The MUC1-ST/Siglec-9 innate check point axis in cancer: prevalence, prognostic significance and potential therapeutics.

Virtually all proteins carried on the surface of cells are decorated with sugars; these combined structures are known as glycoproteins. These sugars, or glycans, are fundamental to the function of the protein and are involved in cell:cell and cell:environment interactions. Importantly the sugars on these glycoproteins change as cancers develop and so, in cancer, the glycoproteins can interact with a new set of proteins and cells. We have previously shown that one particular glycoprotein, MUC1, expressed by breast cancers carries aberrant glycans that allow it to bind to specific immune cells, called monocytes and macrophages that infiltrate the tumour. Once the binding takes place the monocytes and macrophages change: the monocytes secrete factors that induce tumour growth and recruit more immune cells, and the macrophages become "tumour-associated macrophages" a type of cell that is associated with invasion and a poor prognosis.

In this project we plan to:
a) Determine how common these particular monocytes and macrophages are in breast cancer and to confirm that they are associated with a particular glycoform of MUC1.
b) Determine if the genes expressed by these macrophages induced by MUC1 is associated with prognosis.
c) Determine if inhibiting the interaction between MUC1 and the monocytes and macrophages can be used as a potential therapy for breast and other cancer patients. The interaction of MUC1 and the receptor on monocytes and macrophages acts as a "checkpoint" to inhibit the innate immune response. We hypothesize that blocking the interaction should release the brakes and allow for the immune system to recognise the tumour.

We plan to use of series of in vitro techniques to address these aims and our results should pave the way for preclinical testing of this new therapeutic approach.

Tumour-associated macrophages are some of the most abundant immune cells found in tumours, their presence correlating with metastasis and poor prognosis. Changes in the post-translational modification glycosylation, is a hallmark of cancer and the glycosylation of MUC1 is dramatically altered in breast cancer. We have shown that a tumour-associated glycoform of MUC1 (MUC1-ST) binds to the lectin Siglec-9 expressed by monocytes and macrophages. This interaction results in the release of factors associated with tumour growth, modulation of the microenvironment and the induction of a tumour-associated macrophage (TAM) phenotype.

In this project we will confirm our in vitro data in vivo using primary breast cancers, define the transcriptome of the MUC1-ST induced macrophages and apply it to public databases to determine prognostic significance, and investigate therapeutic applications of blocking the MUC1-ST/Siglec-9 interaction using human in vitro models

We will phenotype macrophages infiltrating into breast cancer by 1) immunohistochemistry and RNAscope 2) flow cytometric analysis, and correlate this with the MUC1 glycoform (MUC1-ST) that binds Siglec-9. Using matched sera and tumour samples we will also determine if there is a correlation between MUC1-ST expressed by the tumours and the presence of factors secreted by myeloid cells after binding to MUC1-ST.
The transcriptome of macrophages differentiated from monocytes by MUC1-ST will be determined by RNAseq allowing us to develop a 'MUC1-ST macrophage' signature, which we will apply to large tumour datasets to understand the impact of these cells on disease.
We will also study the therapeutic potential of using antibodies to Siglec-9 to block the migration of labelled monocytes induced by MUC1-ST educated monocytes using in vitro assays and an organotypic breast cancer culture model. The effect on angiogenesis will also be investigated in vitro.

This research will further our understanding of an entirely novel mechanism used by tumours to modulate their microenvironment and evade the host immune response. This may lead to new therapeutic approaches for the treatment of breast and other cancers. The importance of glycobiology in research is not well recognised in the UK compared to other countries, especially the US, where considerable funding streams are dedicated to this field. The project will raise the profile of glycobiology both nationally and internationally.

The beneficiaries of this research fall into the following categories:
1. Contribution to the nations health: The research has high potential to lead to a new therapeutic approach for the treatment of breast and other cancers. The proposal includes ex vivo work to determine the therapeutic potential of antibodies that prevent the interaction that leads to immune suppression. This will generate important preclinical data which will inform further work with animal models and, ultimately, clinical trials.
2. Clinicians will benefit from having additional therapeutic options to recommend to their patients.
3. Fostering the global economy: King's College London has filed a patent application on the research we performed leading up to this proposal (application number of GB1611535.4, entitled "Methods and Compositions for Treating Cancer with Siglec-9 Activity Modulators", filing date 1 July 2016). King's have licensed the rights under this patent to a company. The Siglec family of lectins have been described as the checkpoint of the innate immune system in ours and others work. Adaptive Immune Checkpoint Inhibitors are estimated to be a multi-billion dollar market over the coming decade. During the period 2015 - 2025, forecast suggests that the market will witness an annualized growth rate of 27.7% (Roots Analysis, Business Research and Consulting). Our research will provide further evidence that Siglecs, in particular Siglec-9, can act as innate immune checkpoints and therefore be excellent immunotherapeutic targets.
4. Public engagement including patients and young people: We feel patients benefit from a greater understanding of their disease and we hope to inspire young people giving them an interest in science and how it can be translated into benefits for society.
5. Academics within King's College and Leeds University, and the national and international academic community will benefit from the research. This has been discussed in detail in the academic beneficiary section.

Timescale:
Short term
Academics and young people should benefit from the research being conducting during at least year 2 and 3 of the project
Longer term:
Contribution to the nation's health: The idea of blocking interactions to enhance naturally occurring adaptive neoantigen immune responses is an idea that is moving fast in oncology as seen with the relatively rapid introduction of immune checkpoint inhibitors as therapeutic options for multiple cancers. It is hoped that clinical trials to block Siglec-9/MUC1 interactions as a single therapy and in combination with other agents will enter clinical trials in five years.
Fostering the global economy: This will happen within 10 years if the clinical trials are positive.