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Patients with triple-negative breast cancer (TNBC) often develop metastases in visceral organs including the liver, but the detailed molecular mechanisms of TNBC liver metastasis is not clearly understood. In this study, we tried to dissect the process of pre-metastatic niche formation in the liver by using patient-derived xenograft (PDX) models of TNBC with different metastatic propensity. RNA sequencing of TNBC PDX models that successfully metastasized to liver showed upregulation of the Cx3cr1 gene in the liver microenvironment. In syngeneic breast cancer models, the Cx3cr1 upregulation in liver preceded the development of cancer cell metastasis and was the result of recruitment of CX3CR1-expressing macrophages. The recruitment was induced by the CX3CL1 production from the liver endothelial cells and this CX3CL1–CX3CR1 signaling in the pre-metastatic niche resulted in upregulation of MMP9 that promoted macrophage migration and cancer cell invasion. In addition, our data suggest that the extracellular vesicles derived from the breast cancer cells induced the TNFα expression in liver, which leads to the CX3CL1 upregulation. Lastly, the plasma CX3CL1 levels in 155 patients with breast cancer were significantly associated with development of liver metastasis. Implications: Our data provides previously unknown cascades regarding the molecular education of pre-metastatic niche in liver for TNBC.

OBJECTIVE We investigated genetic risk of cardiovascular disease (CVD) by age at type 2 diabetes (T2D) diagnosis. RESEARCH DESIGN AND METHODS We compared incident CVD events by age at T2D diagnosis using UK Biobank (N = 12,321) and the Seoul National University Hospital (SNUH) cohort (N = 1,165). Genetic risk was quantified using polygenic risk score (PRS). RESULTS Individuals with earlier T2D diagnosis had higher CVD risk. In UK Biobank, the effect size of coronary artery disease (CAD) PRS on incident CAD was largest in individuals diagnosed with T2D at ages 30–39 years (hazard ratio 2.25; 95% CI 1.56–3.26) and decreased as age at diagnosis increased: ages 40–49 (1.51; 1.30–1.75), 50–59 (1.36; 1.24–1.50), and 60–69 years (1.30; 1.14–1.48) (Pinteraction = 0.0031). A similar trend was observed in the SNUH cohort. This increased genetic risk associated with earlier T2D diagnosis was largely mitigated by a healthy lifestyle. CONCLUSIONS Individuals with an earlier T2D diagnosis have a higher genetic risk of CAD, and this information could be used to tailor lifestyle interventions.

Background Triple negative breast cancer (TNBC) is a breast cancer subtype with increased risk of distant metastasis, especially to lung and liver. In addition to the intrinsic characteristics of tumor cells, the molecular features of the tumor microenvironment cells can also regulate the metastasis process of TNBC. In this study, we tried to characterize the transcriptomic features of microenvironment fibroblasts associated with the distant metastasis of TNBC. Method We established PDX models by obtaining tumor tissues from 26 triple-negative breast cancer patients and transplanting them into the mammary fat pads of immunodeficient mice. When the PDX tumors grew sufficiently in the fat pads, we excised them and saved the mice to assess whether each PDX tumor was capable of developing spontaneous distant metastasis. We used bulk RNA sequencing and single cell RNA sequencing to determine the transcriptomic features associated with the distant metastasis. Results We classified the 26 TNBC PDX models based on their in vivo metastasis capacity and the clinical outcomes of the corresponding patients. Non-metastatic PDX models were defined as the lack of developing distant metastasis in either mouse experiment or clinical follow-up data (n=5). PDX models were defined as metastatic models when they developed distant metastasis in both mouse and human (n=4). We performed single cell RNA sequencing using the nine PDX tumors and observed a substantial gene expression differences of fibroblasts in the tumor microenvironment between the metastatic and non-metastatic PDX models. The fibroblasts of the metastatic PDX models showed up-regulation of genes involved in the response to hypoxia, inflammatory response, neutrophil chemotaxis, cellular response to IL-1, positive regulation of cell proliferation, and negative regulation of apoptotic process. Moreover, this difference of gene expression between metastatic and non-metastatic TNBC PDX models were not identified by the bulk RNA sequencing data. Conclusion By using the single cell RNA sequencing using the TNBC PDX models, we identified key features of fibroblasts in the tumor microenvironment that contributes to the metastasis capacity of TNBC. Further studies are on-going to elucidate the underlying mechanisms. Citation Format: Woo Hang Heo, Woochan Lee, SONGBIN LI, Jong-Il Kim, Hyeong-Gon Moon. The relationship between the gene expression difference of fibroblasts in the tumor microenvironment and metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-06.

