Abstract Background The molecular landscape of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been extensively characterized through single-modality studies. However, the interplay between molecular modalities and their collective influence on treatment response and outcomes remains poorly understood. Methods We integrated genomic, epigenomic, transcriptomic, and ex vivo drug response data from 1231 patients diagnosed with BCP-ALL. Using Multi-Omics Factor Analysis, we identified signatures explaining key aspects of the integrative molecular landscape, referred to as cross-modal elements (CMEs). The CME-derived signatures were introduced into pathway and intermodal network analyses, while their impact on patient outcomes was assessed through survival modeling. Results Pathway and network analyses annotate the resulting integrative CMEs, linking them to key biological processes, including disease development, cellular regulatory processes, metabolic pathways, and drug response. By leveraging correlations between DNA methylation and ex vivo response to doxorubicin, we stratify patients with hyperdiploidy into subgroups that differ in relapse-free survival. These signatures are independent of clinical variables. Survival models incorporating CME-selected ex-vivo drug responses combined with clinical data improve risk prediction compared to clinical models alone (FDR < 0.05), demonstrating the potential of integrative multiomics in refining risk stratification. Conclusions Our study highlights the importance of multimodal data integration in BCP-ALL to provide biological insights with potential relevance for precision medicine.

Ex vivo drug response profiling is emerging as a valuable tool for identifying drug resistance mechanisms and advancing precision medicine in hematological cancers. However, the functional impact of dysregulation of the epigenome and transcriptome in this context remains poorly understood. In this study, we combined ex vivo drug sensitivity profiling with transcriptomic and epigenomic analyses in diagnostic samples from 597 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Ex vivo resistance to antimetabolites (e.g., cytarabine, thioguanine), glucocorticoids (e.g., dexamethasone, prednisolone), and doxorubicin was independently associated with reduced relapse-free survival (P < 0.05). Molecular profiling identified pretreatment DNA methylation and gene expression patterns distinguishing resistant from sensitive cases, revealing key drug resistance signatures. These included aberrant expression of genes related to heme metabolism (e.g., ATPV06A) and KRAS signaling (e.g., GS02). Notably, we also observed atypical expression of genes usually restricted to T cells and other immune cells (e.g., ITK) in resistant BCP-ALL cells. Our findings demonstrate that ex vivo drug response patterns are predictive of clinical outcomes and reflect intrinsic molecular states associated with drug tolerance. This integrative multi-omics approach highlights potential therapeutic targets and underscores the value of functional precision medicine in identifying treatment vulnerabilities in pediatric ALL.

Environmental pollutants are commonly present in low concentrations, often as complex mixtures that can lead to various interaction phenomena, including synergism, antagonism, or additive effects. These interactions can alter the overall toxicity or biological impact of the mixture when compared to the effects of individual pollutants. While regulatory agencies typically assess the safety of individual pollutants, the cumulative and interactive effects of pollutant mixtures are less well understood. This study aims to address this gap by evaluating the interactions of eight common pollutants (BPA, BADGE, DBP, DEHP, PFOA, 4MBC, OMC, TCEP) at their no observed effect level (NOEL) concentrations. The study specifically focuses on identifying potential synergistic effects on cell viability, transcriptomic changes, and phenotypic outcomes in two biological models: Drosophila and mammalian cell lines. Nearly 400 combinations of pollutants were tested, and their effects were compared to those of individual pollutants and appropriate controls. The results highlighted significant findings, including detrimental effects of the BPA-DBP-OMC mixture on neuronal differentiation and the BADGE-OMC-TCEP mixture's negative impact on muscle differentiation on Drosophila and mammalian cell lines. Cytotoxic synergy effects were also found between bisphenol derivatives and phthalates. These findings emphasize the need to consider the risks associated with pollutant mixtures.

