The cGAS-STING pathway is a central regulator of anti-tumor immunity, sensing cytosolic double-stranded DNA and inducing type I interferon responses. However, whether cGAS-STING directly coordinates DNA damage response in non-immune cells and thereby contributes to cell-intrinsic prevention of oncogenesis is not known. Using mouse models with deletion of ribonucleotide excision repair in the intestinal epithelium ( H2b ΔIEC ) and additional knock-out of STING ( H2b ΔIEC / STING -/- , H2b/STING ΔIEC ) we show that STING deficiency compromises DNA damage induced signalling via ATM and impairs homologous recombination repair, thereby promoting chromosomal instability and driving formation of spontaneous intestinal adenocarcinoma. Mechanistically, STING loss disrupts the NBS1-ATM axis and attenuates DNA damage signalling through enhanced phosphatase-mediated signal termination. Using single strand sequencing, we show that epithelial loss of STING promotes chromosomal instability and accumulation of mutations. By employing kinase screening, we further show that STING deficiency abrogates p53-dependent checkpoint control via overactivation of Cyclin-dependent kinase 1 (CDK1). CDK1 inhibitors lead to a STING-specific sensitization of tumor organoids and colorectal cancer cell lines, pointing towards a STING-specific vulnerability of tumors to CDK1 inhibition. Together, these data identify STING as an epithelial, interferon-independent genome-integrity checkpoint that couples DNA damage signaling to cell-cycle control, thereby restraining chromosomal instability, tumor evolution and revealing a therapeutically exploitable vulnerability in colorectal cancer.

Cardiogenic shock continues to have a high mortality rate over the past decade. A machine learning-based mortality risk score could aid ICU triage, deepen the understanding of cardiogenic shock, and guide intervention strategies based on predictive outcomes. This study aimed to develop and externally validate a machine learning algorithm for predicting mortality in cardiogenic shock patients using daily-measured routine parameters. We utilized a dataset from our cardiogenic shock registry, comprising 147 features, to compare various classifiers for 24-hour mortality prediction. Among the tested classifiers, the XGBoost classifier demonstrated the highest performance. The 25 most important features were then selected based on the feature importance scores. A machine-learning pipeline (Figure 1) was trained on patient day formatted data from three different databases including patients with cardiac admission diagnoses, lactate levels ≥2 mmol/L, and vasopressor treatment (SCAI C-E). We applied a novel clustering technique to distinguish data with high and low predictive accuracy, designating them as "high/low confidence subgroups". Subsequently, multiple models were developed to predict mortality over different timeframes (24 hours, 2 days, 3 days, up to 15 days). External validation was performed on three additional datasets extracted from separate publicly available databases to assess model generalizability. The training set included 23,925 patient days, while the test set comprised 24,037 patient days. For the 24-hour mortality prediction in the training dataset, the high confidence subgroup contained 12,695 patient days, and the low confidence subgroup had 11,230 observations. The model achieved an AUROC of 0.97 in the high confidence subgroup and 0.84 in the low confidence subgroup, resulting in an overall AUROC of 0.92. In external validation, the high confidence subgroup included 11,594 patient days, with the low confidence subgroup having 12,443 observations. The classifier demonstrated an AUROC of 0.92 in the high confidence subgroup and 0.73 in the low confidence subgroup, leading to an overall AUROC of 0.84. Despite a decline in AUROC over time, the precision-recall area under the curve (PRAUC) improved, likely due to label rebalancing (Figure 2). To enhance interpretability, SHAP waterfall plots and DICE_ML counterfactual reasoning were employed to clarify individual predictions, possibly providing actionable insights for clinicians in the future. A user-friendly interface was developed for clinical application. This study successfully predicted mortality for patients with cardiogenic shock across multiple timeframes, demonstrating strong AUROC performance, especially in the newly-identified high confidence subgroup.Figure 1Figure 2

Vascular function is impaired in patients with aortic valve stenosis (AS). The impact of transcatheter aortic valve implantation (TAVI) on endothelial function is inconclusive so far. Therefore, we sought to assess the short-term influence of TAVI on endothelial dysfunction in patients with AS. We recruited 47 patients (76.6 % male, 80.04 years old) with AS scheduled for TAVI. Endothelial function was assessed by fingertip reactive hyperemia peripheral arterial tonometry (RH-PAT). Measurements were conducted one day before and three days after TAVI. Patients were grouped according to RH-PAT change after TAVI. Overall, RH-PAT measurements did not significantly improve after TAVI (Reactive Hyperemia Index: 1.5 vs 1.6, p = 0.883; logarithm of the Reactive Hyperemia Index: 0.44 vs. 0.49, p = 0.523). Interestingly, patients with no RH-PAT improvement after TAVI displayed a more severe AS and had lower blood pressure after TAVI. This might be due to a more disturbed blood flow in patients with a smaller aortic valve area and higher peak aortic valve velocity. The relationship between AS severity, endothelial dysfunction and TAVI has to be investigated in future research that apply longitudinal study designs.

