REQUISITOS DE LOS CANDIDATOS

Previous work in the Brenton Laboratory (the PhD project of Siru Virtanen) identified phenotypic subpopulations within HGSOC, and presented a putative cancer stem cell popu- lation distinguished by the epithelial surface adhesion marker EpCAM. Sorting of EpCAM+ and EpCAM− cells from HGSOC patient samples followed by growth in vitro showed no proliferation from EpCAM+ _{cells, while EpCAM}− _{cells were able to proliferate, and give}

thermore, this EpCAM− subpopulation could be further subdivided by the marker surface marker podoplanin. The subpopulation positive for podoplanin showed the same ability to proliferate in in vitro assays, while the podoplanin negative subpopulation showed no proliferation, much like the EpCAM+_{subpopulation.}

Attempts to replicate this experiment in vivo struggled at first, as none of the subpop- ulations appeared able to initiate tumours in vivo in isolation. In contrast, mixed cell populations were able to initiate tumours in the same context with far fewer cells. Based on the hypothesis that this result indicated that EpCAM− TICs are dependent on cell-cell interactions with bulk EpCAM+_{tumour cells, a second experiment was devised. Cells were}

sorted into subpopulations as before and, in each case, a single subpopulation labelled us- ing a constitutive lentivirus expressing the fluorophore zsGreen (seeFigure 1.2). When the EpCAM−/Podoplanin+ subpopulation was labelled, the majority of the resulting tumour showed green fluorescence labelling. Approximately 70% of the cells in the resultant tumour were labelled, which corresponds well with the ∼70% efficiency of the labelling process. When either the EpCAM−/Podoplanin−, or the EpCAM+_{subpopulation were labelled, the}

resulting tumour did not show detectable fluorescence. In all cases the resulting tumour was composed of a heterogeneous mix of all three subpopulations. This indicates that these in vivo tumours had arisen from the EpCAM−/Podoplanin+ _{subpopulation, and that this}

subpopulation had given rise to a phenotypically mixed tumour.

Given this evidence, a cancer stem cell (CSC) model was proposed (seeFigure 1.3). This hypothesises that in a HGSOC tumour, the EpCAM−/Podoplanin+ _{cells represent a pool}

of indefinitely cycling CSCs which not only maintain and grow their own numbers, but are also the origin of the EpCAM+ _{bulk tumour. The EpCAM}+ _{cells in this hypothesis are}

not proliferative, and their numbers are instead replenished by the EpCAM−/Podoplanin+ CSCs which can divide asymmetrically.

This replenishment may also occur through Transit-Amplifying Cells (TAs). In this variant on the CSC model, CSCs divide very slowly, giving rise to TA cells which can divide rapidly, but only for a limited number of cycles, before they themselves become quiescent. It is not clear what phenotype these TA cells would have since they would not be detected by the experiments previously described, since all proliferation is ultimately originating from the CSCs (since any transplanted TAs would be expended within a handful of division cycles). One further observation made by Siru was that treatment of HGSOC cells with cisplatin chemotherapy both in vitro and in vivo appeared to selectively enrich for the TICs. This would suggest that these cells have some means of preferential survival under chemotherapy conditions. This finding might tend to favour a TA-based model, since this would allow for slow-cycling CSCs which would naturally be expected to be less affected by platinum chemotherapy. In this model the rapid division occurs within the TA fraction. These cells would be quickly killed off by chemotherapy, but could be repopulated by slow-growing CSCs. Alternatively, it is possible that the CSCs are faster cycling, but protected from chemotherapy by intrinsic resistance mechanisms (drug efflux or upregulated DNA repair

Figure 1.2: Previous Brenton Laboratory experiments studying tumour initiation. Top panel shows in vitro sphere-forming and colony-forming assays. EpCAM− cells produce spheroids and colonies while EpCAM+ _{do not appear to proliferate. Bottom panel shows labelling}

experiments in which each subpopulation in turn was labelled with an HIV-zsGreen consti- tutive marker. When the EpCAM− subpopulation was labelled, the resulting tumour was marked. When EpCAM+ _{was labelled, the tumour was unmarked. Resulting spheroids,}

colonies, and xenografts all showed mixed heterogeneity, suggesting the EpCAM− subpop- ulation drives proliferation.

Figure 1.3: The cancer stem cell hypothesis. Top panel shows a model CSC hierarchy; bottom panel shows how such a hierarchy would explain the results summarised inFigure 1.2

mechanisms as discussed previously).

This was however a relatively minor experiment, and as a result the data are limited. In addition, although the EpCAM+_{fraction is much reduced, there is only a small increase in}

relative EpCAM−/PDPN+ _{proportion. This suggests that the bulk of the relative increase}

may be in the EpCAM−/PDPN− fraction, which is not demonstrated to have tumour- initiating capacity.

In a 2015 paper, Janzen et al. reported a strong inverse correlation between CA-125 and TIC capacity (Janzen et al.,2015). The CA-125−subpopulation appears to be considerably more efficient in generating xenograft tumours (as well as resulting in larger tumour size and reduced latency). The conclusions of this paper are supported by previous literature sug- gesting that CA-125 may be downregulated in cells undergoing epithelial-to-mesenchymal transition (Comamala et al., 2011). Furthermore, this may explain why CA-125, used as a clinical marker for diagnosis of HGSOC, is suboptimal as a marker of relapse; if disease regrows from a chemoresistant subpopulation deficient in CA-125, this clinical test would only show a meaningful rise once these cells had given rise to bulk cells high in CA-125. Interestingly some tumours did still grow from the CA-125+ _{fraction, though it is not clear}

if this represents CA-125 being imperfect for discriminating TICs, or simply the technical limits of separation within the experiment. The authors performed equivalent viral labelling experiments to those performed by Siru, and produced similar but not identical results. When the CA-125− subpopulation was labelled, the resulting tumours were heterogeneous but highly labelled, and similar conclusions can be drawn from this as can be drawn from Siru’s work, suggesting that the CA-125− subpopulation and the EpCAM− subpopulation identified by Siru may be one and the same. However, when they labelled the CA-125+

subpopulation, the resulting tumours were far less strongly labelled, and predominantly in only the CA-125+ _{subpopulation, suggesting they may have successfully marked unipotent}

progenitors. This CA-125−_{population also shows enrichment in response to platinum ther-}

apy (again in accordance with Siru’s work), and the authors build on this, demonstrating evidence to suggest upregulation of DDR factors, quicker resolution of γH2AX foci, and suppression of pro-apoptotic factors in favour of anti-apoptotic ones, including the cIAP proteins which inhibit caspase activation (although the rigour of the transcriptional data has been called into question as errors in the analysis have some to light (Janzen et al.,

2016) ). Furthermore they then go on to test the cIAP inhibiting agent Birinapant which when combined with platinum therapy shows a potent ability to deplete HGSOC xenograft tumours. Treatment with platinum alone results in a residual CA-125− population, but co-treatment appears to eliminate this population too, eliminating near to all tumour mass after 3 weeks of treatment. No tumour regrowth was seen in these samples, versus singly treated xenografts which were all observed to relapse – however, data for this follow-up is shown for only 3-4 weeks, so this may well simply represent a slightly longer latency to relapse rather than complete elimination of the disease.