Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing.publishedVersio

Method for the classification of brain cancer treatment's responsiveness via physical parameters of DCE-MRI data

Tumors have several important hallmarks; anomalous and heterogeneous behaviors of their vascular structures, and high angiogenesis and neovascularization. Tumor tissue presents high blood flow (F) and extraction ratio (E) of contrast molecules. Consequently there is growing interest in non invasive methods for characterizing changes in tumor vasculature. Toft's model has been extensively used in the past in order to calculate Ktrans maps which take into consideration both F and E. However, in this thesis we argue that for accurate tumor characterization we need a model able to compute both F and E in tissue plasma. This project has been developed as part of a larger project, working toward building a Clinical Decision Support System (CDSS): an interactive expert computer software, that helps doctors and other health professionals make decisions regarding patient treatment progress. Using the Gamma Capillary Transit Time (GCTT) pharmacokinetic model we calculate F and E separately in a more realistic framework; unlike other models it takes into account the heterogeneity of the tumor, which depends on parameter a-1. a-1 is the width of the distribution of the capillary transit times within a tissue voxel. In more detail, a-1 expresses the heterogeneity of tissue microcirculation and microvasculature. We studied 9 patients pathologically diagnosed with glioblastoma multiforme (GBM), a common malignant type of brain tumor. Several physiological parameters including the blood flow and extraction ratio distributions were calculated for each patient. Then we investigated if these parameters can characterize early the patients' responsiveness to current treatment; we assessed the classification potential based on the actual therapy outcome. To this end, we present a novel analysis framework which exploits the new parameter a-1 and organizes each voxel into four sub-region. Our results indicate that early characterization of response based on GCCT can be significantly improved by focusing on tumor voxels from a specific sub-region

Comparative study of fluorescence spectroscopy of release by cancer cells of different formulation of photosensitizers, foscan and fospeg.

123 σ.O καρκίνος του προστάτη θεωρείται θεραπεύσιμη ασθένεια, αν και οι σύγχρονες θεραπείες συνήθως προκαλούν δυσάρεστες παρενέργειες στους ασθενείς. Η λύση του παρόντος προβλήματος έρχεται με μία σχετικά καινούρια θεραπεία στην αντιμετώπιση της νόσου του καρκίνου, την φωτοδυναμική θεραπεία. Αρκετές φωτοευαίσθητες ουσίες χρησιμοποιούνται στη φωτοδυναμική θεραπεία, οι οποίες διαφέρουν σε αρκετά χαρακτηριστικά τους. Υπάρχουν πολυάριθμες μελέτες για τα χαρακτηριστικά της φωτοευαίσθητης ουσίας, όμως ελάχιστες έρευνες ασχολούνται με τη σύγκριση αυτών σε καρκινική κυτταρική σειρά ώστε να συμβάλουν στην επιλογή της βέλτιστης φωτοδραστικής ουσίας για συγκεκριμένες εφαρμογές. Ένας πιθανός τρόπος για τη βελτίωση της δραστικότητας μίας φωτοευαίσθητης ουσίας είναι η εγκλεισμένη λιποσωμιακή μορφή της. Στη συγκεκριμένη διπλωματική εργασία σκοπός είναι η συγκριτική μελέτη της αποδέσμευσης από κύτταρα καρκινικής σειράς του προστάτη LNCaP της ελεύθερης και της εγκλεισμένης λιποσωμιακής μορφής της m-THPC (foscan και fospeg αντίστοιχα). Η συγκεκριμένη εργασία προσδιορίζει την ενδοκυττάρια συγκέντρωση της φωτοευαίσθητης ουσίας σαν συνάρτηση του χρονικού διαστήματος μετά από την απομάκρυνση της φωτοευαίσθητης ουσίας από την καρκινική σειρά, με αρχικές συγκεντρώσεις 0.15μg/ml και 1.2μg/ml και την μεταξύ τους σύγκριση. Συγκεκριμένα, θα καθοριστεί ποια από τις δύο ουσίες αποδεσμεύεται πιο γρήγορα από τα κύτταρα. Από την επεξεργασία των αποτελεσμάτων, προκύπτει ότι η εγκλεισμένη λιποσωμιακής μορφής της m-THPC (fospeg) ενισχύει την πρόσληψη της φωτοευαίσθητης ουσίας από τα καρκινικά κύτταρα, δηλαδή έχει περισσότερη απορρόφηση από την καρκινική σειρά. Επίσης είναι φανερό ότι αυξάνει το ρυθμό της αποδέσμευσης από τα καρκινικά κύτταρα. Με τα παραπάνω χαρακτηριστηκά μπορούμε να αντιληφθούμε ότι η φωτοευαίσθητη ουσίας της εγκλεισμένης λιποσωμιακής μορφής m-THPC fospeg οδηγεί στη μείωση της δόσης της φωτοευαίσθητης ουσίας και με τη γρήγορη αποδέσμευση της, αυτό συμβάλει σημαντικά στην εξάλειψη της φωτοευαισθησίας μετά την φωτοδυναμική θεραπεία.Prostate cancer is considered a curable condition, even though current treatments can cause unpleasent side effects. The solution to this problem is provided by a considerably new treatment in dealing with cancer, called photodynamic therapy (PDT). Multiple photosensitizers are employed in this therapy, differing in many of their charactiristics. Although, countless in vitro studies on the attributes of photosensitizers do exist, a direct comparison of these substances on one cell line are rare and may contribute to the choice of the optimal photoactive substance for a specific application. One way for improvement is the encapsulation of liposomal formulation; using liposomes as transport agents offers plenty of advantages. In this thesis project the main purpose is the comparative study of the release, from cells of the prostate cancer cell lines LNCaP, of the free and the liposomal encapsulated form of m-THPC (foscan and fospeg respectively). The experiments in this work identify the intracellular concentration of the photosensitive substance as a function of time, after the removal of the photosensitive substance from the original tumor cell lines with concentrations of 0.15ìg/ml and 1.2ìg/ml and their comparison. Specifically, we will determine which of the two substances are released faster than the cells After processing the results, it was conluded that the liposomal encapsulated form of m-THPC (fospeg) enhances the absorbtion of the photosensitive substance by tumor cell lines. It also became obvious that it was increased the rate of release cells. With the aforementioned features we can deduct that the photosensitizers of the encapsulated liposomal formulation m-THPC (fospeg) lead to dose reduction of the photosensitive substance and with its quick release it contribute significantly to the elimination of photosensitivity after photodynamic therapy.Γεωργία Β. Κανλ

