Development and Characterization of Human Tumor Models by Orthotopic Implantation

Titelblatt, Inhaltsverzeichnis, Lebenslauf Einleitung Literaturübersicht Material und Methoden Etablierung der orthotopen Modelle Angangsrate und Wachstumsgeschwindigkeit Koerpergewichtsveraenderung Infiltration und Metastasierung Histologie und Zytologie Immunhistochemie Vaskulaere Permeabilitaet Chemosensibilitaet auf Standardsubstanzen Literaturverzeichnis Asziteszellinie PRCL PC3MAS Vergleich orthotop und subkutaner Xenograft Vergleich orthotoper Xenograft und Patient Bedeutung für die Substanzentwicklung Zusammenfassung summary Abkuerzungsverzeichnis AnhangIn der vorliegenden Arbeit wurden vier subkutan auf der Nacktmaus wachsende humane Tumorxenografts orthotop in die Nacktmaus implantiert. Bei zwei weiteren untersuchten orthotopen Modellen stammte das Spendermaterial von orthotop implantierten Xenografts ab. Im Einzelnen handelte es sich um das Kolonkarzinom CXF 1103T, das Blasenkarzinom BXF 1299T, die Prostatakarzinome PRXF PC3T, PC3MT und PC3MMT sowie das Aszitesmodell PRXF PC3MAS. Die Angangsraten lagen mit Ausnahme des PRXF PC3(T) bei beiden Implantationsarten im gleichen Größenbereich, während die Verdoppelungszeiten nach orthotoper kürzer waren als nach subkutaner Implantation. Diese Beobachtung bestätigte die Untersuchungen anderer Arbeitsgruppen. Das abweichende Verhalten des PRXF PC3(T) war auch im Vergleich mit anderen Arbeiten nicht zu klären. Daß humane Tumorzellen nur dann metastasieren, wenn sie in ihr Ursprungsorgan implantiert werden, wurde in dieser Arbeit bestätigt. Im orthotopen Modell lag die mediane Metastasierungsrate bei 74%. Die Metastasierungsmuster von Xenograft und Patiententumor waren in weiten Teilen identisch. Sowohl Übereinstimmung als auch Abweichungen vom Verhalten im Patienten konnten bei Durchsicht der Literatur bestätigt, die Gründe für die Abweichung aber nicht erklärt werden. Aus den vorliegenden Daten ergibt sich die Hypothese, daß das Metastasierungsverhalten in der Maus von Einflüssen abhängig ist, denen das Spendermaterial seit der Entnahme aus dem Patienten unterlegen war. Der histologische Aufbau der Xenografts war unabhängig von ihrer Lokalisation. Ebenso waren die histologischen Befunde des Patiententumors und des korrespondierenden Xenografts identisch. Immunhistochemisch wurde in allen Tumoren humanes Zytokeratin unabhängig vom Implantationsort nachgewiesen. Daher eignete sich diese Methode zur Identifikation fraglicher Mikrometastasen. Das Kolonkarzinom CXF 1103(T) exprimierte humanes karzinoembryogenes Antigen (CEA) unabhängig von seiner Lokalisation. Da im Serum des betreffenden Patienten keine erhöhten CEA-Werte nachgewiesen worden waren, lag der Verdacht nahe, daß es sich bei CXF 1103(T) um einen sogenannten "low secreter" handelte, eine Geschwulst, die CEA zwar exprimiert aber nicht oder nur in sehr geringen Mengen in den Blutkreislauf sezerniert. Die Konstanz histologischer und immunhistologischer Eigenschaften beim Vergleich zwischen Patiententumor und Xenograft einerseits sowie den beiden Implantationsmöglichkeiten andererseits wurde in der Literatur bereits erwähnt. Die Beobachtung anderer Arbeitsgruppen, daß der Implantationsort den histologischen Aufbau des Xenograft beeinflusst, konnte in dieser Arbeit nicht bestätigt werden. Die zytologischen Untersuchungen der Brust- und Bauchhöhlenergüsse des Aszites-Modelles führten zu dem Schluß, daß sowohl die Tumorzellkonzentration als auch der relative Anteil der einzelnen Zelltypen an der Gesamtheit unabhängig von der injizierten Zellzahl und von der Herkunft der injizierten Zellen war. Der Einfluss des Implantationsortes auf die Beschaffenheit der tumoreigenen Gefäße wurde in der Literatur widersprüchlich behandelt. Der in dieser Arbeit verwendete Parameter der vaskulären Permeabilität zeigte sich unabhängig vom Implantationsort. Im Gegensatz dazu waren die Gefäße der Metastasen durchlässiger als diejenigen der Primärtumore. Dieses Phänomen wurde anhand des selektiven Charakters des Metastasierungsvorganges erklärt. Die Chemotherapieversuche mit BXF 1299T und PRXFT bestätigten, daß es prinzipiell möglich ist, eine für solche Experimente ausreichende Tumorzahl zu transplantieren und deren Wachstumsverlauf in situ zu verfolgen. Eine Bestimmung von Metasasierungsrate und Tumorgewicht am Ende des Versuches blieb im Rahmen dieser Arbeit aber unerlässlich für die Bestimmung der Wirksamkeit einer Substanz. Im Gegensatz zu Beobachtungen anderer Arbeitsgruppen blieb die Ansprechbarkeit gegenüber einer Chemotherapie prinzipiell vom Implantationort unbeeinflußt. Allerdings waren die orthotopen Modelle etwas empfindlicher als die subkutanen. Die Auswahl der Spendertumore, die im subkutanen Modell ein dem Patienten sehr ähnliches Chemosensibilitätsprofil aufwiesen, gewährleistete diese Übereinstimmung der beiden Modelle. Folglich macht es Sinn, Substanzen, bevor sie am aufwendigen orthotopen Modell untersucht werden, am entsprechenden subkutanen Xenograft zu untersuchen. Die in dieser Arbeit verwendeten Zytostatika hemmten, sofern sie eine hemmende Wirkung auf das Tumorwachstum hatten, in statistisch