Cellular Distribution Pattern Research Articles

Abstract 3648: The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL

Abstract Point process analyses are widely used in ecology and geography to understand the impact and relevance of spatial distribution of points in a complex system. To date, only few studies have explored point process analyses in the context of tumor heterogeneity. Malignancies often show variable patterns of cellular distribution, and the relationship of these topographic variables with underlying biological processes and clinical outcomes is not well understood. Here, we performed spatial point process analyses in diffuse large B-cell lymphoma (DLBCL), leveraging advances in multiplexed immunohistochemistry and cellular phenotyping, with downstream multi-omic and clinicopathological analyses. We have demonstrated that survival in DLBCL is strongly associated with the fraction of malignant cells showing co-expression of the oncogenes MYC and BCL2 in the absence of BCL6 (M+2+6-). These cells display non-random spatial organization within tumor infiltrates, deviating from a poisson distribution. We therefore modeled the spatial organization of M+2+6- cells within DLBCL, using x-y coordinate information from multiplexed fluorescent immunohistochemistry (mfIHC) in four independent cohorts (N=449 patients). We derived spatial point patterns, upon which Geyers point process models were applied. We observed that patients could be divided into two groups based on these: one group showed “clustered” spatial organization, while the other displayed a “dispersed” M+2+6- cell distribution. Cases with predominantly “dispersed” M+2+6- cells showed shorter overall survival in all analyzed cohorts (P < 0.05 in 4/4 cohorts). We performed multi-omic analyses to identify potential biological explanations for this association between topography and clinical outcome. Using digital spatial profiling of the transcriptome from CD20, CD3 and CD68 compartments in 47 DLBCL samples, we observed that lymphomas with an M+2+6- ‘dispersed’ phenotype had an immunologically cold microenvironment enriched in Tregs and exhausted CD4+ and CD8+ T cells. Through an integration with single-cell transcriptomic analyses (N=22 samples) we observed that malignant B-cells from cases with M+2+6- ‘dispersed’ phenotype expressed genes implicated in cell migration and adhesion, but also in potentially targetable immune checkpoints such as LAG3. In summary, by integrating multiplexed immunophenotyping and point process analysis with molecular profiling in the setting of DLBCL, we show that a dispersed pattern of distribution of the M+2+6- subclonal fraction uniquely confers poor prognosis, and associates with a potentially reversible immune cold microenvironment composition. This represents the first demonstration that the spatial distribution of malignant cell subpopulations in lymphoma can embody biological and clinical significance. Citation Format: Shruti Shridar, Michal Marek Hoppe, Patrick Jaynes, Gayatri Kumar, Siddham Jasoria, Ziwei Meng, Vaibhav Rajan, Kasthuri Kannan, Claudio Tripodo, Anand Devaprasath Jeyasekharan. The spatial organization of cells expressing MYC and BCL2 affects immune microenvironment composition and prognosis in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3648.

Is bladder outlet obstruction rat model to induce overactive bladder (OAB) has similarity to human OAB? Research on the events in smooth muscle, collagen, interstitial cell and telocyte distribution

BackgroundCellular and cytoskeletal events of overactive bladder (OAB) have not been sufficiently explored in human bladder due to different limitations. Bladder outlet obstruction (BOO) had been induced in different animal models with different methods to induce (OAB). Similarity of the animal models of BOO to the human OAB is postulated but has not been confirmed. The interstitial cells of Cajal (ICCs), and telocytes (TCs) are an important players in smooth muscles conductivity, they had not been well investigated in the previous BOO models. Objectives are to investigate the morphological pattern of cellular, cytoskeleton and telocytes distribution in BOO rat model and to match the events in two time periods and compare it to the findings in real-world human OAB.MethodsFemale Sprague-Dawley rats (Rattus norvegicus) were randomly divided into: sham (n = 10), BOO 6 W (n = 10), BOO 8 W (n = 10). Operative procedure to Induce BOO was done under anesthesia with intraperitoneal Ketamine administration. The Effect of induction of BOO was evaluated after 6 and 8 weeks. The rats were anesthetized, and the urinary bladder was removed, while the rat was unconscious under anaesthesia it was transferred to the inhalation anaesthesia cage for euthanasia, rats were sacrificed under light anesthesia using isoflurane. Care of animals, surgical procedure, and euthanasia adhered to Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines for the Euthanasia of Animals. The retrieved bladder was processed for examination with histopathology, immunohistochemistry (IHC), and transmission electron microscopy (EM).ResultsHistological examination of the bladder shows thinner urothelium, condensation of collagen between muscle bundles. IHC with c-kit shows the excess distribution of ICCs between smooth muscle bundles. EM shows frequent distribution of TCs that were situated between collagen fibers. Finings in BOO 6 W group and BOO 8 W group were comparable.ConclusionThe animal model study demonstrated increased collagen/ smooth muscle ratio, high intensity of ICCs and presence of TCs. Findings show that a minimally invasive procedure to induce BOO in rats had resulted in an OAB that has morphological changes that were stable in 6 & 8 weeks. We demonstrated the distribution of TCs and ICCs in the rat animal model and defined them. The population of TCs in the BOO rat model is described for the first time, suggests that the TCs and ICCs may contribute to the pathophysiology of OAB. Similarity of animal model to human events OAB was demonstrated. These findings warrant further study to define the role of TCs in OAB.Clinical trial registryThe study does not require a clinical trial registration; it is an experimental animal study in basic science and does not include human subjects.

