) as an adjuvant treatment modality for local tumor control in various cancer entities. It combines the application of electric pulses to the tumor tissue with the administration of chemotherapeutic agents, rendering the cell membrane permeable to otherwise impermeant or poorly permeant anticancer drugs ,25,26,272+-ATPases to transport calcium out of the cells, and reduced production of ATP due to disruption in the mitochondrial membrane potential by the high intracellular calcium concentrations [Calcium electroporation (CaEP) is a prospective anticancer treatment where high concentrations of calcium are introduced into the cytosol following the application of electric pulses. The high intracellular concentrations of calcium ions can lead to an adenosine triphosphate (ATP) deficit ,29,30,31trations ,33,34,35Preclinical investigations suggest that this acute and severe ATP depletion leads to necrotic, apoptotic, or autophagic cell death ,29,30,31To reproduce the heterogenic characteristics of uveal melanoma, four UM cell lines with different genetic profiles were analyzed in the present study: two UM cell lines, UM92.1 and Mel270, and two uveal primary melanoma (UPM) cell lines, UPMD2 and UPMM3. The genetic and morphologic characteristics are summarized in All cell lines were authenticated by short tandem repeat profiling according to published data ,42,43,442).The UM cell lines UM92.1 and Mel270 were maintained in RPMI 1640 medium . The UPM cell lines UPMD2 and UPMM3 were cultivated in Ham/F12 medium . The cell medium was supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) and was exchanged two times per week. The cell cultures were incubated in a humidified incubator  in 100 µL of the respective cell culture medium. The spheroid cultures were incubated in a humidified chamber  for one week until electroporation.The 3D spheroids were generated by seeding 5 × 105 cells were resuspended in 400 µL Hepes buffer containing 0, 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin.The UM cells were trypsinized and transferred into Ingenio cuvettes  for the formation of monolayer cell cultures. A total of 5 × 10Seven days after generating the UM spheroids, the medium was gently removed and replaced with 100 µL Hepes buffer containing 0, 0.55 mg/mL, 1.11 mg/mL, 2.78 mg/mL, 5.55 mg/mL, and 8.33 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The spheroids were treated at higher concentrations of calcium chloride because preliminary tests showed no effect after treatment at the concentrations used on the single-cell model (data not shown).The monolayer cell cultures and the spheroids were then electroporated using a Genedrive voltage pulse generator  with an electroporator adapter for cuvettes and a two parallel aluminum electrode (4 mm apart) respectively. In this study, cells and spheroids were electroporated with eight rectangular pulses  of a 1000 V/cm pulse strength according to the European Standard Operating Procedure on Electrochemotherapy (ESOPE) alone or in combination with the aforementioned calcium and bleomycin concentrations.4 cells were transferred to white opaque-walled multi-well plates  in 100 µL medium (RPMI 1640) or Ham/F12 medium, supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) per well. Changes in cellular ATP concentrations  were analyzed using the CellTiter-Glo 2.0 assay  according to the manufacturer’s instructions. Equal portions of CellTiter-Glo 2.0 and cell suspension were mixed and placed in the centrifuge (750 rpm) for two minutes. After a further 15 min of incubation in a dark chamber at room temperature, the luminescence was recorded using the plate reader ClarioStar Plus . The ATP luminescence was given in RLU (relative light units).Following electroporation of monolayer cell cultures, the cell suspensions, approx. 10n = 5–8 each condition) was assessed using the CellTiter-Glo 3D (Promega) according to the manufacturer’s instructions. Equal amounts of CellTiter-Glo 3D and spheroid cultures were mixed by pipetting up and down for 30 s to ensure complete lysis of the cells and the release of ATP. The mixture was transferred to white opaque-walled multi-well plates. After five minutes of incubation in the centrifuge (750 rpm) and a further 25 min in a dark chamber at room temperature, the luminescence was measured using a reader ClarioStar Plus. The ATP luminescence was given in RLU.The cellular ATP level in UM and UPM spheroids  were seeded on flat bottomed 96-well plates  with 104 cells in 100 µL medium per well. To determine cell viability and growth, the cells were fixated 24 h, 3 days, and 7 days after electroporation with 50 µL 10% trichloroacetic acid  for one hour at 4 °C and then stained with 50 µL 0.4% Sulforhodamine B solution . After approximately three washing steps with 1% acetic acid, the minoxanthene dye was then dissolved with 150 µL 10 mM Tris buffer (pH 