relevanter Weise auch die Bildung von Metastasen. Damit erwiesen sich die untersuchten orthotopen Modelle als geeignet für die Testung antimetastatisch wirkender Substanzen und bestätigten damit gleichlautende Aussagen der Literatur. Die Bedeutung orthotop implantierter humaner Xenografts in der Tumorforschung wurde durch die vorliegende Arbeit unterstrichen. Insbesondere für die Entwicklung antimetastatisch wirkender Substanzen sowie lokaler Therapieformen ist dieses Modell geeignet. Durch die starke Anlehnung an die klinischen Verhältnisse wird es darüber hinaus möglich, wichtige Mechanismen der Tumorbiologie, insbesondere des Metastasierungsvorganges zu studieren, um daraus Konsequenzen für eine bessere Krebstherapie ziehen zu können.In this study the following human tumor xenografts were implanted orthotopically in nude mice: colon carcinoma CXF 1103T, bladder carcinoma BXF 1299T, prostate carcinomas PRXF PC3T, PC3MT, PC3MMT and the ascites model PRXF PC3MAS. In case of four models implanted tumor material derived from xenografts growing subcutaneously in nude mice. For implantation of PRXF PC3MT and PRXF PC3MAS orthotopically growing xenografts were used as donor material. With the exception of PRXF PC3(T), take rates of orthotopic and ectopic models were similar. However, xenografts implanted into the orthotopic site grew faster than the xenografts at the heterotopic site. These results confirm with the examinations of other groups. Low take rate of PRXF PC3T could not be explained by going through literature. The fact that human tumor cells only metastasize when implanted in the corresponding organ was beared out in the present study. Median metastasis rate amounted to 74 %. Distribution of secondary lesions was almost similar to the situation in the corresponding patient. Correspondance as well as divergence from the patient tumor has been described but not explained before. Data presented in this study allow to suppose a correlation between the metastastic behavior of the tumor in the mouse and the different influences on the resected patient specimen before implantation. Histologically, primary tumor and metastatic lesions were similar when human tumors were implanted in the corresponding organ of the nude mouse. The histology was not influenced by the transplantation site. Even the specimen obtained from the patient had histologically high similarities with the xenograft growing in nude mice. Thus, histological structure was not influenced by the tumor environment or host biology. The expression of human cytokeratin was determined by immunohistochemistry. Since the expression was independent of localization of the tumor, measurement of cytokeratin was a feasible method to detect micrometastases. For colon carcinoma CXF 1103T the expression of carcinoembryogenic antigen (CEA) was determined. This xenograft did express the tumor marker irrespective of the implantation site. In patient serum however CEA-levels were not detectable. Originally, CXF 1103T was probably a low secreter, which means a tumor expressing CEA but not secreting it. The results of histological and immunohistological examinations correlated well with the results of other groups. Cytological examination of the ascites model showed no relation between tumor cell concentration or relative tumor cell number found in the pleural and peritoneal cavity on one hand and the number of injected tumor cells or their origin on the other hand. The influence of the implantation site on structural quality of tumor vessels was discussed contrarily. Vascular permeability was not influenced by the implantation site. Apart from visceral metastases, these tumors retained much more dye than the primary tumors. Thus, the selective process of metastasis enhanced porosity of the tumor vessels. For BXF 1299T and PRXF PC3MT chemosensitivity against standard anticancer agents was tested. Orthotopic implanted tumors turned out to be a feasible model for anticancer drug testing in vivo. In contrast to observations of other authors, chemosensitivity was not influenced by the localization of the tumor. The orthotopic models showed a slightly enhanced responsivness in comparison to their subcutaneous counterpart. This phenomenon could be explained by the fact that the chemosensitivity profile of the selected xenografts showed even in the subcutaneous model high correspondence with the patient tumor. Thus, testing anticancer drugs in the subcutaneous model before turning to the large-scale orthotopic model seamed to be a appropriate strategy. So far as the tested anticancer drugs showed antitumoral activity they also inhibited the development of metastases. Therefore, the examinated orthotopic models turned out to be suitable for development of antimetastatic drugs. This study illuminated the importance of human orthotopic models in oncology. Developing antimetastastic drugs as well as testing of local therapies seem to be the most important application for the models described in this thesis. In addition, they allow to study different aspects of tumor biology and will facilitate the development of new anticancer strategies

