Timing of Imaging after D-Luciferin Injection Affects the Longitudinal Assessment of Tumor Growth Using In Vivo Bioluminescence Imaging

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring

Characterization of an Orthotopic Rat Model of Glioblastoma Using Multiparametric Magnetic Resonance Imaging and Bioluminescence Imaging

Glioblastoma multiforme (GBM) is a lethal and incurable disease. The C6 rat model of GBM shares several similarities to human GBM and longitudinal non-invasive imaging may allow tumour features to be studied. In this thesis, a multimodality imaging framework, consisting of bioluminescence imaging (BLI) and multiparametric magnetic resonance imaging (mpMRI), was applied to the C6 rat model to characterize the growth of orthotopic tumours. BLI signal, a measure of cell viability, tended to increase and then decrease in the majority of animals, whereas tumour volume (from MRI) continually increased. Cellular viability and tumour volume did not correlate across all days, highlighting the value of using complimentary imaging modalities. Apparent diffusion coefficient maps and immunohistochemistry suggests decreases in BLI signal are in part due to decreased tumour cellularity (i.e. necrosis). This is the first use of BLI and mpMRI to characterize this model, and highlights the inter-subject variability in tumour growth

Multimodality Imaging of Tumour Pathophysiology and Response to Pharmacological Intervention

This thesis describes the need for imaging the tumour pathophysiological microenvironment in order to understand response to treatment. Specifically looking at tumour vascularisation in in vivo murine xenograft models of disease, response to treatment with vascular disruption is assessed via photoacoustic tomography (PAT) and magnetic resonance imaging (MRI). Photoacoustic imaging is a novel imaging modality based on the detection of ultrasound waves created by the absorption of nano-second pulsed laser energy within tissue chromophores. It has the spectral specificity of optical techniques whilst also achieving the high resolution of ultrasound. Haemoglobin is the main chromophore found in biological tissue and this modality is therefore ideally suited to imaging tumour vascularisation. Using a Fabry-Perot interferometer this thesis demonstrates for the first time the feasibility of using PAT for re-clinical research and the characterisation of typical tumour vascular features in a non-invasive non-ionising manner. Response to different concentrations of a vascular disrupting drug is then demonstrated, with novel insights in to how tumours recover from vascular damage observed. MRI of response to vascular disruption is also presented. As MRI is widely used in the clinic it can serve as a translational tool of novel imaging biomarkers, and serves to further understand the differences in response of pathologically vascularised of tumours. This thesis looks at markers associated with disruption of haemodynamics, using apparent diffusion (ADC) to elucidate onset of necrosis, increase in haemoglobin concentration (R2*) as indication of impaired flow, and arterial spin labelling (ASL) as a marker of tumour blood perfusion. This is shown in both subcutaneous and clinically relevant liver metastasis models. Taken as whole, the results from this thesis indicate that whilst understanding the response of the tumour vasculature to pharmacological intervention is complex, novel imaging techniques can provide invaluable translational information on the pathophysiology of tumours

Investigating the Mechanisms of Breast Cancer Metastasis Using Multimodality Molecular Imaging

Introduction: Breast cancer recurrence continues to be a significant challenge in the clinic. Despite successful removal and/or treatment of the original tumour, many patients experience relapse in the breast or at distant sites. Furthermore, the diagnosis of metastatic disease often occurs too late for effective treatment. Methods: In this thesis, we combine iron-based cellular MRI and longitudinal BLI to noninvasively track the fate of cancer cells into overt tumours in the mouse brain. We then apply this imaging model to study the effect of a primary breast tumour on the growth of secondary metastases in an immune competent mouse model. Finally, we utilized dual-luciferase BLI to investigate the potential of self-homing circulating tumour cells (CTCs) as a novel cancer theranostic in both orthotopic and metastatic models of breast cancer. Results: BLI complemented our cellular MRI technologies well by providing longitudinal measures of cancer cell viability. Using in vivo BLI/MRI, we demonstrated the presence of a 4T1 primary tumour significantly enhances total brain tumour burden. Finally, using dual-luciferase BLI, we demonstrated the ability of experimental CTCs to home to and treat primary tumours and disseminated breast cancer lesions. Conclusion: MRI and BLI are complementary technologies to noninvasively study the fate of breast cancer cells, as well as the mechanisms contributing to metastasis including CTR/CTE and tumour self-homing. Furthermore, we provide evidence that CTCs are a novel theranostic platform for the visualization and treatment of pre-established tumour sites throughout the body

Noninvasive biophotonic imaging for studies of infectious disease

According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo

Estabelecimento e caracterização de modelos resistentes e matastaticos de osteossarcoma humano in vitro e in vivo

