Microenvironmental control of malignant growth

The tumor microenvironment (TME) comprises a complex milieu of different cell types, including cancer associated fibroblasts (CAFs) and immune cells, blood vessels, and the extracellular matrix. Through its interaction with cancer cells, it plays an essential role in cancer invasion and metastasis. The inherent complexity of the TME presents a challenge to study it within experimental model systems. It underscores the importance of complementing such research with observation from human tumor tissues, wherein this intricate complexity is preserved. In Paper IV, we introduce a new software designed to explore the Human Protein Atlas, an online database that includes image data on the protein expression across normal and cancerous tissues from immunohistochemically (IHC) stained tissues. In Paper I, we use this software to identify 12 novel proteins expressed in cancerassociated fibroblasts, four revealing connections to Rho-kinase signaling. We contrast their expression across various tumors and against normal tissue fibroblasts, uncovering expression variability among cancer types and confirm their similarities with the myofibroblastic phenotype. In Paper II, we explore the expression of the proteoglycan Decorin, abundantly present in normal connective tissue and having tumor inhibitory properties, showing its downregulation in the connective tissue surrounding tumors. In Paper III, based on our observations in Paper I of the connection of Rhosignaling in CAFs, we study the effects of knocking out the related RhoA in fibroblasts both in vitro and in vivo models. We demonstrate that the knockout fibroblasts compromise their tumor inhibitory capacity, enhancing cancer cell growth, migration, and metastasis. In Paper VI, we develop a new method for analyzing the extensive data within the Human Protein Atlas by developing a deep-learning-based image classifier. Utilizing a limited training image set, we classify all images available for the prostate, identifying 44 new markers of prostate basal cells. In Paper IV, we explore the influence of the TME on cancer cells by systematically analyzing 20 pancreatic cancer patient samples utilizing an IHC panel. We define shifts in cancer cell phenotype relative to tissue localization, including a transition to a more indolent cancer phenotype, an effect on cancer cell proliferation, and a tendency to normalize the cancer cell phenotype. In conclusion, we developed two new methods that enable us to study protein expression in normal and cancerous tissues by enhancing the capabilities of the HPA. We identified new markers of CAFs and revealed a connection to Rhosignaling. Knocking out the related RhoA in experimental systems resulted in the fibroblasts losing their cancer inhibitory capacity. Finally, we show the remarkable plasticity of cancer cells, demonstrating that their phenotype undergoes significant alterations based on their spatial localization within normal tissue

Interactions between the neuromodulatory systems and the amygdala: exploratory survey using the Allen Mouse Brain Atlas.

Neuromodulatory systems originate in nuclei localized in the subcortical region of the brain and control fundamental behaviors by interacting with many areas of the central nervous system. An exploratory survey of the cholinergic, dopaminergic, noradrenergic, and serotonergic receptor expression energy in the amygdala, and in the neuromodulatory areas themselves was undertaken using the Allen Mouse Brain Atlas. The amygdala was chosen because of its importance in cognitive behavior and its bidirectional interaction with the neuromodulatory systems. The gene expression data of 38 neuromodulatory receptor subtypes were examined across 13 brain regions. The substantia innominata of the basal forebrain and regions of the amygdala had the highest amount of receptor expression energy for all four neuromodulatory systems examined. The ventral tegmental area also displayed high receptor expression of all four neuromodulators. In contrast, the locus coeruleus displayed low receptor expression energy overall. In general, cholinergic receptor expression was an order of magnitude greater than other neuromodulatory receptors. Since the nuclei of these neuromodulatory systems are thought to be the source of specific neurotransmitters, the projections from these nuclei to target regions may be inferred by receptor expression energy. The comprehensive analysis revealed many connectivity relations and receptor localization that had not been previously reported. The methodology presented here may be applied to other neural systems with similar characteristics, and to other animal models as these brain atlases become available

Single neuron transcriptomics identify SRSF/ SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing.

We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log(2)1.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently

Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

BACKGROUND: Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling. METHODS AND RESULTS: Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO(2 )= 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-β, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media. CONCLUSION: Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ

Glioblastoma: development of new diagnostic tools based on EV-associated proteins

Glioblastomas multiforme (GBM) are the most common malignant primary brain tumors in adults. They are highly aggressive and have an overall survival of <15 months despite maximal surgical resection and chemoradiation (Ostrom, 2019). GBMs are typically heterogeneous with a wide range of genetic and epigenetic variations among tumor cells. Extracellular vesicles (EVs) represent one of the plausible ways through which can be obtained a better understanding of the heterogeneous subpopulations of GBM / molecular signatures. EVs hold promise for the discovery of potential tumours biomarkers useful in clinical managment for GBM patient diagnosis and follow-up. Isolating EVs from body fluids and screening their protein content may serve as a complementary approach to assess the heterogeneous molecular landscape of GBM as tumors evolve. Many reports support the idea that Hsps are implicates in the pathogenesis and in the progression of different human neoplasms, by uncertain metabolic mechanism. Athough Hsps perform their canonical “chaperoning” functions in both prokariotic and eukaryotic cells, they have also acquired, probly during evolution, “extra-chaperoning” roles. Among these roles, there are some involved in the mechanism of cancerogenesis. In this study, we evaluate the exspression of some Hsps (in particular Hsp10, Hsp27, Hsp60, Hsp70, and Hsp90) through experiments of immnohistochemistry in samples of GBM and healthy controls, and also by immunofluorescence analysis on priamry and secondary cell lines of GBM. We also focused to research these proteins in EVs isolated from plasma obatained from patients with GBM, before and after surgery. The isolation was followed by a morphological and biochemical characterization of the EVs, in order to better study the characteristics of these potential natural carriers for the tool development for diagnostic and, possibly, also follow-up biopsy.

