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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.

Adding irinotecan to neoadjuvant chemoradiotherapy (nCRT) in locally advanced rectal cancer (LARC) increases the pathologic complete response (pCR) rate but brings more toxicities. Robust biomarkers to predict response to irinotecan-based nCRT are extremely necessary for selecting the right patients. Our previous study suggests that patient-derived tumor organoids (PDTOs) sensitivity to chemoradiotherapy matches patient responses. In this study, we investigated whether PDTOs sensitivity to irinotecan can predict complete response (CR) and survival. Eligible patients receiving irinotecan-based nCRT between April 5, 2017 and December 11, 2020 were enrolled in the training cohort (n = 91) for response prediction and survival analysis. Patients receiving nCRT between February 21, 2021 and September 17, 2021 were included in the validation cohort (n = 27). Predictive performances of irinotecan organoid size ratio (OSR) for CR or pCR were evaluated. The irinotecan-sensitive groups had higher response rates compared with the insensitive groups (training cohort: 71.8% vs 24.4%, P

Traumatic brain injury refers to brain injury caused by mechanical impact often leading to severe morbidity and mortality. Despite increasing awareness, there are no effective treatments strategies. Therefore, there is a need to develop new effective treatments for this injury. Forsythiaside A is a monomer of phenylethanolglucoside extracted from Forsythia, which has a wide range of pharmacological properties including protective effects on brain tissue. Herein, using a rat model of traumatic brain injury, we have shown that forsythiaside A can improve nerve function and brain tissue injury in rats with traumatic brain injury, and reduce brain inflammation and neuronal apoptosis. We have further shown that forsythiaside A regulates toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B signaling pathway. This opens the possibility of a potentially promising therapeutic drug for the treatment of traumatic brain injury.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

Background Gallbladder carcinoma (GBC) is one of the most lethal malignancies which do not have a targeted drug in the clinic. Patient-derived primary cell lines (PDCs) are useful in assessment of cancer complexity and heterogeneity, drug-sensitivity tests, and personalized-drug-selection guidance. The aim of this study is to establish GBC PDCs and characterize their biological features. Methods The characterization of PDCs was defined by morphology, growth kinetics, chromosomal analysis, short tandem repeat (STR) analysis, RNA-seq and tumorigenicity. Glycosylation of PDCs derived from GBC was first studied, and the PDC model’s performance were also tested and evaluated using seven molecular target inhibitors. Results Three novel GBC cell lines from three GBC patients were successfully established and denoted as JXQ-3D-902R4, JXQ-3D-4494R, and JXQ-3D-4786R. These cell lines demonstrated the heterogeneous characteristics of tumor morphology and phenotypes which are consistent with primary GBC, such as irregular cell shape, varied chromosomal numbers, and different STR patterns. Moreover, the growth activity and tumorigenicity ability varied among the cell lines, of which JXQ-3D-4494R exhibited the best growth rate. Furthermore, glycan profiling of whole proteins were detected and characterized. Unique N-glycans of each PDC were identified, JXQ-3D-902R4, JXQ-3D-4494R and JXQ-3D-4786R contained ten, four and seven unique glycans, respectively. The epithelial origins of three PDCs were confirmed using RNA-seq based on the highly expressed typical epithelial marker genes. Moreover, the drug-sensitivity results demonstrated that the three PDCs exhibited different responses to the seven-most commonly used targeted medicines belonging to three groups: cell-cycle inhibitors, PI3K/AKT/mTOR signaling-pathway inhibitors, and ErbB inhibitors. JXQ-3D-4494R was sensitive to most of the inhibitors, JXQ-3D-4786R was sensitive to ErbB inhibitors, and JXQ-3D-902R4 was sensitive to PI3K/AKT/mTOR inhibitors. Conclusions These results indicate that PDCs may be efficient preclinical models for further investigation of the biological behaviors and potential targeted therapies of human GBC.

Epithelial ovarian carcinoma (EOC) is one of the most common gynecologic malignancies with a high mortality rate. Serum biomarkers and imaging approaches are insufficient in identifying EOC patients at an early stage. This study is to set up a combination of proteins from serum small extracellular vesicles (sEVs) for the diagnosis of early-stage EOC and to determine its performance. A biomarker for early-stage ovarian cancer (BESOC) cohort was used as a Chinese multi-center population-based biomarker study and registered as a Chinese Clinical Trial ChiCTR2000040136. The sEV protein levels of CA125, HE4, and C5a were measured in 299 subjects. Logistic regression was exploited to calculate the odds ratio and to create the sEV protein model for the predicted probability and subsequently receiver-operating characteristic (ROC) analysis. The combined sEV marker panel of CA125, HE4, and C5a as a sEV model obtained an area under curve (AUC) of 0.912, which was greater than the serum model (0.809), by ROC analysis to identify EOC patients from the whole cohort. With the cutoff of 0.370, the sensitivity and specificity of the sEV model were 0.80 and 0.89, which were much better performance than the serum markers (sensitivity: 0.55~0.66; specificity: 0.59~0.68) and the risk of ovarian malignancy algorithm (ROMA) index approved by the U.S. Food and Drug Administration (sensitivity: 0.65; specificity: 0.61), to identify EOC patients from patients with benign ovarian diseases or other controls. The sEV levels of CA125 significantly differed among early-stage and late-stage EOC (p < 0.001). Moreover, the AUC of ROC to identify early-stage EOC patients was 0.888. Further investigation revealed that the sEV levels of these 3 proteins significantly decreased after cytoreductive surgery (CA125, p = 0.008; HE4, p = 0.025; C5a, p = 0.044). In summary, our study showed that CA125, HE4, and C5a levels in serum sEVs can identify EOC patients at the early stage, elucidating the possibility of using a sEV model for the diagnosis of early-stage EOC.

