Thank you Aakriti! 🙏

Thank you Dimitris 🙏

Thank you for all your help with single cell imaging approaches! A fab collaborator as ever ♥️

Couldn't have done it without you!! ♥️

Thanks Dylan!

Thank you Michalis ♥️

Heterogeneity is rife in all tumours and we’re excited to explore drug distribution with other drug/cancer combinations, and the implications for efficacy and resistance. Finally, a huge thank you to the Fets Lab members, all collaborators and of course the patients. We’d love to hear any feedback!

By contrast, Olaparib isn’t a weak base so shouldn’t accumulate in lysosomes. Sure enough, when we measured intracellular concentration of all three drugs, Olaparib was not displaced by increased lysosomal pH, but in accordance with our GeoMx data, both Niraparib and Rucaparib were.

But why does Rucaparib accumulate in lysosomes to begin with? Rucaparib (and also niraparib!) are weak bases, becoming protonated and therefore more hydrophilic at the low lysosomal pH. This creates a gradient that favours more drug across the cell

…and in fact, the more rucaparib can accumulate in the lysosomes, the higher the signal in the nuclear compartment, where it interacts with its target proteins PARP1 and PARP2!

But how does this impact activity? We found that high-Rucaparib cells showed increased DNA damage (γH2AX) and reduced proliferation after treatment, suggesting the lysosomal drug pool isn’t ‘trapped’, but contributes to efficacy…

Importantly, the more acidic the lysosome (modulated with V-ATPase inhibitors or activators), the more rucaparib accumulated within the cell, showing that this organelle really was a driver of intracellular rucaparib concentration

In fact, the punctate distribution of rucaparib co-localised with lysosomes, and the content of lysosomes per cell was highly correlated to rucaparib content!

As well as imaging, we could FACS sort cells that accumulate high rucaparib and compare to low drug cells. By doing this as early as one hour after treatment to minimise drug responses, we could better understand what was driving differential accumulation, which again revealed lysosomal signatures…

….and shows heterogenous accumulation even within cell line models— a perfect tool to dig deeper.

The PDE system is powerful, but takes 24h for the drugs to reach steady-state throughout, making it tricky to unravel the factors driving differential drug accumulation from concentration-dependent drug responses. BUT! Rucaparib is naturally fluorescent…

Using GeoMx Spatial Transcriptomics on adjacent slices for rucaparib and niraparib-treated PDEs, we compared high and low drug regions. High drug regions were enriched in apoptotic signatures, and intriguingly, we also found an association with #lysosomal signatures!

Dosing ex-vivo, we could image drug distribution independently of tumour vasculature, to explore cell-intrinsic differences in drug accumulation. Mass Spec Imaging, with expert tutelage of @zoehall-icl.bsky.social, revealed particularly striking heterogeneity in rucaparib and niraparib-treated PDEs

We asked whether intra-tumour heterogeneity affects drug distribution, and if this impacts drug response. To answer this, Carmen set up a multimodal imaging pipeline using PARP inhibitor-dosed patient derived explants (PDEs) from HGSOC tumours (big thanks to Paula Cunnea&Christina Fotopoulou!)