Background Malignant phyllodes tumour (MPT) is a rare breast malignancy with epithelial and mesenchymal features. Currently, there are no appropriate research models or effective targeted therapeutic approaches for MPT. Methods We collected fresh frozen tissues from nine patients with MPT and performed whole-exome and RNA sequencing. Additionally, we established patient-derived xenograft (PDX) models from patients with MPT and tested the efficacy of targeting dysregulated pathways in MPT using the PDX model from one MPT. Results MPT has unique molecular characteristics when compared to breast cancers of epithelial origin and can be classified into two groups. The PDX model derived from one patient with MPT showed that the mouse epithelial component increased during tumour growth. Moreover, targeted inhibition of platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) by imatinib mesylate and PKI-587 showed in vivo tumour suppression effects. Conclusions This study revealed the molecular profiles of MPT that can lead to molecular classification and potential targeted therapy, and suggested that the MPT PDX model can be a useful tool for studying the pathogenesis of fibroepithelial neoplasms and for preclinical drug screening to find new therapeutic strategies for MPT.

Supplementary Table 1. Information about patients derived xenograft models and derived patient. Supplementary Table 2. Ontology of differentially expressed genes in metastasis-harboring liver of PDX models. Supplementary Table 3. Differentially expressed genes in liver and lung of metastasis-harboring PDX models. Supplementary Table 4. Human ratio in lung and liver of additional PDX models identified by RNA sequencing. Supplementary Table 5. Gene ontology of co-expressed genes with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 6. Top ranked genes having significant correlation with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 7. Demographic and clinical characteristics of all patients. Supplementary Table 8. Information about used primer sequences for quantitative real-time PCR Supplementary Figure 1. Liver and lung H&E staining pictures of eight nonmetastatic PDX models a. Microscopic images of non-metastasis harboring PDX mouse model's liver (left) and lung (right) taken at 400x magnification. Supplementary Figure 2. Immunohistochemistry about CX3CR1 in liver and lung of breast cancer metastasis patients. Supplementary Figure 3. Hepatic metastasis after four weeks after orthotopic injection of 4T1 breast cancer cells identified by H&E staining. Supplementary Figure 4. Identification of pre-metastatic niche in liver tissues of 4T1 bearing mice a. mRNA expression of inflammation associated genes identified by real-time PCR in liver tissues of 4T1 bearing mice. Supplementary Figure 5. Identification of CX3CR1 positive cell population. Supplementary Figure 6. FACS analysis with anti-CX3CR1 of Raw264.7 macrophage cell lines. Supplementary Figure 7. Trans-well migration assay of BMDM with or without CX3CL1. Supplementary Figure 8. Mmp9 mRNA expression having significant correlation with Cx3cr1 mRNA expression in liver of PDX models. Supplementary Figure 9. MMP9 expression of macrophages with CX3CL1 treatment. Supplementary Figure 10. Signaling pathway of CX3CL1 treated macrophages. Supplementary Figure 11. Splenic injection of 4T1 cells inducing hepatic metastasis within two weeks in most cases. Supplementary Figure 12. mRNA expression about Cx3cr1 and Mmp9 in liver of vehicle or AZD8797 treated BALB/C mice identified by real-time PCR. Supplementary Figure 13. Correlation plot of Tnfa and Cx3cr1 mRNA expression in liver and lung of PDX models. Supplementary Figure 14. EVs isolation of CD63-mRuby-4T1.

FPKM expression values of 2223 genes deregulated in response to treatment in at least one condition compared to control (P < 0.05)

Supplementary Figure S1. Expression of BCL2L1 and BCL2 in CRC cell lines; Supplementary Figure S2. Estimated BCL2L1 copy numbers of CRC patients by droplet digital PCR (ddPCR); Supplementary Figure S3. Screening of MCL1-modulating agents for synergistic effect with ABT- 263 in KRAS-mutated and BCL2L1-amplified CRC cells (SW620); Supplementary Figure S4. Screening of MCL1-modulating agents for synergistic effect with ABT- 263 in KRAS wild and BCL2L1-diploid CRC cells (SNU1684); Supplementary Figure S5. Effect of ABT-263 and YM-155 combination treatment in CRC cell lines with different KRAS/BRAF mutation status and BCL2L1 copy numbers; Supplementary Figure S6. Effect of ABT-263 and YM-155 in siBCL2L1-treated SW620 cells; Supplementary Figure S7. The quantification of MCL1 protein expression and levels of cleaved caspase 3 and cleaved PARP from Figure 2; Supplementary Figure S8. The quantification of MCL1 protein expression and levels of cleaved caspase 3 and cleaved PARP from Supplementary Figure S5; Supplementary Figure S9. Estimation of apoptosis induced by combination of ABT-263 and YM- 155; Supplementary Figure S10. The quantification of protein expression levels from Figure 3; Supplementary Figure S11. The quantification of protein expression levels from Figure 4; Supplementary Figure S12. The quantification of protein expression levels from Figure 5; Supplementary Figure S13. In vivo efficacy of ABT-263 and YM-155 treatment in CRC cell line xenograft models.