e15598 Background: 5-FU-based regimens are the mainstay treatment for colorectal cancer. However, the limited response rate to 5-FU-based regimens hampered the increase in the treatment efficacy of locally advanced rectal cancer. During the drug screening in the rectal cancer organoid biobank (n = 106), we found out that more than half of the organoids that were resistant to 5-FU monotherapy and sensitive to irinotecan maintained sensitive status to the treatment of 5-FU combined with irinotecan (combination-sensitive), while a quite proportion of them turn to become remarkably resistant to 5-FU plus irinotecan (combination-resistant). Some studies suggested the mechanism of 5-FU-based resistance was associated with the activation of some oncogenic pathways. However, the mechanism of the addition of 5-FU promoting the resistance to irinotecan is still unclear. Methods: To investigate the relationship between the combination-resistant organoids and corresponding patient responses, we collected patient clinical responses and survival outcomes. RNA sequencing and ATAC sequencing were performed to identify differential genes and their enrichment pathways. Moreover, small molecule inhibitors were used to try to reverse the resistance induced by 5-FU. Results: Twenty-one organoids showed sensitivity to irinotecan and resistance to 5-FU. Among them, 8 organoids (38.1%) were combination-resistant and 13 organoids (61.9%) showed sensitivity to combination treatment. Combination-resistant organoids corresponding patients had worse pathologic tumor regression grades (pTRGs) distribution (8/8, 100% had TRGs of 2-3 versus 100% had TRGs of 0-1 in combination-sensitive patients; p < 0.05) and disease-free survival (DFS) rates (HR:11.57, 95% IC:1.025-130.5; p = 0.0047) than patients whose organoids were combination-sensitive. Combination-resistant organoids had higher expression of Ki-67 and CD44 and reduced cleaved-caspase 3 in the combination treatment group and 5-FU group compared with the irinotecan group and control. Furthermore, we identified hedgehog pathway activation after adding 5-FU to irinotecan in combination-resistant organoids through RNA sequencing and ATAC sequencing. GANT-61, an inhibitor of the hedgehog pathway, increased sensitivity to the combination of 5-FU and irinotecan in combination-resistant organoids. Conclusions: We found that organoids which are sensitive to irinotecan turn to become resistant to 5-FU combined with irinotecan. Moreover, corresponding patients had worse responses and a lower DFS rate. The hedgehog pathway is activated in these organoids after the addition of 5-FU to irinotecan, and GANT-61 could reverse the resistance caused by 5-FU. However, more organoid-associated trials with large-scale patients are warranted to reverse the 5-FU-based resistance in the future.

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.

Gallbladder carcinoma (GBC) is a lethal biliary tract malignant neoplasm. Patient-derived primary cancer cell lines (PDPCs) are appropriate models to explore biological characteristics and potential therapeutics; however, there is a lack of PDPCs in GBC. In this study, we aimed to establish and characterize the GBC PDPCs, and further investigated the intra-tumor heterogeneity (ITH). Multi-region sampling (3–9 regions) of the operable tumor tissue samples was used to establish PDPCs. Short tandem repeat genotyping for cell authentication and karyotyping was performed, followed by whole-exome sequencing and RNA sequencing to assess the ITH at the genetic and transcriptional levels, respectively. Thirty-eight PDPCs were successfully established from seven GBC patients and characterized. ITH was observed with a median of 38.3% mutations being heterogeneous (range, 26.6–59.4%) across all patients. Similar with other tumor types, TP53 mutations were always truncal. In addition, there were three genes, KMT2C, CDKN2A, and ARID1A, with truncal mutations in at least two patients. A median of 370 differentially expressed genes (DEGs) was identified per patient. Distinct expression patterns were observed between major histocompatibility complex (MHC) class I and II genes. We found the expression of MHC class II genes in the PDPC samples was closely regulated by CIITA, while that of MHC class I genes were not correlated with CIITA expression. The PDPCs established from GBC patients can serve as novel in vitro models to identify the ITH, which may pave a crucial molecular foundation for enhanced understanding of tumorigenesis and progression.

