The role of the peripheral nervous system on tumor progression: an anatomic investigation

There is a growing appreciation for the interplay between the nervous system and cancer progression. Accordingly, neuromodulation of adrenergic signaling to tumors is a promising new avenue for cancer therapy. The purpose of this study was to characterize the neural anatomy for orthotopic liver tumors in a rat model of hepatocellular carcinoma and evaluate the potential role for studying neuromodulation on disease progression in this tumor model.

Materials and Methods:

An HCC model was generated by orthotopic liver implantation of McA RH7777 (ATCC) hepatoma cells in Buffalo rats. Injection sites were then dried with gauze to prevent leakage to surrounding tissue and the circulation. After the tumors reached 10mm in size, the rats were deeply anesthetized with isoflurane and transcardially perfused with ice-cold physiological saline followed by 10% buffered formalin. A dual tracing experiment was performed by injecting the tracers into spleen and liver with Alexa FluoroVR (AF)-conjugated cholera toxin subunit B (CTb). Six days after injection, thoracic organs were removed and verified with a fluorescent stereomicroscope and only animals with visible ganglia fluorescence were further included in tissue dissection and analysis. Ganglia were removed under microscopy and fixed in 10% buffered formalin and embedded in OCT. Liver tumors were harvested and cryopreserved followed by staining with primary antibodies targeting nerve tissue including anti-tyrosine hydroxylase (rabbit), NF-H chicken Polyclonal Antibody, ChAT recombinant mouse monoclonal antibody, and VGAT polyclonal antibody. Tissue slices were then examined using a Leica SP5 confocal microscope.

Results:

The cholera toxin subunit tracer injected into the spleen and liver tissue were able to successfully localize to the splanchnic ganglia innervating these organs. Migration of this tracer from its injection site to ganglia cell bodies was confirmed by colocalization with TH staining on confocal microscopy. Furthermore, confocal microscopy demonstrated a large quantity of neural tissue within the tumor, with target-to-background ratios > 3:1 relative to adjacent non-malignant liver tissue. This was true for markers of cholinergic (ChAT) and GABAergic (VGAT) neurotransmission.

Conclusion:

We have identified the relevant splanchnic ganglia innervating the liver in spleen and also confirmed the abundance of neural tissue within orthotopic HCC tumors.  Neuromodulation of these targets may have therapeutic implications on tumor progression and is the subject of future studies.