Caperucita azul

2.1 Aims

The extensive intratumoral heterogeneity of glioblastoma renders it susceptible to evolution when exposed to selective pressures. Such pressures as imposed by the tumour microenvironment and therapeutic regimens can lead to the expansion or contraction of various subclones depending on the relative fitness of each subclone as determined by its sensitivity to a given challenge. For example, a certain drug could remove all sensitive clones and thus provide the opportunity for resistant clones to expand quickly to grow a recurrent tumour due to competitive release. In this way, selective pressures drive evolution and contribute to tumour recurrence, progression, and treatment failure. While the role of chemotherapy in treatment- induced evolution has been studied, the impact of radiotherapy alone on glioblastoma evolution has not been determined.

As outlined in this thesis, I will endeavor to describe the effect of ITH on intrinsic radiosensitivity to determine whether spatially distinct regions of the same tumour may have different sensitivities. Then I will discuss whether radiation alone can drive the evolution of GSC-initiated orthotopic xenografts and whether it leads to the emergence of resistant subclones. In this context, the GSC-initiated xenograft model will also be proposed as a potential model system for studying recurrent GB and reirradiation protocols. Finally, I will report on biocompatibility and efficacy studies for a new hydrogel delivery system , which could provide immediate and local delivery of targeted combinatorial therapeutics, including radiosensitisers. Ultimately, by better characterizing the impact of radiation on glioblastoma evolution and by studying recurrent GB and retreatment strategies, we can harness insights related to recurrent glioblastoma biology and radiation biology for the production of more effective systemic and local therapies to improve outcomes for glioblastoma patients. With this thesis, I would like to answer the following questions: (1) Does ITH translate into differences in intrinsic radiosensitivities between patient-derived cell lines derived from distinct tumour fragments? (2) Does radiation alone drive glioblastoma evolution? (3) If radiation drives glioblastoma evolution, does it lead to the emergence of radioresistant clones? (4) Can the GSC-initiated xenograft model be used to study recurrent GB treatment strategies? (5) Are hyaluronic acid-based hydrogels biocompatible and effective at local drug delivery?

2.2 Hypotheses

(1) Does ITH translate into differences in intrinsic radiosensitivities between patient-derived cell lines derived from distinct tumour fragments?

Hypothesis: I hypothesised that patient-derived cell lines derived from spatially distinct tumour fragments would display intratumoral heterogeneity. Furthermore, I hypothesised that the heterogeneity between cell lines would extend to radioresponse such that different cell lines from the same tumour would display different intrinsic radiosensitivities.

After deriving cell lines from multiple spatially distinct tumour fragments from several patients, we examined differences in radioresponse through standard radiation biology techniques. Similarities or differences in radioresponse and extent of ITH were further interrogated through genomic analysis of these patient-derived cell lines and their corresponding tumour samples. If there are major differences in radioresponse and genetic alterations between cell lines derived from distinct regions of the same tumour, then it would suggest that multiple therapeutic targets are needed for holistic treatment of GB.

(2) Does radiation alone drive glioblastoma evolution?

Hypothesis: I hypothesised that, similar to other therapeutic modalities, radiation is capable of providing selective pressure sufficient to drive glioblastoma evolution.

GSC-initiated orthotopic xenograft models treated with fractionated radiotherapy were utilised to examine the survival, phenotypic, and genomic consequences of radiation. Due to the variability in tumour growth patterns and inconsistency in growth rates of the patient- derived cell lines utilised for Aim 1, those lines were deemed inappropriate for examining the effects of a therapeutic challenge. Therefore, GSC lines which have previously been shown to provide reproducible in vivo tumours with consistent radioresponse were selected for the study of radiation-induced evolution. Morphologic and histological analysis characterised potential

changes in recurrent GB biology. Viral integration site analysis (VISA) examined the impact of radiation on clonal diversity, while WES highlighted any changes in mutation patterns of GSC-initiated tumours before and after treatment with radiation. These measures were employed to provide evidence for radiation-driven GB evolution.

(3) If radiation drives glioblastoma evolution, does it lead to the emergence of radioresistant clones?

Hypothesis: As mentioned above, I predicted that radiation drives glioblastoma evolution. Furthermore, I hypothesised that such radiation-induced evolution would lead to the emergence of resistant clones which would contribute to the regrowth of a radioresistant recurrent tumour.

GSC-initiated orthotopic xenograft models were again applied to study the functional implications of possible radiation-induced evolution. Tumour cells were isolated from morbid control and irradiated tumours and were put back into culture. These xenograft-derived cell lines were subsequently tested for evidence of differential radioresponse through clonogenic survival assays and a reimplantation survival study. Such studies may provide valuable information on the potential functional implications of radiation-induced GB evolution.

(4) Can the GSC-initiated xenograft model be used to study recurrent GB treatment strategies?

Hypothesis: I predicted that through regular in vivo tumour growth monitoring, GSC-initiated xenografts could serve as models of recurrent tumour growth and could be successfully reirradiated to provide a clinically relevant model of recurrence and retreatment.

GSC-initiated xenografts underwent a first course of fractionated radiation. After treatment, the irradiated mice were followed by serial imaging to monitor for tumour regrowth. Once regrowth was noted, the same fractionated radiation protocol was reapplied. Survival analysis was performed to determine the in vivo radioresponse of recurrent tumours after

reirradiation. Such studies may not only demonstrate whether such models would be useful for studying recurrent treatment regimens, but may also serve as a more clinically relevant approach for determining functional implications of radiation-induced evolution.