Intestinal stem cells (ISCs) promote tissue repair after genotoxic or immune-mediated injury. However, ISCs are particularly sensitive to various stressors and primary targets of overwhelming immune responses, such as interferon γ (IFNγ)-mediated killing. In mouse models of radiation therapy-induced gut damage and in biopsies from patients who underwent allogeneic hematopoietic stem cell transplantation, we observed IFNγ expression by intestinal Treg cells. Treg cells leverage combined IFNγ and interleukin 10 (IL-10) stimulation of ISCs to nurture the growth of intestinal organoids through the activation of the mTORC1 and Myc pathways. Similarly, Treg cells or the combined addition of recombinant IFNγ and IL-10 promoted the regeneration of organoids after irradiation, and both cytokines were essential for ensuring epithelial regeneration following acute intestinal tissue injury in vivo. The exposure of organoids to growth factor-free culture conditions revealed distinct EGF-like properties of IFNγ and Wnt-like properties of IL-10. While IFNγ rapidly induced epithelial proliferation, it depleted the pool of ISCs in vitro. Only the combination of IFNγ and IL-10 led to epithelial proliferation and organoid growth while simultaneously ensuring ISC maintenance over time. Our results reveal a context-dependent role of inflammatory signaling in ISCs, through which Treg cells promote epithelial repair following therapy-induced injury.

PURPOSE:Combining immune checkpoint inhibitors (ICIs) with radiation therapy (RT) has led to significant advancements in cancer treatment. However, evidence from clinical and experimental studies suggests that this combination may increase hematopoietic and lymphatic toxicity. This study aims to investigate the effects of the concurrent application of ICIs (anti-PD-1 and anti-CTLA-4) on radiation-induced hematopoietic and lymphatic injuries under standardized and controlled experimental conditions. METHODS AND MATERIALS:We used various experimental models in C57BL/6 and BALB/c mice to evaluate the impact of ICIs combined with RT on the hematopoietic system. These models involved different RT doses, regimens, and target sites in both healthy and tumor-bearing mice. RESULTS:Our findings showed that the concurrent use of ICIs did not meaningfully affect post-RT pancytopenia kinetics or the regeneration of specific blood cell lineages over time. Consistently, combining RT with ICIs did not significantly enhance DNA damage in immune cells within the bloodstream. This outcome was comparable across different RT doses, regimens, and target sites and was reproducible in both tumor-bearing and nontumor-bearing mice. Additionally, there were no significant increases in late side effects, including reductions in bone marrow cell counts or megakaryocyte numbers, after combined radioimmunotherapy. CONCLUSIONS:These findings suggest that combining ICIs with RT does not exacerbate hematological toxicity. This information is valuable for interpreting adverse events in clinical trials involving radioimmunotherapy and for predicting potential hematological side effects in cancer patients receiving these treatments.

Induction of apoptosis in murine PDAC and human lung adenocarcinoma Mavs-defective tumor cells in response to CAR T cell-mediated killing