Zebrafish patient-derived xenograft models predict lymph node involvement and treatment outcome in non-small cell lung cancer

Background Accurate predictions of tumor dissemination risks and medical treatment outcomes are critical to personalize therapy. Patient-derived xenograft (PDX) models in mice have demonstrated high accuracy in predicting therapeutic outcomes, but methods for predicting tumor invasiveness and early stages of vascular/lymphatic dissemination are still lacking. Here we show that a zebrafish tumor xenograft (ZTX) platform based on implantation of PDX tissue fragments recapitulate both treatment outcome and tumor invasiveness/dissemination in patients, within an assay time of only 3 days. Methods Using a panel of 39 non-small cell lung cancer PDX models, we developed a combined mouse-zebrafish PDX platform based on direct implantation of cryopreserved PDX tissue fragments into zebrafish embryos, without the need for pre-culturing or expansion. Clinical proof-of-principle was established by direct implantation of tumor samples from four patients. Results The resulting ZTX models responded to Erlotinib and Paclitaxel, with similar potency as in mouse-PDX models and the patients themselves, and resistant tumors similarly failed to respond to these drugs in the ZTX system. Drug response was coupled to elevated expression of EGFR, Mdm2, Ptch1 and Tsc1 (Erlotinib), or Nras and Ptch1 (Paclitaxel) and reduced expression of Egfr, Erbb2 and Foxa (Paclitaxel). Importantly, ZTX models retained the invasive phenotypes of the tumors and predicted lymph node involvement of the patients with 91% sensitivity and 62% specificity, which was superior to clinically used tests. The biopsies from all four patient tested implanted successfully, and treatment outcome and dissemination were quantified for all patients in only 3 days. Conclusions We conclude that the ZTX platform provide a fast, accurate, and clinically relevant system for evaluation of treatment outcome and invasion/dissemination of PDX models, providing an attractive platform for combined mouse-zebrafish PDX trials and personalized medicine

Genetic determinants of gut microbiota composition and bile acid profiles in mice.

The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions

Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes

Capturing complex tumour biology in vitro : histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1 alpha. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.Peer reviewe

Mutations in SPAG1 Cause Primary Ciliary Dyskinesia Associated with Defective Outer and Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders

Mutations in SPAG1 Cause Primary Ciliary Dyskinesia Associated with Defective Outer and Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders