Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

Plateletcrit is predictive of clinical outcome and prognosis for early-stage breast cancer: A retrospective cohort study based on propensity score matching.

PURPOSE: Breast cancer (BC) is diagnosed as the most common cancer in women in 2022 according to the American Cancer Society. It is essential to detect early and treat early. Several studies have shown that some blood parameters have important predictive value for BC. In this study, we aim to explore whether some immune-associated blood parameters are relevant to disease-free survival (DFS) in early-stage BC. METHODS: A single-center, regression cohort study of 1490 patients with early-stage BC in Shanghai Cancer Center was conducted from January 2008 to December 2016. The patients were matched according to the ratio of 1:1 based on Propensity Score Matching (PSM). All patients who experienced disease progression were matched successfully. Thus, 58 pairs of subjects were obtained. Matched blood parameters were evaluated by paired samples t-test or Wilcoxon signed-rank test. Factors with statistical difference were further evaluated by stratified COX regression model. RESULTS: Univariate analysis showed differences in platelet-related parameters (PLT, PCT, and PLR) and NLR between the two matched groups. However, stratified COX regression analysis, which ruled out the confounding effects of multiple factors, found that only PCT had prognostic value in early BC patients at baseline and study endpoint. Meanwhile, platelet-related parameters (PLT, MPV) and NLR were different in the progressive group by self before and after comparison. However, the multiple-factor analysis showed that only the NLR had prognostic value. ROC curve analysis indicated that the best sensitivity (65.45%) and specificity (78.18%) were obtained when the baseline PCT was 0.225. The optimal sensitivity (70.91%) and specificity (65.45%) were obtained when the PCT of disease progression was 0.215. The Kaplan-Meier curve was used to calculate the DFS rate based on the critical values of the two groups. CONCLUSIONS: Some blood parameters have value to predict DFS in early-stage BC patients, especially platelet-associated parameters.

Outcomes with and without postmastectomy radiotherapy for pT3N0-1M0 breast cancer: An institutional experience.

AIM: The objective of this study is to comprehensively evaluate the therapeutic efficacy of postmastectomy radiotherapy (PMRT) in treating patients with pT3N0-1M0 breast cancer within the context of modern therapeutic strategies. METHODS: Clinical data from patients with pT3N0-1M0 breast cancer who underwent mastectomy from January 2005 to December 2018 at our institution were retrospectively analyzed. RESULTS: The study involved a total of 222 participants, with 112 individuals undergoing PMRT and 110 individuals not receiving it. The median follow-up duration was 77 months (range: 6-171 months). The entire cohort demonstrated 5-year disease-free survival (DFS) and overall survival (OS) rates of 85.1% and 91.0%, respectively, along with a locoregional recurrence (LRR) rate as low as 7.2%. The PMRT group showed significantly better 5-year DFS (90.2% vs. 80.0%, p = 0.02) and OS (95.5% vs. 86.4%, p = 0.012) rates, as well as a lower LRR rate (4.5% vs. 10.0%, p = 0.122), compared to the group without PMRT. Cox regression analysis confirmed the independent prognostic significance of PMRT for both DFS (p = 0.040) and OS (p = 0.047). Following propensity score matching (PSM), the analysis included 100 matched patients, revealing an improved prognosis for those who received PMRT (DFS: p = 0.067; OS: p = 0.043). CONCLUSIONS: Our study reveals favorable prognoses for pT3N0-1M0 breast cancer patients treated within contemporary therapeutic approaches. The pivotal role of PMRT in this context is evident. However, due to the retrospective design of our study and the relatively limited sample size, further investigation is imperative to validate and enhance these initial findings.

Interleukin-33 Potentiates TGF-ß Signaling to Regulate Intestinal Stem Cell Regeneration After Radiation Injury.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal radiation injury. Accumulating evidence indicates that the interleukin family members play critical roles in intestinal stem-cell-mediated epithelial regeneration. However, little is known about the relationship between interleukin 33 (IL-33)/ST2 axis and intestinal regeneration after radiation injury. We demonstrate here that IL-33 expression significantly increased after radiation treatment. Deficiency of IL-33/ST2 promotes intestinal epithelial regeneration, resulting in a reduction of mortality during radiation-induced intestine injury. Using ex vivo organoid cultures, we show that recombinant IL-33 promotes intestinal stem cell differentiation. Mechanistically, the effects of IL-33 were mediated by activation of transforming growth factor-ß signaling. Our findings reveal a fundamental mechanism by which IL-33 is able to regulate the intestinal crypt regeneration after tissue damage.

