MOC2-7 cells are HNSCC cancer cells derived from a CXCR3 null mouse on a C57BL/6 background (Judd et al., 2012). Their implantation in male mice results in dense innervation of tumors with nociceptor nerves (Restaino et al., 2023). To define the timing of tumor innervation, C57BL/6 wildtype male mice were orthotopically implanted (oral cavity) with MOC2-7 cells, and tumors were collected on days 4-, 10-, and 20- post-implantation. Western blot analysis of whole tumor lysate indicated expression of Tau, a neuronal marker (Georgieff et al., 1993), as early as day four post-tumor implantation, which increased over time (Figure 1A, Figure 1—source data 1, Figure 1—source data 2, Figure 1—figure supplement 1). A similar increase in the neuronal marker doublecortin (DCX) was noted (Figure 1B, Figure 1—source data 3, Figure 1—source data 4, Figure 1—figure supplement 2). While DCX is well-known for its expression in immature neurons, it is also expressed in adult peripheral neurons, including dorsal root ganglia (Dellarole and Grilli, 2008), and in non-neuronal tissues (Bernreuther et al., 2006). The DCX signal did not originate from the tumors, as MOC2-7 cell lysate was negative for this protein (Figure 1B, Figure 1—source data 3, Figure 1—source data 4). Consistent with this, immunohistochemical staining of these tumors with the neuronal marker β-III tubulin (β3T), demonstrated the increasing presence of nerves beginning on day 4 post-tumor implantation (Figure 1C–E).

To map the origin of tumor-infiltrating nerves and define the circuits they converge upon, mice with palpable oral MOC2-7 tumors (approximately day 15 post-tumor implantation) were intra-tumorally injected with wheat germ agglutinin, WGA, a neural tracer conjugated to a fluorophore (n=10 mice). WGA is a lectin molecule that specifically binds to sialic acid residues present ubiquitously on neuronal membranes. It has been used extensively to map neuronal circuits centrally and in the periphery (Borges and Sidman, 1982; van der Want et al., 1997) and is a known transganglionic and transynaptic neuronal tracer (McNicholas and Michael, 2017; Dumas et al., 1979; Sillitoe, 2016) making it ideal for mapping neural circuits (Tabuchi et al., 2000; Levy et al., 2017; Itaya, 1987; Itaya et al., 1986; Carson and Mesulam, 1982). Following tracer injection, tumor growth was permitted for an additional 3–7 days to allow time for tracer labeling to occur. Animals were then euthanized, and tumors, trigeminal (TGM) ganglia, and brains were harvested and analyzed by microscopy. Microscopic examination of tumors revealed nerves with robust WGA signals (Figure 1F) as well as the V3 branch of the ipsilateral, but not the contralateral, TGM ganglion (Figure 1G and H). Consistent with the restricted labeling of tumor-infiltrating nerves, the tracer did not diffuse outside the tumor mass (Figure 1I).

Examination of brains from MOC2-7 tumor-bearing animals revealed tracer⁺ neurons in specific regions including spinal nucleus of the trigeminal (SpVc), parabrachial nucleus (PBN), and central amygdala (CeA; Figure 1J). Sections of brain from these regions show the presence of tracer-positive neurons (Figure 1K). Tracer injections of equivalent volume and concentration into the oral cavities of control non-tumor-bearing animals did not label the TGM ganglia nor areas in the brain (data not shown). A larger volume (10 μl) of tracer injected into non-tumor bearing mice resulted in tracer labeling of ipsilateral TGM neurons and brain (Figure 1L). These control studies indicate that the nerve density and distribution present in the tumor bed are higher than in control mice, with the consequence that a small volume (2 μl instead of 10 µl) of tracer is sufficient to result in nerve labeling. The mapped circuit encompasses pre-existing connections to brain areas that regulate pain and affect (Yeh et al., 2018; Hu, 2016; Zhu et al., 2022; Russo and Nestler, 2013). These data indicate that the nerve infiltration into the tumor extends this circuit.

