Dissecting the role of extra centrosomes-induced exosome secretion in PDAC microenvironment

Despite progress, cancer is still a serious challenge in medicine. This is particularly true for pancreatic cancer, which has a dismal 5-year survival rate of <5%. This highly aggressive behaviour is cause in part caused by fibrosis, which forms a barrier around the tumour that hinders drug delivery and efficacy. This barrier is cause by changes in a cell population, named stellate cells, induced by the tumour. Combating fibrosis in pancreatic cancer is thus considered to be an important area for therapeutic intervention.

We recently found that a population of cancer cells that carries amplification of a cellular organelle called centrosome is able to activate stellate cells. Centrosome amplification, an aberration that exists in cancer cells, was described in later 1800s as a possible cause of tumourigenesis. We now know that these abnormalities are a common feature of human tumours and can actively contribute to tumour formation. Published work from our lab was the first to identify a novel function of these abnormalities in influencing the surrounding cells (Arnandis et al., 2018). This work demonstrates that cancer cells with amplified centrosomes have a broader role in tumourigenesis than previously anticipated by communicating and changing other cells within tumours. In particular our new unpublished data shows that this communication between cancer cells with extra centrosomes and pancreatic stellate cells leads to their activation, which could contribute to fibrosis in PDAC. This is mediated via the release of small vesicles from cells with extra centrosomes, exosomes, that are packed with factors that will instruct stellate cells to become activated. Thus, understanding how cells with extra centrosomes activate stellate cells could help understanding fibrosis and lead to therapeutic strategies to treat pancreatic cancer patients.

In this research proposal we aim to determine how centrosome amplification induces the release of exosomes and in turn how these vesicles activate stellate cells. We will use a combination of approaches, including live cell imaging, to identify the mechanisms responsible for exosome release. Furthermore, using well-established methods to identify the factors contained in these exosomes, we propose to uncover the molecules responsible for stellate cell activation. We will test if blocking the identified factors prevents stellate activation and fibrosis using 3-D culture models that mimic the interaction between cancer and stellate cells in tumours. Changes in signalling induced by exosomes in stellate cells will also be studied to understand the mechanisms of activate. This could be important to identify signatures of stellate cell activation and fibrosis in pancreatic tumours and will provide another opportunity to identify potential therapeutic targets to block fibrosis. Finally, we will use mouse models to test our findings in vivo. This will provide relevant pre-clinical models to assess the importance of our findings and test response to therapy. Thus, our work will not only contribute to understand for the first time the role of centrosomes amplification in tumour microenvironment but could also have a major impact in developing therapeutic strategies that target fibrosis to treat patients that suffer from pancreatic cancer.

Centrosome abnormalities, in particular centrosome amplification, are common features of human tumours. Work from our lab showed for the first time that cancer cells containing supernumerary centrosomes promote non-cell autonomous invasion of surrounding cells (Arnandis et al., 2018). This is mediated by an oxidative stress response induced by extra centrosomes that culminates with secretion of pro-invasive factors. These findings underscore a novel role for these abnormalities in cancer and open exciting questions regarding how they influence tumours and microenvironment. Our unpublished data demonstrates that centrosome amplification is sufficient to induce secretion of small extracellular vesicles, such as exosomes. Moreover, these exosomes can activate pancreatic stellate cells (PSCs). Both exosome secretion and PSC activation are a result of oxidative stress, suggesting that our findings could have a broader relevance in cancer as many oncogenic events induce an oxidative stress response. This is particularly important in pancreatic cancer, where PSC activation-induced fibrosis constitutes a major barrier to drug delivery and efficacy. Here, we propose to use a combination of approaches, such as microscopy, RNA-seq, proteomics and mouse models to: 1) determine the role of extra centrosomes and oxidative stress in exosome biogenesis and secretion; 2) Identify the exosomal factors responsible for PSC activation and the signalling pathways they regulate; 3) characterise fibrosis in vitro and in vivo and assess the role of exosome-induced PSC activation in pancreatic cancer progression and fibrosis. We will also test if preventing PSC activation impacts response to Gemcitabine in vivo. Our work will be the first demonstration for a role for centrosome amplification in tumour microenvironment, will elucidate mechanisms leading to exosome release in cancer and can lead to the development of therapeutic strategies that target fibrosis.

Centrosome amplification is a common feature of human tumours. However, the contribution of extra centrosomes to tumourigenesis is largely unclear. Our recent work uncovered a novel and unexpected role for centrosome amplification in changing surrounding tumour and non-tumour cells. These findings suggest that extra centrosomes have a broader role in tumourigenesis than previously anticipated Here we propose to investigate the role of extra centrosomes in exosome secretion and in turn how these exosomes impact tumour microenvironment, in particular fibroblast activation.

- We predict that this project will generate major interest not only in the fields of centrosomes and cancer, but also the field of extracellular vesicles. By dissecting the mechanisms of exosomal secretion and the components required to activate PSC cells, we will for the first time describe a function for centrosome amplification in regulating tumour microenvironment. This project has the potential to uncover novel signalling pathways that are regulated by exosome incorporation in PSC cells and how these contribute to PSC activation, an aspect poorly understood.

- In addition to the scientific community, our work will also impact the wider community through the public engagement program at Barts Cancer Institute. Our press office to the local and international media will advertise the work generated in this study. Our laboratory participates in lab tours for the public that provides another opportunity to engage with the general public.

- Because centrosome amplification is a common feature of human tumours, these findings could have a broader impact in understanding how centrosome amplification impacts cancer in general. In addition, we intend to translate our data to develop new exosome-based biomarkers and therapies that target exosome-induced PSC activation and fibrosis. In particular non-invasive of exosome-based and fibrosis signatures that can be used to develop biomarkers for disease progression and therapeutic intervention. Fibrosis also promotes a highly immunosuppressive tumour environment in PDAC, leading to a failure in immunotherapy approaches. Thus, in the future we will also test how blocking exosome-mediated PSC activation changes the immune landscape to PDAC and response to immune checkpoint inhibitors.

- Our previous work on HSET, a kinesin important for the survival of cells with extra centrosomes, opened a new venue for the development of selective cancer therapies, with several independent inhibitors against HSET already developed. By demonstrating a broader impact of centrosome amplification in cancer, our work could further motivate testing such drugs in the clinic.

- If we succeed, our work can have a major impact in the clinic. We will develop early clinical trials with the help of the Experimental Cancer Medicine Centre and our collaborator Hemant Kocher at BCI. Hemant Kocher is a pancreatic surgeon and scientist and the lead of clinical trials for pancreatic cancer. His help will be crucial in designing and implementing such trials.