Supplementary Table S1. List of previously reported MCL1-modulating agents.

Supplementary Table S3. List of 737 genes significantly altered in combination treatment of ABT-263 and YM-155 compared to other samples (P < 0.05).

Functionally enriched Gene Ontology biological processes in combination treatment of ABT-263 and YM-155 compared to other samples (P

PurposeThe value of the genomic profiling by targeted gene-sequencing on radiation therapy response prediction was evaluated through integrated analysis including clinical information. Radiation response prediction model was constructed based on the analyzed findings.Materials and MethodsPatients who had the tumor sequenced using institutional cancer panel after informed consent and received radiotherapy for the measurable disease served as the target cohort. Patients with irradiated tumor locally controlled for more than 6 months after radiotherapy were defined as the durable local control (DLC) group, otherwise, non-durable local control (NDLC) group. Significant genomic factors and domain knowledge were used to develop the Bayesian Network model to predict radiotherapy response.ResultsAltogether, 88 patients were collected for analysis. Of those, 41 (43.6%) and 47 (54.4%) patients were classified as the NDLC and DLC group, respectively. Somatic mutations of NOTCH2 and BCL were enriched in the NDLC group, whereas, mutations of CHEK2, MSH2, and NOTCH1 were more frequently found in the DLC group. Altered DNA repair pathway was associated with better local failure–free survival (hazard ratio, 0.40; 95% confidence interval, 0.19 to 0.86; p=0.014). Smoking somatic signature was found more frequently in the DLC group. Area under the receiver operating characteristic curve of the Bayesian network model predicting probability of 6-month local control was 0.83.ConclusionDurable radiation response was associated with alterations of DNA repair pathway and smoking somatic signature. Bayesian network model could provide helpful insights for high precision radiotherapy. However, these findings should be verified in prospective cohort for further individualization.

Female pattern hair loss (FPHL), the most common cause of alopecia in adult women, is classified into two subtypes: early onset and late onset (or postmenopausal). Little is known about the clinical features and genetic characteristics of early onset female pattern hair loss (eFPHL).To investigate the clinical features and genetic characteristics of eFPHL.Patients with eFPHL and controls without eFPHL were prospectively recruited. The demographic and clinical features were collected. Single nucleotide polymorphisms (SNPs) located around the selected 30 candidate genes potentially associated with eFPHL were evaluated.eFPHL patients (n = 63) manifested a decreased hair shaft density and cross-sectional area of the hair shaft compared to the control group (n = 341). eFPHL is associated with androgen-related features, including scalp greasiness, folliculitis, hirsutism, and polycystic ovary syndrome. Scalp pain and itching have been reported more frequently in patients with eFPHL. Forty-nine SNPs located around PPARGC1A, ABCC4, CYP11B2, FSHB, and CYP19A1 were found to be significant for eFPHL, including two PPARGC1A-associated SNPs: rs186530605 and rs192713767 (p = 3.94 × 10This study provided clinical features and genetic variants for eFPHL, which could provide insight into the underlying pathologic etiology. Considering the limited number of patients, a large-scale study is required in the future.

Supplementary Figure from Comprehensive Analysis of Alternative Splicing in Gastric Cancer Identifies Epithelial–Mesenchymal Transition Subtypes Associated with Survival

Supplementary Data from Comprehensive Analysis of Alternative Splicing in Gastric Cancer Identifies Epithelial–Mesenchymal Transition Subtypes Associated with Survival

Supplementary Table from Comprehensive Analysis of Alternative Splicing in Gastric Cancer Identifies Epithelial–Mesenchymal Transition Subtypes Associated with Survival

Alternatively spliced RNA isoforms are a hallmark of tumors, but their nature, prevalence, and clinical implications in gastric cancer have not been comprehensively characterized. We systematically profiled the splicing landscape of 83 gastric tumors and matched normal mucosa, identifying and experimentally validating eight splicing events that can classify all gastric cancers into three subtypes: epithelial-splicing (EpiS), mesenchymal-splicing (MesS), and hybrid-splicing. These subtypes were associated with distinct molecular signatures and epithelial–mesenchymal transition markers. Subtype-specific splicing events were enriched in motifs for splicing factors RBM24 and ESRP1, which were upregulated in MesS and EpiS tumors, respectively. A simple classifier based only on RNA levels of RBM24 and ESRP1, which can be readily implemented in the clinic, was sufficient to distinguish gastric cancer subtypes and predict patient survival in multiple independent patient cohorts. Overall, this study provides insights into alternative splicing in gastric cancer and the potential clinical utility of splicing-based patient classification.Significance:This study presents a comprehensive analysis of alternative splicing in the context of patient classification, molecular mechanisms, and prognosis in gastric cancer.