Mebendazole (Mbz), a well‑known anthelminthic drug, has demonstrated anticancer properties in tumor models and patients, and is thus under consideration for repositioning into an anticancer drug. Mbz is directly cytotoxic in cell lines by various mechanisms and acts indirectly via immunomodulation. In the present study, the anticancer effects of Mbz, alone and in combination with cytotoxic drugs, were further characterized using primary cultures of patient tumor cells ex vivo and the murine colon cancer cell line, CT26, in vitro and in vivo. Patient‑derived tumor cells from acute myeloid leukemia (AML) and ovarian, colorectal and renal cancer were exposed to Mbz alone and, for solid tumors and the CT26 cell line, in combination with irinotecan, cisplatin or gemcitabine (patient cells only). Cytotoxicity was assessed using the fluorometric microculture cytotoxicity assay. In vivo, the antitumor effects of Mbz and irinotecan, alone and in combination, were evaluated in the BALB/c CT26 colon cancer mouse model by tumor growth measurements and flow cytometric analysis of tumor immune cell infiltration. In the patient cell samples, Mbz showed modest single‑agent cytotoxicity, with the AML samples being the most sensitive, and displayed enhanced effects when combined with cytotoxic drugs, particularly irinotecan. CT26 cells showed modest dose‑independent sensitivity to Mbz, which enhanced the effect of both cisplatin and irinotecan. In vivo, Mbz and irinotecan both inhibited tumor growth, but the combination did not significantly outperform Mbz alone. Flow cytometry of the resected mouse tumors indicated that Mbz promoted macrophage polarization from the M2 to M1 phenotype, suggesting that immune modulation may contribute to its anticancer effect. Mbz has features making it a candidate for repositioning into an anticancer drug and part of its effect may be mediated by macrophage modulation.

Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27 327 cells are freely available to extend to future scRNA-seq methodological comparisons.

To facilitate the translation of immunotherapies from bench to bedside, predictive preclinical models are essential. We developed the in vivo immuno-oncology Hollow Fiber Assay (HFA) to bridge the gap between simpler cell-based in vitro assays and more complex mouse models for immuno-oncology drug evaluation. The assay involves co-culturing human cancer cell lines or primary patient-derived cancer cells with human immune cells inside semipermeable hollow fibers. Implanted intraperitoneally in mice, the fibers captured treatment-induced immune cell-mediated cancer cell killing following treatments with aCD3 and/or IL-2, demonstrating the feasibility of the assay. Traditional models require lengthy observation periods to monitor tumor growth and treatment response. The immuno-oncology HFA enables a rapid initial in vivo evaluation of immunological agents on cancer and immune cells of human origin, addressing two of the 3Rs — reduction and refinement — in animal research.

