Tumor-infiltrating nerves functionally alter brain circuits and modulate behavior in a mouse model of head-and-neck cancer

Reviewer #1 (Public Review):

Summary:

Using a mouse model of head and neck cancer, Barr et al show that tumor-infiltrating nerves connect to brain regions via the ipsilateral trigeminal ganglion, and they demonstrate the effect this has on behavior. The authors show that there are neurites surrounding the tumors using a WGA assay and show that the brain regions that are involved in this tumor-containing circuit have elevated Fos and FosB expression and increased calcium response. Behaviorally, tumor-bearing mice have decreased nest building and wheel running and increased anhedonia. The behavior, Fos expression, and heightened calcium activity were all decreased in tumor-bearing mice following nociceptor neuron elimination.

Strengths:

This paper establishes that sensory neurons innervate head and neck cancers and that these tumors impact select brain areas. This paper also establishes that behavior is altered following these tumors and that drugs to treat pain restore some but not all of the behavior. The results from the experiments (predominantly gene and protein expression assays, cFos expression, and calcium imaging) support their behavioral findings both with and without drug treatment.

Weaknesses:

Study suggests that the effects of their tumor models of mouse behavioral are largely non-specific to the tumor as most behaviors are rescued by analgesic treatment. So, most of the changes were likely due to site-specific pain and not a unique signal from the tumor.

Reviewer #2 (Public Review):

Summary:

Cancer treatments are not just about the tumor - there is an ever-increasing need for treating pain, fatigue, and anhedonia resulting from the disease as patients are undergoing successful but prolonged bouts with cancer. Using an implantable oral tumor model in the mouse, Barr et al describe neural infiltration of tumors, and posit that these nerve fibers are transmitting pain and other sensory signals to the brain that reduce pleasure and motivation. These findings are in part supported by anatomical and transcriptional changes in the tumor that suggest sensory innervation, neural tracing, and neural activity measurements. Further, the authors conduct behavior assays in tumor-bearing animals and inhibit/ablate pain sensory neurons to suggest the involvement of local sensory innervation of tumors in mediating cancer-induced malaise.

Strengths:

• This is an important area of research that may have implications for improving the quality of life of cancer patients.

• The studies use a combination of approaches (tracing and anatomy, transcriptional, neural activity recordings, behavior assays, loss-of-function) to support their claims.

• Tracing experiments suggest that tumor-innervating afferents are connected to brain nuclei involved in oral pain sensing. Consistent with this, the authors observed increased neural activity in those brain areas of tumor-bearing animals. It should be noted that some of these brain nuclei have also been implicated in cancer-induced behavioral alterations in non-head and neck tumor models.

• Experiments are for the most part well-controlled, and approaches are validated.

• The paper is well-written and the layout was easy to follow.

Weaknesses:

• The main claim is that tumor-infiltrating nerves underlie cancer-induced behavioral alterations, but the experimental interventions are not specific enough to support this. For example, all TRPV1 neurons, including those innervating the skin and internal organs, are ablated to examine sensory innervation of the tumor. Within the context of cancer, behavioral changes may be due to systemic inflammation, which may alter TRPV1 afferents outside the local proximity of tumor cells. A direct test of the claims of this paper would be to selectively inhibit/ablate nerve fibers innervating the tumor or mouth region.

• Behavioral results from TRPV1 neuron ablation studies are in part confounded by differing tumor sizes in ablated versus control mice. Are the differences in behavior potentially explained by the ablated animals having significantly smaller tumors? The differences in tumor sizes are not negligible. One way to examine this possibility might be to correlate behavioral outcomes with tumor size.

Reviewer #3 (Public Review):

Summary:

The authors have tested for and demonstrated a physical (i.e., sensory nerves to the brain) connection between tumors and parts of the brain. This can explain why there is an increase in depressive disorders in HNSCC patients. While connections such as this have been suspected, this is a novel demonstration pointing to sensory neurons that is accompanied by a remarkable amount of complementary data.

Strengths:

There is substantial evidence provided for the hypotheses tested. The data are largely quite convincing.

Weaknesses:

The authors mention in their Discussion the need for additional experiments. Could they also include / comment on the potential impact on the anti-tumor immune system in their model?

Minor:

The authors mention the importance of inflammation contributing to pain in cancer but do not clearly highlight how this may play a role in their model. Can this be clarified?

The tumor model apparently requires isoflurane injection prior to tumor growth measurements. This is different from most other transplantable types of tumors used in the literature. Was this treatment also given to control (i.e., non-tumor) mice at the same time points? If not, can the authors comment on the impact of isoflurane (if any) in their model?

The authors emphasize in several places that this is a male mouse model. They mention this as a limitation in the Discussion. Was there an original reason why they only tested male mice?