The observation of nerves infiltrating the tumor mass prompted us to ask whether they undergo alterations by their mere presence within this ‘foreign’ environment. Consistent with this, we previously found that melanoma-infiltrating neurons have a unique transcriptome (Balood et al., 2022). To test whether this is also the case in HNSCC, we orthotopically implanted MOC2-7 cells into the oral cavity of male wildtype mice. After 14 days, TGM neurons were harvested and analyzed by qPCR using a commercial array for neural transmission and membrane trafficking genes. Compared to control TGM (non-tumoral) neurons, ipsilateral TGM neurons from tumor-bearing animals harbored increased expression (>fourfold) in genes involved in neuronal signaling/receptors (Gabrg1, Gabra4, Grin2c, Grm3) and synaptic transmission (Gria2; Figure 2A; Supplementary file 1; Michetti et al., 2019). The gene demonstrating the highest increase in expression, Fus, was of particular interest; it increases in expression within DRG neurons following nerve injury and contributes to injury-induced pain (Han et al., 2023; Du et al., 2022). Of note, we purposefully used whole trigeminal ganglia rather than FACS-sorted tracer-positive dissociated neurons to avoid artificially imposing injury and altering the transcript levels of these cells (Liu et al., 2022b; Malin et al., 2007). Thus, significantly elevated expression of Fus by ipsilateral TGM neurons from tumor-bearing animals suggests the presence of neuronal injury induced by the malignancy. This is consistent with our previous findings (Inyang et al., 2024) and those of others (Horan et al., 2022) showing that tumor-infiltrating nerves harbor higher expression of nerve-injury transcripts and neuronal sensitization.

Given this transcriptomic alteration, we tested whether tumor-infiltrating neurons exhibit functional alterations. Thus, MOC2-7 tumor-bearing mice were intra-tumorally injected with the fluorophore-conjugated WGA tracer. Five days later, tracer⁺ TGM ganglia were cultured, and responsiveness to noxious stimuli was analyzed by calcium microscopy. Neuronal Ca⁺² responses to capsaicin (300 nM), which binds and activates TRPV1 channels, were measured by Fluo-4AM fluorescence microscopy. We found that tracer⁺ tumor-infiltrating ipsilateral TGM neurons show increased responsiveness to capsaicin compared to tracer negative contralateral neurons (amplitude, Figure 2B–D; the area under the curve, Figure 2E). Similar to contralateral TGM neurons, non-tumor-bearing TGM neurons elicited a normal response to capsaicin (Figure 2F).

This heightened sensitivity could reflect increased TRPV1 expression and/or its phosphorylation. We tested whether this was the case using western blotting and discovered an overexpression of the sigma 1 receptor (σ1R) and increased TRPV1 phosphorylation in the TGM ganglia from tumor-bearing animals (Figure 2G, Figure 2—source data 1, Figure 2—source data 2, Figure 2—source data 3, Figure 2H and I). Of note, the σ1R is an endoplasmic reticulum chaperone protein that directly interacts with TRPV1 and regulates its membrane expression (Ortíz-Rentería et al., 2018), while protein kinase C phosphorylation of serine 502 and 800 of TRPV1 reduces the receptor’s activation threshold (Bhave et al., 2003; Numazaki et al., 2003; Numazaki et al., 2002; Bhave et al., 2002). Taken together, our data indicate that HNSCC-infiltrating nerves have a unique transcriptome and a heightened sensitivity to noxious stimuli characterized by an increased TRPV1 expression and phosphorylation consistent with TRPV1 sensitization secondary to oral cancer (Scheff et al., 2022).

The transcriptional and functional changes evident in tracer⁺ tumor-infiltrating neurons could result in alterations in central target neurons. To assess this, brain sections from MOC2-7 tumor-bearing and non-tumor-bearing animals were immunofluorescently stained for cFos and ΔFosB, two markers of neuronal activity with different courses of expression (Chung, 2015; Hudson, 2018; Nestler et al., 2001). ΔFosB expression was increased in several brain regions of tumor-bearing animals, while cFos expression was also increased in the PBN (Figure 3A–F). Predominant differences in the long-lived ΔFosB were expected (Nestler et al., 1999), as those with the short-lived cFos changes are more challenging to capture using single time point assessment.