Purpose:Genomic and transcriptomic alterations during metastasis are considered to affect clinical outcome of colorectal cancers, but detailed clinical implications of metastatic alterations are not fully uncovered. We aimed to investigate the effect of metastatic evolution on in vivo treatment outcome, and identify genomic and transcriptomic alterations associated with drug responsiveness.Experimental Design:We developed and analyzed patient-derived xenograft (PDX) models from 35 patients with colorectal cancer including 5 patients with multiple organ metastases (MOMs). We performed whole-exome, DNA methylation, and RNA sequencing for patient and PDX tumors. With samples from patients with MOMs, we conducted phylogenetic and subclonal analysis and in vivo drug efficacy test on the corresponding PDX models.Results:Phylogenetic analysis using mutation, expression, and DNA methylation data in patients with MOMs showed that mutational alterations were closely connected with transcriptomic and epigenomic changes during the tumor evolution. Subclonal analysis revealed that initial primary tumors with larger number of subclones exhibited more dynamic changes in subclonal architecture according to metastasis, and loco-regional and distant metastases occurred in a parallel or independent fashion. The PDX models from MOMs demonstrated therapeutic heterogeneity for targeted treatment, due to subclonal acquisition of additional mutations or transcriptomic activation of bypass signaling pathway during tumor evolution.Conclusions:This study demonstrated in vivo therapeutic heterogeneity of colorectal cancers using PDX models, and suggests that acquired subclonal alterations in mutations or gene expression profiles during tumor metastatic processes can be associated with the development of drug resistance and therapeutic heterogeneity of colorectal cancers.

Supplementary Materials and Methods, Supplementary Figures S1-S10 and Supplementary Tables S1-S6

Objective: We investigated genetic risk for cardiovascular disease (CVD) by type 2 diabetes (T2D) diagnosis age. Research Design and Methods: We compared incident CVD events by T2D diagnosis age using UK Biobank (N=12,321) and Seoul National University Hospital (SNUH) cohort (N=1,165). Genetic risk was quantified using polygenic risk score (PRS). Results: People with earlier T2D diagnosis had higher CVD risk. In UK Biobank, effect size of coronary artery disease (CAD) PRS on incident CAD was largest in people diagnosed of T2D at ages 30-39 (hazard ratio 2.25, 95% CI 1.56-3.26) and decreased for later-onset: 1.51 (1.30-1.75) at 40-49, 1.36 (1.24-1.50) at 50-59, and 1.30 (1.14-1.48) at 60-69 (Pinteraction=0.0031). Similar trend was observed in SNUH cohort. This increased genetic risk associated with earlier T2D diagnosis was largely mitigated by healthy lifestyle. Conclusion: People with earlier T2D diagnosis have higher CAD genetic risk, and this could be used to tailor lifestyle intervention.

We present an in-depth single cell atlas of in vitro multi-culture systems on human primary airway epithelium derived from normal and diseased lungs of 27 individual donors. Our large-scale single cell profiling identifies new cell states and differentiation trajectories of rare airway epithelial cell types in human distal lungs. By integrating single cell datasets of human lung tissues, we discover immune primed subsets enriched in lungs and organoids derived from patients with chronic respiratory disease. To demonstrate the full potential of our platform, we further illustrate transcriptomic responses to various respiratory virus infections in in vitro airway models. Our work constitutes a single-cell roadmap for the cellular and molecular characteristics of human primary lung cells in vitro and their relevance to human tissues in vivo.

Dysembryoplastic neuroepithelial tumor (DNET) is a low-grade brain tumor commonly associated with drug-resistant epilepsy. About half of DNETs are accompanied by tiny nodular lesions separated from the main mass. The existence of these satellite lesions (SLs) has shown a strong association with tumor recurrence, suggesting that they are true tumors. However, it is not known whether SLs represent multiple foci of progenitor tumor cell extension and migration or a multifocal development of the main DNET. This study was designed to elucidate the histopathology and pathogenesis of SLs in DNETs. Separate biopsies from the main masses and SLs with DNET were analyzed. We performed comparative lesion sequencing and phylogenetic analysis. FGFR1 K656E and K655I mutations or duplication of the tyrosine kinase domain was found in all 3 DNET patients and the main masses and their SLs shared the same FGFR1 alterations. The phylogenic analysis revealed that the SLs developed independently from their main masses. It is possible that the main mass and its SLs were separated at an early stage in oncogenesis with shared FGFR1 alterations, and then they further expanded in different places. SLs of DNET are true tumors sharing pathogenic mutations with the main masses. It is plausible that multifocal tumor development takes place in the dysplastic cortex containing cells with a pathogenic genetic alteration.