The BET Protein Inhibitor JQ1 Decreases Hypoxia and Improves the Therapeutic Benefit of Anti-PD-1 in a High-Risk Neuroblastoma Mouse Model

Anti-programmed death 1 (PD-1) is a revolutionary treatment for many cancers. The response to anti-PD-1 relies on several properties of tumor and immune cells, including the expression of PD-L1 and PD-1. Despite the impressive clinical benefit achieved with anti-PD-1 in several cancers in adults, the use of this therapy for high-risk neuroblastoma remains modest. Here, we evaluated the therapeutic benefit of anti-PD-1 in combination with JQ1 in a highly relevant TH-MYCN neuroblastoma transgenic mouse model. JQ1 is a small molecule inhibitor of the extra-terminal domain (BET) family of bromodomain proteins, competitively binding to bromodomains. Using several neuroblastoma cell lines in vitro, we showed that JQ1 inhibited hypoxia-dependent induction of HIF-1α and decreased the expression of the well-known HIF-1α downstream target gene CA9. Using MRI relaxometry performed on TH-MYCN tumor-bearing mice, we showed that JQ1 decreases R2* in tumors, a parameter associated with intra-tumor hypoxia in pre-clinical settings. Decreasing hypoxia by JQ1 was associated with improved blood vessel quality and integrity, as revealed by CD31 and αSMA staining on tumor sections. By analyzing the immune landscape of TH-MYCN tumors in mice, we found that JQ1 had no major impact on infiltrating immune cells into the tumor microenvironment but significantly increased the percentage of CD8+ PD-1+, conventional CD4+ PD-1+, and Treg PD-1+ cells. While anti-PD-1 monotherapy did not affect TH-MYCN tumor growth, we showed that combinatorial therapy associating JQ1 significantly decreased the tumor volume and improved the therapeutic benefit of anti-PD-1. This study provided the pre-clinical proof of concept needed to establish a new combination immunotherapy approach that may create tremendous enthusiasm for treating high-risk childhood neuroblastoma

Early-life influenza A (H1N1) infection independently programs brain connectivity, HPA AXIS and tissue-specific gene expression profiles

Abstract Early-life adversity covers a range of physical, social and environmental stressors. Acute viral infections in early life are a major source of such adversity and have been associated with a broad spectrum of later-life effects outside the immune system or “off-target”. These include an altered hypothalamus–pituitary–adrenal (HPA) axis and metabolic reactions. Here, we used a murine post-natal day 14 (PND 14) Influenza A (H1N1) infection model and applied a semi-holistic approach including phenotypic measurements, gene expression arrays and diffusion neuroimaging techniques to investigate HPA axis dysregulation, energy metabolism and brain connectivity. By PND 56 the H1N1 infection had been resolved, and there was no residual gene expression signature of immune cell infiltration into the liver, adrenal gland or brain tissues examined nor of immune-related signalling. A resolved early-life H1N1 infection had sex-specific effects. We observed retarded growth of males and altered pre-stress (baseline) blood glucose and corticosterone levels at PND42 after the infection was resolved. Cerebral MRI scans identified reduced connectivity in the cortex, midbrain and cerebellum that were accompanied by tissue-specific gene expression signatures. Gene set enrichment analysis confirmed that these were tissue-specific changes with few common pathways. Early-life infection independently affected each of the systems and this was independent of HPA axis or immune perturbations

Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing