Patient-derived tumor organoids predict responses to irinotecan-based neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer.

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 < .0001; validation cohort, 81.8% vs 18.8%, P = .002). Moreover, the irinotecan-sensitive group had higher rates of 3-year disease-free survival (DFS: 71.6% vs 55.5%, P = .034) and distant metastasis-free survival (DMFS, 77.9% vs 57.2%, P = .015) than the irinotecan-insensitive group. 5-FU and irradiation sensitivities failed to predict 3-year DFS (5-FU: 65.4% vs 61.9%, P = .643; irradiation: 84.8% vs 57.8%; P = .072). Performances of irinotecan OSR to predict CR or pCR were good in the training cohort (CR: AUC = 0.828; 95% CI = 0.723-0.932; pCR: AUC = 0.864; 95% CI = 0.759-0.961). The validation showed robust predictive ability (CR: AUC = 0.796, 95% CI = 0.5974-0.9952; pCR: AUC = 0.917, 95% CI = 0.7921-1.0000). Irinotecan sensitivity in PDTOs was a predictive and prognostic factor in LARC.

Cell-Type-Specific Gene Inactivation and In Situ Restoration via Recombinase-Based Flipping of Targeted Genomic Region.

Conditional gene inactivation and restoration are powerful tools for studying gene functions in the nervous system and for modeling neuropsychiatric diseases. The combination of the two is necessary to interrogate specific cell types within defined developmental stages. However, very few methods and animal models have been developed for such purpose. Here we present a versatile method for conditional gene inactivation and in situ restoration through reversibly inverting a critical part of its endogenous genomic sequence by Cre- and Flp-mediated recombinations. Using this method, we generated a mouse model to manipulate Mecp2, an X-linked dosage-sensitive gene whose mutations cause Rett syndrome. Combined with multiple Cre- and Flp-expressing drivers and viral tools, we achieved efficient and reliable Mecp2 inactivation and restoration in the germline and several neuronal cell types, and demonstrated phenotypic reversal and prevention on cellular and behavioral levels in male mice. This study not only provides valuable tools and critical insights for Mecp2 and Rett syndrome, but also offers a generally applicable strategy to decipher other neurologic disorders.SIGNIFICANCE STATEMENT Studying neurodevelopment and modeling neurologic disorders rely on genetic tools, such as conditional gene regulation. We developed a new method to combine conditional gene inactivation and restoration on a single allele without disturbing endogenous expression pattern or dosage. We applied it to manipulate Mecp2, a gene residing on X chromosome whose malfunction leads to neurologic disease, including Rett syndrome. Our results demonstrated the efficiency, specificity, and versatility of this new method, provided valuable tools and critical insights for Mecp2 function and Rett syndrome research, and offered a generally applicable strategy to investigate other genes and genetic disorders.

Clinical research on ultrasonically guided intrahepatic injections of HAS in interventional treatment of liver carcinomas.

PURPOSE: To study the clinical value of intrahepatic injections of Highly Agglutinative Staphylococcin (HAS) in the interventional treatment of liver carcinomas. METHODS: Under ultrasonic guidance, intrahepatic injections of HAS were administered in 22 cases of pathologically diagnosed liver carcinomas, 3 days, 7 days, 30 days, 3 months, 6 months, 9 months, and 12 months after microwave coagulation therapy. The dose of each injection was 2000U. RESULTS: Immunohistochemical staining of the sample from the tumor site after HAS administrations demonstrated a significant increase in the number of antitumor immune cells compared with that before the injections (p<0.01) and an improvement in local immune status. One-year survival rate and recurrence rate, which were determined by Kaplan- Meier method, were 93.8% and 81.9% respectively. CONCLUSIONS: As a new route of administration, intrahepatic injections of HAS are a safe and effective procedure and deserves further clinical research and discussion.

Low-dose radiotherapy synergizes with iRGD-antiCD3-modified T cells by facilitating T cell infiltration.

BACKGROUND AND PURPOSE: Poor penetration of transferred T cells represents a critical factor impeding the development of adoptive cell therapy in solid tumors. We demonstrated that iRGD-antiCD3 modification promoted both T cell infiltration and activation in our previous work. Interest in low-dose radiotherapy has recently been renewed due to its immuno-stimulatory effects including T cell recruitment. This study aims to explore the synergistic effects between low-dose radiotherapy and iRGD-antiCD3-modified T cells. MATERIALS AND METHODS: Flow cytometry was performed to assess the expression of iRGD receptors and chemokines. T cell infiltration was evaluated by immunohistofluorescence and in vivo real-time fluorescence imaging and antitumor effects were investigated by in vivo bioluminescence imaging in the gastric cancer peritoneal metastasis mouse model. RESULTS: We found that 2 Gy irradiation upregulated the expression of all three iRGD receptors and T-cell chemokines. The addition of 2 Gy low-dose irradiation boosted the accumulation and penetration of iRGD-antiCD3-modified T cells in peritoneal tumor nodules. Combining 2 Gy low-dose irradiation with iRGD-antiCD3-modified T cells significantly inhibited tumor growth and prolonged survival in the peritoneal metastasis mouse model with a favorable safety profile. CONCLUSION: Altogether, we demonstrated that low-dose radiotherapy could improve the antitumor potency of iRGD-antiCD3-modified T cells by promoting T cell infiltration, providing a rationale for exploring low-dose radiotherapy in combination of other adoptive T cell therapies in solid tumors.