Patient-Derived Organoids from Colorectal Cancer with Paired Liver Metastasis Reveal Tumor Heterogeneity and Predict Response to Chemotherapy.

There is no effective method to predict chemotherapy response and postoperative prognosis of colorectal cancer liver metastasis (CRLM) patients. Patient-derived organoid (PDO) has become an important preclinical model. Herein, a living biobank with 50 CRLM organoids derived from primary tumors and paired liver metastatic lesions is successfully constructed. CRLM PDOs from the multiomics levels (histopathology, genome, transcriptome and single-cell sequencing) are comprehensively analyzed and confirmed that this organoid platform for CRLM could capture intra- and interpatient heterogeneity. The chemosensitivity data in vitro reveal the potential value of clinical application for PDOs to predict chemotherapy response (FOLFOX or FOLFIRI) and clinical prognosis of CRLM patients. Taken together, CRLM PDOs can be utilized to deliver a potential application for personalized medicine.

Assay establishment and validation of a high-throughput organoid-based drug screening platform.

BACKGROUND: Organoids are three-dimensional structures that closely recapitulate tissue architecture and cellular composition, thereby holding great promise for organoid-based drug screening. Although growing in three-dimensional provides the possibility for organoids to recapitulate main features of corresponding tissues, it makes it incommodious for imaging organoids in two-dimensional and identifying surviving organoids from surrounding dead cells after organoids being treated by irradiation or chemotherapy. Therefore, significant work remains to establish high-quality controls to standardize organoid analyses and make organoid models more reproducible. METHODS: In this study, the Z-stack imaging technique was used for the imaging of three-dimensional organoids to gather all the organoids' maximum cross sections in one imaging. The combination of live cell staining fluorescent dye Calcein-AM and ImageJ assessment was used to analyze the survival of organoids treated by irradiation or chemotherapy. RESULTS: We have established a novel quantitative high-throughput imaging assay that harnesses the scalability of organoid cultures. Using this assay, we can capture organoid growth over time, measure multiple whole-well organoid readouts, and show the different responses to drug treatments. CONCLUSIONS: In summary, combining the Z-stack imaging technique and fluorescent labeling methods, we established an assay for the imaging and analysis of three-dimensional organoids. Our data demonstrated the feasibility of using organoid-based platforms for high-throughput drug screening assays.

CBP/P300 Inhibitors Mitigate Radiation-Induced GI Syndrome by Promoting Intestinal Stem Cell-Mediated Crypt Regeneration.

PURPOSE: Radiation-induced gastrointestinal syndrome (RIGS) is currently the main cause of death for people exposed to a high dose of irradiation during nuclear incidents, and there is currently no approved effective therapy. Here, we found that CBP/P300 inhibitors, with high efficacy and low toxicity, might be promising radiation mitigators that can cure RIGS. METHODS AND MATERIALS: Ex vivo 3D organoid cultures derived from mouse jejunum and human ileum and colon were used to examine the radio-mitigative effects of CBP/P300 inhibitors. The radio-mitigative effect was evaluated by quantifying the survival rate and size of organoids after radiation. SGC-CBP30 (50 mg/kg body weight), an inhibitor of CBP/P300, was intraperitoneally injected into C57B/6J mice 24 hours after subtotal-body irradiation or whole-body irradiation. The regenerated crypts and animal survival were determined by microcolony assay and the Kaplan-Meier method, respectively. Lgr5-lacZ mice were used to evaluate the survival of intestinal stem cells after treatments. RESULTS: We found that CBP/P300 inhibitors were effective mitigators that could be used to treat RIGS. CBP/P300 inhibition promoted the regeneration of intestinal organoids in vitro and of crypts in vivo. Remarkably, the administration of CBP/P300 inhibitors to mice 24 hours after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, resulting in improved mouse survival. Moreover, our data show that CBP/P300 inhibitors rescued irradiated mice from RIGS by delaying intestinal epithelial cell cycle progression after radiation. CONCLUSIONS: These data demonstrate that CBP/P300 inhibitors are effective medical countermeasures to mitigate gastrointestinal toxicity from radiation.

SIRT1 inhibitors mitigate radiation-induced GI syndrome by enhancing intestinal-stem-cell survival.