Recent advancements in spatial biology have revolutionized our understanding of the organization and functional dynamics of cells and tissues. In this study, we applied Molecular Pixelation (MPX), a single-cell spatial proteomics assay, to investigate the modulation of the cell surface proteome in an in vitro drug screening model using the ETV6::RUNX1 acute lymphoblastic leukemia (ALL) cell line, Reh . Specifically, we focused on the in vitro response to fludarabine, a chemotherapeutic agent used prior to allogenic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy in high-risk, refractory, or relapsed ALL patients. Using MPX, we quantified changes in protein abundance, spatial distribution, and colocalization of 76 targeted cell surface proteins in Reh cells before and after fludarabine treatment. Our analysis revealed 25 proteins with altered abundance, 24 proteins with increased polarity, and 138 protein pairs with modified colocalization following treatment. Notably, the tetraspanins CD82 and CD53, which are known for their roles in chemotherapy resistance, exhibited increased abundance, polarization, and colocalization post-treatment, suggesting their potential as a therapeutic scaffold. These findings underscore the unique ability of spatially resolved single-cell proteomics to uncover nuanced cellular responses that would otherwise remain undetected. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Fludarabine, a purine nucleotide analogue, is integral to preparative regimens for CAR-T cell therapy and allo-SCT in treating B-cell pediatric acute lymphoblastic leukemia (B-ALL). By disrupting DNA synthesis and repair, fludarabine induces apoptosis in proliferating leukemia cells. It also plays a vital role in immunosuppression, lymphodepletion, and tumor burden reduction, optimizing the efficacy of subsequent therapies. Despite its critical role, the molecular impact of fludarabine on leukemia cells and its efficacy factors remain inadequately understood. Methods: We utilized a cutting-edge single-cell spatial proteomics assay, Molecular Pixelation (MPX, Karlsson et al. Nat Methods 2024), an optics-free DNA sequencing-based method to analyze the cell surface interactome in response to fludarabine. The glucocorticoid-resistant ETV6::RUNX1 cell line REH and the BCR::ABL1 cell line SUP-B15 were incubated with and without fludarabine for 72 hours at varying concentrations, with repeated technical repetitions. Approximately 1,000 surviving single cells per condition were analyzed using the MPX assay (Pixelgen Technologies), wherein antibody-oligonucleotide conjugates generated nanometer-sized molecular pixels, read out by sequencing on a NovaSeq X Plus instrument (Illumina). Spatial proteomics networks for 76 proteins were computationally reconstructed from DNA-sequencing reads by inferring the relative locations of antibody-oligonucleotide conjugates, creating ~1,000 spatially connected neighborhoods per cell. Orthogonal immunocytochemistry (ICC) data were generated to validate the top protein interactions. Results: We have previously shown that ex-vivo drug screening in combination with single-cell transcriptome sequencing (scRNA-seq) readout can elucidate transcriptional effects of fludarabine (Gezelius et al. NAR Genomics and Bioinformatics 2024). This approach confirmed fludarabine's disruption of DNA synthesis and repair in dividing cells, and in addition revealed previously unknown extensive dysregulation of transcripts encoding cell surface proteins. The scRNA-seq data do not adequately resolve the spatial nor functional dynamics of the cell surface proteome, leaving this important aspect of response to fludarabine unexplored. In this study, we take advantage of recent advancements in single-cell spatial proteomics profiling to spatially elucidate how the cell surface proteome organization and function is modulated during fludarabine treatment. Spatial statistical analysis revealed novel patterns in cell surface organization during fludarabine treatment. The MPX assay identified 27 proteins with significantly altered abundance post-treatment (adj. Wilcoxon p < 0.01), consistent with the increased abundance of cell-surface proteins observed in our previous scRNA-seq data. Additionally, 26 proteins exhibited changes in polarity (adj. Wilcoxon p < 0.01) after fludarabine treatment, with most proteins shifting from a dispersed to a polarized state. We also detected 138 protein pairs with modified co-localization patterns. Notably, fludarabine enhanced the abundance, polarization, and co-localization of cadherin proteins CD82 and CD53, suggesting a potential therapeutic scaffold. This finding was further validated through orthogonal ICC experiments. Conclusions: This study advances our understanding of the spatial organization of the cell surface proteome in leukemia cells following fludarabine exposure. The findings provide new insights into fludarabine's effects at the single-cell level and may inform the development of more effective targeted therapies.

Background It has become evident in the field of oncology that the outcome of medical treatment is influenced by the combined effect exerted on both cancer- and immune cells. Therefore, we evaluated potential immunological effects of 46 standard anticancer agents and 22 commonly administered concomitant non-cancer drugs. Methods We utilized a miniaturized in vitro model system comprised of fluorescently labeled human colon and lung cancer cell lines grown as monocultures and co-cultured with activated peripheral blood mononuclear cells (PBMCs). The Bliss Independence Model was then applied to detect antagonism and synergy between the drugs and activated immune cells. Results Among the standard anticancer agents, tyrosine kinase inhibitors (TKIs) stood out as the top inducers of both antagonism and synergy. Ruxolitinib and dasatinib emerged as the most notably antagonistic substances, exhibiting the lowest Bliss scores, whereas sorafenib was shown to synergize with activated PBMCs. Most concomitant drugs did not induce neither antagonism nor synergy. However, the statins mevastatin and simvastatin were uniquely shown to synergize with activated PBMC at all tested drug concentrations in the colon cancer model. Conclusion We utilized a miniaturized tumor-immune model to enable time and cost-effective evaluation of a broad panel of drugs in an immuno-oncology setting in vitro. Using this approach, immunomodulatory effects exerted by TKIs and statins were identified.