Newly developed 3D in vitro models to study tumor-immune interaction.

Immunotherapy as a rapidly developing therapeutic approach has revolutionized cancer treatment and revitalized the field of tumor immunology research. 3D in vitro models are emerging as powerful tools considering their feature to maintain tumor cells in a near-native state and have been widely applied in oncology research. The novel 3D culture methods including the co-culture of organoids and immune cells, ALI culture, 3D-microfluidic culture and 3D-bioprinting offer new approaches for tumor immunology study and can be applied in many fields such as personalized treatment, immunotherapy optimizing and adoptive cell therapy. In this review, we introduce commonly used 3D in vitro models and summarize their applications in different aspects of tumor immunology research. We also provide a preliminary analysis of the current shortcomings of these models and the outlook of future development.

Quality traits analysis of 153 wheat lines derived from CIMMYT and China.

In order to understand the difference of quality for Chinese and CIMMYT wheat varieties (lines), we selected 153 wheat germplasm from both China and CIMMYT to explore the contribution relationship of different allelic variation combinations to wheat quality through genotyping and phenotyping, including grain hardness, polyphenol oxidase (PPO) activity, lipoxygenase (LOX) activity, yellow pigment (YP) content and protein content. In terms of flour milling quality, Chinese wheat varieties were mainly carrying Pina-D1a/Pinb-D1b, accounting for 32.0% of the total tested varieties, while the CIMMYT wheat lines were mainly carrying Pina-D1b/Pinb-D1a with 45.8% of the total collection. The distribution frequencies of subunit 1/2* and 5 + 10 were 47.0% and 42.5%, respectively, in CIMMYT varieties, however they were only 31.4% and 13.7% respectively of the Chinese wheat tested varieties. In addition, the proportion of phytoene synthase (PSY) allele, PPO allele and LOX active allele were roughly the same between Chinese and CIMMYT varieties. Based on the present study, we found that Pina gene had a greater impact on grain hardness value than Pinb gene; The influence of PPO-A1 gene on polyphenol oxidase activity was more significant than PPO-D1 gene. The high protein content of varieties mostly containing hardness genes and 1/2*/5 + 10 subunit combinations. Based on the present study, we found that the quality gene distribution of Chinese and CIMMYT varieties was quite different, for instance, the high-quality HMW-GS subunits of Chinese varieties were lower than CIMMYT lines. It will be much useful for Chinese wheat breeders to develop good quality wheat variety by crossing with 3 good strong gluten CIMMYT wheat lines by molecular marker-assisted selection.

Application of imaging techniques in pancreaticobiliary maljunction.

Imaging techniques are useful tools in the diagnosis and treatment of pancreaticobiliary maljunction (PBM). PBM is a precancerous lesion often relative to the disease of the pancreas and biliary tract, for example, cholecystolithiasis, protein plugs, and pancreatitis. For patients with PBM, early diagnosis and timely treatment are highly important, which is largely dependent on imaging techniques. The continuous development of imaging techniques, including endoscopic retrograde cholangiopancreatography, magnetic resonance cholangiopancreatography, computed tomography, ultrasound, and intraoperative cholangiography, has provided appropriate diagnostic and therapeutic tools for PBM. Imaging techniques, including non-invasive and invasive, have distinct advantages and disadvantages. The purpose of this paper is to review the application of various imaging techniques in the diagnosis and treatment of PBM.

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

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

Genome-wide Association Study for Starch Pasting Properties in Chinese Spring Wheat.

In order to understand the genetic basis of starch pasting viscosity characteristics of Chinese spring wheat, we assessed the genetic variation of RVA parameters determined by the Rapid Visco Analyser in a panel of 192 Chinese spring wheat accessions grown in Er'shi, Shihezi and Zhaosu during 2012 and 2013 cropping seasons. A genome-wide association study with 47,362 single nucleotide polymorphism (SNP) markers was conducted to detect marker-trait associations using mixed linear model. Phenotypic variations of RVA parameters ranged from 1.6 to 30.7% and broad-sense heritabilities ranged from 0.62 to 0.91. Forty-one SNP markers at 25 loci were significantly associated with seven RVA traits in at least two environments; among these, 20 SNPs were located in coding sequences (CDS) of 18 annotation genes, which can lead to discovering novel genes underpinning starch gelatinization in spring wheat. Haplotype analysis revealed one block for breakdown (BD) on chromosome 3B and two blocks for pasting temperature (T) on chromosome 7B. Cultivars with superior haplotypes at these loci showed better starch pasting viscosity than the average of all cultivars surveyed. The identified loci and associated markers provide valuable sources for future functional characterization and genetic improvement of starch quality in wheat.

