Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank’s Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose–response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.</jats:p

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank’s Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose–response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation

A 107-gene Nanostring assay effectively characterizes complex multiomic gastric cancer molecular classification in a translational patient-derived organoid model.

4049 Background: Multi-omics profiling of gastric cancer (GC) has produced numerous molecular classification systems. However, widespread clinical implementation and testing of molecular subtypes are currently limited. Here, we develop, validate and implement a custom Nanostring assay capable of allocating GC molecular subtypes to clinical specimens in a translational patient-derived organoid model. Methods: Using publicly available whole-transcriptome data, machine learning models were developed to predict GC molecular subtypes from 376 Cancer Genome Atlas (TCGA) and 1797 Tumour Microenvironment Score (TME) patients. Models were generated using feature selection with 10-fold nested cross-validation. GC biopsies from 10 local patients were preserved in paraffin (tumour) and established as an organoid culture (organoid). Gene expression was measured using Nanostring. The allocation of molecular subtypes was internally and externally validated using gold-standard reference features in public databases comprising 2202 GC patients and 10 tumour-organoid pairs, respectively. We evaluated the concordance of tumour-organoid molecular subtypes and explored the correlation between subtype scores and in-vitro chemotherapy response. Results: Classification models for TCGA (57 genes) and TME (50 genes) predicted subtypes with an accuracy ± standard deviation of 89.46% ± 0.04 and 89.33% ± 0.02, respectively. Subtype assignment of microsatellite instability (MSI) in reference to capillary electrophoresis was found to have 99.3% [95% CI 97.4-99.9, n = 277] internal and 100% [95% CI 83.2-100, n = 20] external accuracy. In reference to Epstein-Barr Virus (EBV) in-situ hybridization, EBV type internal and external accuracy was 98.7% [95% CI 97.4-99.5, n = 552] and 100% [95% CI 83.2-100, n = 20], respectively. TCGA Genomically Stable (GS) scores followed a previously characterized enrichment of diffuse-type histology compared to intestinal-type in internal and external cohorts (Dunn’s Test, p &lt; 0.0001 and p &lt; 0.05, n = 1471 and n = 15, respectively). Statistically similar subtype scores (Paired Wilcoxon, p &gt; 0.05) were found for tumour-organoid pairs. Discordance occurred in three tumour-organoid pairs. In-vitro Drug Sensitivity Score was not statistically efficacious in any molecular subtype, but Pearson correlation identified increasing efficacy with increasing EBV and MSI scores. Conclusions: Patient-derived organoids generally recapitulate the molecular subtype of parent tumours; however, in specific cases, subtype discordance occurs. Although additional external validation is required, our 107 gene assay effectively captures multi-omics classification systems in GC and allows future inquiry into the prognostic and therapeutic implications of these molecular subtypes. </jats:p

The sympathetic nervous system regulates the release of anti-inflammatory peptides from salivary glands (93.18)

Abstract Chronic and acute stress have profound effects on inflammation. In rats, allergic inflammation is regulated by the sympathetic nervous system acting on salivary glands. Human asthma is frequently accompanied by salivary gland inflammation. Salivary gland dysfunction in stressed individuals could enhance asthma severity. Salivary gland prohormone SMR1 (submandibular rat-1) is cleaved into two peptides that are anti-inflammatory in rats, mice, dogs, sheep, cats, and human cells in pulmonary inflammation, food allergy, septic shock, pancreatitis, and spinal cord injury. We hypothesized that modulation of the autonomic nervous system would change the expression, processing, and secretion of SMR1 and its peptides. Rats were injected with saline, isoproterenol, or pilocarpine, or the superior cervical ganglion was excised. Saliva, blood, and tissues were collected and analyzed for SMR1. Adrenergic stimulation caused the majority of SMR1 into be secreted into saliva in 60 min. Removal of the superior cervical ganglion that innervates the salivary glands changed SMR1 protein levels in the salivary glands. SMR1 secretion into saliva in response to acute stress may provide a large pool of SMR1-derived peptide products that mediate anti-inflammatory responses locally and systemically. This research is funded by AllerGen NCE.</jats:p