Imaging in pre-clinical cancer research : applied to bone metastases

The aim of this work was to develop methods to measure structural changes in the skeleton using MicroCT. In addition, these new methods should be able to quantify biologically relevant changes. In order to do this, normalized methods to analyse MicroCT scans and perform quantitative measurements within these datasets are described in this thesis. These techniques were combined with a biological angiogenesis assay and used as research tools in a study comparing various different combination treatments of bone metastases.KWF KankerbestrijdingUBL - phd migration 201

Dual inhibition of the vicious cycle and tumor growth in breast and prostate cancer bone metastasis – evaluation of various therapeutic approaches in experimental mouse models

Bone metastases affect the vast majority of advanced breast and prostate cancer patients with metastatic disease. Since the bone microenvironment can help cancer cells to resist treatment, it is important to find treatment strategies that could disturb this intimate relationship. We studied different treatment strategies in breast and prostate cancer mouse models to test the efficacy and clarify the mode-of-action of the tested agents. It was found that the anti-neoplastic agent sagopilone effectively inhibits bone resorption, and that alpha-radiation emitting radium-223 is able to affect both tumor growth and tumor-induced bone reaction. Radium-223 was shown to be actively incorporated into the newly formed bone by osteoblasts, providing rationale for the efficacy in osteoblastic bone metastases. However, in a striking discovery, we observed radium-223 to be efficacious in both types of bone metastases: in osteoblastic prostate cancer as well as in osteolytic breast cancer, reducing both tumor burden and the bone reaction. These effects combined contributed to increased survival, whereas zoledronic acid, inhibiting only bone resorption, and doxorubicin, affecting only tumor growth, did not increase the survival in our breast cancer bone metastasis model. The effects of a sequential combination of doxorubicin followed by zoledronic acid were investigated in an osteolytic breast cancer model, but synergistic effects were not observed. Taken together, it was shown that effectively disrupting multiple steps of the vicious cycle can result in markedly improved treatment results. However, efficacy varies between study settings, models and drug combinations, suggesting the need of careful optimization for maximal therapeutic effects

Automated analysis and visualization of preclinical whole-body microCT data

In this thesis, several strategies are presented that aim to facilitate the analysis and visualization of whole-body in vivo data of small animals. Based on the particular challenges for image processing, when dealing with whole-body follow-up data, we addressed several aspects in this thesis. The developed methods are tailored to handle data of subjects with significantly varying posture and address the large tissue heterogeneity of entire animals. In addition, we aim to compensate for lacking tissue contrast by relying on approximation of organs based on an animal atlas. Beyond that, we provide a solution to automate the combination of multimodality, multidimensional data.* Advanced School for Computing and Imaging (ASCI), Delft, NL * Bontius Stichting inz Doelfonds Beeldverwerking, Leiden, NL * Caliper Life Sciences, Hopkinton, USA * Foundation Imago, Oegstgeest, NLUBL - phd migration 201

Harnessing label-free Raman spectroscopy for metastatic cancer diagnosis and biologic development

Optical spectroscopy is unique amongst experimental techniques in that it can be performed in near-physiological conditions, achieve high molecular specificity, and explore dynamics on timescales ranging from nanoseconds to days. In particular, Raman spectroscopy has emerged in the last two decades as a uniquely versatile method to investigate the structures and properties of molecules in diverse environments through interpreting vibrational transitions. In this thesis, we present four interconnected biomedical and biopharmaceutical applications of Raman spectroscopy that exploit its exquisite molecular specificity, non-perturbative nature, and near real-time measurement capability. In the first presented study, we harness spontaneous Raman spectroscopy in conjunction with multivariate analysis to rapidly and quantitatively determine antibody-drug conjugate aggregation with the goal of eventual application as an in-line tool for monitoring protein particle formation. By exploring subtle, but consistent, differences in spectral vibrational modes of various monoclonal antibodies (mAb) aggregations, a support vector machine-based regression model is developed which is able to accurately predict a wide range of protein aggregation. In addition, the investigation of these spectral vibrational modes also offers new insights into mAb product-specific aggregation mechanisms. Second, leveraging surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR), we present a design of plasmonic nanostructures based on rationally structured metal-dielectric combinations, which we call composite scattering probes (CSP). Specifically, we design CSP configurations that have several prominent resonance peaks enabling higher tunability and sensitivity for self-referenced multiplexed analyte sensing. The CSP prototypes were used to demonstrate differentiation of subtle changes in refractive index (as low as 0.001) as well as acquire complementary untargeted plasmon-enhanced Raman measurements from the biospecimen’s compositional contributors. In the third study, we demonstrate that Raman spectroscopy offers vital biomolecular information for early diagnosis and precise localization of breast cancer-colonized bone alterations. We show that as early as two weeks after intracardiac injections of breast cancer cells in mouse models, Raman measurements in femur and spine uncover consistent changes in both bone matrix and mineral composition. This research effort opens the door for improved understanding of breast metastatic tumor-related bone remodeling and establishing a non-invasive tool for detection of early metastasis and prediction of fracture risk. In parallel with this effort, we also seek to identify the differences between organ-specific isogenic metastatic breast cancer cells. By interpreting the informative spectral bands, we are able to unambiguously identify these isogenic cell lines as unique biological entities. Our spectroscopic study and corresponding metabolic research indicate that tissue-specific adaptations generate biomolecular alterations on cancer cells