BACKGROUND Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, but there is a shortage of effective biomarkers for its diagnosis. AIM To explore blood exosomal micro ribonucleic acids (miRNAs) as potential biomarkers for HCC diagnosis. METHODS T RESULTS The principal component analysis suggested that daily alcohol consumption could alter the blood exosomal miRNA profiles of hepatitis B virus positive non-HCC patients through miR-3168 and miR-223-3p. The miRNA profiles also revealed the tumor stages of HCC patients. High expression of miR-455-5p and miR-30c-5p, which significantly correlated with better overall survival in tumor tissues, could also be detected in blood exosomes. Two pairs of miRNAs (miR-584-5p/miR-106-3p and miR-628-3p/miR-941) showed a 94.1% sensitivity and 68.4% specificity to differentiate HCC patients from non-HCC patients. The specificity of the combination was substantially influenced by alcohol consumption habits. CONCLUSION This study suggested that blood exosomal miRNAs can be used as new non-invasive diagnostic tools for HCC. However, their accuracy could be affected by tumor stage and alcohol consumption habits.

Multidrug resistance-related protein 1 (MRP1) is involved in the biological transport of several molecules with diverse structural characteristics outside of the cell. In addition to its transport activity, MRP1 exhibits multiple defense mechanisms in vivo. MRP1 is highly expressed in normal lung tissues and plays a protective role in the process of chronic obstructive pulmonary disease. In the present study, human bronchial epithelial cells (16HBE14o-cells) were stimulated by cigarette smoke extract (CSE) in vitro to simulate a smoking environment. On this basis, the mechanism of Allyl isothiocyanate (AITC) administration on the expression of MRP1 in CSE-stimulated 16HBE14o-cells was investigated. The effects of CSE on the viability of 16 HBE14o-cells were investigated by an MTT assay. The changes in the mRNA expression levels of nuclear erythroid factor 2 (Nrf2) and MRP1 were investigated in CSE-stimulated 16HBE14o-cells using western blotting and reverse transcription quantitative PCR (RT-qPCR). Immunofluorescence analysis was used to detect Nrf2 nuclear translocation. Incubation of the cells with 5% CSE for 24 h had minor effects on cell viability and resulted in the activation of the JNK and p38MAPK signaling pathways. AITC activated the JNK pathway, inhibited the activation of the p38MAPK pathway in 16HBE14o-cells stimulated by 5% CSE and upregulated the expression levels of Nrf2 and MRP1 in a time-dependent manner. The upregulation of Nrf2, MRP1 and of Nrf2, and MRP1 mRNA expression levels in CSE-stimulated cells was inhibited by pretreatment with SP600125 (a JNK pathway inhibitor). Furthermore, the fluorescence intensity in the nucleus was significantly enhanced following AITC pretreatment and the analysis indicated nuclear translocation of Nrf2 in the cells. These results indicated that Nrf2 and MRP1 expression levels in CSE-stimulated cells were altered following AITC pretreatment. Thus demonstrating that the primary mechanism may be associated with activation of the JNK pathway, while the p38MAPK pathway may not be involved.

Tissue sampling of biliary tract carcinomas (BTCs) for molecular characterization is challenging. The aim of this study was to investigate the possibility of identifying individual actionable mutations derived from bile cell-free DNA (cfDNA) using targeted deep sequencing. Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in the present study. Using targeted deep sequencing with a panel of 150 tumor-related genes, paired bile cfDNA and tumor DNA were analyzed for mutational variants individually and then compared. The present study, to the best of our knowledge, is the first to reveal that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case for plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. When comparing bile cfDNA and tumor DNA for single nucleotide variation (SNV)/insertion and deletion (Indel), the results using targeted deep sequencing revealed high sensitivity (94.7%) and specificity (99.9%). Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. The present study revealed that targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. These preliminary results may shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

Background: Colon signet-ring cell carcinoma (SRCC) is a rare type of malignant dedifferentiated adenocarcinomas, and associated with poor survival. However, an in-depth study of the biological features of colon SRCC is hampered by lack of in vitro model. Our study was designed to establish and characterize cell cultures from SRCC for further translational and biological research. Methods: By harnessing the power of organoid culture system, we established a human colon SRCC organoid line from surgical sample of one patient with colon SRCC. The organoid line was characterized at the morphology, histology, ultrastructure, chromosome stability level and its tumorigenic ability was verified by xenografts. Capture-based targeted DNA sequencing combining with drug screen based on a bespoke 88 compound library identified the possible target and the treatment response was confirmed by in vivo organoid system and in vitro xenografts. Findings: Our data showed that the colon SRCC organoid line, YQ-173, resemble histological and growth characteristics of the original tumor cells and have high tumor formation rate. DNA sequencing indicated JAK2 might be the treatment target. In vitro drug screen found that AT9283 and Pacritinib could be the effective JAK2 inhibitors, which was consistent with in vivo xenografts' response. Interpretation: We report, for the first time, the establishment of SRCC organoid line allowing in-depth study of SRCC biology, as well as a strategy to assess in vitro drug testing in a personalized fashion. Funding: This work was supported by National Natural Science Foundation of China (31470826, 31670858, 81672374), Science and Technology Commission of Shanghai Municipality (16411966300), and Shanghai Sailing Program (19YF1409500). Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval: The tissue was obtained with informed consent for experimentation with the patient and this study was approved by the ethical committee of Fudan University Shanghai Cancer Center. All animal experiments were performed according to the guidelines for the care and use of laboratory animals and were approved by Institutional Animal Care and Use Committee of Fudan University.

