Revealing calcium channel function in neuroendocrine cancers by integrating electrophysiology and 3D super-resolution microscopy

Background. Neuroendocrine (NE) cancers are tumours whose cells express features of neuroendocrine cells.

They include some of the most aggressively invasive and lethal cancers, for example small-cell lung cancer (SCLC) and neuroblastoma.

In addition to the many types of primary NE tumours, breast, prostate and other cancers can evolve towards a small-cell NE phenotype during progression. Improved understanding of NE cancers and new therapeutic strategies are urgently needed. Both normal and cancerous NE cells are electrically excitable and secretory.

Previous work indicates that calcium-permeable voltage-gated and receptor ion channels play key roles in the electrical excitation, secretion and motility of NE cells, and in autoimmune complications of NE cancer. Aims. We aim to reveal calcium channel function in NE tumours through super-resolved optical electrophysiology.

A novel 3D super-resolution microscope will be developed, customised to image at 100 μm depth, 30 nm 3D resolution and millisecond time resolution.

Single functional calcium channels and nanodomain calcium signalling will be resolved in 3D cultures and sliced live tumour tissue, in concert with patch-clamp electrophysiology.

We will determine how different types of calcium channels are activated during invasive growth, and in parallel, we will investigate how targeting calcium channels in vivo can inhibit invasion of NE cancers.

Methods. 3D tumouroids of cell lines from genetically-engineered mouse models and human NE cancer cell lines, and acute live tumour tissue slices from intrasplenic transplantation will be used.

Extended-depth super-resolution will be achieved through beam-shaping techniques using microlens arrays and phase masks that establish axial asymmetry. This will be combined with whole-cell patch-clamp, incorporating fluorescent calcium indicators.

Single active calcium channel localization will be achieved by imaging channel-associated calcium influx, complemented by immunolabelling. Fluorescent membrane-bound probes will be combined with capacitance measurement to localize and quantify secretion. In vivo, metastasis of NE cells will be modelled by seeding of cells in organotypic tissue slice cultures.

In vivo, intrasplenic transplantation assays will be used to test the effects of targeting calcium channels genetically and pharmacologically.

How the results of this research will be used. 3D super-resolution live-cell microscopy integrated with electrophysiology will provide an unprecedented level of fundamental knowledge about how membrane potential and calcium channel activation determine calcium signalling in NE cancer cells, This will provide a scientific basis for future therapies targetting ion channel and calcium-related signalling in neuroendocrine cancers.