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The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. We used a primary tissue culture system to generate a murine p53 null gastric tissue model containing both epithelium and mesenchymal stroma. To characterize the microenvironment and niche signaling, we used single cell RNA sequencing (scRNA-Seq) to determine the transcriptomes of 4,391 individual cells. Based on specific markers, we identified epithelial cells, fibroblasts and macrophages in initial tissue explants during organoid formation. The majority of macrophages were polarized towards wound healing and tumor promotion M2-type. During the course of time, the organoids maintained both epithelial and fibroblast lineages with the features of immature mouse gastric stomach. We detected a subset of cells in both lineages expressing Lgr5, one of the stem cell markers. We examined the lineage-specific Wnt signaling activation, and identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that this primary tissue culture system enables one to study gastric tissue niche signaling and immune response in vitro.

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type–bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring.

The stomach is composed of multiple cell types that include epithelial and surrounding stromal components. The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis as well as tumor development. However, little is know about the mediators of intercellular communication among these different cell types in the stomach. To recapitulate features of the gastric cellular microenvironment and intercellular signaling in vitro, we applied a system to maintain primary gastric tissue in an air-liquid interface. Referred to as organoids, this method of primary tissue culture replicates the critical microenvironment of different cellular lineages such as epithelium and endogenous mesenchymal stroma. We analyzed the transcriptomes of over 5,000 individual cells from this primary gastric tissue culture. Based on gene expression of cell type specific markers, we identified epithelial cells, fibroblasts and macrophages that makeup the gastric tissue at the early stage of organoid formation. Importantly, we found that macrophages in the tissue explant microenvironment were polarized towards wound healing and tumor promotion. During the course of time, the gastric organoids maintained both epithelial and fibroblast lineages. We detected a subset of cells in both lineages expressing stem cell marker Lgr5, the lineage-specific Wnt signaling activation, and identified that Rspo3, a Wnt agonist, was specifically expressed in the mesenchymal fibroblast lineage. Wnt4 was significantly enriched in the epithelial cells. Thus, these genomic results implicated potential growth factors involved in cell lineage crosstalk in the local microenvironment. These results suggested that the fibroblast cells in stroma provide an endogenous source of the R-spondin to activate Wnt signaling in tumor cells. Our results implicate RSPO3 as a part of the tumor-promoting microenvironment and a potential therapeutic target.

Food consumption and preferences may be shaped by exposure to stressful environments during sensitive periods in development, and even small changes in consumption can have important effects on long term health. Adolescence is increasingly recognized as a sensitive period, in which adverse experiences can alter development, but the specific programming effects that may occur during adolescence remain incompletely understood. The current study seeks to explore the effects of stress during late adolescence on consumption of a palatable, high-fat, high-sugar food in adulthood—under basal conditions, as well following acute stress. Male Long-Evans rats were exposed to a regimen of variable stress for seven days in late adolescence (PND 45–51). During the stress regimen, stressed animals gained significantly less weight than control animals, but weight in adulthood was unaffected by adolescent stress. Palatable food consumption differed between experimental groups, and the direction of effect depended on context; stressed rats ate significantly more palatable food than controls upon first exposure, but ate less following an acute stressor. Leptin levels and exploratory behaviors did not differ between stressed and non-stressed groups, suggesting that other factors regulate preference for a palatable food. Altered food consumption following adolescent stress suggests that rats remain sensitive to stress during late adolescence, and that adult feeding behavior may be affected by previous adverse experiences. Such programming effects highlight adolescence as a period of plasticity, with the potential to shape long term food consumption patterns and preferences.

Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients. Citation Format: Christina M. Wood-Bouwens, Christine Handy, Billy Lau, Hanlee Ji. Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2017-2715

Follicular lymphoma (FL) is a generally incurable B-cell malignancy which has the potential to transform into highly aggressive lymphomas. Genomic studies indicate it is often a small subpopulation rather than the dominant population in the FL that gives rise to the more aggressive subtype.To resolve the underlying transcriptional networks of follicular B-cell lymphomas at single molecule and cell resolution, we leveraged droplet-based barcoding technology for highly parallel single cell RNA-Seq. We analyzed the transcriptomes from tens of thousands of cells derived from five primary FL tumors (average > 5,000 cells/sample). Simultaneously, we conducted multi-dimensional flow cell sorting to validate our characterizing of cellular lineages and critical expressed proteins. For each tumor, we identified multiple cellular subpopulations, matching known hematopoietic lineages. Despite some common features, such as MYC and BCLoverexpression, distinct transcriptional patterns and regulatory programs were evident among the different tumors. Within each tumor, subpopulations of malignant cell transcripts were compared to matched normal B-cells (Figure 1). Transcriptome analysis of >13,500 B-cells across 5 samples reveals sample-specific and joint clusters of B-cells. Each dot represents the t-SNE projection of a single cell's transcriptome profile. Single cells are colored according to sample origin (control PBMC samples 1 & 2; follicular lymphoma samples 1, 2 & 3). Normal B-cells within follicular lymphoma samples cluster together with PBMC derived B-cells. Malignant B-cells cluster according to sample origin, except for the cycling tumor cells (upper left cell cluster). Malignant B-cells were characterized by expression of restricted immunoglobulin light chain type (either kappa or lambda), BCL2 expression, and CD20 expression. We show evidence for the coexistence of small, malignant subpopulations alongside the dominant FL population in the majority of tumors. These smaller subpopulations harbored several transcriptional changes that were absent from the dominant population, including upregulation of MHC class II expression, downregulation of β-2-microglobulin, and exclusive expression of chemokines (CCL22 and CCL17), that would have resulted in alteration in antigen presentation and of the T cell immune system. In addition, we characterize the transcriptomes of the infiltrating immune cell populations that are characteristic of this disease. Our findings provide an unprecedented resolution of distinct immune lineages as seen by transcriptionally characterized cellular diversity. Figure 1 Assignment of 13.5K B-cells to normal and malignant phenotypes. Figure 1. Assignment of 13.5K B-cells to normal and malignant phenotypes. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.