Purpose: Biliary tract cancer (BTC) is a kind of malignant digestive system tumors without a targeted drug approved in the clinic. A high proportion of gene mutations and abnormal expressions of ERBB pathway have been reported in BTC. However, there are few studies on targeting ERBB in BTC. In this study, we aim to explore the role of targeting ERBB and its potential resistance mechanisms in BTC. Materials and Methods: We used the tumor tissue samples of BTC patients to establish patient-derived cell lines (PDCs), and perform the biological characteristics of PDCs. At the first, Western Blot was used to select the PDCs with different ERBB2 expressions as in vitro models. Secondly, colony formation assay was to verify the sensitivity of inhibitors and Western blot was to confirm the change of signaling pathways in PDCs. Then, whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) were integrated to explore the potential resistance mechanisms of ERBB inhibitors. Moreover, stable PDCs with overexpression and knockdown established by lentiviral vectors were used. Finally, PDC xenografts were used to further confirm the hypothesis from the in vitro study. Results: Twenty-two BTC PDCs were successfully established in this study. According to ERBB2 expression, nine PDCs were selected for the sensitivity evaluation of ERBB inhibitors. Four PDCs of them are sensitive to three ERBB inhibitors, while the other five PDCs are not. Furthermore, colony formation assay has demonstrated the difference of colonies between sensitive cell lines and resistant cell lines for all three ERBB inhibitors. Meanwhile, Western Blot showed that inhibition activity is negatively correlated with phosphorylation of ERBB2 in PDCs. These results demonstrated that decreasing ERBB activity could inhibit the proliferation of BTC PDCs. Further analysis showed that all four sensitive PDCs highly expressed ERBB2, while four resistant PDCs carried with low ERBB2 expression levels. HCC783 was the only one PDC with high ERBB2 expression as well as resistance to ERBB inhibitors and was considered as a primary resistant PDC for follow-up study. RNA-seq analysis results showed that SMARCA1 was significantly lower (p < 0.01) and the gene transcriptional spectrum were greatly changed in resistant PDCs compared with sensitive PDCs, suggesting that SMARCA1 may have been involved in resistance mechanisms of ERBB inhibitors in BTC. Importantly, stable PDCs were established with overexpression and knockdown of SMARCA1 and the colony formation assay with them confirmed this hypothesis in vitro. The results demonstrated that a resistant cell line by overexpression of SMARCA1 could make sensitive to ERBB inhibitors, while a sensitive cell line by knockdown of SMARCA1 could become relatively resistant to ERBB inhibitors. The exploration of resistance mechanisms triggered by SMARCA1 and the in vivo validation with PDC xenografts are undergoing. Conclusions: In this study, the PDC model, drug screening and colony formation assay were integrated to verify that ERBB inhibitors could prevent the proliferation of BTC cells for the first time. Furthermore, WES and RNA-seq combined with stable PDC models were used to discover and validate that SMARCA1 is one of the potential resistance mechanisms of ERBB inhibitors in BTC. Citation Format: Feiling Feng, Qingbao Cheng, Dadong Zhang, Chen Liu, Xiaoya Xu, Bin Li, Huizhen Wang, Yong Yu, Bin Li, Xiaobing Wu, Jun Zhou, Kaijian Chu, Zhenghua Xie, Qingxiang Gao, Lei Xiong, Fugen Li, Bin Yi, Xiaoqing Jiang. Study on the role of targeting ERBB and its potential resistance mechanism in biliary tract cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1912.

