PARA PRÁCTICAS CLÍNICAS (CURSO 2004-2005)

At present, the greatest challenge in the field of translational research into HGSOC is the limited numbers of tractable therapeutic approaches. The deployment of PARP inhibitor drugs in routine clinical practice in recent years represents a notable success story and an example of how an improved understanding of fundamental biology can be exploited to the clinical benefit of patients (Konecny & Kristeleit,2016). However, these developments are only of benefit for a small percentage of patients, and further work is urgently required to improve the dismal prospects of the majority of HGSOC sufferers (Bowtell et al.,2015). A number of promising concepts have been proposed which may offer additional angles for development of new therapeutic approaches. These include various approaches to stratify- ing HGSOC by genomic features, an approach which has already proven its value in breast cancer (Curtis et al.,2012). This is more complex in HGSOC as the disease is not driven by point mutations but rather by more complex genomic copy-number changes, which them- selves are complex, diverse, and multifactorial (Mittempergher, 2016). Recent efforts are beginning to elucidate prognostic features within this complex ‘genomic chaos’, which with further development will provide additional tools to inform treatment. This type of work potentially opens up additional tractable therapeutic angles through the identification of dysfunctional pathways (e.g. PI3K–AKT signalling), defects in cellular processes (e.g. cell- cycle abnormalities) and routes to genomic dysfunction (e.g. breakage-fusion-bridge events) (Macintyre et al.,2018), which may be exploited for future translational advances.

Other efforts at stratification have focused not on categorising different tumours, but rather the cells found within each tumour population. The aim of these studies is to identify conserved forms of heterogeneity within these populations, and to determine how any sub-

populations interact in case any of these offer therapeutically tractable targets. The cancer stem cell (CSC) hypothesis is a particularly attractive one in this context as it proposes a clear hierarchy of cell types, with distinct proliferative characters, in which decapita- tion of the key cancer stem cell fraction would theoretically prevent disease regrowth, and would allow the remainder of the tumour to be eliminated with conventional chemotherapy. The attractiveness of the hypothesis is also increased by the convincing evidence of cancer stem cells in other malignant tissues such as glioblastoma (Chen et al., 2012), melanoma (Driessens et al.,2012), and intestinal adenoma (Kozar et al.,2013).

Therapeutic exploitation of cancer stem cells in HGSOC is dependent on developing a clear profile of these cells and then identifying exploitable targets for development of pharmaco- logical intervention. As outlined extensively in subsection 1.3.3, research in this area has proved challenging, and a decade of studies have led to a plethora of proposed profiles, with many studies showing conflicting findings, and most finding that their proposed non-CSC fractions remain tumour initiating, albeit at a somewhat reduced rate.

This study sheds light on these findings by presenting data on proliferation dynamics within HGSOC tumours grown in vivo, along with empirical tumour growth rates, which indicate that proliferation within HGSOC is widespread across the tumour, and not isolated to a small fraction of cells. Examination of the parameters of these data through modelling of various hypothetical situations indicates that a CSC model would require significantly different parameters and suggests that the CSC hypothesis is wholly incompatible with the observations made here. This conclusion completely undermines the therapeutic rationale for interest in CSCs as it refutes the idea that there is a distinct subpopulation which if eliminated would render the tumour unable to regrow. As such it offers a basis on which to re-evaluate the focus of research in this area, rather than investing further time and resources in the isolation and characterisation of a subpopulation whose elusiveness now appears to be because it does not exist.

Beyond the generation of evidence to refute a prevailing hypothesis, this work also offers some initial clues into potential future avenues for study. The apparent stochastic nature of HGSOC proliferation outlined in this work, along with the slow-cylcing subpopulation identified, provides an elegantly simple explanation for disease reccurence observed in clinical practice, as the proportion of slow-cycling cells waxes and wanes in response to treatment and disease regrowth. This work indicates (although does not conclusively demonstrate) that the emergence of the slow-cycling population as the major population as a result of chemotherapy may well be due to the lower frequency at which these cells enter cell-cycle (as these cells are ‘vulnerable’ far less frequently). This outlines a possible biological vulnerability of the wider tumour cell population. If the rate of entry into cell-cycle is correlated with the rate of attrition as a result of chemotherapy, then further research into this area may prove clinically valuable. For example this hypothesis would suggest that extending the window over which patients are treated with carboplatin chemotherapy would kill a higher proportion of tumour cells and result in more effective debulking. Whether better debulking would

improve outcomes, and how the additional patient toxicity could be mitigated are open questions, but this research provides the necessary foundation for subsequent translational research.

Chapter 2

Materials and methods

The following list of materials and methods details final methodologies for each experiment described. Initial methodological approaches, and the optimisation process are described in more detail in later chapters.

2.1

Xenograft generation

2.1.1

Tissue processing

Patient solid tumour samples were obtained from Addenbrooke’s Hospital as part of the OV04 study. Samples were collected by staff of the Addenbrooke’s Hospital Human Research Tissue Bank who used a variety of methods, including imprinting methods and sections cut from samples snap-frozen in liquid nitrogen, to assess them (as discussed further in Chapter 3). Histological assessment and cellularity estimates were then (typically) made by a Consultant Pathologist. Where these samples were confirmed to be HGSOC, or where a complete assessment was not available, they were made available to the Brenton Lab. Confirmed non-HGSOC samples, and those determined to be benign were not used. Ascites samples (liquid build-up in the peritoneum containing single and clustered tumour cells) were also collected as part of the wider OV04 study.