Next, we assessed the neuronal activity of tumor-bearing animal brains using stereotaxically injected AAV1-Syn-GCaMP6f, a viral vector encoding a neuron-specific synapsin-driven calcium sensor (Chen et al., 2013b), into the PBN. Two weeks after intra-cranial injection of the virus, mice were orally implanted with MOC2-7 cells. Approximately 18 days post-tumor inoculation, the animals were euthanized, and the neuronal calcium activity was recorded in ex vivo brain slices using a mini scope. While baseline fluorescence was similar between tumor-bearing and control animals, that recorded upon stimulation (KCl; 30 mM) was significantly higher in neurons of tumor-bearing animals (Figure 3G, H, I and J, Figure 3—figure supplements 1–3). These functional and Fos staining data indicate that central neurons within the tumor-brain circuit are functionally altered compared to their healthy brain counterparts.

Cancer patients, and even more notably, survivors, experienced poor mental health (Carreira et al., 2018; Niedzwiedz et al., 2019; Utley et al., 2022; Ji et al., 2021). The neural connection between tumor and brain, together with our finding that TRPV1-expressing nociceptor neurons within this circuit become functionally altered, might contribute to these changes. To test whether this is the case, we genetically engineered mice with ablated nociceptor neurons (TRPV1-Cre::Floxed-DTA). TRPV1-Cre::Floxed-DTA animals lack TRPV1-expressing cells (including neurons) as well as many other nociceptive neurons. These nociceptor-neuron-ablated mice have been previously characterized and lack the expected sensitivity to temperature as well as itch and pain reactions to chemical mediators such as capsaicin (Mishra et al., 2011). First, we confirmed the absence of TRPV1⁺ neurons in the TGM of these ablated animals (Figure 4A). Fos immunostaining of brains from nociceptor ablated and control (C57BL/6) mice show no significant differences indicating that, in the absence of a malignancy, the neurons in these regions are not differentially activated (Figure 4—figure supplements 1–3). The absence of nociceptor neurons in tumor-bearing animals, however, decreased cFos and ΔFosB in the PBN, and ΔFosB in the SpVc (Figure 4B and C). Other tested regions were not impacted (Figure 4D). These data suggest that the tumor-brain communication was disrupted in the absence of nociceptor neurons.

To evaluate the effects of this disruption on cancer-induced behavioral changes, we assessed the animals’ general well-being through nesting behavior (Neely et al., 2019) and anhedonia using the cookie test (Liu et al., 2018; Eliwa et al., 2021), as well as body weight and food disappearance as surrogates for oral pain and/or loss of appetite. Nociceptor-neuron-ablated mice showed increased nesting performance (Figure 4E) and decreased anhedonia (Figure 4F) compared to intact mice. This was accompanied by smaller tumor growth (Figure 4G) and increased survival (Figure 4H). Given the impact of nociceptor neuron ablation on tumor growth, we wondered whether differences in tumor volume contributed to the behavioral differences we noted. Thus, the behavior data were graphed as a function of tumor volume. A simple linear regression model was used to fit the data. In the case of nesting scores, the linear regression did not fit the data points very well making it difficult to assess nesting scores at a given tumor volume (Figure 4—figure supplement 4). However, the linear regression model fit the time to interact data better. Here, the data suggest that tumor volume did influence behavior as at any given tumor volume the time to interact with the cookie is generally smaller in TRPV1-Cre::Floxed-DTA animals as compared to C57BL/6 animals (Figure 4—figure supplement 5). While both groups showed similar body weight loss (Figure 4I), nociceptor ablated animals show a transient (Wk 1, 4, 5) decrease in food disappearance (Figure 4J). Significant effects for all statistical analyses in Figure 4 are presented in Supplementary file 2.