Abnormal interneuron development in disrupted-in-schizophrenia-1 L100P mutant mice

BACKGROUND: Interneuron deficits are one of the most consistent findings in post-mortem studies of schizophrenia patients and are likely important in the cognitive deficits associated with schizophrenia. Disrupted-in-Schizophrenia 1 (DISC1), a strong susceptibility gene for schizophrenia and other mental illnesses, is involved in neurodevelopment, including that of interneurons. However, the mechanism by which DISC1 regulates interneuron development remains unknown. In this study, we analyzed interneuron histology in the Disc1-L100P single point mutation mouse, that was previously shown to have behavioral abnormalities and cortical developmental defects related to schizophrenia. RESULTS: We sought to determine whether a Disc1-L100P point mutation in the mouse would alter interneuron density and location. First, we examined interneuron position in the developing mouse cortex during embryonic days 14–16 as an indicator of interneuron tangential migration, and found striking migration deficits in Disc1-L100P mutants. Further analysis of adult brains revealed that the Disc1-L100P mutants have selective alterations of calbindin- and parvalbumin-expressing interneurons in the cortex and hippocampus, decreased GAD67/PV co-localization and mis-positioned interneurons across the neocortex when compared to wild-type littermates. CONCLUSION: Our results are consistent with the anomalies seen in post-mortem schizophrenia studies and other Disc1 mutant mouse models. Future research is required to determine the specific mechanisms underlying these cellular deficits. Overall, these findings provide further evidence that DISC1 participates in interneuron development and add to our understanding of how DISC1 variants can affect susceptibility to psychiatric illness

Distribution of Tyrosine Hydroxylase-Expressing Interneurons with Respect to Anatomical Organization of the Neostriatum

We have recently shown in vitro that striatal tyrosine hydroxylase-expressing interneurons identified in transgenic mice by expression of enhanced green fluorescent protein (TH-eGFP) display electrophysiological profiles that are distinct from those of other striatal interneurons. Furthermore, striatal TH-eGFP interneurons show marked diversity in their electrophysiological properties and have been divided into four distinct subtypes. One question that arises from these observations is whether striatal TH-eGFP interneurons are distributed randomly, or obey some sort of organizational plan as has been shown to be the case with other striatal interneurons. An understanding of the striatal TH-eGFP interneuronal patterning is a vital step in understanding the role of these neurons in striatal functioning. Therefore, in the present set of studies the location of electrophysiologically identified striatal TH-eGFP interneurons was mapped. In addition, the distribution of TH-eGFP interneurons with respect to the striatal striosome–matrix compartmental organization was determined using μ-opioid receptor (MOR) immunofluorescence or intrinsic TH-eGFP fluorescence to delineate striosome and matrix compartments. Overall, the distribution of the different TH-eGFP interneuronal subtypes did not differ in dorsal versus ventral striatum. However, striatal TH-eGFP interneurons were found to be mostly in the matrix in the dorsal striatum whereas a significantly higher proportion of these neurons was located in MOR-enriched domains of the ventral striatum. Further, the majority of striatal TH-eGFP interneurons was found to be located within 100 μm of a striosome–matrix boundary. Taken together, the current results suggest that TH-eGFP interneurons obey different organizational principles in dorsal versus ventral striatum, and may play a role in communication between striatal striosome and matrix compartments

A Single Cell Transcriptomics Map of Paracrine Networks in the Intrinsic Cardiac Nervous System

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease

Assessment of histopathological methods of evaluating response to neoadjuvant therapy in oesophageal and gastric adenocarcinoma

Upper gastrointestinal tract (GIT) cancers usually receive neoadjuvant therapy prior to surgery. The histological assessment of this response and if this can be predicted on the pre-treatment biopsy are the subject of this thesis. The first study assessed the inter- and intra-observer variation amongst pathologists in evaluating the degree of regression using the Mandard scoring system. The results showed that the reproducibility of this system was only fair to moderate in both cases of inter- and intra-observer testing. The second study examined the levels of expression of selected tumour markers before and after neoadjuvant chemotherapy. These included markers monitoring apoptosis (p53 and bcl-2), proliferation (Ki-67), angio- and lymphangio-genesis (VEGF, CD-31 and LYVE-1). The levels of expression in these markers were measured in the pre-treatment biopsies, to monitor if they could predict the response to neoadjuvant therapy. It was found that when the panel of chosen markers being used together, delivered a much higher power of prediction rather than adopting only one marker, where the collective power of prediction was 80.6%, whereas individually, the power of prediction ranged between 24.6% (VEGF) and 60.7% (Ki-67). The third study explored the use of digital image analysis in assessing the response to neoadjuvant therapy. It was found that while this technique paralleled the Mandard scoring system, it delivered a more objective and reproducible assessment. On the basis of these results I suggest that image analysis should be used to assess tumour regression especially in the context of clinical trials. In this retrospective study it has been shown that the pre-treatment biopsy can predict the degree of regression