Immune cellular patterns of distribution affect outcomes of patients with non-small cell lung cancer

Studying the cellular geographic distribution in non-small cell lung cancer is essential to understand the roles of cell populations in this type of tumor. In this study, we characterize the spatial cellular distribution of immune cell populations using 23 makers placed in five multiplex immunofluorescence panels and their associations with clinicopathologic variables and outcomes. Our results demonstrate two cellular distribution patterns—an unmixed pattern mostly related to immunoprotective cells and a mixed pattern mostly related to immunosuppressive cells. Distance analysis shows that T-cells expressing immune checkpoints are closer to malignant cells than other cells. Combining the cellular distribution patterns with cellular distances, we can identify four groups related to inflamed and not-inflamed tumors. Cellular distribution patterns and distance are associated with survival in univariate and multivariable analyses. Spatial distribution is a tool to better understand the tumor microenvironment, predict outcomes, and may can help select therapeutic interventions.

Discovery of cellular and genetic signatures of immune tolerance in kidney transplant recipients through single cell RNA sequencing analysis.

The objective of this study was to uncover distinct cellular and genetic signatures of transplant operational tolerance (TOT) in kidney transplant recipients (KTRs) through single cell RNA sequencing (scRNA-seq) using peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 12 KTRs, including those with TOT (TOT, n = 4), stable allograft function on maintenance immunosuppression (STA, n = 4) and biopsy-proven allograft rejection (BPAR, n = 4). ScRNA-seq of PBMCs was analyzed using 20 cell surface marker antibody sequencing to annotate clusters and 399 immune response panel to identify gene expression. Differences in cellular distribution and gene expression were compared among the three groups. Heatmap hierarchical clustering showed that overall cellular distribution pattern was distinct in TOT in comparison with those in the other two groups, with the proportion of B cells being higher in TOT, attributed to immature B cell fraction (TOT vs. STA vs. BPAR: 4.61% vs. 1.27% vs. 2.53%, p = 0.01). Transcript analysis of B cells revealed that genes involved in allo-immune pathway were downregulated in TOT. In T cell subset analysis, the proportion of naïve T cells and regulatory T cells (Tregs) was increased. In transcript analysis, genes associated with inflammation were decreased, while expression levels of CCR6 in Tregs were increased in TOT. Proportions of NKT and NK cells were increased in TOT than in the other two groups. This study showed that TOT has distinct cellular and genetic signatures such as increases of immature B cells, naïve T cells and Tregs and high expression levels of CCR6 in Tregs.

Single-cell multi-element analysis reveals element distribution pattern in human sperm.

Multiple elements in human sperm have been demonstrated to play significant roles in the reproductive process, but their simultaneous detection in single cells remains challenging. We propose a novel analytical procedure using single-cell inductively coupled plasma-time of flight-mass spectrometry (scICP-TOF-MS) to simultaneously quantify multiple elements of individual sperm cells. A promising label-free cell identification strategy based on the endogenous element was developed to obtain valid data. The element contents exhibited varied degrees of heterogeneity in single cells. Machine learning-based analysis of the multi-dimension dataset indicated different distribution patterns and physiological roles among the simultaneously detected elements.