High-dose radiation exposure induces gastrointestinal (GI) stem cell death, resulting in denudation of the intestinal mucosa and lethality from GI syndrome, for which there is currently no effective therapy. Studying an intestinal organoid-based functional model, we found that Sirtuin1(SIRT1) inhibition through genetic knockout or pharmacologic inhibition significantly improved mouse and human intestinal organoid survival after irradiation. Remarkably, mice administered with two doseages of SIRT1 inhibitors at 24 and 96 h after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, with improved mouse survival (88.89% of mice in the treated group vs. 0% of mice in the control group). Moreover, our data revealed that SIRT1 inhibition increased p53 acetylation, resulting in the stabilization of p53 and likely contributing to the survival of intestinal epithelial cells post-radiation. These results demonstrate that SIRT1 inhibitors are effective clinical countermeasures to mitigate GI toxicity from potentially lethal radiation exposure.

Metformin chlorination byproducts in drinking water exhibit marked toxicities of a potential health concern.

Metformin (MET), a worldwide used drug for type 2 diabetes, has been found with the largest amount by weight among all drugs in aquatic environment, including the drinking water systems where this emerging micropollutant is inevitably transformed during chlorination process. Whether MET chlorination byproducts Y (C4H6ClN5) and C (C4H6ClN3) exist in drinking water remains unknown. Although MET has health-promoting properties, whether or how its chlorination byproducts affect health is still uncharacterized. Here we reveal that MET and byproduct C are present in worldwide drinking water with the highest doses detected for MET and C as 1203.5 ng/L and 9.7 ng/L respectively. Under simulated chlorination conditions, we also demonstrate that both byproducts can be increasingly produced with increment of MET concentration, suggesting a hidden threat on the safety and sustainability of global water supply. Through systematic evaluations, we demonstrate that MET chlorination byproducts Y and C exhibit toxicities instead of genotoxicity to live worms and human HepG2 cells at millimolar doses. Moreover, both byproducts are harmful to mice and particularly Y at 250 ng/L destroys the mouse small intestine integrity. Unprecedentedly, we unveil boiling and activated carbon adsorption as effective alternative solutions that may be in urgent demand globally for removing these byproducts from drinking water.

Bach2 Deficiency Promotes Intestinal Epithelial Regeneration by Accelerating DNA Repair in Intestinal Stem Cells.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2-/- crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.

Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling.

BACKGROUND: Dachshund homologue 1 (DACH1) is highly expressed in LGR5+ intestinal stem cells and colorectal tumours. However, the roles of DACH1 in intestinal cell stemness and colorectal tumorigenesis remain largely undefined. METHODS: We used immunohistochemistry, western blotting and quantitative real-time PCR to analyse DACH1 expression in colorectal cancer (CRC) samples. CRISPR/Cas9 gene editing and lentiviral vector-mediated overexpression and shRNA-mediated knockdown of DACH1 were utilized to modulate DACH1 expression in cell lines and organoids. An intestinal organoid-based functional model was analysed, and cancer cell colony formation, sphere formation assays and murine xenotransplants were performed to reveal the role of DACH1 in CRC cell proliferation, stemness and tumorigenesis. Immunofluorescence, co-immunoprecipitation, RNA interference and microarray data analyses were conducted to demonstrate the association between DACH1 and the bone morphogenetic protein (BMP) signalling pathway. FINDINGS: DACH1 is specifically expressed in discrete crypt base cells, and increased DACH1 expression was found in all stages of CRC. Moreover, the high expression of DACH1 independently predicted poor prognosis. In colon cancer cells, shRNA-mediated suppression of DACH1 inhibited cell growth in vitro and in vivo. By studying the intestinal organoid-based functional model, we found that depletion of DACH1 reduced the organoid formation efficiency and tumour organoid size. DACH1 overexpression stimulated both colonsphere formation and tumour organoid formation in the context of dysregulated BMP signalling. Mechanistic characterizations indicated that overexpression of DACH1 affects a subset of stem cell signature genes implicated in stem cell proliferation and maintenance through the suppression of BMP signalling via SMAD4. INTERPRETATION: Together, our study highlights DACH1 as an integral regulator of BMP signalling during intestinal tumorigenesis, and DACH1 could be a potential prognostic marker and therapeutic target for colorectal cancer patients.