Purpose: Lung cancer is the most common malignancy with the highest morbidity and mortality worldwide. Low-dose computed tomography (LDCT) is one of the main tools for lung cancer screening and diagnosis. However, high false positive, high cost, over-diagnosis and radiation exposure are major drawbacks to screen pulmonary nodules by LDCT. Thus, a noninvasive diagnostic approach with high specificity and accuracy is badly needed to enhance LDCT. Liquid biopsy represent a valuable non-invasive approach when biopsy or resection is not the first choice. Till now, it is rare to see the studies on exosome-derived miRNAs as early diagnosis biomarkers to distinguish benign and malignant pulmonary nodules using small RNA sequencing. Here, we aimed to explore the diagnostic value of a panel of significantly differential expressed plasma exosomal miRNAs between benign and malignant pulmonary nodule samples in Chinese cohorts. Materials and Methods: This study was registered at Chinese Clinical Trial Registry (www.chictr.org.cn) with registration number ChiCTR1800019877. Forty-five patients including twenty-six lung adenocarcinoma and nineteen benign nodules with various pathological characteristics were enrolled as a training cohort. A test cohort consisted of sixty-two patients with twenty-four benign nodules patients similar to training cohort and thirty-eight lung adenocarcinoma. Exosomes were precipitated from the plasma, and small RNA sequencing was performed to identify the differential expressed miRNAs. A statistical model consisting of a panel of exosomal miRNAs was trained to discriminate benign nodules from cancerous ones. The model was validated in the independent test cohort, and re-confirmed in an external dataset from another Chinese cohort. Enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also performed. Results: Characteristic proteins and morphology of exosomes were characterised by western blotting, nanoparticle tracking analysis and scanning electron microscopy. Five differential expressed miRNAs (let-7b-3p, miR-101-3p, miR-125b-5p, miR-150-5p, and miR-3168) with median expression > 50 were selected by LASSO-penalized regression as a linear model to classify samples into benign or maligant groups with 10-fold cross-validation to determine the model parameters. When the specificity set 94.7% and 91.7% for the training and test cohorts, respectively, the model had 57.7% and 57.9% sensitivity in both cohorts. The model was also confirmed in an external dataset with 87.5% specificity and 53.1% sensitivity. The expression of each biomarker in benign, adenocarcinoma in situ/microinvasive adenocarcinoma and invasive adenocarcinoma nodules were gradually altered. Four of the five biomarkers were gradually increased, whereas one miRNA was gradually decreased. GO and KEGG analysis demonstrated that biological process and pathways of the genes targeted by five biomarkers were associated with tumor development. Conclusions: This study using small RNA sequencing identified five plasma exosome-derived differentially expressed miRNAs as a diagnosis model to distinguish benign and malignant pulmonary nodules, which provides insights into the feasibility of exosomal miRNAs as a novel early diagnosis approach for lung adenocarcinoma. Citation Format: Di Zheng, Yang Yang, Chunyan Wu, Huizhen Wang, Jiyang Zhang, Shiyi Liu, Xiaoya Xu, Hao Chen, Dadong Zhang, Fugen Li, Jian Ni, Gening Jiang, Jianfang Xu. A panel of plasma exosomal miRNAs as diagnosis biomarker to distinguish benign and malignant nodules in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 756.

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.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and current treatments exhibit limited efficacy against advanced HCC. The majority of cancer-related deaths are caused by metastasis from the primary tumor, which indicates the importance of identifying clinical biomarkers for predicting metastasis and indicating prognosis. Patient-derived cells (PDCs) may be effective models for biomarker identification. In the present study, a wound healing assay was used to obtain 10 fast-migrated and 10 slow-migrated PDC cultures from 36 HCC samples. MicroRNA (miRNA) signatures in PDCs and PDC-derived exosomes were profiled by microRNA-sequencing. Differentially expressed miRNAs between the low- and fast-migrated groups were identified and further validated in 372 HCC profiles from The Cancer Genome Atlas (TCGA). Six exosomal miRNAs were identified to be differentially expressed between the two groups. In the fast-migrated group, five miRNAs (miR-140-3p, miR-30d-5p, miR-29b-3p, miR-130b-3p and miR-330-5p) were downregulated, and one miRNA (miR-296-3p) was upregulated compared with the slow-migrated group. Pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were significantly enriched in the ‘focal adhesion’ pathway, which is consistent with the roles of these miRNAs in tumor metastasis. Three miRNAs, miR-30d, miR-140 and miR-29b, were significantly associated with patient survival. These findings indicated that these exosomal miRNAs may be candidate biomarkers for predicting HCC cell migration and prognosis and may guide the treatment of advanced HCC.

Background While it is known that irradiation can induce local and systemic bone loss over time, how focal irradiation induces systemic bone complications remains unclear. Immune cells are thought to be crucial to bone homeostasis, and abnormal immune cells lead to serious disruption of bone homeostasis, such as in acute lymphoblastic leukaemia. This disruption primarily occurs due to inhibition of the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Methods In this study, we detected local and systemic bone loss in trabecular bone by micro-computed tomography (micro-CT) and measurement of peroxisome proliferator-activated receptor gamma (PPARγ) and runt-related transcription factor 2 (RUNX2) expression in BMSCs using real-time polymerase chain reaction and western blotting. Additionally, changes in lymphocytes (B cells and CD4+ and CD8+ T cells) in the peripheral blood and bone marrow were analysed by flow cytometry. BMSC-derived osteoblasts and adipocytes, cultured in osteogenic or adipogenic media or co-cultured with lymphocytes, were detected by BCIP/NBT, Alizarin Red S and Oil Red O staining. Results Focal irradiation induced local and systemic bone loss in trabecular bone. Increased PPARγ expression and decreased RUNX2 expression were observed, accompanied by upregulated adipogenesis and downregulated osteogenesis of BMSCs. B cells and CD8+ T lymphocytes were increased in the blood and bone marrow after irradiation, while CD4+ T lymphocytes were decreased in the blood. Inhibition of RUNX2 expression and reduction of alkaline phosphatase activity and mineralization deposits were observed in lymphocyte-co-cultured BMSCs, accompanied by an increase in PPARγ expression and in the number of lipid droplets. Conclusions Focal irradiation induced local and systemic bone loss in trabecular bone. Increased B cells and CD8+ T lymphocytes led to systemic bone loss by decreasing BMSC osteogenesis.