Patients with peritoneal metastasis from colorectal cancer (PMCRC) may have a chance of cure when treated with cytoreductive surgery (CRS) combined with heated intraperitoneal chemotherapy (HIPEC)1–5. Choice of chemotherapy for HIPEC has been based on knowledge of its systemic effects, pharmacokinetics, technical feasibility, hyperthermic efficacy enhancement, and tolerance6–8. Selection of cancer drugs for treatment based on phenotypical assessment of patient cancer cell drug sensitivity ex vivo is one approach to personalized cancer treatment. One technique for this is the fluorometric microculture cytotoxicity assay (FMCA) that has been used in drug development and for the development of personalized cancer medicine9–16. This study investigated whether ex vivo assessment of drug sensitivity by the FMCA provides predictive information in terms of peritoneal recurrence-free survival (PRFS) and overall survival (OS) in patients treated with CRS and HIPEC for isolated PMCRC. The patient cohort for this study was from a prospectively maintained institutional database at the Uppsala University Hospital, a tertiary care unit for PMCRC in Uppsala, Sweden. Patients with PMCRC treated with CRS and HIPEC have been registered since 2003. Ex vivo drug sensitivity testing using the FMCA started in April 2007. Thus, consecutive patients treated with CRS and HIPEC for PMCRC from April 2007 to October 2018 were considered for providing data for this report. Patients underwent HIPEC with either single-drug oxaliplatin, mitomycin C, or irinotecan, or a combination of oxaliplatin and irinotecan. HIPEC was performed in an open manner according to the coliseum method. Single-drug oxaliplatin was dosed at 350–460 mg/m2, and oxaliplatin and irinotecan combined at 360 mg/m2 for both drugs. These treatments lasted for 30 min. Mitomycin C was dosed at 35 mg/m2 divided into three injections with 50 per cent given at time 0, 25 per cent at 30 min, and 25 per cent at 60 min from the start of HIPEC for a total of 90 min. Follow-up data on PRFS and OS were collected for the final analysis. The Uppsala University ethical committee approved the study (Dnr 2007/237 for tumour sampling and ex vivo assessment of drug activity, and Dnr 2013/203 for clinical data collection).

PDF - 105K, This supplement contains three parts. The first presents a motivation/derivation of the cell proliferation model used in the main text. The second gives the theroetical details about the omnibus statistical test used in the main text. The third part presents an extended discussion regarding two topics breifly mentioned at the end of the main text.

Mebendazole (Mbz), a well-known anthelminthic drug, has been demonstrated to have anti-cancer properties in tumor models and patients. The therapeutic effect of Mbz was suggested to be due to microtubule inhibition, but more recently we reported that Mbz switches macrophages from the M2 to the tumor suppressive M1 phenotype. The aims of this study were to further investigate in tumor models the cytotoxic and immunomodulatory effects of Mbz alone, in combination with cytotoxic drugs and PD-1 antibody. Tumor samples from patients with solid or hematological malignancies undergoing diagnostic or therapeutic procedures were obtained in accordance with ethical permission. The samples were then prepared to isolated tumor cells for ex vivo testing. Ex vivo activity of Mbz alone and in combination with standard cytotoxic drugs was assessed using the fluorometric microculture cytotoxic assay. Survival index and synergy scores were calculated using GraphPad and SynergyFinder. In vivo antitumor activity of Mbz, irinotecan, a PD-1 inhibitor and their combinations were examined in a murine BALB/c model with inoculated syngeneic colon cancer cells (CT26). Tumor growth was measured by caliper and tumors were retrieved and assessed by flow cytometry for immune cell infiltration. Mbz alone showed some modest cytotoxic activity against patient tumor cells ex vivo, with hematological tumor cells being slightly more sensitive. The cytotoxic effect of Mbz was generally additive to that of other drugs but synergistic when combined with irinotecan. In the murine model, Mbz, irinotecan and the PD-1 antibody alone inhibited tumor growth. Combined with irinotecan Mbz had an additive effect, but was synergistic when combined with the PD-1 antibody. In line with the latter observation, flow cytometry showed an increase of M1 macrophages and decrease of M2 macrophages in tumors from animals treated with the combination. Mbz show promising features making it suitable for repositioning into an anti-cancer drug, most notably as a modulator of the macrophage phenotype that may enhance and the effect of check-point inhibitors.