AIMS:Data on the prognostic value of left- and right-atrial strain after transcatheter aortic valve replacement (TAVR) for severe aortic stenosis (AS) are limited. Aim of this study was to evaluate outcomes of patients undergoing TAVR stratified by left- and right-atrial strain. METHODS AND RESULTS:Using data from a high-volume academic centre, left- and right-atrial reservoir strain (LASr and RASr) was obtained in patients who underwent TAVR for severe AS from 2018 until 2021. Patients were stratified into groups with normal atrial function (LASr and RASr normal), uni-atrial strain impairment (LASr or RASr impaired), and bi-atrial strain impairment (LASr and RASr impaired). Endpoints were 3 year survival, symptomatic improvement as assessed by New York Heart Association functional class (NYHA class) as well as technical and device success defined by the Valve Academic Research Consortium composite endpoints. The study included 1888 patients at a mean age of 81.0 ± 7.8 years (44.3% women). Mean LASr and RASr were 16.5 ± 9.4% and 21.6 ± 12.4%, respectively. Optimized cut-offs for mortality prediction were 15.5% for LASr and 15.0% for RASr. LASr and RASr were normal in 751 patients (39.8%). Impairment of either right-atrium (RA) or left-atrium (LA) strain was observed in 633 patients (33.5%) and 504 patients (26.7%) presented with reduced LA and RA strain. While impairment of either LASr or RASr was associated with a 1.7-fold increased risk of 3 year all-cause mortality after adjustment for multiple confounders (95% confidence interval [CI] 1.2-2.5, P = 0.005), bi-atrial strain impairment exhibited an even higher 3 year mortality risk (Hazard ratio 2.5, 95% CI 1.7-3.6, P < 0.001). CONCLUSION:Pre-procedural assessment of atrial strain is associated with increased 3 year mortality and might facilitate outcome prediction and patient selection in patients undergoing TAVR for severe AS.

Despite the remarkable success of chimeric antigen receptor (CAR) T cells in certain hematologic malignancies, only modest responses have been achieved in solid tumors. Defective cell death pathways have recently been suggested as a tumor-intrinsic form of resistance to CAR T-cell treatment. In this study, we showed that insufficient activity of the innate RNA-sensing receptor system retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling protein (MAVS) leads to tumor cell-inherent resistance to CAR T-cell attack. Active RIG-I/MAVS signaling in tumor cells primed intrinsic mitochondrial apoptosis pathways and expression of cell death receptors, which funneled into CAR T-cell-triggered cell death. CAR T-cell reliance on tumor-intrinsic RIG-I signaling was observed in various murine and human cancer types, independent of the CAR construct used, and the dependence was most pronounced under conditions with low target antigen expression or low effector/target ratios. RIG-I-induced proapoptotic priming of CAR T-cell susceptibility involved auto-/paracrine type-I IFN signaling loops and could spread to bystander tumor cells. Strong tumor-intrinsic RIG-I/MAVS signaling imprinted an activated cytolytic phenotype on tumor-interacting CAR T cells. Agonist-mediated targeting of the RIG-I pathway in the tumor microenvironment rendered murine melanoma susceptible to CAR T-cell therapy in vivo with enhanced infiltration of active CAR T cells. Together, these data identify insufficient RIG-I/MAVS activity and associated impaired cell death signaling in malignant cells as a resistance mechanism to CAR T cells. Targeting tumor-intrinsic RIG-I is a potential strategy to sensitize solid tumors to CAR T-cell treatment.Significance: Insufficient activity of the RIG-I/MAVS pathway is a tumor intrinsic resistance mechanism to CAR T cells, providing the rationale for targeting RIG-I to optimize CAR T efficacy in patients with solid cancers.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with neoadjuvant radio(chemo)therapy failing in approximately 70 % of cases due to high tumor heterogeneity, and intrinsic radioresistance. Patient-derived organoids (PDOs) closely recapitulate appearance, and functionality as the original tissue and have potential to explore novel therapies for personalized radiooncology.In this study, the radioresponse of PDAC PDO lines was determined after irradiation (RT). PDOs were immunohistochemically characterized by γ-H2AX, HIF-1α and Ki-67 staining. RNA sequencing data were analyzed by gene set enrichment analyses to investigate underlying mechanisms of radioresistance. Preclinical findings were correlated with clinical data from the corresponding patients.PDOs showed a significant heterogeneity in response to radiation and were classified into radiosensitive, intermediate, and radioresistant subgroups. A correlation between radiosensitivity and enhanced proliferation and decreased hypoxia was observed. OXPHOS-related gene signatures were significantly overexpressed in the radioresistant phenotype. Translationally, radioresistance in PDOs was associated with significantly poorer survival of patients.Our platform demonstrated heterogeneity in radioresponse reflecting the clinical situation and correlation with clinical outcomes. Immunohistochemical staining and transcriptomic profiling identified molecular signatures, including HIF-1α and OXPHOS-related pathways, associated with radioresistance. Implementing PDO-based radioresponse profiling in clinical workflows may enable patient stratified treatment approaches. Overall, our findings suggest that functionalizing PDOs for radioresponse might extend PDO-informed precision oncology.