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

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

Sinistral portal hypertension associated with pancreatic pseudocysts - ultrasonography findings: A case report.

BACKGROUND: Sinistral portal hypertension associated with pancreatic pseudocysts is rare, often caused by extrinsic compression of splenic vein, the follow-up examinations by ultrasonography for early diagnosis are quietly necessary since haematemesis, a life-threatening condition. Few studies have reported the ultrasonography findings of sinistral portal hypertension. CASE SUMMARY: A 52-year-old man presented with acute abdominal pain after drinking, steatorrhea, weight loss and accidentally melena in the past 2 mo. He underwent ultrasound-guided fine needle aspiration in other hospital and diagnosed with pancreatic pseudocysts. Ultrasonography imaging, in our department, appeared as cystic heterogeneous hypoechoic area with the size of 4.7 cm × 3.8 cm that located posterior to the body and tail of pancreas, adjacent to splenic vein associated with thrombosis resulted from compression. Spleen incrassated to approximately 7.3 cm, but no dilation of main portal vein was presented. Color Doppler Flow Imaging demonstrated the formation of splenic venous collateral, nevertheless no significantly flow signals was observed in splenic vein. Pulsed Doppler revealed that the peak velocity of splenic venous collateral was 18.4 cm/s with continuous waveform. Laparotomy confirmed sinistral portal hypertension associated with pancreatic pseudocysts, subsequently distal pancreatectomy combined with splenectomy and partial gastrectomy was performed. CONCLUSION: It's important clinically to know the ultrasound appearance of sinistral portal hypertension associated with pancreatic pseudocysts for sonographer and physician.

A novel allele of L-galactono-1,4-lactone dehydrogenase is associated with enhanced drought tolerance through affecting stomatal aperture in common wheat.

In higher plants, L-galactono-1,4-lactone dehydrogenase (GLDH) plays important roles in ascorbic acid (AsA) biosynthesis and assembly of respiration complex I. Here we report three homoeologous genes (TaGLDH-A1, -B1 and -D1) encoding common wheat GLDH isozymes and a unique allelic variant (TaGLDH-A1b) associated with enhanced drought tolerance. TaGLDH-A1, -B1 and -D1 were located on chromosomes 5A, 5B and 5D, respectively, and their transcripts were found in multiple organs. The three homoeologs each conferred increased GLDH activity when ectopically expressed in tobacco. Decreasing TaGLDH expression in wheat significantly reduced GLDH activity and AsA content. TaGLDH-A1b differed from wild type allele TaGLDH-A1a by an in-frame deletion of three nucleotides. TaGLDH-A1b was biochemically less active than TaGLDH-A1a, and the total GLDH activity levels were generally lower in the cultivars carrying TaGLDH-A1b relative to those with TaGLDH-A1a. Interestingly, TaGLDH-A1b cultivars showed stronger water deficiency tolerance than TaGLDH-A1a cultivars, and TaGLDH-A1b co-segregated with decreased leaf water loss in a F2 population. Finally, TaGLDH-A1b cultivars generally exhibited smaller leaf stomatal aperture than TaGLDH-A1a varieties in control or water deficiency environments. Our work provides new information on GLDH genes and function in higher plants. TaGLDH-A1b is likely useful for further studying and improving wheat tolerance to drought stress.

Robotic surgery twice performed in the treatment of hilar cholangiocarcinoma with deep jaundice: delayed right hemihepatectomy following the right-hepatic vascular control.

OBJECTIVE: To explore and find a new method to treat hilar cholangiocarcinoma with deep jaundice assisted by Da Vinci robot. METHODS: A hilar cholangiocarcinoma patient of type Bismuch-Corlette IIIa was found with deep jaundice (total bilirubin: 635 µmol/L). On the first admission, we performed Da Vinci robotic surgery including drainage of left hepatic duct, dissection of right hepatic vessels (right portal vein and right hepatic artery), and placement of right-hepatic vascular control device. Three weeks later on the second admission when the jaundice disappeared we occluded right-hepatic vascular discontinuously for 6 days and then sustained later. On the third admission after 3 weeks of right-hepatic vascular control, the right hemihepatectomy was performed by Da Vinci robot for the second time. RESULTS: The future liver remnant after the right-hepatic vascular control increased from 35% to 47%. The volume of left lobe increased by 368 mL. When the total bilirubin and liver function were all normal, right hemihepatectomy was performed by Da Vinci robot 10 weeks after the first operation. The removal of atrophic right hepatic lobe with tumor in bile duct was found with no pathologic cancer remaining in the margin. The patient was followed up at our outpatient clinic every 3 months and no tumor recurrence occurs by now (1 y). CONCLUSIONS: Under the Da Vinci robotic surgical system, a programmed treatment can be achieved: first, the hepatic vessels were controlled gradually together with biliary drainage, which results in liver's partial atrophy and compensatory hypertrophy in the other part. Then a radical hepatectomy could be achieved. Such programmed hepatectomy provides a new treatment for patients of hilar cholangiocarcinoma with deep jaundice who have the possibility of radical heptolobectomy.