Doutoramento em BiologiaOsteossarcoma é uma doença rara, sendo o tipo mais comum de tumor maligno do osso. O pico de incidência ocorre durante a adolescência e desenvolve-se principalmente nos ossos longos. Os tratamentos atuais incluem quimioterapia antes e após a cirurgia e a ressecção cirúrgica de todos os locais envolvidos (tumor primário e metástases quando presente). As metástases, principalmente nos pulmões, são um grande problema no diagnóstico (20-30% dos pacientes) e durante a história natural do osteossarcoma (cerca de 30% de recaída), afetam uma percentagem considerável de pacientes e são considerados os maiores problemas desta doença. Biologicamente, os osteossarcomas são um dos tumores mais complexos observados nas crianças, no que diz respeito à heterogeneidade, anomalias moleculares e cromossómicas e ao seu microambiente específico. A resistência aos agentes quimioterápicos utilizados no tratamento do osteossarcoma também é um fator prognóstico de alto risco de recaída, independentemente da quimioterapia utilizada. É urgente compreender os mecanismos relacionados a esses fenómenos e desenvolver novos quimioterápicos para superar esses problemas e aumentar a taxa de sobrevivência do paciente. O desenvolvimento de novos fármacos requer múltiplos modelos pré-clínicos adequados para mimetizar a complexidade genómica do osteossarcoma que se desenvolve-se num microambiente ósseo e metastático nos pulmões, apesar dos tratamentos quimioterápicos habituais. Nesta tese, foram desenvolvidos e caracterizados diferentes modelos pré-clínicos clinicamente relevantes in vitro e in vivo, incluindo modelos resistentes bioluminescentes, de modo a melhor compreender esta doença e alguns dos mecanismos de resistência relacionados. Desenvolvemos, em primeiro lugar, dois modelos ortotópicos xenotransplantados derivados de linhas celulares (CDX) bioluminescentes (Luc/mKate2), capazes de desenvolver metástases espontaneamente. As células bioluminescentes foram injetadas ortotopicamente, em diferentes contextos: imune (estirpes de ratinhos de laboratório - nude e NSG) e ósseo (intratibial e paratibial com ativação do periósteo). O sistema IVIS SpectrumCT, combinando tomografia computadorizada longitudinal (TC) e bioluminescência, foi utilizado para acompanhar o crescimento primário do tumor e a disseminação metastática em tempo real. O contexto imune murino, o contexto genético dos dois modelos CDX e o contexto ósseo (intratibial ou paratibial) influenciaram o enxerto tumoral, o crescimento primário do tumor e o comportamento agressivo local (osteocondensação e osteólise), bem como a disseminação metastática para os pulmões, ossos e baço (uma localização incomum em seres humanos). Observou-se também que a estirpe de ratinhos NSG e a injeção intratibial apresentam melhores características para o desenvolvimento de modelos que a injeção paratibial ou a estirpe de ratinhos nude. Seguidamente, desenvolvemos modelos resistentes bioluminescentes in vitro, aos principais medicamentos utilizados no osteossarcoma, nomeadamente metotrexato (5modelos) e doxorrubicina (1modelo), por exposição contínua a esses medicamentos. Realizando o mesmo procedimento, não foi obtida resistência à mafosfamida. Investigamos os mecanismos da resistência adquirida relacionados com estas drogas e observamos comportamentos diferenciais in vitro e in vivo (com modelos CDX ortotópicos bioluminescentes) das linhas resistentes e respetivas linhas parentais. Um mecanismo de resistência na linha celular resistente à doxorrubicina foi observado, nomeadamente a indução da proteína PgP. Mostramos diferentes mecanismos de resistência adquirida ao metotrexato de acordo com o backgroud genético das linhas celulares, que afetam a expressão génica e provocam alterações no número de cópias ao nível dos cromossomas. Foram observados diferentes comportamentos dos modelos resistentes bioluminescentes ortotópicos (CDX) in vivo em comparação com as respetivas linhas parentais. Finalmente, utilizando amostras de osteossarcoma humano provenientes de biópsias de pacientes em recidiva após a quimioterapia habitual, foram desenvolvidos modelos resistentes xenotransplantados derivados do paciente (PDX), quer subcutaneamente quer ortotopicamente (no osso). A caracterização desses modelos está em curso, em particular a comparação das características moleculares destes (sequenciamento completo do exoma e sequenciamento do ARN) com as do tumor do paciente na recaída e do mesmo no diagnóstico. Todos esses modelos desenvolvidos em diferentes contextos in vitro e in vivo trazem informações complementares para outros tipos de modelos de osteossarcoma já existentes. Estes modelos são necessários para obter mais informações sobre os diferentes processos que envolvem o desenvolvimento inicial, a progressão e a sensibilidade/resistência ao tratamento no osteossarcoma. Permitem ajudar ainda a avaliação de novos quimioterápicos, de modo a encontrar soluções para a atual falta de terapias eficientes no osteossarcoma.Osteosarcoma is a rare disease and the most common type of malignant bone tumor. The peak incidence occurs during the adolescence and the disease develops mainly in long bones. Current treatments include chemotherapy before and after surgery and surgical resection of all the involved sites (primary tumor and metastasis when present). Metastases mainly in the lungs are a major challenge at diagnosis (20-30% of the patients) and during the natural history of osteosarcoma (around 30% of relapse, most being metastatic), affect a considerable percentage of patients with osteosarcoma, being considered the biggest problem of this disease. Biologically, osteosarcomas are one of the most complex tumours in children in regard to tumour heterogeneity, molecular and chromosomal abnormalities, and their specific microenvironment. Resistance to the chemotherapeutic agents used in osteosarcoma is also a prognostic factor of high risk of relapse, whatever the chemotherapy used. It is urgent to understand the mechanisms related with these phenomena and develop new drugs in order to overcome these challenges and increase patient survival. New drug development requires suitable multiple pre-clinical models to better mimic the genomic complexity of osteosarcoma which develops in a bone microenvironment and in a metastatic setting in the lungs, despite usual chemotherapeutic treatments. In this thesis, we developed and characterised different and clinically relevant in vitro and in vivo preclinical models, including bioluminescent resistant models in order to understand better this disease and some of the resistant mechanism related. First, two bioluminescent (Luc/mKate2) cell line derived xenograft (CDX) models were developed in an orthotopic bone setting able to spontaneously metastasize. Bioluminescent cells were injected orthotopically, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system, combining longitudinal computed tomography (CT) and bioluminescence, was used to follow primary tumor growth and metastatic spread in real-time. The murine immune context, the genetic background of the two CDX-models, and the bone context (intratibial or paratibial) influenced tumor engraftment, primary tumor growth and local aggressive behavior (osteocondensation and osteolysis) as well as metastatic spread to lungs, bone, and spleen (an unusual localization in humans). It was also observed that intratibial injection in NSG mice showed better characteristics for model development than paratibial injection or nude mice recipient. We further developed in vitro bioluminescent models that were resistant to the main drugs used in osteosarcoma, methotrexate (5 models) and doxorubicin (one model), by continuous exposure to these drugs. With the same technique no resistance was obtained for mafosfamide. We explored the mechanism of the acquired resistance to these drugs and observed the differential in vitro and in vivo behaviors (with CDX bioluminescent orthotopic models) of the resistant lines and their parental counterpart. A multidrug phenomenon by PgP induction was observed in the doxorubicin resistant cells. We show different mechanisms of acquired resistance to methotrexate according to the genetic background of the cell lines affecting either gene expression and copy number abnormalities. Different in vivo behavior of the resistant bioluminescent orthotopic CDX models compared to their parental counterparts were observed. Finally, using human biopsy samples of osteosarcoma relapsing after usual anti-osteosarcoma chemotherapy were developed resistant patient-derived xenograft (PDX) models, either in subcutaneous as in orthotopic bone setting. The characterization of these models are still ongoing, in particular the comparison of their molecular characteristics, i.e. using whole exome and RNA sequencing, in comparison with the patient tumor at relapse and with the same patient tumor at diagnosis. All these multiple models developed in different in vitro and in vivo contexts bring complementary information to other types of existing osteosarcoma models. They are needed to get more insight into the different processes involving osteosarcoma initiation, progression and in particular treatment sensitivity/resistance. They will further help drug testing to find solution to the current lack of efficient new drugs in osteosarcoma