PURPOSE:The identification of internal mammary lymph node metastases and the assessment of associated risk factors are crucial for adjuvant regional lymph node irradiation in patients with breast cancer. The current study aims to investigate whether tumor contact with internal mammary perforator vessels is associated with gross internal mammary lymph node involvement. METHODS AND MATERIALS:We included 297 patients with primary breast cancer and gross internal mammary (IMN+) and/or axillary metastases as well as 230 patients without lymph node metastases. Based on pretreatment dynamic contrast-enhanced magnetic resonance imaging, we assessed contact of the tumor with the internal mammary perforating vessels (IMPV). RESULTS:A total of 59 patients had ipsilateral IMN+ (iIMN+), 10 patients had contralateral IMN+ (cIMN+), and 228 patients had ipsilateral axillary metastases without IMN; 230 patients had node-negative breast cancer. In patients with iIMN+, 100% of tumors had contact with ipsilateral IMPV, with 94.9% (n = 56) classified as major contact. In iIMN- patients, major IMPV contact was observed in only 25.3% (n = 116), and 36.2% (n = 166) had no IMPV contact at all. Receiver operating characteristic analysis revealed that "major IMPV contact" was more accurate in predicting iIMN+ (area under the curve, 0.85) compared with a multivariate model combining grade of differentiation, tumor site, size, and molecular subtype (area under the curve, 0.65). Strikingly, among patients with cIMN+, 100% of tumors had contact with a crossing contralateral IMPV, whereas in cIMN- patients, IMPVs to the contralateral side were observed in only 53.4% (iIMN+) and 24.8% (iIMN-), respectively. CONCLUSIONS:Tumor contact with the IMPV is highly associated with risk of gross IMN involvement. Further studies are warranted to investigate whether this identified risk factor is also associated with microscopic IMN involvement and whether it can assist in the selection of patients with breast cancer for irradiation of the internal mammary lymph nodes.

Right ventricular (RV) dysfunction in patients undergoing transcatheter aortic valve implantation (TAVI) for aortic stenosis (AS) has long been disregarded. We aimed to assess the predictive value of RV to pulmonary artery coupling (RV/PAc), defined as tricuspid annular plane systolic excursion to systolic pulmonary artery pressure, on mortality in different flow types of AS after TAVI. All patients undergoing TAVI for AS at our centre between 2018 and 2020 were assessed; 862 patients were analysed. The cohort was dichotomized using a ROC analysis (cut-off 0.512 mm/mmHg), into 429 patients with preserved and 433 patients with reduced RV/PAc. Reduced RV/PAc was associated with male sex and a higher rate of comorbidities. Short-term VARC-3 endpoints and NYHA classes at follow-up were comparable. Reduced RV/PAc was associated with higher 2-year all-cause mortality (35.0

BACKGROUND:Knowledge about atrial functional tricuspid regurgitation (afTR) in transcatheter aortic valve replacement (TAVR) patients is scarce. OBJECTIVES:The aim of the study was to analyze the association between the entity and the development of tricuspid regurgitation (TR) in patients undergoing TAVR for aortic stenosis and concomitant TR. METHODS:We analyzed patients undergoing TAVR for severe aortic stenosis from January 2013 to December 2020 and concomitant at least moderate TR at baseline. afTR was defined as enlargement of the right atrium in relation to the right ventricle. TR development after TAVR and 3-year all-cause mortality were evaluated. RESULTS:Out of 3,474 TAVR patients, we identified 420 patients with concomitant at least moderate TR. A total of 363 patients were included in the study, with 178 patients stratified in the afTR and 185 in the non-afTR group based on a receiver-operating characteristic curve cutoff of 1.132 of the right atrial/right ventricular area ratio. TR improvement after TAVR was observed in significantly less patients with afTR compared with non-afTR (31.1% vs 60.6%; P < 0.001). Multivariate regression analysis confirmed afTR as independent predictor for TR persistence (adjusted OR: 2.80; 95% CI: 1.66-4.76; P < 0.001). Moreover, afTR was associated with aggravation of TR after TAVR (17.0% vs 6.8%; P = 0.013). Three-year all-cause mortality was significantly higher in patients with persistence compared with patients with improvement of TR (P < 0.001). CONCLUSIONS:In TAVR patients, afTR is an independent predictor for TR persistence. Moreover, TR persistence is associated with increased 3-year all-cause mortality.

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT ( n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase ( BCoAT , which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.