BACKGROUND:High-throughput screening (HTS) of small molecule drug libraries has greatly facilitated the discovery of new cancer drugs. However, most phenotypic screening platforms used in the field of oncology are based solely on cancer cell populations and do not allow for the identification of immunomodulatory agents. METHODS:We developed a phenotypic screening platform based on a miniaturized co-culture system with human colorectal cancer- and immune cells, providing a model that recapitulates part of the tumor immune microenvironment (TIME) complexity while simultaneously being compatible with a simple image-based readout. Using this platform, we screened 1,280 small molecule drugs, all approved by the Food and Drug Administration (FDA), and identified statins as enhancers of immune cell-induced cancer cell death. RESULTS:The lipophilic statin pitavastatin had the most potent anti-cancer effect. Further analysis demonstrated that pitavastatin treatment induced a pro-inflammatory cytokine profile as well as an overall pro-inflammatory gene expression profile in our tumor-immune model. CONCLUSION:Our study provides an in vitro phenotypic screening approach for the identification of immunomodulatory agents and thus addresses a critical gap in the field of immuno-oncology. Our pilot screen identified statins, a drug family gaining increasing interest as repurposing candidates for cancer treatment, as enhancers of immune cell-induced cancer cell death. We speculate that the clinical benefits described for cancer patients receiving statins are not simply caused by a direct effect on the cancer cells but rather are dependent on the combined effect exerted on both cancer and immune cells.

Introduction While novel approaches for treatment of acute lymphoblastic leukemia (ALL) in children have resulted in significant improvements in survival rates, survivors are faced with potentially life-long challenges as a result of the highly toxic treatment and the outcome for patients who relapse remains poor. The current risk-adapted treatment for ALL in children includes multiple drugs administered sequentially or in combination, with little variation in timing or intensity. Factors that affect varying treatment response between patients and cellular mechanisms that underly response to individual drugs are still under exploration. In this study, we investigate the relationship between ex-vivo drug resistance, transcriptomes, epigenomes and clinical outcome, with the aim of better understanding underlying determinants of treatment response. Methods Pediatric patients diagnosed with B-ALL between 1992 and 2008 in the Nordic countries were included in the study (n = 598). For these patients, ex-vivo drug response to ten clinically used drugs was determined using the fluorometric microculture cytotoxicity assay (FMCA). Clinical data, including follow-up information (overall survival and relapse free survival) was available for all patients. Additionally, DNA-methylation (450K array, n = 383) and gene expression profiling (RNA-seq, n = 119) was performed on a subset of diagnostic patient samples. The patients were stratified into low, medium and high resistance groups based on ex-vivo drug response. The low and high resistance groups were compared with regard to differentially methylated CpG sites or expressed genes associated with individual drug response profiles. Results Overall survival was significantly lower for patients with higher ex-vivo drug resistance to dexamethasone, doxorubicin and thioguanine compared to patients that displayed sensitivity to these drugs (p < 0.05). Similarly, relapse free survival was significantly lower for patients with ex-vivo resistance to cytarabine, dexamethasone, doxorubicin, prednisolone and thioguanine (p < 0.05). After adjusting for sex and risk group, according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocol, in a Cox proportional hazards model, higher hazard ratios for relapse were observed in the highly resistant group across all drugs. We identified distinct DNA methylation and gene expression patterns in patients with low and high resistance, indicating the presence of predetermined molecular resistance fates. These resistance patterns included 665 genes that were differentially expressed (absolute log2FC > 1, Benjamini-Hochberg adjusted p value < 0.05) and 1 422 CpG sites with differential methylation status (absolute mean β-value difference > 0.2, Benjamini-Hochberg adjusted p value < 0.05) in resistant compared to sensitive groups, for one or multiple drugs. With the aim of identifying the genes with the largest impact on drug resistance, we selected the most differentially expressed genes and the genes with the largest difference in methylation status between resistant and sensitive samples. Among these, we identified ~20 putative genes of interest for future studies into resistance mechanisms based on potential functional prioritization. Conclusion Our findings confirm that drug response profiles are associated with outcome in pediatric ALL, reflecting response in vivo. We show molecular evidence for differentiating low- and high-resistant types based on diagnostic gene expression and DNA methylation profiles, suggesting contribution of certain molecular signatures to drug response. Our data integrating molecular data with drug response profiles may help identifying such changes of importance in drug resistance mechanisms.