The developmental ablation of TRPV1 neurons can lead to unintended effects on tumor innervation and behavior. Thus, we chemoablated TRPV1 neurons in 4-week-old juvenile C57BL/6 mice using resiniferatoxin (RTX) (Liu et al., 2022a; Mishra and Hoon, 2010). We examined nesting behavior in RTX and vehicle-treated C57BL/6 male mice orthotopically implanted with MOC2-7 cells. An additional control group consisted of age-matched C57BL/6 mice without tumors. Although all groups displayed similar nesting scores initially, the scores in vehicle-treated mice declined significantly faster compared to RTX-treated mice (Figure 5A). Body weight decreased in all tumor-bearing mice, but the extent of weight loss was similar between vehicle and RTX-treated mice (Figure 5B). Similar to the developmental ablation of TRPV1 neurons, their chemoablation post-development resulted in a similar decrease in tumor growth compared to controls (Figure 5C).

Since HNSCC tumors cause oral pain and both nesting and the cookie test require the use of the mouth, we also evaluated the effect of nociceptor ablation on voluntary wheel running to gain additional insights into cancer-associated fatigue, a surrogate for depressive-like behaviors in tumor-bearing mice (Norden et al., 2015a; Zombeck et al., 2013; Grossberg et al., 2018; Bower et al., 2011; Norden et al., 2015b). To assess the potential influence of cancer-associated pain, 1 week post tumor inoculation, groups of mice were treated with carprofen (a non-steroidal anti-inflammatory), extended-release buprenorphine (opioid), or vehicle. We found that wheel running decreased as tumors progressed, an effect partially alleviated by carprofen and buprenorphine (Figure 6A and B). The nesting behavior (Figure 6C), body weight (Figure 6D), and food disappearance (Figure 6E) were also alleviated by the painkillers. Neither carprofen nor buprenorphine impacted MOC2-7 tumor growth compared to vehicle-treated animals (Figure 6F), but buprenorphine increased tumor growth compared to carprofen-treated mice. Significant effects for all statistical analyses in Figure 6 are presented in Supplementary file 3. These findings suggest that choosing pain management drugs in the context of cancer can potentially influence tumor growth adversely. Furthermore, the data reveal that behaviors associated with the tumor site are adversely affected by cancer-associated pain. Pain-induced anhedonia is mediated by changes in the reward pathway. Specifically, in the context of pain, dopaminergic neurons in the ventral tegmental area (VTA) become less responsive to pain and release less serotonin. This decreased serotonin results in disinhibition of GABA release; the resulting increased GABA promotes an increased inhibitory drive leading to anhedonia (Markovic et al., 2021) and, when extreme, anorexia. Carprofen and buprenorphine treatments completely reversed nesting behavior and significantly improved eating. Inflammation (Bekhbat et al., 2022) and opioids (Le Merrer et al., 2009) directly influence reward processing and though our tracing studies did not indicate that the tumor-brain circuit includes the VTA, this brain region may be indirectly impacted by tumor-induced pain in the oral cavity. Thus, an alternative interpretation of the data is that the effects of carprofen and buprenorphine treatments on nesting and food consumption may be due to inhibition of anhedonia (and anorexia) rather than, or in addition to, relieving oral pain (Campos et al., 2017). However, merely alleviating pain (or inhibiting anhedonia) does not fully restore all behaviors, as evidenced by persistent issues in activities like wheel running.

All animal studies were performed with approval from the Institutional Animal Care and Use Committee at Sanford Research and were within institutional guidelines and complied with all relevant ethical regulations. Sanford Research has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (assurance number: A-4568–01) and is accredited by AAALAC, Intl. Sanford Health is also a licensed research facility under the authority of the United States Department of Agriculture (USDA) with USDA certificate number 46 R-011.

All studies utilizing animals were performed with approval from the appropriate ethical bodies to ensure sufficient protection was in place (Sanford IACUC protocol# 2023–0105). This study was a collaborative, multi-disciplinary, multi-institutional effort with contributions from researchers in academic positions.