Ciliary movements within the human airway are essential for maintaining a clean lung environment. Motile cilia have a characteristic ciliary beat frequency (CBF). However, CBF measurement with current video microscopic techniques can be error-prone due to the use of the single-point Fourier transformation, which is often biased for ciliary measurements. Herein, we describe a new video microscopy technique that harnesses a metric of motion-contrast imaging and image correlation for CBF analysis. It can provide objective and selective CBF measurements for individual motile cilia and generate CBF maps for the imaged area. The measurement performance of our methodology was validated with in vitro human airway organoid models that simulated an actual human airway epithelium. The CBF determined for the region of interest (ROI) was equal to that obtained with manual counting. The signal redundancy problem of conventional methods was not observed. Moreover, the obtained CBF measurements were robust to optical focal shifts, and exhibited spatial heterogeneity and temperature dependence. This technique can be used to evaluate ciliary movement in respiratory tracts and determine whether it is non-synchronous or aperiodic in patients. Therefore, our observations suggest that the proposed method can be clinically adapted as a screening tool to diagnose ciliopathies.

Introduction: Although previous genome-wide association studies (GWASs) have identified genetic susceptibility loci for abdominal adiposity, GWASs on Asian samples remain scarce. Therefore, we performed a GWAS for abdominal adipose tissue depots in a Korean population. Methods: A total of 1,937 Korean men were included in the study. Areas of abdominal fat were quantified by computed tomography. We performed a GWAS analysis under an additive model, and a replication study was conducted on 480 additional Korean adult men. Results: In the discovery step, we identified a total of 10 single-nucleotide polymorphisms (SNPs) associated with adiposity indicators (p < 1 × 10−5). The top SNP, rs1028014, for visceral adipose tissue (VAT) was located in the ZMAT4 gene and remained significant after adjustment for body mass index (BMI). Three additional SNPs were also associated with VAT-adj-BMI and located within the SLC26A10, FAM155A, and COL4A1-COL4A2 genes, respectively. In addition, we identified a SNP (rs4668224) of the MYO3B gene for visceral-to-subcutaneous fat ratio. For subcutaneous adipose tissue and total adipose tissue, two (rs6585735 and rs363527) and three SNPs (rs1487892, rs9357565, and rs1985358) were found, respectively. Overall, eight SNPs were used in the replication study; however, none of the SNPs reached our level of significance for replication (p < 0.0063). Nevertheless, rs4773144 of COL4A1-COL4A2 for VAT-adj-BMI was the most interesting SNP identified in previous GWASs for coronary artery disease (based on the same risk allele “G”), along with functional effects. Conclusion: This study suggests for the first time that an SNP (rs4773144) of COL4A1-COL4A2 may contribute to the increase in VAT level, especially in adult Korean men.

Alternatively spliced RNA isoforms are a hallmark of tumors, but their nature, prevalence, and clinical implications in gastric cancer have not been comprehensively characterized. We systematically profiled the splicing landscape of 83 gastric tumors and matched normal mucosa, identifying and experimentally validating eight splicing events that can classify all gastric cancers into three subtypes: epithelial-splicing (EpiS), mesenchymal-splicing (MesS), and hybrid-splicing. These subtypes were associated with distinct molecular signatures and epithelial–mesenchymal transition markers. Subtype-specific splicing events were enriched in motifs for splicing factors RBM24 and ESRP1, which were upregulated in MesS and EpiS tumors, respectively. A simple classifier based only on RNA levels of RBM24 and ESRP1, which can be readily implemented in the clinic, was sufficient to distinguish gastric cancer subtypes and predict patient survival in multiple independent patient cohorts. Overall, this study provides insights into alternative splicing in gastric cancer and the potential clinical utility of splicing-based patient classification. Significance: This study presents a comprehensive analysis of alternative splicing in the context of patient classification, molecular mechanisms, and prognosis in gastric cancer.

The spatial tumor shape is determined by the complex interactions between tumor cells and their microenvironment. Here, we investigated the role of a newly identified breast cancer‐related gene, calsequestrin 2 (CASQ2), in tumor–microenvironment interactions during tumor growth and metastasis. We analyzed gene expression and three‐dimensional tumor shape data from the breast cancer dataset of The Cancer Genome Atlas (TCGA) and identified CASQ2 as a potential regulator of tumor–microenvironment interaction. In TCGA breast cancer cases containing information of three‐dimensional tumor shapes, CASQ2 mRNA showed the highest correlation with the spatial tumor shapes. Furthermore, we investigated the expression pattern of CASQ2 in human breast cancer tissues. CASQ2 was not detected in breast cancer cell lines in vitro but was induced in the xenograft tumors and human breast cancer tissues. To evaluate the role of CASQ2, we established CASQ2‐overexpressing breast cancer cell lines for in vitro and in vivo experiments. CASQ2 overexpression in breast cancer cells resulted in a more aggressive phenotype and altered epithelial–mesenchymal transition (EMT) markers in vitro. CASQ2 overexpression induced cancer‐associated fibroblast characteristics along with increased hypoxia‐inducible factor 1α (HIF1α) expression in stromal fibroblasts. CASQ2 overexpression accelerated tumorigenesis, induced collagen structure remodeling, and increased distant metastasis in vivo. CASQ2 conferred more metaplastic features to triple‐negative breast cancer cells. Our data suggest that CASQ2 is a key regulator of breast cancer tumorigenesis and metastasis by modulating diverse aspects of tumor–microenvironment interactions., CASQ2 is a potential regulator of the tumor–microenvironment interaction. CASQ2 was identified through correlation analysis of tumor three‐dimensional shapes. Overexpression of CASQ2 in breast cancer cells resulted in more aggressive phenotypes and dysregulation of intracellular signaling pathways, along with increased tumorigenesis and remodeling of collagen structures. In vivo, CASQ2 was linked to HIF‐1 signaling and increased metastatic colonization. Thus, CASQ2 conclusively conferred breast cancer cells with more metaplastic features.