Patient-Derived Organoids Predict Chemoradiation Responses of Locally Advanced Rectal Cancer.

Accumulating evidence indicates that patient-derived organoids (PDOs) can predict drug responses in the clinic, but the ability of PDOs to predict responses to chemoradiation in cancer patients remains an open question. Here we generate a living organoid biobank from patients with locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiation (NACR) enrolled in a phase III clinical trial. Our co-clinical trial data confirm that rectal cancer organoids (RCOs) closely recapitulate the pathophysiology and genetic changes of corresponding tumors. Chemoradiation responses in patients are highly matched to RCO responses, with 84.43% accuracy, 78.01% sensitivity, and 91.97% specificity. These data imply that PDOs predict LARC patient responses in the clinic and may represent a companion diagnostic tool in rectal cancer treatment.

Inhibition of SIRT1 promotes taste bud stem cell survival and mitigates radiation-induced oral mucositis in mice.

Taste loss is one of the debilitating complications in radiation-induced oral mucositis (RIOM), as occurs in head and neck cancer patients undergoing radiotherapy. We report here a radio-mitigation effect of Sirtuin 1 (SIRT1) inhibitors in taste bud organoids and a mouse model of radiation-induced taste bud injury. The organoids, developed from circumvallate (CV) papilla, were irradiated with single dose of X-rays and inhibitors of SIRT1 or SIRT2 were added 24 h later. The survival was evaluated by measuring the number and size of regenerated organoids after irradiation (IR). Oral mucositis (OM) was induced by IR of the oral region of Lgr5-lacZ transgenic mice. The surviving Lgr5+ taste bud stem cells were identified after lacZ-staining and the mucosal ulceration on tongue dorsal surface was determined by histological methods. Results showed that SIRT1 inhibitors (nicotinamide, EX527, salermide and sirtinol), but not SIRT2 inhibitors, significantly improve taste bud organoid survival after IR. Remarkably, administration of nicotinamide (NAM), a recognized inhibitor of SIRT1 to mice 24 h after IR promotes the survival of Lgr5+ taste bud stem cells, resulting in alleviated tongue mucositis. In conclusion, SIRT1 inhibitors promote Lgr5+ taste bud stem cell survival and mitigate RIOM in mice. These observations have important implications for efforts to develop therapeutic strategies against taste dysfunction and mucosal ulceration in RIOM.

Activated B Lymphocyte Inhibited the Osteoblastogenesis of Bone Mesenchymal Stem Cells by Notch Signaling.

Estrogen is very important to the differentiation of B lymphocytes; B lymphopoiesis induced by OVX was supposedly involved in osteoporosis. But the effects of B lymphocytes on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not clear. In this study, we detected bone quality and bone loss in a trabecular bone by electronic universal material testing machine and microcomputed tomography (micro-CT) in OVX and splenectomized-ovariectomy (SPX-OVX) rats. Additionally, changes in lymphocytes (B lymphocyte, CD4+ and CD8+ T lymphocytes, and macrophages) in the bone marrow were analyzed by flow cytometry. The osteogenesis of BMSCs cocultured with normal and LPS-pretreated B lymphocytes was detected by BCIP/NBT and Alizarin red S staining. Measurement of the Notch2, Notch4, Hey1, Hey2, Hes1, and runt-related transcription factor 2 (Runx2) expression in BMSCs cocultured with B lymphocytes was done using real-time PCR. The effects of dexamethasone and DAPT (inhibitor of Notch signaling) on osteogenesis of BMSCs were detected by BCIP/NBT, Alizarin red S staining, and real-time PCR. Osteoporosis happened in OVX rats, more serious in SPX-OVX rats, B lymphocytes increased in OVX rats, and sharply higher in SPX-OVX rats. Osteoporosis did not happen in SPX rats which is still companied with a high increase of B lymphocytes. LPS-pretreated B lymphocytes suppressed the osteogenesis of BMSCs, but the normal B lymphocytes could not. The LPS-pretreated B lymphocytes upregulated the expression of Notch4, Hes1, and Hey2 and downregulated the expression of Runx2 in BMSCs. Dexamethasone and DAPT could downregulate the high expression of Notch4, Hes1, Hey2 and upregulate the low expression of Runx2 in BMSCs which cocultured with LPS treated B lymphocytes, the inhibited ALP and Alizarin red staining in BMSCs which cocultured with LPS treated B lymphocytes also partly restored.