C57BL/6-derived MOC2-7 cells (RRID:CVCL_ZD34; Judd et al., 2012 previously known as MOC7)(Onken et al., 2014) were a kind gift from Dr. Ravindra Uppaluri (Dana-Farber Cancer Institute, Boston, MA). They were authenticated by STR profiling (ATCC Cell Line Authentication Service) and tested as mycoplasma negative by a Sanford Research Core facility. They were maintained in DMEM with 10% fetal calf serum and cultured at 37 °C and 5% CO_2; with culture medium refreshed every 3 days. MOC2-7 tumors have been characterized as non-inflamed and poorly immunogenic (Moore et al., 2016b; Moore et al., 2016a; Shah et al., 2016). These cells can be obtained through Kerafest (https://www.kerafest.com).

All animal experiments were performed in the Sanford Research Animal Resource Center which is a specific pathogen-free facility. All mice were maintained in IVC Tecniplast Green line Seal Safe Plus cages which were opened only under aseptic conditions in an animal transfer station. All cages were changed every other week using aseptic technique. All cages had individual HEPA filtered air. Animal rooms were maintained at 75 °F, 30–70% humidity, with a minimum of 15 air changes per hour, and a 14:10 light/dark cycle. Corncob bedding, which was autoclaved prior to use, was maintained in all cages. Irradiated, sterile food (Envigo) and acidified water (pH 2.8–3.0) were available ad libitum. There was a maximum of 5 mice/cage. All animals were observed daily for abnormal behavior, signs of illness or distress, the availability of food and water and proper husbandry. Animals injected with murine tumor cells were 10-week-old C57BL/6 or TRPV1-Cre::Floxed-DTA mice (The Jackson Laboratory) weighing approximately 24 g. Investigators were blinded to the groups when assessing animals (e.g. measuring tumors). Animals were numbered by ear punch and cage number. For all studies, animals were randomized into groups.

Nociceptor-neuron-ablated TRPV1-Cre::Floxed-DTA animals were generated by crossing ROSA26-DTA (diphtheria toxin A, B6.129P2-Gt(ROSA)26Sor ^tm1(DTA)Lky/J)(Jax#009669) and TRPV1-Cre (B6.129-Trpv1^tm1(cre)Bbm /J) (Cavanaugh et al., 2011) (Jax# 017769) mice; the progeny (TRPV1-Cre::Floxed-DTA) express DTA (diphtheria toxin fragment A) under control of the TRPV1 promoter, thereby genetically ablating all TRPV1 expressing cells (including TRPV1-expressing neurons) throughout development. Absence of TRPV1-expressing sensory neurons was validated by IHC staining of TGM ganglia for TRPV1.

Chemoablation of TRPV1-expressing neurons post-development was accomplished with resiniferatoxin (RTX), a naturally occurring potent TRPV1 agonist. RTX treatment results in the specific ablation of TRPV1 neurons (post-development), while sparing other TRPV1 expressing (non-neuronal) cells. RTX treatment in mice consisted of three injections of RTX given 24 hr apart. The injections are given in the flank and were: 30 µg/kg on day 1, 70 µg/kg on day 2, and 100 µg/kg on day 3. Just prior to the first RTX injection, mice are treated with 0.1 mg/kg of buprenorphine, given intraperitoneally, as the first injection causes pain. Subsequent injections do not require buprenorphine. It takes 4 weeks for TRPV1 nerve ablation to occur. Control mice received injections with vehicle solution (DMSO with Tween80 in PBS). The tail flick test was used to confirm nerve ablation.

Prior to testing, mice are acclimated to the room for 30 min. A water bath is pre-warmed to 52 °C and a thermometer is used to constantly monitor the temperature of the water. The mice are picked up using a fresh paper towel so that only their tail is outside the paper towel. Once the mouse is relaxed in the tester’s hand, the mouse’s tail is lowered into the water so that a quarter of the tail is submerged. The time for the tail to be flicked out of the water is recorded. If no response has occurred by 20 s, the mouse’s tail is removed from the water to prevent tissue damage. A response time latency greater than 10 s signifies denervation.