10.5). Finally, the absorbance was recorded at 560 nm using the reader ClarioStar Plus . Based on these data, the cell line-specific growth rate (SGR) in % per day was calculated by using the following Equation (1).After the electroporation procedure, the UM and UPM cells , on day 3 (3 d), as well as on day 7 (7 d) after treatment using a Zeiss Primovert bright-field microscope at 4× magnification. The image and morphological analysis of the spheroids was carried out with a Zeiss Axiocam 105 and ZENcore software . The optic density  and the cross-sectional area were measured to evaluate the mass density of the spheroids. Due to frequent detachment of cells from the main spheroid core due to low adhesion and density following treatment, the Feret’s diameter was calculated. This value represents the mean diameter of the not dissociated spheroid as well as the mean distance between the most distant spheroid compartments. All measurements were performed using the open-source software Fiji ImageJ 1.53c .n = 3–6 each condition) were diluted in medium (RPMI or Ham’s F12) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) and seeded on Cellstar 6-well plates  with 2 × 103 cells in 3 mL medium per well.UM and UPM cell suspensions of the monolayer cell cultures  with 2 × 103 cells in 3 mL medium per well.One week after the electroporation procedure, spheroids treated the same were pooled and seeded on 6-well plates  with 2 × 102), the medium was removed, and the cells were stained with 4% crystal violet solution. To assess the survival fraction (SF) of proliferating cancer cells, colonies containing 50 or more cells were counted using a Leica S6D Greenough stereo microscope . The plating efficiency ) and, therefore, the survival fraction ) were calculated in percentage and statistically analyzed.After a two-week for monolayer and a three-week incubation phase for spheroids in a humidified chamber . A value of To assess the cytotoxicity of CaEP compared to ECT with bleomycin in UM and UPM cells and spheroids, an ATP assay was performed 24 h after treatment . The anaThe ATP levels of UM and UPM spheroids were only marginally affected following calcium chloride and bleomycin administration without EP A–D. Elecp < 0.005). The reduction in cell viability and the cytotoxic effect of the tested conditions were not statistically significant in the UPMM3 cells (The sulforhodamine B (SRB) assay was used for cell density determination and, therefore, as a cytotoxicity screening of the tested agents. The tumor growth was quantified by calculating the specific growth rate (SGR). The cytotoxic effect of 2.5 µg/mL bleomycin and EP alone in the UM92.1 A and MelM3 cells G. NevertOne week following CaEP, a significant reduction in the cell viability with a mean specific growth rate B of 17.7The image documentation was conducted before and on days 3 and 7 following treatment . The masThe survival fraction in the monolayer cell lines was significantly decreased in all tested cell lines for all tested conditions . A statiAn overall reduction in the survival fraction and, therefore, the colony formation capacity of the tested spheroids was documented for all cell lines following CaEP and ECT A–D. The 2+) plays an important role as a second messenger by maintaining cytoplasmic Ca2+ concentration low at rest and by mobilizing Ca2+ in response to stimulus, which in turn activates the cellular reaction [2+ level in concentrations about 20 to 40 nM [ECT is a well-established electroporation-based treatment modality with high response rates in various tumor entities ,37,38,46reaction . In addito 40 nM .Despite positive results in vitro and in vivo, the application of CaEP is mainly restricted to research purposes ,52,53,54UM92.1 cells show a more significant response than Mel270 in regard to the ATP reduction in 2D cell cultures, whereas the ATP levels in the spheroids are comparable for both cell lines. The difference between the two tested models could be accredited to a more accurate simulation of morphological and biochemical features of the original tissue cells in the spheroids since 3D cell cultures offer investigative conditions that closely resemble the in vivo situation. In respect of the cell viability, a significant reduction was detected with concentrations of calcium chloride higher than 0.28 mg/mL. For the UM92.1 and Mel270 cell lines, a similarly significant effect regarding cell viability and specific growth rate reduction could be achieved in monolayer cell cultures after ECT with both bleomycin concentrations and following CaEP only after application of 1.1 mg/mL calcium chloride. Analyzing the image documentation of the spheroids, a more evident size reduction and detachment manifest in the UM92.1 and Mel270 cell lines in comparison to the UPM spheroids after both treatment modalities and independently from the administered