Abstract LSD1 has emerged as a potential therapeutic target to increase the effectiveness of cancer immunotherapy. We have developed a series of novel small molecules, exemplified by the lead substance BEA-17, that modulates LSD1 via binding to an allosteric site, without directly inhibiting its enzymatic activity. In cells, BEA-17 induces a reduction of LSD1 levels. In addition, BEA-17 upregulates the expression of endogenous retroviral genes and T cell-attractant chemokines and does so in an LSD1-dependent manner. In a co-culture of HeLa and PBMCs, BEA-17 increases cell kill of cancer cells by immune effector T cells, also in an LSD1-dependent manner. In a CT26 syngeneic animal model of colon cancer, BEA-17 potentiates the activity of anti-PD1 inhibitors. Finally, in a syngeneic GL261 animal model of glioblastoma, BEA-17 increases the effectiveness of standard-of-care temozolomide + radiation. Citation Format: Wei B. Emond, Rajiv Sawant, Matthis Geitmann, Johan Winquist, Peter Brandt, Ulf Bremberg, Per Källblad, Vendela Parrow, Claes Andersson, Kristin Blom, Nasrin Najafi, Tobias Bergström, Fredrik J. Swartling, Mats Hellström, Konrad F. Koehler. Potentiation of immunotherapy by LSD1 modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 705.

