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Brain neurons form functionally relevant excitatory glutamatergic synapses with metastatic cells of non-neural cancers

Jan 15, 2024 | Neuroscience (Featured)

In this study, the authors from Germany investigated whether synaptic contacts exist between neurons and brain metastatic cells of non-neural cancers, at which stage of the brain metastatic cascade synapses between neurons and individual metastatic cancer cells are formed, and whether these synaptic contacts support metastasis and cancer progression. 

The high-grade gliomas form synapses that hijack electrical signals from healthy nerve cells to drive their growth. The expression of Ca2+ permeable glutamate AMPA receptors in the glioma cells is a key feature of neuron-glioma synapses. AMPA receptors are heterotetrameric complexes composed of subunits GluR 1-4. These glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. The researchers pointed out that neuron-tumor synapses were not detected in brain tumors of lower malignancy (oligodendrogliomas or meningiomas). However, very aggressive, incurable, primary, or secondary brain tumors receive neuronal synaptic input, driving a disease progression.

High morbidity and mortality are associated with brain metastases, most often originating from breast and lung cancer, and melanoma. It has been reported that the formation of excitatory synapses between presynaptic neurons and postsynaptic cancer cells in certain cancer types of neural origin stimulates tumor growth and invasion (Venkataramani, V. et al. Nature 2019; 573, 532–538. https://www.nature.com/articles/s41586-019-1564-x)

 

 

 

About the study

Mice (>8 weeks old) were anesthetized and injected with 500,000 human brain metastatic cells in the left ventricle. Female mice were used for the breast cancer model of brain metastases, and, male mice for the melanoma model. The brains of mice were separated and prepared for cryo- or vibratome sectioning. To conduct intravital imaging, a chronic cranial window with a titanium ring was administered to the mice, at least three weeks before intracardial injection of human brain metastatic cells.

The authors assessed whether and at what stage of the brain metastatic cascade, synapses between neurons and individual metastatic cancer cells were formed. To identify the earliest stages of brain seeding, researchers performed intravital correlative microscopy through a cranial window in mice, followed by three-dimensional electron microscopy. To investigate synaptic connections between neurons and brain metastatic cells, the researchers performed patch-clamp recordings of single breast cancer or melanoma cells during early growth in the perivascular niche. 

 

The results

When circulating breast cancer and melanoma cells from tumor models left the blood vessel and extravasated into the mouse brain, they were consistently found in a perivascular niche. These findings suggested that this niche has a survival-promoting function for non-neural, metastatic cancer cells. Importantly, Ca2+ transients were detected in vivo during metastatic seeding of the perivascular niche and early proliferation. Moreover, the increase in growth coincided with Ca2+ activity in breast cancer micrometastases, which contrasted with brain metastases that were Ca2+ silent.

In 20% of single breast cancer cells or micrometastases, clear synapses between neurons and individual metastatic cancer cells were detected in the perivascular niche. In addition, electron microscopy revealed synapses between neurons and individual metastatic cancer cells in the micrometastatic stage of two models of melanoma brain metastases.

Since neuronal stimulation can increase the concentration of Ca2+ in the cytosol, the researchers examined the potential impact of neuronal activity on metastatic cancer cells. In mice with metastatic breast cancer or melanoma, intravital microscopy showed that Ca2+ transients in brain cancer cells were significantly reduced when the mice were anesthetized with ketamine/xylazin, compared to the Ca2+ transients recorded in the same brain regions when these mice were awake. Importantly, Ca2+ transients recorded in cancer cells, associated with neuronal activity in a crucial metastatic niche, provided the first evidence of functional communication between neurons and metastatic cancer cells. The inability to induce action potentials in brain metastatic cells suggests that these cells cannot generate regenerative potentials. Instead, they were the receivers of unidirectional synaptic input from neurons.

The patch-clamp recordings of single breast cancer and melanoma cells during early growth in the perivascular niche demonstrated that synapses between neurons and brain metastatic cancer cells generate postsynaptic currents mediated by AMPA receptors. The presence of spontaneous excitatory postsynaptic currents (sEPSCs) in a subset of tumor cells confirmed that synapses between presynaptic neurons and postsynaptic tumor cells were functional. These currents demonstrated a fast rise time and exponential decay, the hallmark features of AMPA receptor-mediated sEPSCs. In breast cancer and melanoma cells, the sEPSCs were completely blocked by cyanquixaline, a specific antagonist of AMPA receptors, confirming that AMPA receptors functionally contribute to synapses between neurons and metastatic cancer cells.

To further confirm the role of AMPA receptors on the growth of brain metastases in vivo, the scientists pharmacologically inhibited the AMPA receptor function with perampanel, a selective and noncompetitive antagonist of AMPA receptors and FDA-approved drug for epilepsy treatment. Importantly, the administration of perampanel resulted in a lower metastatic burden and fewer brain metastases per mouse.

 

 

Conclusion

This study has shown, for the first time, that neurons can form physiologically relevant excitatory glutamatergic synapses with metastatic cells of non-neural cancers. This started at very early stage after extravasation into the brain parenchyma, during the residence of cancer cells in the perivascular niche, which is a critical step for their survival.

It is important to note that cancer cells always harbored the post-synapse and never showed pre-synaptic features. In addition, the vast majority of synapses between neurons and individual metastatic cancer cells were indeed direct synapses between presynaptic neurons and postsynaptic cancer cells, without a non-malignant neuronal structure co-located on the postsynaptic side. These findings indicate de novo synaptogenesis in brain metastases. This is opposite to results observed in glioma cancer cells, which demonstrated a frequent hijacking of pre-existing brain synapses.

The neuron-cancer synapses generated excitatory postsynaptic currents mediated by AMPA glutamate receptors in cancer cells, with cancer cells on the postsynaptic (receiving) side of the synapses. These AMPAergic synapses not only support and stimulate cancers of neural origin, like gliomas but also brain-seeding of metastatic breast cancer and melanoma cells. The discovery of such Ca2+ transients, that depend on neuronal activity in breast cancer and melanoma cells, suggests a common mechanism by which synaptic interactions between neurons and cancer cells can be translated into growth-promoting signals.

The pharmacologic inhibition of these neuron-metastatic synapses with an approved antiepileptic drug perampanel strongly reduced metastatic burden and opened up the possibility for a new, clinically actionable concept of metastasis prevention. The authors concluded that the discovery that neurons form synapses with metastatic cells of tumors originating outside the nervous system was unexpected, but opened a new chapter in the cancer neuroscience field.

The results of the study have been published on a preprint server and are currently being peer-reviewed.

Journal Reference

Venkataramani V, Karreman MA, Nguyen LC, et al. Direct excitatory synapses between neurons and tumor cells drive brain metastatic seeding of breast cancer and melanoma. BioRxiv preprint.  https://doi.org/10.1101/2024.01.08.574608

 

 

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