Background Post-Therapy-Pneumonitis (PTP) is a critical side effect of both, thoracic radio(chemo)therapy (R(C)T) and immune checkpoint inhibition (ICI). However, disease characteristics and patient-specific risk factors of PTP after combined R(C)T + ICI are less understood. Given that RT-triggered PTP is strongly dependent on the volume and dose of RT, driven by inflammatory mechanisms, we hypothesize that combination therapy of R(C)T with ICI influences the dose-volume-effect correlation for PTP. This study focuses on the development of a method for evaluation of alterations in the dose-volume-effect correlation of PTP after R(C)T with and without ICI. Methods and materials PTP volumes were delineated on the follow-up diagnostic Computed Tomography (CT) and deformably matched to the planning CT. Dose data was converted to 2-Gy equivalent doses (EQD2) and dosimetrically analyzed. The method was exemplarily tested on an internal patient cohort including 90 patients having received thoracic R(C)T + ICI (39) and R(C)T (51). Additionally, data on previous chemotherapy and RT, smoking status and pulmonary co-morbidity was conducted. An exploratory analysis has been performed and a matched pair analysis with regard to planning target volumes (PTV) was conducted for curative intended (definitive) and palliative patient cohorts individually. Results The presented method was able to demonstrate differences in the dose-volume-effect-correlation of PTP for the different therapies. The dosimetric analysis revealed large volumetric fractions (55%) of the PTP volumes to be located outside of high dose (EQD2 < 40 Gy) regions for R(C)T + ICI. There was a non-significant trend towards increased AUC values for R(C)T + ICI compared to R(C)T only (3743.6 Gy∙% vs. 2848.8 Gy∙%; p -value = 0.171). In contrast to the data for the palliative intended treatment group, for definitive R(C)T + ICI, data tended towards increased volumes with higher doses. Conclusions The proposed method was capable to demonstrate dosimetric differences in the dose-volume-effect relationship of PTP for patients with R(C)T + ICI and patients with R(C)T only. In this exploratory analysis, the patient cohorts were too small and inhomogeneous to reveal statistically significant dosimetric differences within PTP volumes for the different groups. However, our observations suggest, that for safe application of thoracic R(C)T + ICI, further careful investigation of dosimetric prescription and analysis concepts with larger and conformer study groups is recommendable.

Aims Studies have shown a so-called off-hour effect for many different diseases, but data are scarce concerning cardiogenic shock. We therefore assessed the association of off-hour vs. on-hour intensive care unit admission with 30-day mortality in patients with cardiogenic shock.Methods and results In total, 1720 cardiogenic shock patients (666 admitted during off-hours) from two large university hospitals in Germany were included in retrospect. An admission during off-hours was associated with increased 30-day mortality compared to an admission during on-hours [crude mortality 48% vs. 41%, HR 1.17 (1.03-1.33), P = 0.017]. This effect remained significant after propensity score matching (P = 0.023). Neither patients with a combined SCAI stage D and E (P = 0.088) or C (P = 0.548) nor those requiring cardiopulmonary resuscitation (P = 0.114) had a higher mortality at off-hour admission. In contrast, those without veno-arterial extracorporeal membrane oxygenation [HR 1.17 (1.00-1.36), P = 0.049], without acute myocardial infarction [HR 1.27 (1.02-1.56), P = 0.029] or a with combined SCAI stage A and B [HR 2.23 (1.08-4.57), P = 0.025] had an increased mortality at off-hour admission.Conclusion Our study showed an increased mortality in patients with cardiogenic shock admitted during off-hours, especially in those with a milder onset of disease. This stresses the importance of a thorough workup of each patient, especially at times of limited resources, the menace of underestimating the severity of cardiogenic shock, and the need for an improved 24x7 available risk stratification. Graphical Abstract