concentrations. The detachment is more prominent after CaEP in comparison to ECT for both UM92.1 and Mel270, whereas ECT has a lower effect in concern to the spheroid detachment and a higher impact on the spheroid size, especially in the UM92.1 cell line. The low cohesion and peripheral cell distribution seen in the spheroid imaging explains the increase in the mean gray value following ECT in both cell lines. The greater response and the robust growth development of the UM92.1 and Mel270 spheroids may be associated with the origin of the cell lines. Mel270 cells arose from a large recurrent tumor after prior irradiation . The UM9Calcium electroporation is postulated to have a limited impact on normal cells compared to malignant cells, as shown in 3D spheroid models and in vivo ,52,53,54Furthermore, the temperature and the time of administration prior to or after electroporation have been discussed as possible factors with an impact on the efficacy of CaEP . In thisThe present study aims to evaluate the cytotoxicity, the morphological changes of tumor spheroids, the effect on the cell viability, the specific growth rate, and the survival fraction following CaEP compared to ECT in 2D monolayer cell cultures as well as in 3D tumor spheroids in four UM cell lines. A dose-dependent reduction in ATP levels, cell viability, and specific growth rate with varying sensitivity among the tested cell lines was documented. The limitation of our study is the short lifespan of spheroids, which affects the size of the treated tumor as well as the central cell necrosis in the tumor organoids. CaEP is a novel treatment modality, and little knowledge has been generated up to now regarding its pathomechanism, and the optimal therapeutic settings and further dependent variables remain to be explored. The use of 3D spheroid models imitates an in vivo setting with higher precision, delivering more representative results for optimization of CaEP settings for targeted treatment. Our in vitro results indicate that CaEP may constitute an efficient and inexpensive therapeutic option for the local tumor control of UM and the prolongation of life expectancy in patients with progressive disease.


Patients with esophageal malignancy have a 5-year survival rate of only 14% in Canada. This high mortality rate is due to three factors: late diagnosis, difficulty in surgically removing the tumor because of its localization, and treatment resistance. Resistance can be developed after prolonged exposure to anti-cancer drugs and/or radiation. Indeed 30% of patients will not respond to treatment or will relapse. Resistance has been mainly ascribed to the presence of cancer stem cells (CSCs) inside the tumor. However, no treatment specifically directed against CSCs is available to patients.Thus, targeting CSCs is a promising strategy to improve the survival of patients with esophageal squamous cell carcinoma (ESCC), the most common type of esophageal cancer worldwide. Therefore, a better understanding of the molecular mechanisms occurring during long-term exposure to cancer treatments is imperative.Herein, we developed an unbiased approach to identify new players in chemotherapy and radiotherapy resistance development in ESCC. We established radio- (R), chemo- (C), and radiochemo-resistant (RC) human ESCC cell lines using prolonged exposure to radiation and/or chemotherapeutic agent 5-FU, respectively.high cells and CD24high/CD44high cells in flow cytometry. We then used a proteomic approach to identify new players in treatment resistance. Interestingly, pathway analysis pointed out to alterations in energy metabolism as well as amino acid metabolism. Seahorse assays showed that resistant cell lines have a lower respiration rate than control cells, while glycolysis remains unchanged. To further characterize these metabolic changes, we performed an unbiased metabolomic study and confirmed a decrease in amino acid levels such as proline, in resistant cell lines. Recently, metabolic regulation has been linked to a new post-translational modification, lactylation. Proteomic data were re-analyzed looking for lactylated protein and found, amongst others, PYCRL, an enzyme implicated in proline biosynthesis, as one of the most differentially lactylated proteins in treated cell lines compared to control. PYCRL lactylation was confirmed using immuno-fluorescence colocalization and immuno-precipitation. Lastly, using AlphaFold, preliminary results point toward the importance of PYCRL lactylation impairing PYCRL homomultimerization.The enrichment in CSCs in all treated cell lines was demonstrated by an increase of ALDH1To conclude, our results suggest an important role of proline metabolism following long-term treatment in ESCC. This study is a first step toward the identification of new targets to fight treatment resistance in ESCC patients.CAG, CIHR, OtherFRQNone Declared