Next-generation tissue microarray (ngTMA) increases the quality of biomarker studies: an example using CD3, CD8, and CD45RO in the tumor microenvironment of six different solid tumor types

Background Tissue microarray (TMA) technology revolutionized the investigation of potential biomarkers from paraffin-embedded tissues. However, conventional TMA construction is laborious, time-consuming and imprecise. Next-generation tissue microarrays (ngTMA) combine histological expertise with digital pathology and automated tissue microarraying. The aim of this study was to test the feasibility of ngTMA for the investigation of biomarkers within the tumor microenvironment (tumor center and invasion front) of six tumor types, using CD3, CD8 and CD45RO as an example. Methods Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. The most representative H&E slide was scanned and uploaded onto a digital slide management platform. Slides were viewed and seven TMA annotations of 1 mm in diameter were placed directly onto the digital slide. Different colors were used to identify the exact regions in normal tissue (n = 1), tumor center (n = 2), tumor front (n = 2), and tumor microenvironment at invasion front (n = 2) for subsequent punching. Donor blocks were loaded into an automated tissue microarrayer. Images of the donor block were superimposed with annotated digital slides. Exact annotated regions were punched out of each donor block and transferred into a TMA block. 420 tissue cores created two ngTMA blocks. H&E staining and immunohistochemistry for CD3, CD8 and CD45RO were performed. Results All 60 slides were scanned automatically (total time < 10 hours), uploaded and viewed. Annotation time was 1 hour. The 60 donor blocks were loaded into the tissue microarrayer, simultaneously. Alignment of donor block images and digital slides was possible in less than 2 minutes/case. Automated punching of tissue cores and transfer took 12 seconds/core. Total ngTMA construction time was 1.4 hours. Stains for H&E and CD3, CD8 and CD45RO highlighted the precision with which ngTMA could capture regions of tumor-stroma interaction of each cancer and the T-lymphocytic immune reaction within the tumor microenvironment. Conclusion Based on a manual selection criteria, ngTMA is able to precisely capture histological zones or cell types of interest in a precise and accurate way, aiding the pathological study of the tumor microenvironment. This approach would be advantageous for visualizing proteins, DNA, mRNA and microRNAs in specific cell types using in situ hybridization techniques.

Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors

Loss of p53 is considered to allow progression of colorectal tumors from the adenoma to the carcinoma stage. Using mice with an intestinal epithelial cell (IEC)-specific p53 deletion, we demonstrate that loss of p53 alone is insufficient to initiate intestinal tumorigenesis but markedly enhances carcinogen-induced tumor incidence and leads to invasive cancer and lymph node metastasis. Whereas p53 controls DNA damage and IEC survival during the initiation stage, loss of p53 during tumor progression is associated with increased intestinal permeability, causing formation of an NF-κB-dependent inflammatory microenvironment and the induction of epithelial-mesenchymal transition. Thus, we propose a p53-controlled tumor-suppressive function that is independent of its well-established role in cell-cycle regulation, apoptosis, and senescence.

miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation

Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

Regression of the inflammatory microenvironment of the peritoneal cavity in women with endometriosis by GnRHa treatment.

OBJECTIVE To investigate the effect of gonadotropin-releasing hormone analogues (GnRHa) on the peritoneal fluid microenvironment in women with endometriosis. STUDY DESIGN Peritoneal fluid was collected from 85 women with severe endometriosis (rAFS stage III and IV) during laparoscopic surgery during the proliferative phase. Prior to surgery clinical data were collected. The concentrations of specific markers for endometriosis in the peritoneal fluid were determined using an ELISA and a comparison between peritoneal fluid markers in women using GnRHa and no hormonal treatment was performed using a non-parametric Mann-Whitney U test. RESULTS The study included peritoneal fluid from 39 patients who had been administered GnRHa (Zoladex(®)) in the three months prior to surgery and 46 from women with no hormonal treatment in this period. Concentrations of IL-8, PAPP-A, glycodelin-A and midkine were significantly reduced in the GnRHa treatment group compared to women receiving no hormonal treatment. RANTES, MCP-1, ENA-78, TNF-α, OPG, IP-10 and defensin showed no significant change between the two groups. CONCLUSIONS GnRHa mediate a significant regression in the inflammatory nature of the peritoneal microenvironment in women with endometriosis.

Factors controlling decomposition rates of fine root litter in temperate forests and grasslands

Background and aims Fine root decomposition contributes significantly to element cycling in terrestrial ecosystems. However, studies on root decomposition rates and on the factors that potentially influence them are fewer than those on leaf litter decomposition. To study the effects of region and land use intensity on fine root decomposition, we established a large scale study in three German regions with different climate regimes and soil properties. Methods In 150 forest and 150 grassland sites we deployed litterbags (100 μm mesh size) with standardized litter consisting of fine roots from European beech in forests and from a lowland mesophilous hay meadow in grasslands. In the central study region, we compared decomposition rates of this standardized litter with root litter collected on-site to separate the effect of litter quality from environmental factors. Results Standardized herbaceous roots in grassland soils decomposed on average significantly faster (24 ± 6 % mass loss after 12 months, mean ± SD) than beech roots in forest soils (12 ± 4 %; p < 0.001). Fine root decomposition varied among the three study regions. Land use intensity, in particular N addition, decreased fine root decomposition in grasslands. The initial lignin:N ratio explained 15 % of the variance in grasslands and 11 % in forests. Soil moisture, soil temperature, and C:N ratios of soils together explained 34 % of the variance of the fine root mass loss in grasslands, and 24 % in forests. Conclusions Grasslands, which have higher fine root biomass and root turnover compared to forests, also have higher rates of root decomposition. Our results further show that at the regional scale fine root decomposition is influenced by environmental variables such as soil moisture, soil temperature and soil nutrient content. Additional variation is explained by root litter quality.

Heuristic decomposition and LP-based scheduling in make-and-pack production

In this paper, we are concerned about the short-term scheduling of industrial make-and-pack production processes. The planning problem consists in minimizing the production makespan while meeting given end-product demands. Sequence-dependent changeover times, multi-purpose storage units with finite capacities, quarantine times, batch splitting, partial equipment connectivity, material transfer times, and a large number of operations contribute to the complexity of the problem. Known MILP formulations cover all technological constraints of such production processes, but only small problem instances can be solved in reasonable CPU times. In this paper, we develop a heuristic in order to tackle large instances. Under this heuristic, groups of batches are scheduled iteratively using a novel MILP formulation; the assignment of the batches to the groups and the scheduling sequence of the groups are determined using a priority rule. We demonstrate the applicability by means of a real-world production process.