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail

Characterization of embryonic stem cell transplantation immunobiology using molecular imaging

Given their self-renewing and pluripotent capabilities, embryonic stem cells (ESCs) are well-poised as a cellular source for tissue regeneration therapy. Successful in vitro differentiation of both mouse (m) and human (h) ESCs into multiple somatic cell types has been reported, including cardiomyocytes, neurons and pancreatic islet cells. However, the host immune response against transplanted ESCs is not well characterized. In fact, controversy remains as to whether ESCs have immune-privileged properties. The scope of the current thesis is to gain insight into immunological aspects of transplantation of embryonic stem cells or their differentiated progeny by using molecular imaging techniques to follow cell fate. Specifically, this thesis presents evidence that: (1) molecular imaging can be used to quantify organ and ESC survival following transplantation and non-invasively follow donor graft fate; (2) ESCs express MHC and co-signaling molecules that are upregulated upon differentiation; (3) mESCs and hESCs can trigger potent cellular and humoral immune responses following allogeneic and/or xenogeneic transplantation, leading to rejection; and (4) immunosuppressive drugs can significantly mitigate the host immune response to prolong hESC survival in immunocompetent mice. These results clearly indicate that ESC immunogenicity is a significant hurdle that must be overcome before successful clinical application can be accomplished.UBL - phd migration 201