Cellular Distribution Pattern of tjp1 (ZO-1) in Xenopus laevis Oocytes Heterologously Expressing Claudins

Epithelial barriers constitute a fundamental requirement in every organism, as they allow the separation of different environments and set boundaries against noxious and other adverse effectors. In many inflammatory and degenerative diseases, epithelial barrier function is impaired because of a disturbance of the paracellular seal. Recently, the Xenopus laevis oocyte has been established as a heterologous expression model for the analysis of transmembrane tight junction protein interactions and is currently considered to be a suitable screening model for barrier effectors. A prerequisite for this application is a physiological anchoring of claudins to the cytoskeleton via the major scaffolding protein tjp1 (tight junction protein 1, ZO-1). We have analyzed the oocyte model with regard to the interaction of heterologously expressed claudins and tjp1. Our experiments have revealed endogenous tjp1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin 1 (CLDN1) to claudin 5 (CLDN5). The amphibian cell model can therefore be used for the analysis of claudin interactions.Graphical

Sonic Hedgehog Induces Mesenchymal Stromal Cell Senescence-Associated Secretory Phenotype and Chondrocyte Apoptosis in Human Osteoarthritic Cartilage.

Hedgehog (HH) signaling plays a critical role in osteoarthritis (OA) pathogenesis, but the molecular mechanism remains to be elucidated. We show here that Sonic Hedgehog (SHH) gene expression is initiated in human normal cartilage stromal cells (NCSC) and increased in OA cartilage mesenchymal stromal cells (OA-MSCs) during aging. Manifesting a reciprocal cellular distribution pattern, the SHH receptors PTCH1 and SMO and transcription factors GLI2 and GLI3 are expressed by chondrocytes (OAC) in OA cartilage. SHH autocrine treatment of osteoarthritis MSC stimulates proliferation, chondrogenesis, hypertrophy, and replicative senescence with elevated SASP gene expression including IL1B, IL6, CXCL1, and CXCL8. SHH paracrine treatment of OAC suppresses COL2A1, stimulates MMP13, and induces chondrocyte apoptosis. The OA-MSC conditioned medium recapitulates the stimulatory effects of SHH on OAC catabolism and apoptosis. SHH knock-down in OA-MSC not only inhibits catabolic and senescence marker expression in OA-MSC, but also abolishes the effect of the OA-MSC conditioned medium on OAC catabolism and apoptosis. We propose that SHH is a key mediator between OA-MSC and OA chondrocytes interaction in human OA cartilage via two mechanisms: (1) SHH mediates MSC growth and aging by activating not only its proliferation and chondrogenesis, but also low-grade inflammation and replicative senescence, and (2) SHH mediates OA-MSC-induced OAC catabolism and apoptosis by creating a pro-inflammatory microenvironment favoring tissue degeneration during OA pathogenesis.

Methods to Determine and Analyze the Cellular Spatial Distribution Extracted From Multiplex Immunofluorescence Data to Understand the Tumor Microenvironment.

Image analysis using multiplex immunofluorescence (mIF) to detect different proteins in a single tissue section has revolutionized immunohistochemical methods in recent years. With mIF, individual cell phenotypes, as well as different cell subpopulations and even rare cell populations, can be identified with extraordinary fidelity according to the expression of antibodies in an mIF panel. This technology therefore has an important role in translational oncology studies and probably will be incorporated in the clinic. The expression of different biomarkers of interest can be examined at the tissue or individual cell level using mIF, providing information about cell phenotypes, distribution of cells, and cell biological processes in tumor samples. At present, the main challenge in spatial analysis is choosing the most appropriate method for extracting meaningful information about cell distribution from mIF images for analysis. Thus, knowing how the spatial interaction between cells in the tumor encodes clinical information is important. Exploratory analysis of the location of the cell phenotypes using point patterns of distribution is used to calculate metrics summarizing the distances at which cells are processed and the interpretation of those distances. Various methods can be used to analyze cellular distribution in an mIF image, and several mathematical functions can be applied to identify the most elemental relationships between the spatial analysis of cells in the image and established patterns of cellular distribution in tumor samples. The aim of this review is to describe the characteristics of mIF image analysis at different levels, including spatial distribution of cell populations and cellular distribution patterns, that can increase understanding of the tumor microenvironment.

D-dopachrome tautomerase activates COX2/PGE2 pathway of astrocytes to mediate inflammation following spinal cord injury

BackgroundAstrocytes are the predominant glial cell type in the central nervous system (CNS) that can secrete various cytokines and chemokines mediating neuropathology in response to danger signals. D-dopachrome tautomerase (D-DT), a newly described cytokine and a close homolog of macrophage migration inhibitory factor (MIF) protein, has been revealed to share an overlapping function with MIF in some ways. However, its cellular distribution pattern and mediated astrocyte neuropathological function in the CNS remain unclear.MethodsA contusion model of the rat spinal cord was established. The protein levels of D-DT and PGE2 synthesis-related proteinase were assayed by Western blot and immunohistochemistry. Primary astrocytes were stimulated by different concentrations of D-DT in the presence or absence of various inhibitors to examine relevant signal pathways. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.ResultsD-DT was inducibly expressed within astrocytes and neurons, rather than in microglia following spinal cord contusion. D-DT was able to activate the COX2/PGE2 signal pathway of astrocytes through CD74 receptor, and the intracellular activation of mitogen-activated protein kinases (MAPKs) was involved in the regulation of D-DT action. The selective inhibitor of D-DT was efficient in attenuating D-DT-induced astrocyte production of PGE2 following spinal cord injury, which contributed to the improvement of locomotor functions.ConclusionCollectively, these data reveal a novel inflammatory activator of astrocytes following spinal cord injury, which might be beneficial for the development of anti-inflammation drug in neuropathological CNS.

Identification of distinct immune landscapes using an automated nine-color multiplex immunofluorescence staining panel and image analysis in paraffin tumor tissues

Immune profiling is becoming a vital tool for identifying predictive and prognostic markers for translational studies. The study of the tumor microenvironment (TME) in paraffin tumor tissues such as malignant pleural mesothelioma (MPM) could yield insights to actionable targets to improve patient outcome. Here, we optimized and tested a new immune-profiling method to characterize immune cell phenotypes in paraffin tissues and explore the co-localization and spatial distribution between the immune cells within the TME and the stromal or tumor compartments. Tonsil tissues and tissue microarray (TMA) were used to optimize an automated nine-color multiplex immunofluorescence (mIF) panel to study the TME using eight antibodies: PD-L1, PD-1, CD3, CD8, Foxp3, CD68, KI67, and pancytokeratin. To explore the potential role of the cells into the TME with this mIF panel we applied this panel in twelve MPM cases to assess the multiple cell phenotypes obtained from the image analysis and well as their spatial distribution in this cohort. We successful optimized and applied an automated nine-color mIF panel to explore a small set of MPM cases. Image analysis showed a high degree of cell phenotype diversity with immunosuppression patterns in the TME of the MPM cases. Mapping the geographic cell phenotype distribution in the TME, we were able to identify two distinct, complex immune landscapes characterized by specific patterns of cellular distribution as well as cell phenotype interactions with malignant cells. Successful we showed the optimization and reproducibility of our mIF panel and their incorporation for comprehensive TME immune profiling into translational studies that could refine our ability to correlate immunologic phenotypes with specific patterns of cells distribution and distance analysis. Overall, this will improve our ability to understand the behavior of cells within the TME and predict new treatment strategies to improve patient outcome.

Functionally defined CD164 epitopes are expressed on CD34+ cells throughout ontogeny but display distinct distribution patterns in adult hematopoietic and nonhematopoietic tissues

AbstractThree distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34+ cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34+ cells throughout ontogeny, in particular on CD34+ cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34+ hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133hiCD34hiCD38lo/− cell population, they also occur on most cord blood Lin−CD34lo/−CD38lo/− cells, which are potential precursors for the AC133hiCD34hiCD38lo/− subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I–positive but class II–negative, while epithelia are weakly CD164 class II–positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II–positive, while lymphoid cells are CD164 class I–positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.

Functionally defined CD164 epitopes are expressed on CD34(+) cells throughout ontogeny but display distinct distribution patterns in adult hematopoietic and nonhematopoietic tissues.

Three distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34(+) cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34(+) cells throughout ontogeny, in particular on CD34(+ )cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34(+) hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133(hi)CD34(hi)CD38(lo/-) cell population, they also occur on most cord blood Lin(-)CD34(lo/-)CD38(lo/- )cells, which are potential precursors for the AC133(hi)CD34(hi)CD38(lo/-) subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I-positive but class II-negative, while epithelia are weakly CD164 class II-positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II-positive, while lymphoid cells are CD164 class I-positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.