Background: Most clinical studies failed to elicit a strong antitumor immune response and subsequent systemic tumor regression after radiation therapy (RT), even in combination with the immune checkpoint inhibitors (ICI) anti-CTLA4 or anti-PD1. Mechanistically, type I interferon (IFN-I) activation is essential for the development of such abscopal effects (AE); however, mechanisms driving or limiting IFN-I activation are ill defined. Groundbreaking discoveries have shown that antibiotics (ABx) can affect oncological outcomes and that microbiotaderived metabolites can modulate systemic antitumor immunity. Recent studies have demonstrated that the bacterial metabolites desaminotyrosine (DAT) and indole-3-carboxaldehyde (ICA) can enhance IFN-I activation in models of inflammatory diseases. Materials and Methods: The subcutaneous bilateral MC38 tumor model is a widely used experimental tool to study the AE in mice. We applied it to explore the influence of broad-spectrum ABx, DAT and ICA on the AE after radioimmunotherapy (RIT). We performed 1x8 Gy of the primary tumor +/- anti-CTLA4 or anti-PD1, and +/- daily oral application of ABx or metabolites. Result: Combinatory ABx had neither a significant effect on tumor growth of the irradiated tumor nor on tumor progression of the abscopal tumor after RIT with anti-CTLA4. Furthermore, DAT and ICA did not significantly impact on the AE after RIT with anti-CTLA4 or anti-PD1. Surprisingly, ICA even appears to reduce outcomes after RIT with anti-CTLA4. Conclusion: We did not find a significant impact of combinatory ABx on the AE. Experimental application of the IFN-I-inducing metabolites DAT or ICA did not boost the AE after combined RIT. Additional studies are important to further investigate whether the intestinal microbiota or specific microbiota-derived metabolites modulate the AE.

Purpose We investigated the involvement of endoplasmic reticulum (ER) stress signaling in cancer cell responses to chemo- and radiotherapy, focusing on three main ER stress mediators, the transcription factors ATF4, XBP1 and ATF6.Methods Public cancer genome datasets were assessed for alterations in ER stress mediators. Surgically resected colorectal cancer tissues were tested by flow cytometry and used to generate patient-derived organoids. Human cell lines and organoids were characterized under oxaliplatin treatment, alone or combined with pharmacological inhibitors of the three ER stress branches, or X-ray irradiation, for cytotoxicity, activation of ER stress and proteome changes. To monitor ER stress in real time, stable HEK293 kidney epithelial cell lines were established expressing ATF4, XBP1, or ATF6, fused with a fluorophore.Results Genomic amplification of ATF6, but not ATF4 or XBP1, was frequent in solid tumor entities like breast, lung and colorectal cancer and significantly associated with reduced disease-free survival. In colorectal cancer, increased ATF6 was associated with genetic instability. Basal ER stress mediator expression was correlated to chemoresistance in colorectal cancer cell lines, and generally high in cancer cells compared to HEK293 cells. With proteomics and live HEK293-based reporter lines, we noted that oxaliplatin treatment induced ER stress in a remarkably different way from the canonical ER stress inducer thapsigargin. Moreover, modulation of ER stress signaling by exogenous expression of the stress mediators positively affects chemoresistance, and pharmacological inhibition of ATF6 sensitizes ER-stressed HCT116 colorectal cancer cells to chemotherapy. Of note, cellular stress responses was strongly dependent on the individual transcription factor: XBP1-driven response appeared multi-functional, involved in ribosome biogenesis stress and associated with oxaliplatin resistance. ATF6-dependent stress signaling was involved in DNA damage repair, and was essential for radioresistance. Moreover, chemoresistance in HCT116 cancer cells was impaired by pharmacological ATF6 inhibition.Conclusion Activation of the ER stress signaling may be critically involved in acquired chemo- and radioresistance. Due to their apparent cytoprotective roles, ATF6 and XBP1 could be attractive predictive biomarkers and putative therapeutic targets.SUMMARY To address their roles in the clinical context, genomic alterations of ATF4, XBP1 and/or ATF6 in human solid tumors were assessed with respect to prognosis and genomic instability. Moreover, surgically resected CRC patient tissues were tested for expression of ER stress markers by flow cytometry and associated with clinical characteristics. In addition, a panel of human cell lines and patient-derived colon organoids were characterized under therapeutic conditions for expression and activation of ER stress proteins, and resulting cytotoxicity was determined. To monitor and modulate ER stress activation in live cells with subcellular resolution, stable reporter cell lines expressing ATF4, sXBP1 or ATF6 proteins fused with a fluorophore were established. These lines were tested for gene or protein expression and cytotoxicity assays to analyze how activation or inhibition of ER stress proteins affects the cellular responses to oxaliplatin treatment or X-ray irradiation. Finally, mass spectrometric proteome analysis was performed to obtain an unbiased readout on the cellular responses to chemotherapy driven by the activation of the ER stress proteins.### Competing Interest StatementThe authors have declared no competing interest.