The cells injected into animals were cultured in media containing 10% fetal calf serum. When cells are harvested for tumor injections, they are first washed two times with PBS and then trypsinized to detach the cells from the plate. Cells are collected, washed again with PBS and resuspended with DMEM without serum; this is what is injected into animals. We harvest cells in this way in order to eliminate any serum being injected into mice. Ketamine (87.5 mg/kg)/xylazine (10 mg/kg) were used to anesthetize mice prior to oral cavity tumor implantation. Tumors were initiated as follows: using a 25-gauge needle, cells (5x10⁴ cells in a total of 50 μl) were implanted orthotopically in the oral cavity of male C57BL/6 mice. Specifically, the needle was placed proximal to the crease of the mouse cheek, inserted at approximately a 45° angle, bevel deep, and tumor cells injected into this location of the cheek pouch. Mice were allowed to recover from anesthesia on a heating blanket and returned to their home cage. Control (non-tumor) mice were treated the same way only their injection contained media alone (no cells). Tumor growth was monitored weekly by caliper measurements of isoflurane anesthetized animals and tumor volume calculated was using the formula: ½ (length*(width^2)). Non-tumor control mice were also anesthetized with isoflurane each time that tumor-bearing animals were anesthetized for tumor measurements.

Criteria for euthanasia in our IACUC approved protocol include maximum tumor volume of 1000 mm³, edema, extended period of weight loss progressing to emaciation, impaired mobility or lesions interfering with eating, drinking or ambulation, rapid weight loss (>20% in 1 week), as well as weight loss at or more than 20% of baseline. In addition to tumor size and weight loss, we use the body condition score to evaluate the state of animals and to determine euthanasia.

Carprofen (Pivetal) was provided in the drinking water (10 mg/kg which is equivalent to 0.067 mg/ml) which was refreshed every 7 days. Buprenorphine-ER (Fidelis Animal Health), a 72 hr extended-release formulation, was given by subcutaneous injection (3.25 mg/kg) every 72 hr. Vehicle-treated animals were treated with subcutaneous injection of saline. Drug treatments commenced on day 7 post-tumor implantation in all groups. Given that buprenorphine treatment requires a subcutaneous injection every 3 days, mice in the other groups (vehicle and carprofen) received a subcutaneous injection of saline at the same volume (60 µl) as the buprenorphine-treated animals. In this way, all animal stress from handling and injection was the same.

Nest building is an innate behavior performed in rodents of both sexes and is a general indication of well-being (Neely et al., 2019); it also measures the motivation to perform a goal-directed task. To assess nesting behavior, individually housed mice were given a nesting square (Ancare) overnight. The following morning, nests were scored by two independent scorers who were blinded to the conditions (Deacon, 2006; Kraeuter et al., 1916). The scoring system is 0–4 and based on the percentage of the nestlet that is shredded and the height of the nest. Two baseline measurements were conducted prior to tumor implantation followed by weekly testing. Exclusion criteria of an average baseline score of 3 or greater was used to ensure all mice were good nest builders at baseline (n=3 C57BL/6 mice and n=7 TRPV1-Cre::Floxed-DTA mice were excluded for this reason).

The cookie test is a variation of the sucrose preference test, a test of anhedonia (the inability to experience enjoyment or interest in previously rewarding activities). This test assesses the animal’s endogenous inclination for sweet tastes and the notion that it derives pleasure from consuming sweets. One hour prior to testing, mouse food is removed and the animals are acclimated to the brightly lit testing room. Following the acclimation period, mice were given a piece of cookie (approximately 1.5 g). The time it took the mouse to bite the cookie from its placement in the cage was measured to determine time-to-interact. Each mouse was acclimated to the cookie every other day for 2 weeks prior to tumor implantation with a baseline measurement followed by weekly testing. Mice were individually housed. One mouse from the C57BL/6 group was excluded due to incomplete data (mouse died before completion of the study).