Background Many previous studies have focused on the molecular landscape of pediatric acute lymphoblastic leukemia (ALL) with the aim to identify genetic changes related to disease development and outcome. However, the functional role of non-coding RNAs (ncRNAs) in leukemogenesis and treatment response is not fully understood. A group of ncRNAs known as small nucleolar RNAs (snoRNAs) regulates ribosomal biosynthesis, RNA splicing, and performs microRNA-like functions that affect post-translational gene expression. These functionalities can be dysregulated in cancer. The aim of this study was to explore the relationship between snoRNA expression and ex-vivo drug resistance in a cohort of pediatric ALL patients enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocols. Methods We analyzed 119 B-ALL patients who were treated according to two consecutive NOPHO protocols (1992 and 2000). B-cells (CD19+) and T-cells (CD3+) from healthy blood donors were used as controls (n = 10). We stratified patients into low-, medium-, and high-resistance groups by ex-vivo drug screening using 10 commonly used chemotherapeutic drugs with the fluorometric microculture cytotoxicity assay (FMCA). RNA sequencing (RNA-seq) data were generated for all patients. Pairwise comparisons (Benjamini-Hochberg (BH) corrected Mann-Whitney U test) were conducted between the low and high resistance groups to detect differentially expressed genes. We selectively retained the differentially expressed snoRNAs for downstream analysis. BH corrected Kruskal-Wallis H and Mann-Whitney U tests were performed to compare the snoRNA expression levels for the three drug resistance groups, and between the ALL and control samples, respectively. Results We identified a set of 23 unique significantly upregulated snoRNAs (p-adjusted value < 0.05 and absolute log2FC>1) in the high-resistance group for one or multiple drugs: amsacrine (ams, n = 10), etoposide (eto, n = 10), tioguanine (thio, n = 8) and mitoxantrone (mito, n = 1). Among these genes, five snoRNAs ( SNORA30, SNORA33, SNORA36C, SNORA62 and SNORA66) were overexpressed in patients resistant to ams and eto, and one snoRNA ( SNORD27) in patients resistant to ams and mito. Gene annotation revealed 22 significantly differentially expressed snoRNAs residing within the intronic region of 20 host genes, either protein coding (including ribosomal genes) or long ncRNAs. Three snoRNAs ( SNORD22-eto, SNORD26-ams and SNORD27-ams and mito) arise from the same host gene ( SNHG1). Of the 23 differentially expressed snoRNAs, the majority (n = 18) were not differentially expressed between the ALL and control samples (BH corrected Mann-Whitney U test p-value ≥ 0.05). The expression of the host genes did not differ among the resistance groups (BH corrected Kruskal-Wallis H test p-value ≥ 0.05). However, we found that 14 of the host genes were differentially expressed between the ALL samples and controls (BH corrected Mann-Whitney U test p-value < 0.05). Conclusions This study is one of the first to focus on the role of snoRNAs and ex-vivo drug resistance in pediatric ALL. Our findings highlight substantial variations in snoRNA expression across the three resistance groups, underscoring the potential role of snoRNAs in modulating drug response. Importantly, our analysis indicates that the differential expression of snoRNAs in the resistance groups cannot be solely attributed to host gene expression, implying that targeting pathways involving host genes might not be the most effective approach. Rather than concentrating on pathways involving host genes, our results suggest that understanding the mechanisms of action of snoRNAs could provide promising avenues for developing novel therapeutic targets to enhance drug response in pediatric ALL.

Cancer patients often suffer from cancer symptoms, treatment complications and concomitant diseases and are, therefore, often treated with several drugs in addition to anticancer drugs. Whether such drugs, here denoted as 'concomitant drugs', have anticancer effects or interact at the tumor cell level with the anticancer drugs is not very well known. The cytotoxic effects of nine concomitant drugs and their interactions with five anti-cancer drugs commonly used for the treatment of colorectal cancer were screened over broad ranges of drug concentrations in vitro in the human colon cancer cell line HCT116wt. Seven additional tyrosine kinase inhibitors were included to further evaluate key findings as were primary cultures of tumor cells from patients with colorectal cancer. Cytotoxic effects were evaluated using the fluorometric microculture cytotoxicity assay (FMCA) and interaction analysis was based on Bliss independent interaction analysis. Simvastatin and loperamide, included here as an opioid agonists, were found to have cytotoxic effects on their own at reasonably low concentrations whereas betamethasone, enalapril, ibuprofen, metformin, metoclopramide, metoprolol and paracetamol were inactive also at very high concentrations. Drug interactions ranged from antagonistic to synergistic over the concentrations tested with a more homogenous pattern of synergy between simvastatin and protein kinase inhibitors in HCT116wt cells. Commonly used concomitant drugs are mostly neither expected to have anticancer effects nor to interact significantly with anticancer drugs frequently used for the treatment of colorectal cancer.

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.

Understanding the immunological effects of chemotherapy is of great importance, especially now that we have entered an era where ever-increasing pre-clinical and clinical efforts are put into combining chemotherapy and immunotherapy to combat cancer. Single-cell RNA sequencing (scRNA-seq) has proved to be a powerful technique with a broad range of applications, studies evaluating drug effects in co-cultures of tumor and immune cells are however scarce. We treated a co-culture comprised of human colorectal cancer (CRC) cells and peripheral blood mononuclear cells (PBMCs) with the nucleoside analogue trifluridine (FTD) and used scRNA-seq to analyze posttreatment gene expression profiles in thousands of individual cancer and immune cells concurrently. ScRNA-seq recapitulated major mechanisms of action previously described for FTD and provided new insight into possible treatment-induced effects on T-cell mediated antitumor responses.