Expression of β-catenin in Minor Salivary Glands Adjacent to Oral Squamous Cell Carcinoma

Objective: This study aims to evaluate the expression pattern, localization, and stain intensity of β-catenin in minor salivary glands adjacent to surgically excised oral squamous cell carcinoma (OSCC). 
 Methods: A retrospective study was held with 16 samples of formalin-fixed paraffin-embedded blocks with minor salivary glands adjacent to OSCC. Sections were stained and evaluated immunohistochemically
 with β-catenin. The staining expression was assessed according to cellular localization, stain intensity, and, lastly, the pattern of stain distribution throughout acini.
 Statistical analysis was performed using SPSS version 24.0 software for Windows, and data analyzed by Fisher's exact test. P-value <0.05 was considered as statistically significant.
 Results: All minor salivary glands in the studied sample showed β-catenin staining with different expression in their functional units, as all had ductal and myoepithelial cells staining with a predominant cytoplasmic
 localization. While the mucous acini showed β- catenin expression in 10 cases (62.5%), this marker was significantly less frequently detected in serous acini of two cases of poorly differentiated OSCC (p= .008).
 A highly significant relation was found between the β-catenin cellular localization and stain distribution pattern in mucous and serous acini. 
 Conclusions: β-catenin had altered cytoplasmic expression in all of the minor salivary glands adjacent to OSCC. Furthermore, the diffuse pattern of distribution throughout the acini could identify the multi-patches
 pathological alteration of this area. The current study clarifies that the adjacent clinically normal-appearing salivary glands could harbor genetic aberrations of their subsequent malignant transformation.

Monitoring the Progression of Liver Fluke-Induced Cholangiocarcinoma in a Hamster Model Using Synchrotron FTIR Microspectroscopy and Focal Plane Array Infrared Imaging.

Cholangiocarcinoma (CCA) is a bile duct cancer that originates in the bile duct epithelium. Northeastern Thailand has the highest incidence of CCA, and there is a direct correlation with liver fluke (Opisthorchis viverrini) infection. The high mortality rate of CCA is a consequence of delayed diagnosis. Fourier transform infrared (FTIR) spectroscopy is a powerful technique that detects the absorbance of molecular vibrations and is perfectly suited for the interrogation of biological samples. In this study, we applied synchrotron radiation-FTIR (SR-FTIR) microspectroscopy and focal plane array (FPA-FTIR) microspectroscopy to characterize periductal fibrosis and bile duct cells progressing to CCA induced by inoculating O. viverrini metacercariae into hamsters. SR-FTIR and FPA-FTIR measurements were performed in liver sections harvested from 1-, 2-, 3-, and 6-month post-infected hamsters compared to uninfected liver tissues. Principal component analysis (PCA) of the tissue samples showed a clear discrimination among uninfected and early-stage (1 and 2 months) and cancerous-stage (3 and 6 months) tissues. The discrimination is based on intensity changes in the phosphodiester band (1081 cm-1), amino acid residue (∼1396 cm-1), and C═O stretching carboxylic esters (1745 cm-1). Infected tissues also show definitive bands at ∼1280, 1234, and 1201 cm-1 characteristic of the collagen triplet and indicative of fibrosis. Hierarchical cluster analysis (HCA) was performed on the FPA data and showed a classification into specific cell types. Hepatocyte, fibrotic lesion, and bile duct (cancer) were classified and HCA mapping showed similar cellular distribution pattern compared to Sirius red staining. This study was also extended to less invasive sample analysis using attenuated total reflectance-FTIR (ATR-FTIR) spectroscopy. Sera from O. viverrini-infected and uninfected hamsters were analyzed using multivariate analysis, including principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA). PCA was able to classify spectra of normal, early-stage CCA, and CCA, while the PLS-DA gave 100% accuracy for the validation. The model was established from 17 samples (11 normal, 6 cancer) in the calibration set and 9 samples in the validation set (4 normal, 2 cancer, 3 precancerous). These results indicate that FTIR-based technology is a potential tool to detect the progression of CCA, especially in the early stages of the disease.

Differential anatomical and cellular expression of lysophosphatidic acid receptor 1 in adult mouse brain

Lysophosphatidic acid (LPA) is a bioactive phospholipid that acts as an extracellular signaling molecule through six G-protein-coupled receptors: LPA1-LPA6. Recent studies have demonstrated that LPA signaling via LPA1 receptor plays a crucial role in cognition and emotion. However, because of limited availability of reliable antibodies, it is currently difficult to identify the cell types expressing LPA1 receptor in the brain. The current study explored the cellular distribution pattern of LPA1 receptor in the brain using the LPA1 lacZ-knock-in reporter mice. In situ hybridization and immunohistochemistry revealed that LacZ gene expression in these mice reflected the expression of endogenous LPA1 receptor in the brain. Overall, some brain nuclei contained higher levels of LPA1 receptor than others. The majority of LPA1 receptor-expressing cells were Olig2+ oligodendrocytes. In addition, ALDH1l1+ astrocytes and CD31+ vascular endothelial cells also expressed LPA1 receptor. By contrast, NeuN+ neuron and Iba1+ microglia expressed little or no LPA1 receptor. The current neuroanatomical findings will aid in elucidating a role of brain LPA1 receptor, especially those involved in cognition and emotion.