Collected tumour material was processed according to a standard operating protocol es- tablished for handling of the OV04 solid tumour material. Samples were transported to the laboratory on ice, and processed as soon as possible upon receipt. Several small pieces (0.25 cm3) were cut from each sample. The first was snap-frozen at −80◦C, and stored for DNA extraction for genomic characterisation. The second was fixed for 24 hours in 10% neu- tral buffered formalin (NBF) before being transferred to 70% ethanol for wax embedding and sectioning (for histology and immunohistochemistry) within the institute’s Histopathology

Core Facility. Samples were routinely haematoxylin/eosin stained to confirm a morphology consistent with tumour, and stained for p53 by immunohistochemistry (IHC) as exception- ally strong staining for p53 (due to over-stabilisation and aggregation of the protein) or else its complete abolition is a distinctive characteristic of HGSOC. Selected samples, including all of the tumours used for the in vivo work, were also further characterised, particularly for EpCAM and WT-1 (another distinctive marker of high-grade tumour). Additional tumour pieces were surgically implanted into mice as described in section 2.2, or were placed in freezing media and stored at −80◦C in the same fashion as for single cell solution (described below).

The remaining sample material was then mechanically dissociated (manually using scalpels) until the pieces were small enough to be drawn up in a 25 mm stripette, and then dissociated overnight (16 h) at 37◦C and 80 RPM on a rotary shaker in order to release the cells from any extracellular structures. Dissociation was conducted in a baffled flask using a dissociation mix of 14 ml DMEM:F12 (50:50) media (Gibco), 5 ml of 7.5% bovine serum albumin fraction V (Invitrogen), insulin (Sigma) at 2.5 ng ml−1 total concentration, gentamycin (Gibco) at 50 ng ml−1, collagenase A (Roche) at 1 mg ml−1and 100 U ml−1hyaluronidase (Sigma). The resulting material was centrifuged at 475 g and the supernatant discarded (where samples with a high fat content gave rise to a layer of fatty material, this was pre-emptively aspirated off to avoid it coating the tube and the pelleted sample). Cells were washed with DMEM:F12 and centrifuged again at 210 g to remove non-cellular debris. The pellet was resuspended in 1 ml of 0.25% trypsin (in citrate buffer) (STEMCELL Technologies) for 4 min to break apart clusters of cells, then washed with DMEM:F12 and centrifuged at 475 g. Next the cells were resuspended in 1 ml of 5 U ml−1 dispase (in HBSS) (STEMCELL Technologies), containing DNAse (Sigma) to a total concentration of 0.1 mg ml−1, for another 4 min to further separate cells, and degrade DNA from dead cells to prevent it binding live cells together; cells were then washed and pelleted as previously. Cells were resuspended in a pre-cooled (4◦C) 3:1 mix of 0.8% ammonium chloride solution (STEMCELL Technologies) and DMEM:F12 to lyse red blood cells, then washed and pelleted as previously. The pellet was resuspended in phosphate buffered saline (PBS) and passed through a 100µm filter to remove undigested material. The cell number was estimated using a haemocytometer and a light microscope, and cells were resuspended in the appropriate volume of freezing media (50% heat-inactivated fetal bovine serum (FBS) (Gibco), 44% DMEM:F12, and 6% dimethyl sulphoxide (DMSO)(Fisher Chemical) and frozen at −80◦C in vials of 5 million or 10 million cells in volumes of 1 ml in 1.8 ml cryovials; freezing was performed in ‘Mr Frosty’ containers which use a propan-2-ol filled insulation layer to ensure a smooth temperature transition to maximise cell viability.

2.1.2

Xenograft processing

The protocol used to dissociate primary ovarian tumour samples was adapted and optimised for the dissociation of xenograft tumours. Xenograft bearing mice were killed by a Schedule 1

method (typically cervical dislocation) and their tumours removed using scalpel and forceps. Half of all the previously described volumes (of the same concentration) were used for each of the three enzymatic steps. Tumours were again mechanically dissociated with scalpels (xenograft tumours lack the fibrosis found in primary tumours, so this process is many times faster) and incubated in collagenase A / hyaluronidase dissociation mix for only 2 h. Given the tendency of the digested cells to clump (and possibly limit the enzymes’ access to the cells) the sample was removed from the incubator after one hour and vigorously agitated by hand to break up clumps of material. Where the resulting cells were for immediate experi- mental use rather than culturing, gentamicin and insulin were not added. The centrifugation step at 210 g and the ammonium chloride step were omitted as xenografts have no fat, and minimal fibrosis and vascularisation, making these removal steps unnecessary.

2.1.3

Mechanical digestion

Xenograft tumours were removed as before and minced between two scalpels. In this case they were minced more extensively, with the aim of producing something with the consis- tency of paste rather than pieces ∼1 mm3_{. Once minced, 1.5 ml of DMEM:F12 was added}

and the paste minced again. The liquid was then drawn up by pipette and filtered through a 100µm filter to remove chunks and clumps. The resulting single-cell-solution was then counted, pelleted at 475 g for 5 min and resuspended in an appropriate volume. These cells were then used for subcutaneous implantation in the normal fashion (as described below).