The objective of this study was to evaluate in vitro antimicrobial and anti-biofilm activity of sodium long chain polyphosphate (SLCPP) and effect of dietary supplementation of SLCPP on growth performance, organ characteristics, blood metabolites, and intestinal microflora of broilers. Antimicrobial activities of SLCPP were observed against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica ser. Pullorum, Shigella sonnei, Klebsiella pneumonia, Pseudomonas aeruginosa in agar well diffusion assay. In addition, SLCPP demonstrated good anti-biofilm activity against K. pneumonia and P. aeruginosa. Furthermore, to investigate the dietary effect of SLCPP, a total of 480 1-day-old male Ross 308 broiler chicks were randomly allotted to three dietary treatment groups (4 replicates per group, 40 birds in each replicate): an antibiotic-free corn-soybean meal basal diet (NC); basal diet + enramycin 0.01% (PC); and basal diet + 0.1% SLCPP (SPP). The experiment lasted for 35 days. Results showed that birds fed with SLCPP had higher body weight (BW) and average daily gain (ADG), and lower feed conversion ratio (FCR) during the grower phase (days 7 to 21) (p < 0.05). Except for blood urea nitrogen, all other blood biochemical parameters remained unaffected by the dietary supplementation of SLCPP. Compared to the control group, lengths of the duodenum and ileum in the SPP group were significantly shorter (p < 0.05). Moreover, counts of lactic acid bacteria (LAB), total aerobes, and Streptococcus spp. in jejunum as well as LAB in cecum were increased in the SPP group than in the PC group (p < 0.05). These results suggest that dietary supplementation of SLCPP might promote the growth of broilers in their early growth phase.

We investigated the molecular mechanisms of paclitaxel resistance in TNBC using seven patient-derived xenograft (PDX) models and TNBC cell lines. Among the seven PDX models, four models showed resistance to paclitaxel. Dysregulation of JAK/STAT pathways and JAK2 copy number gains were observed in the four paclitaxel-resistant PDX tumors. In TNBC cell lines, silencing the JAK2 gene showed a significant but mild synergistic effect when combined with paclitaxel in vitro. However, JAK1/2 inhibitor treatment resulted in restoration of paclitaxel sensitivity in two out of four paclitaxel-resistant PDX models and JAK1/2 inhibitor alone significantly suppressed the tumor growth in one out of the two remaining PDX models. Transcriptome data derived from the murine microenvironmental cells revealed an enrichment of genes involved in the cell cycle processes among the four paclitaxel-resistant PDX tumors. Histologic examination of those PDX tumor tissues showed increased Ki67-positive fibroblasts in the tumor microenvironment. Among the four different cancer-associated fibroblast (CAF) subtypes, cycling CAF exhibiting features of active cell cycle was enriched in the paclitaxel-resistant PDX tumors. Additionally, fibroblasts treated with the conditioned media from the JAK2-silenced breast cancer cells showed downregulation of cell cycle-related genes. Our data suggest that the JAK2 gene may play a critical role in determining responses of TNBC to paclitaxel by modulating the intrinsic susceptibility of cancer cells against paclitaxel and also by eliciting functional transitions of CAF subtypes in the tumor microenvironment. KEY MESSAGES : We investigated the molecular mechanisms of paclitaxel resistance in TNBC. JAK2 signaling was associated with paclitaxel resistance in TNBC PDX models. Paclitaxel-resistant PDX tumors were enriched with microenvironment cCAF subpopulation. JAK2 regulated paclitaxel-resistant CAF phenotype transition.