Relationship between thyroid tumor radiosensitivity and nuclear localization of DNA-dependent protein kinase catalytic subunit.

Thyroid tumors are the most common types of endocrine malignancies and are commonly treated with radioactive iodine (RAI) to destroy remaining cancer cells following surgical intervention. We previously reported that the expression levels of double-stranded DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which plays a key role in non-homologous end joining, are correlated with the radiosensitivity of cancer cells. Specifically, cells expressing high levels of DNA-PKcs exhibited radiation resistance, whereas cells expressing low levels were sensitive to radiation treatment. In this study, we observed full-length native DNA-PKcs (460 kDa) in radiation-resistant FRO and KTC-2 cells through western blot analysis using an antibody against the C-terminus of DNA-PKcs. In contrast, cleaved DNA-PKcs (175 kDa) were observed in radiation-sensitive TPC-1 and KTC-1 cells. Almost equal amounts of DNA-PKcs were observed in moderately radiation-sensitive WRO cells. We also describe a simple method for the prediction of radiation therapy efficacy in individual cases of thyroid cancers based on staining for DNA-PKcs in human cancer cell lines. Immunofluorescent staining showed that native DNA-PKcs was localized largely in the cytoplasm and only rarely localized in the nuclei of radiation-resistant thyroid cancer cells, whereas in radiation-sensitive cancer cells a 175-kDa cleaved C-terminal fragment of DNA-PKcs was localized mainly inside the nuclei. Therefore, DNA-PKcs moved to the nucleus after γ-ray irradiation. Our results suggest a new method for classifying human thyroid tumors based on their cellular distribution patterns of DNA-PKcs in combination with their radiosensitivity.

In situ Hybridization for Sipunculus nudus Coelomic Fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells.

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.

Hepatic Cellular Distribution of Silica Nanoparticles by Surface Energy Modification.

The cellular distribution of silica nanoparticles (NPs) in the liver is not well understood. Targeting specific cells is one of the most important issues in NP-based drug delivery to improve delivery efficacy. In this context, the present study analyzed the relative cellular distribution pattern of silica NPs in the liver, and the effect of surface energy modification on NPs. Hydrophobic NP surface modification enhanced NP delivery to the liver and liver sinusoid fFendothelial cells (LSECs). Conversely, hydrophilic NP surface modification was commensurate with targeting hepatic stellate cells (HSCs) rather than other cell types. There was no notable difference in NP delivery to Kupffer cells or hepatocytes, regardless of hydrophilic or hydrophobic NP surface modification, suggesting that both the targeting of hepatocytes and evasion of phagocytosis by Kupffer cells are not associated with surface energy modification of silica NPs. This study provides useful information to target specific cell types using silica NPs, as well as to understand the relationship between NP surface energy and the NP distribution pattern in the liver, thereby helping to establish strategies for cell targeting using various NPs.

Distribution Patterns of Three Molecularly Defined Classes of GABAergic Neurons Across Columnar Compartments in Mouse Barrel Cortex.

The mouse somatosensory cortex is an excellent model to study the structural basis of cortical information processing, since it possesses anatomically recognizable domains that receive different thalamic inputs, which indicates spatial segregation of different processing tasks. In this work we examined three genetically labeled, non-overlapping subpopulations of GABAergic neurons: parvalbumin- (PV+), somatostatin- (SST+), and vasoactive intestinal polypeptide-expressing (VIP+) cells. Each of these subpopulations displayed a unique cellular distribution pattern across layers. In terms of columnar localization, the distribution of these three populations was not quantitatively different between barrel-related versus septal compartments in most layers. However, in layer IV (LIV), SST+, and VIP+, but not PV+ neurons preferred the septal compartment over barrels. The examined cell types showed a tendency toward differential distribution in supragranular and infragranular barrel-related versus septal compartments, too. Our data suggests that the location of GABAergic neuron cell bodies correlates with the spatial pattern of cortical domains receiving different kinds of thalamic input. Thus, at least in LIV, lemniscal inputs present a close spatial relation preferentially to PV+ cells whereas paralemniscal inputs target compartments in which more SST+ and VIP+ cells are localized. Our findings suggest pathway-specific roles for neocortical GABAergic neurons.