Prior to tumor implantation, mice were singly housed with a running wheel maintained in the home cage continuously. The only time that the wheel was removed was when mice were undergoing nesting. The mice were acclimated to the wheel for 2 weeks prior to tumor implantation. This acclimatization period provided the time to stabilize their running performance. Running wheels were maintained in the home cage for the duration of the study. As mice are most active during the dark phase, nighttime running (8pm to 8am) was assessed. Running data were collected in 1 min bins continuously throughout the duration of the experiment.

Animals were provided 100 g of solid food per cage each week. At the end of each week, the remaining food was weighed, and this value was used to calculate the amount of food that disappeared in that week per cage. For all behavioral experiments, mice were singly housed.

When oral tumors reached approximately 5x5 mm in size, neural tracer was injected into the tumor as described below. Ketamine (87.5 mg/kg)/xylazine (10 mg/kg) were used to anesthetize the mice. A 10 µL Hamilton syringe with 30 G needle was loaded with 1% Wheat Germ Agglutinin (WGA) conjugated to either AlexaFluor 594 (Invitrogen) or CF568 (Biotium) in PBS and 2 μl was slowly injected intra-tumorally with the bevel side up. The needle was inserted approximately midway through the tumor then pulled back slightly to reduce pressure and leakage of tracer following injection. The injection of tracer occurred slowly over the course of 10 min. After the tracer was injected, the needle was kept in place for 2 additional minutes before being slowly removed. Mice were placed on a heating pad until recovery from anesthesia. Five to 7 days later, mice were deeply anesthetized and transcardially perfused with ice cold PBS followed by 4% paraformaldehyde and trigeminal ganglia (TGM) were carefully removed and placed in HBSS in a 24-well plate kept on ice. TGM harvesting was completed as follows: euthanized animals were subjected to a midline incision while in the prone position to expose the crown of the skull. The brainstem was separated from the spinal cord by a transverse cut and the top of the skull was removed, exposing the brainstem and TGM. All tissues (tumor, ganglia and brain) were fixed, sectioned, and imaged for WGA labeled nerve fibers and somas under confocal microscopy. WGA injection into control (non-tumor bearing animals) was performed the same way and injection was into the same oral region where tumors were implanted. Volumes of 2 or 10 μl of WGA were used.

Tissues were formalin fixed, paraffin-embedded and cut into 5 µm sections. The BenchMark XT automated slide staining system (Ventana Medical Systems, Inc) was used to optimize antibody dilutions and staining. The Ventana CC1 solution was used to perform the antigen retrieval step (basic pH tris base buffer). Tissues were incubated in primary antibody for 1 hr. The Ventana iView DAB detection kit was used as the chromogen and the slides were counterstained with hematoxylin.

Anti-β-III Tubulin (Abcam, Cat# ab78078, 1:250, RRID: AB_2256751), Anti-TRPV1 (Alomone Labs Cat# ACC-030, 1:400, RRID:AB_2313819).

cFos (Cell Signaling, Cat# 2250, 1:10,000, RRID: AB_2247211), ΔFosB (Abcam, Ab11959, 1:5000, RRID:AB_298732). The following secondary antibodies were used: Alexa568 anti-rabbit (Thermo Fisher, Cat# A-11011, 1:500, RRID:AB_143157), Alexa488 anti-mouse (Thermo Fisher, Cat# A-11001, 1:500, RRID:AB_2534069).

β-actin (Sigma Life Science, Cat#A2228, 1:1000, RRID: AB_476697), Tau (Abcam, Cat# ab75714, 1:500, RRID:AB_1310734), phosphorylated TRPV1 (Thermo Fisher Scientific, Cat# PA5-64860, 1:500, RRID:AB_2663797), Sigma-1R (ProteinTech, Cat# 15168–1-AP, 1:1000, RRID:AB_2301712), Doublecortin (DCX) (Santa cruz, Cat# sc-271390, 1:1000, RRID:AB_10610966).