Chronic inflammation is one of the main symptoms of cancer cachexia, and cyclooxygenase 2 inhibitors, such as celecoxib, may be beneficial in counteracting the major symptoms of this syndrome. In the current study, celecoxib was orally administered to BALB/c male mice with colon 26 adenocarcinoma. Tumor growth, survival rate, body weight and food intake of the mice with cancer cachexia were recorded during the experiments. The host inflammatory response was assessed by morphological observations and hematoxylin-eosin staining. The serum levels of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor, interleukin-6 and tumor necrosis factor-α in mice with cancer cachexia were measured by ELISA. Celecoxib administration attenuated the decline in body weight and food intake of mice with cancer cachexia, and improved the survival rate of cachectic mice. Erythrocyte counts and hemoglobin concentration significantly increased in cachectic mice receiving celecoxib compared with control cachectic mice. Notably, celecoxib administration significantly reduced the serum level of VEGF in mice with colon 26 adenocarcinoma, and the cachectic events were also relieved by treatment with a VEGF antibody. The cyclooxygenase 2 inhibitor celecoxib produced positive therapeutic effects in mice with cancer cachexia. This function was regulated at least partly by downregulation of serum levels of VEGF.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is of high mortality with a shortage of effective non-invasive biomarkers in the diagnosis and prognosis. Without a proper method to identify reasonable target molecules and their references for normalization, robust and accurate blood miRNA biomarkers have not been reported. We aim to explore the exosomal miRNAs as biomarkers for the diagnosis and prognosis of PDAC with sufficient biological insights and robustness. Materials and Methods: miRNA-seq was performed on miRNAs extracted from blood exosomes. 16 non-metastatic PDAC, 11 metastatic PDAC and 15 pancreatitis patients were enrolled in 2017 as the training set. Another 18 non-metastatic PDAC, 12 metastatic PDAC and 18 pancreatitis patients were enrolled in 2018 as the testing set. Bayesian network was built on the levels of miRNAs to identify candidate biomarkers with biological insights. The area under the curve (AUC) of receiver operating characteristic (ROC) was used to measure the accuracy of each pair of miRNAs. Results: The exosomal miRNA from the plasma of these patients with PDAC or pancreatitis has been successfully extracted and sequenced. The Bayesian network consisted of 337 nodes and 713 edges. The quotient of miR-95-3p (a “consequence” of “cancer”) over miR-26-5p (a “cause” of “pancreatitis”) performed 81.5% sensitivity and 93.3% specificity in the training cohort, and 86.7% sensitivity and 100% specificity in the testing cohort respectively. Furthermore, considering either high serum level of CA19-9 or the quotient of miR-95-3p/miR-26-5p as PDAC, the sensitivity increased to 96.5%, and the specificity was maintained at 96.9%. In addition, miR-335-5p level in blood exosome could predict metastasis with a mean accuracy of 77.2%, and showed strong relation with overall survival after surgeries. Conclusions: MiRNAs biomarkers from blood exosomes in differential diagnostics and prognosis are identified and rationale by Bayesian network. Integration of biomarkers of various molecular types could enhance the diagnostic accuracy beyond single molecular types. Blood exosomal miRNAs could be applied to monitor the development of PDAC. Citation Format: Shiwei Guo, Huan Wang, Hao Qin, Dadong Zhang, Zhuo Shao, Yanan Zhang, Bin Song, Xiaoya Xu, Jing Shen, Zishuo Chen, Xiaohan Shi, Suizhi Gao, Yaqi Pan, Fugen Li, Gang Jin. Identifying blood exosomal miRNAs as biomarkers in the diagnosis and prognosis of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1363.