Although FDG-PET is widely used in cancer, its role in gastric cancer (GC) is still controversial due to variable [18F]fluorodeoxyglucose ([18F]FDG) uptake. Here, we sought to develop a genetic signature to predict high FDG-avid GC to plan individualized PET and investigate the molecular landscape of GC and its association with glucose metabolic profiles noninvasively evaluated by [18F]FDG-PET. Based on a genetic signature, PETscore, representing [18F]FDG avidity, was developed by imaging data acquired from thirty patient-derived xenografts (PDX). The PETscore was validated by [18F]FDG-PET data and gene expression data of human GC. The PETscore was associated with genomic and transcriptomic profiles of GC using The Cancer Genome Atlas. Five genes, PLS1, PYY, HBQ1, SLC6A5, and NAT16, were identified for the predictive model for [18F]FDG uptake of GC. The PETscore was validated in independent PET data of human GC with qRT-PCR and RNA-sequencing. By applying PETscore on TCGA, a significant association between glucose uptake and tumor mutational burden as well as genomic alterations were identified. Our findings suggest that molecular characteristics are underlying the diverse metabolic profiles of GC. Diverse glucose metabolic profiles may apply to precise diagnostic and therapeutic approaches for GC.

BACKGROUND: Molecular characterization in pediatric papillary thyroid cancer (PTC), distinct from adult PTC, is important for developing molecularly targeted therapies for progressive radioiodine-refractory ((131)I-refractory) PTC. METHODS: PTC samples from 106 pediatric patients (age range: 4.3–19.8 years; n = 84 girls, n = 22 boys) who were admitted to SNUH (January 1983–March 2020) were available for genomic profiling. Previous transcriptomic data from 125 adult PTC samples were used for comparison. RESULTS: We identified genetic drivers in 80 tumors: 31 with fusion oncogenes (RET in 21 patients, ALK in 6 patients, and NTRK1/3 in 4 patients); 47 with point mutations (BRAF(V600E) in 41 patients, TERT(C228T) in 2 patients [1 of whom had a coexisting BRAF(V600E)], and DICER1 variants in 5 patients); and 2 with amplifications. Fusion oncogene PTCs, which are predominantly detected in younger patients, were at a more advanced stage and showed more recurrent or persistent disease compared with BRAF(V600E) PTCs, which are detected mostly in adolescents. Pediatric fusion PTCs (in patients

OBJECTIVE We evaluated the frequency, risk factors and the follow-up outcomes of thyroid nodules, and genetic alterations in thyroid cancer, in youth with childhood-onset Hashimoto thyroiditis (HT) residing in an iodine-sufficient country. DESIGN A retrospective cohort study. PATIENTS AND MEASUREMENTS A total of 213 patients (194 females, mean age 10.6 years at the time of HT diagnosis) were ultrasonographically evaluated. Thyroid nodules were categorized using the Korean Thyroid Imaging Reporting and Data System (K-TIRADS) and the American College of Radiology Thyroid Imaging Reporting and Data System (ACR-TI-RADS). RESULTS Thyroid nodules were detected in 40 (18.8%) patients over a median follow-up period of 3.4 years, usually after the onset of puberty. A family history of thyroid disease (hazard ratio 2.1, p = .031) was predictive of thyroid nodule detection. Papillary thyroid carcinoma (PTC) was diagnosed in 9 (4.2% of all and 22.5% of nodule-positive patients). The malignant nodules had a higher K-TIRADS or ACR-TI-RADS risk level compared with benign nodules (p

Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.

This study evaluates the in vitro probiotic characteristics of

The global incidence of early-onset colorectal cancer (EO-CRC) is rapidly rising. However, the reason for this rise in incidence as well as the genomic characteristics of EO-CRC remain largely unknown. We performed whole-exome sequencing in 47 cases of EO-CRC and targeted deep sequencing in 833 cases of CRC. Mutational profiles of EO-CRC were compared with previously published large-scale studies. EO-CRC and The Cancer Genome Atlas (TCGA) data were further investigated according to copy number profiles and mutation timing. We classified colorectal cancer into three subgroups: the hypermutated group consisted of mutations in POLE and mismatch repair genes; the whole-genome doubling group had early functional loss of TP53 that led to whole-genome doubling and focal oncogene amplification; the genome-stable group had mutations in APC and KRAS, similar to conventional colon cancer. Among non-hypermutated samples, whole-genome doubling was more prevalent in early-onset than in late-onset disease (54% vs 38%, Fisher’s exact P = 0.04). More than half of non-hypermutated EO-CRC cases involved early TP53 mutation and whole-genome doubling, which led to notable differences in mutation frequencies between age groups. Alternative carcinogenesis involving genomic instability via loss of TP53 may be related to the rise in EO-CRC., Colorectal cancer: tumor genetic classification could help guide treatment Researchers in South Korea have identified distinct types of genetic changes in colorectal cancer (CRC). Groups led by Tae Won Kim at the University of Ulsan, Seoul, and Jong-il Kim at Seoul National University looked for mutations in genes in tumors from 880 patients, including all active genes in 47 patients with early-onset colorectal cancer (EO-CRC). The cancers were classified into three subgroups. A “hypermutated” group had mutations in genes involved in DNA replication and repair. The entire genome was doubled in another group, which included the EO-CRC cases. In this group, TP53 gene function, which is known to be crucial in suppressing tumor formation, was lost. A third group had mutations in two other identified genes. Identifying which subgroups patients belong to may help determine the best treatment option.