Bisphosphonates: from bone-targeted therapies to molecular imaging agents of in vivo bone metabolism

Using bisphosphonates as targeting molecules to generate new drug conjugates and bone imaging probes for the assessment of bone metabolisms and the prevention of bone diseas

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis

Targeting bone-microenvironment-tumour cell interactions : IGF-1 receptor kinase inhibitors.

Bone metastases are a frequent clinical complication associated with cancer. The aim of this PhD thesis was to set up a model system for the study of tumour cell – bone cell interactions in vitro, ex vivo and in vivo and to use this system to test the efficacy of a novel therapeutic agent for the treatment of osteolytic bone disease. Co-culture or conditioned medium studies using human or mouse cancer cell lines were used to develop an in vitro model system of tumour cell – bone cell interactions. This showed that osteolytic tumour cells enhance osteoclast formation, fusion and resorption through the production of various factors that act directly on osteoclasts and their precursors. And in addition, that osteolytic tumour cells also enhance osteoclastogenesis indirectly via increasing the production of RANKL in osteoblasts. Other effects on osteoblasts included reductions in differentiation, migration and adhesion. Successful ex vivo and in vivo models for the study of tumour – induced osteolysis were created using adapted organ cultures and intratibial injection techniques respectively. IGF-1 and its receptor are known to play important roles in both bone metabolism and breast cancer. Therefore a study of the effects of IGF-1 receptor inhibition on tumour cell – bone cell interactions was performed. In vitro studies showed that the novel IGF-1 receptor tyrosine kinase inhibitor PQIP significantly inhibited IGF-1 and breast cancer enhanced osteoclast formation. Western blot analysis suggested this may be due to the inhibition of both IGF-1 and cancer conditioned medium induced PI3k/Akt activation. Moreover, treatment of osteoblasts with PQIP inhibited cancer cell conditioned medium induced increases in RANKL production. Ex vivo studies using human MDA-MB-231 – mouse calvarial organ co-cultures demonstrated that MDA-MB-231 cells caused osteolysis and this was completely prevented by PQIP without affecting cancer cell viability. Furthermore, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following mouse 4T1 breast cancer cell intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in breast cancer-induced osteoclast number and activity. Consistent with the significant inhibition of osteoblast differentiation, spreading, migration and bone nodule formation observed in vitro, PQIP also inhibited osteoblast number and bone formation in vivo. No inhibition of in vivo tumour volume was observed. These findings clearly suggest that oral PQIP treatment reduced the rate of cancer associated bone turnover. In conclusion, this thesis successfully demonstrates a model system for investigating tumour cell-bone cell interactions in vitro, ex vivo and in vivo. Using this model system I showed that pharmacologic inhibition of IGF-1 receptor kinase activity using PQIP inhibits osteoclast and osteoblast changes induced by breast cancer cells in vitro and in vivo and prevents osteolysis ex vivo and in vivo. This indicates that PQIP and its novel derivatives which are now in advanced clinical development may be of value in the treatment of osteolytic bone disease associated with breast cancer