Purpose: Biliary tract cancers (BTCs) are less common, but rapidly lethal tumors. Despite recent improvements in multimodal therapy, the 5-year survival rate is less than 10% in patients with advanced or metastatic BTC. BTC patients lack personalized treatment options, partly because tissue samplings are often inadequate for molecular characterization. Thus, this study examined the use of bile cell-free DNA (cfDNA), which is released by tumor and normal cells, as a potential liquid biopsy able to provide genomic information for the whole tumor. Materials and Methods: Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in this study August 2017 to March 2018. To investigate the use of bile cfDNA as a liquid biopsy in BTC patients, targeted deep sequencing, with a panel of 150 tumor-related genes, was used to analyze paired bile cfDNA and tumor DNA for mutational variants individually and then compared. The sensitivites and specificities of detecting mutations including SNV/Indel and copy number variation (CNV) in bile cfDNA were analyzed. Results: This study is the first to show that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case in plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. The mutational profiles of bile cfDNA showed that of the SNV/Indel mutations, the highest variation was seen in TP53, followed by KRAS, NOTCH1, NOTCH2 and KMT2A. Among the CNV mutations, low recurrent amplified genes, such as CCNE1, ERBB2, CDKN1B, ZNF217, and CDK6, were identified; with CDKN2A shown to have a deep deletion. Among these genes, NOTCH1, NOTCH2, ERBB2 and CDK6 are of interest as potential drug targets. When comparing bile cfDNA and tumor SNV/Indel detection results using targeted deep sequencing, a high sensitivity (94.7%) and specificity (99.9%) were obtained. Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. Conclusions: This study shows that long DNA fragments are prevalent in bile cfDNA samples and targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. This is the first time that a mutational landscape, including SNVs, Indels and CNVs, has been obtained for bile cfDNA from BTC patients. These preliminary results maybe shed light on bile cfDNA as a promising liquid biopsy for BTC patients. Citation Format: Ningjia Shen, Dadong Zhang, Lei Yin, Yinghe Qiu, Jian Liu, Wenlong Yu, Xiaohui Fu, Bin Zhu, Xiaoya Xu, Anqi Duan, Zishuo Chen, Xiang Wang, Xinkai Cao, Teng Zhao, Zisong Zhou, Lianghe Yu, Hao Qin, Zheng Fang, Jing-Yu Li, Yuanjin Liu, Lei Xiong, Bo Yuan, Fugen Li, Yongjie Zhang. Bile cell-free DNA as a powerful liquid biopsy of the somatic variants in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2292.

Bacterial infection is a key factor in airway inflammation. The present study describes the time-dependent changes in the leukocyte counts and cytokine levels of the bronchoalveolar lavage fluid (BALF) following subacute airway inflammation induced by lipopolysaccharide (LPS), a major component of the outer membranes of Gram-negative bacteria. LPS (200 μg/rat) or saline was intratracheally administered to rats which were sacrificed 2, 4 or 7 days after LPS treatment. Airway inflammation was evaluated using hematoxylin and eosin staining, cell counts and proinflammatory cytokine levels in the BALF. Rat airways obtained from the LPS group exhibited marked airway wall thickening and infiltration of inflammatory cells compared with the control group, as well as elevated cell counts (neutrophils, macrophages, lymphocytes) and proinflammatory cytokine levels [(tumor necrosis factor (TNF)-α, interleukin (IL)-1β, cytokine-induced neutrophil chemoattractant (CINC)-1)] in the BALF, which peaked on day 2 and subsequently decreased until the experimental endpoint. Notably, IL-1β levels induced by LPS changed in a similar manner to macrophage cell counts, but not neutrophil and lymphocyte counts. Moreover, TNF-α and CINC-1 levels did not decrease as rapidly as neutrophil counts after peaking. These findings suggest that macrophages may play a significant role in maintaining subacute inflammatory responses induced by LPS in rat airways.

Systemic inflammation might modulate the microenvironment in the lungs and promotes metastasis. E-selectin, an inflammation inducible endothelial cell adhesion molecule, has been reported to play an important role in homing metastatic cancer cells. To study the effects of E-selectin expression induced by systemic inflammation on breast cancer metastasis, we first treated BALB/c mice with lipopolysaccharide (LPS) to induce systemic inflammation. Pulmonary tissues were analyzed by wet/dry ratio, hematoxylin and eosin (H&E) staining and immunohistochemistry. Then 4T1 cells were injected via tail vein. Lung surface metastasis was counted and detected by histological analysis. LPS-induced E-selectin expression and tumor cells adhesion were assessed by western blotting and immunofluorescence. The circulating levels of proinflammatory cytokines in sera were evaluated by ELISA. Our results showed that a significant increase in breast cancer metastasis to lungs was observed in LPS-treated mice vs. the PBS-treated mice, accompanying with an increased E-selectin expression in pulmonary tissue of LPS-treated mice. In vitro studies showed a significant elevation of E-selectin production in MPVECs which enhanced the adhesion activity of 4T1 cells. Treatment with anti-E-selectin antibody significantly reduced the development of metastasis in vivo, and significantly reduced the adhesion of 4T1 cells to MPVECs in vitro. Our results suggest that systemic inflammation may increase the expression of E-selectin which mediated the lung metastasis of breast cancer in mouse model.

