A quadruplex-DNA binder functionalised with a SPECT probe for in vivo imaging

Guanine-rich sequences of DNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target quadruplexes. However, to date, there is very limited information of the biodistribution of quadruplex binders in whole organisms. Here, we report the functionalisation of a well-established G4 DNA binder (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies have shown the overall biodistribution of the G4 DNA binder highlighting its accumulation in the tumour.

CD8+ T cells inhibit metastasis and CXCL4 regulates its function

Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. Results We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. Conclusions CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

Resolving Tumour Clonal Heterogeneity and Spatial Complexity using Nuclear Tandem Epitope Protein (nTEP) Barcoding

ABSTRACTTumours are composed of an array of unique cancer cell clones along with many non-tumour cells such as immune cells, fibroblasts and endothelial cells, which make up the complex tumour microenvironment. To better understand the co-evolution of tumour clones and cells of the tumour microenvironment, we require tools to spatially resolve heterotypic cellular interactions at the single cell level. We present a novel protein-based barcoding technology termed nuclear tandem epitope protein (nTEP) barcoding, which can be designed to combinatorially encode and track dozens to hundreds of tumour clones in their spatial context within complex cellular mixtures using multiplexed antibody-based imaging. Here we provide proof-of-principle of nTEP barcoding and develop the technology, which relies on lentiviral - based stable expression of a nuclear-localised fluorophore that contains unique combinations of protein epitope tags that can be decoded by a limited set of antibodies. By generating a series of cell lines expressing unique nTEP barcodes, we were able to robustly identify and spatially deconvolve specific clones present within highly complex cell mixtures at the single cell level using state-of-the-art iterative indirect immunofluorescence imaging (4i). We define the utility of nTEP-barcoding as a powerful tool for visualising and resolving tumour heterogeneity at the cellular level, and envision its usage in mouse tumour models for understanding how tumour clones modulate and interact with stromal- and immune cells in cancer.