Immune checkpoint inhibitor (ICI) has an emerging role in several types of cancer. However, the mechanisms of acquired resistance (AR) to ICI have not been elucidated yet. To identify these mechanisms, we analyzed the pre- and post-ICI paired tumor samples in patients with AR. Six patients with renal cell carcinoma, urothelial cell carcinoma, or head and neck cancer, who showed an initial response to ICI followed by progression and had available paired tissue samples, were retrospectively analyzed. Whole exome sequencing, RNA sequencing, and multiplex immunohistochemistry were performed on pre-treatment and resistant tumor samples. The median time to AR was 370 days (range, 210 to 739). Increased expression of alternative immune checkpoints including TIM3, LAG3, and PD-1 as well as increased CD8+ tumor-infiltrating lymphocytes were observed in post-treatment tumor than in pre-treatment tumor of a renal cell carcinoma patient. In contrast, CD8+ T cells and immunosuppressive markers were all decreased at AR in another patient with human papillomavirus-positive head and neck squamous cell carcinoma. This patient had an evident APOBEC-associated signature, and the tumor mutation burden increased at AR. Resistant tumor tissue of this patient harbored a missense mutation (E542K) in PIK3CA. No significant aberrations of antigen-presenting machinery or IFN-γ pathway were detected in any patient. Our study findings suggest that the observed increase in immunosuppressive markers after ICI might contribute to AR. Moreover, APOBEC-mediated PIK3CA mutagenesis might be an AR mechanism. To validate these mechanisms of AR, further studies with enough sample size are required.

Vertical sleeve gastrectomy (VSG) is the most commonly performed bariatric/metabolic surgery, exhibiting a high rate of diabetes remission in humans. To elucidate the molecular mechanisms of VSG, we performed transcriptomic analysis of the liver, fat, and muscle in VSG mice. C57BL/6 mice fed a high-fat diet were randomly assigned to sham or VSG surgery. The sham-operated mice were fed ad libitum (sham group) or pair-fed (sham-PF group) matching their food intake to the VSG-operated mice. Comparative transcriptomic analysis of the liver, fat, and muscle using RNA sequencing was performed. VSG reduced body weight and improved glucose tolerance compared to the sham group, but not more than the sham-PF group. Improvement in fatty liver and adipose tissue inflammation was comparable between VSG and sham-PF. However, global gene expression profiles showed distinctive changes in the liver, fat, and muscle of the VSG group compared to both the sham or sham-PF groups. The liver showed the most prominent gene expression changes. Immune response-related pathways were commonly upregulated in the three organs of the VSG group compared to the sham or sham-PF. VSG induces organ-specific gene expression changes in the liver, fat, and muscle, which may play critical roles in metabolic improvements after VSG.

The tumor microenvironment (TME), which the cancer is known to remodel, can interact with cancer in various ways, such as promoting or hindering metastasis. The challenge with comprehending TME is that each patient's individual variance makes it difficult to carry out a consistent analysis. We used the PDX model to analyze the alterations entirely brought about by cancer cells of each patient in the same environment exclusively. In 121 breast cancer patients, we created a PDX model and performed WES and RNA-seq. We repeatedly performed metastatic induction tests on the models of 26 TNBC patients among the PDX models we generated. The replicated experiments confirmed that cancer cells with frequent metastasis usually spread well, while cancer cells with seldom metastases rarely metastasize. For each of the five samples that had the greatest and worst rates of metastasis, the pre-metastatic xenograft tumor was analyzed using scRNA-seq. Metastasis could not be predicted by only cancer cells because there was no difference between the two groups. Still, fibroblasts and macrophages had distinct gene markers before metastasis which can predict metastasis. To explore TME that develops pre-metastatic niches (PMN) in organs that would be metastasis targets, we developed a metastatic model using 4T1 cell lines. Daily lung macrophage RNA-seq, lung WES, and lung scRNA-seq procedures were carried out for four weeks to examine the precise timing of metastasis and the daily changes in the transcriptome. Surprisingly, a few days before the presence of any cancer cells, the gene expression of lung macrophages had already undergone significant modification. Additionally, only injections of tumor-conditioned media that mimicked metastasis could activate PMN in lung macrophages. Citation Format: Woochan Lee, Hyewon Chung, Woohang Heo, Hyeong-Gon Moon, Seung Hyeok Seok, Jong-Il Kim. Identify features of fibroblast and macrophage which regulate metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B019.