Inflammation is a potent promoter of tumor metastasis. The aim of the present study was to explore the function of systemic inflammation in the formation of lung metastasis of breast cancer cells in a mouse model. BALB/c mice were injected intraperitoneally with lipopolysaccharide (LPS) in order to establish an inflammatory animal model and 4T1 murine breast cancer cells were injected through the tail vein to induce lung metastasis. The levels of proinflammatory cytokines were evaluated by ELISA. Metastases on the surface of the lungs were counted and histologically analyzed by hematoxylin and eosin staining. Angiogenesis in the lungs was examined by CD31 immunofluorescence. Mouse pulmonary endothelial cells (MPVECs) were isolated and used to assay endothelial tube formation and determine the protein expression levels of vascular endothelial growth factor (VEGF) in vitro. Serum levels of VEGF and prostaglandin E2 (PGE2), the number and size of metastatic lesions, and the expression levels of cyclooxygenase‑2 were significantly greater in the lungs of LPS‑treated mice, as compared with those in control mice threated with phosphate‑buffered saline. Blood vessel density was also markedly increased in the LPS‑treated mice. These increases were reversed by treatment with celecoxib. In vitro, the protein expression levels of VEGF produced by the PGE2‑treated cells were significantly increased in a concentration‑dependent manner. In addition, the production of VEGF was increased in response to treatment with the PGE2 receptor (EP2) agonist ONO‑AE1‑259‑01; however, this increase was abrogated by treatment with AH6809, an EP2 receptor antagonist. Treatment with PGE2 or VEGF alone promoted the tube formation of MPVECs and this effect was reversed by treatment with celecoxib. These results demonstrated that PGE2 may regulate the release of VEGF by MPVECs through the EP2 receptor pathway and thereby promoted pulmonary angiogenesis and breast cancer metastasis in a mouse model.

Lung is a common site for the occurrence of melanoma metastasis, the mechanism by which primary melanoma affects the lungs before tumor cells arrival is poorly understood. The aim of this study was to explore lung microenvironment response to primary melanoma in the premetastatic phase. Melanoma cells (B16) were implanted into the Balb/c mice back, pulmonary inflammation response was analyzed by wet/dry ratios and H&E staining, the relationship between inflammation cells and metastatic foci was analyzed by bone marrow transplant mouse model, pulmonary vasculature was further analyzed by whole mount staining, and the circulating levels of proinflammatory cytokines in sera were evaluated by mouse cytokine array. In the premetastatic stage, significant inflammation response in lungs was induced by a distant primary melanoma, inflammation cells colonize in premetastatic sites before tumor cells arrived, and the sites of inflammation cells clusters are tumor metastasis sites. VEGF, M-CSF and TNF-α may be the underlying factors responsible for the increased metastasis in the B16-bearing mice. Treatment with celecoxib had effects on inflammation response and reduced cancer metastasis. In the premetastatic phase, the melanoma induces pulmonary inflammation response, changes the lung environment and then facilitates lung metastasis. Thus, inhibition of lung inflammation may provide potential targets for the prevention of metastasis.

The aim of this study was to identify serum protein fingerprints of small cell lung cancer (SCLC) and potential biomarkers related to chemotherapy resistance of SCLC with surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS). A total of 60 SCLC patients and 48 age- and sex-matched healthy individuals were enrolled. The chemotherapy regimen was cisplatin plus etoposide. All patients received two cycles of chemotherapy. Serum protein profiles were detected using SELDI-TOF MS and the spectra were analyzed with support vector machines (SVMs). Western blotting was performed to verify the results of SELDI-TOF MS. Three top scored peaks, at m/z of 6269, 9043 and 13124 Da, were finally selected as potential biomarkers for detection of SCLC. The SVM classifier separated the SCLC from the healthy samples in the blind test, with a sensitivity of 92.4% and a specificity of 92.5%. For the 56 eligible chemotherapy patients, 4 had a complete response (7.14%), 39 patients had a partial response (69.6%), 9 patients had a stable disease (16.1%) and 4 patients had a progressive disease (7.14%). The model constructed using two protein peaks with m/z of 8830 and 10468 Da separated the chemotherapy-resistant group from the chemotherapy-sensitive group with a sensitivity of 80.0% and a specificity of 80.0%. Initial protein database searching identified 10468 Da as S100-A9 which was confirmed by western blotting. The present results suggest that the combination of SELDI-TOF MS with SVM may provide a useful means in the search for serum biomarkers for predicting chemotherapy resistance in patients with SCLC.

To promote bone formation is one of the fundamental strategies in osteoporosis treatment and fractures repair. As one of the stimulators on bone formation, osteogenic growth peptide (OGP) increases both proliferation and differentiation of the osteoblasts in vitro and in vivo, in which osteoprotegerin (OPG) has been suggested being involved. In this study, we evaluated the effects of OGP on bone marrow mesenchymal stem cells (MSCs) from OPG-deficient mice in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase (ALP) activity assay, real-time polymerase chain reaction, and western blot analysis. Results showed that OGP stimulated MSC proliferation and increased the expression of CDK2 and cyclin A in MSCs both at mRNA and protein levels. However, no differentiative effect of OGP was shown as ALP activity and the expression levels of Runx2 and Osterix were not increased significantly by OGP. Our study suggested that OGP may increase the bone formation in OPG-deficient mice by stimulating MSC proliferation rather than differentiation, and probably by triggering CDK2/cyclin A pathway.