Brains were post-fixed for 24 hr. Coronal or sagittal sections were cut at 40 µm using a vibratome. Sequential fluorescent immunolabeling was performed for cFos or ΔFosB on free-floating sections. All sections were blocked with 10% goat serum in 0.3% Tx-100. All antibodies were diluted in PBS containing 0.03% Triton X-100 and 2% normal goat serum. Sections were incubated in Fos antibody for 72 hr. Following washes in 0.1 M phosphate buffered saline, sections were incubated in secondary antibody for 3 hr. Following additional washes, sections were mounted onto glass slides, and cover slipped with cytoseal prior to imaging. The total numbers of Fos-labeled nuclei were counted on 10×2 D images acquired using a laser-scanning confocal microscope (Nikon A1R) and verified as positive if the signal filled the nucleus and stood out clearly compared to surrounding tissue Kee et al., 2007. Nuclei were counterstained with DAPI. Quantification of Fos-labeled cells was performed using digital thresholding of Fos-immunoreactive nuclei. The threshold for detection was set at a level where dark Fos-immunoreactive nuclei were counted, but nuclei with light labeling, similar to background staining, were not. For each animal, 3–4 sections were selected at 120 μm intervals. ImageJ cell counter (v. 1.52) was used to quantify Fos +nuclei per region / section.

To assess transcriptional changes in tumor-infiltrating neurons we utilize a qPCR array (ScienCell, GeneQuery Neural Transmission and Membrane Trafficking, #MGK008). These qPCR-ready 96 well plates enable rapid profiling of 88 key genes important for neuronal functions (listed along with built-in control genes in Supplementary file 1). Mice bearing MOC2-7 oral tumors were injected with tracer (as described above); TGM ganglia were isolated on day 28 post-tumor implantation, their RNA harvested, converted into cDNA and then assayed with the array as per manufacturer’s instructions (ScienCell, GeneQuery arrays). We used N=4 TGM/group from n=4 mice/group; n=4 plates/condition. Control RNA was isolated from TGM ganglia from age-matched non-tumor bearing mice (n=4 mice). Relative gene expression was calculated as per manufacturer’s recommendations.

Tumors were excised from euthanized mice, taking care to eliminate as much non-tumor tissue as possible. Tumors were then placed in approximately 500 µl lysis buffer (50 mM Tris HCl pH 7.4, 100 mM NaCl, 100 mM NaF, 10 mM NaPPi, 2 mM Na₃VO₄, 10% glycerol, HALT protease inhibitor cocktail) with 1% TX-100 and kept on ice for 10 min. The homogenate was sonicated 3 x for 15 s and then incubated on ice for 15 min. Samples were then centrifuged at 2000 × g for 5 min at 4 °C, supernatant was collected and further centrifuged at 12,000 × g for 10 min at 4 °C. Protein concentrations of the supernatants were determined by BCA protein assay.

Trigeminal ganglia were harvested as described above. Ganglia were lysed using a homogenizer in lysis buffer at 4 °C. Homogenates were centrifuged at 10,000 × g for 20 min and protein concentrations determined from the supernatant. Protein concentration was measured by BCA protein assay (Pierce, Cat#23225) and 40 µg of total protein were separated by SDS-PAGE, transferred to PDVF membranes which were then blocked for 30 min at room temperature (RT) in 5% milk in PBS. Membranes were incubated with primary antibody overnight at 4 °C. Following 1xTBST washes, membranes were probed with an HRP-conjugated secondary antibody (1:10,000 dilution) for 1 hr at RT, washed and imaged on a Li-COR Odyssey FC imaging system. Densitometric quantification was used to assess changes in protein expression.

Raw images of western blots were analyzed using ImageJ. Briefly, images were opened and bands of interested were selected by gating with the rectangle selection tool. Densitometry was measured based on grey scale analysis of selected area. Analysis was conducted on proteins of interest as well as the loading control band (β-actin). Relative expression of proteins of interest was determined as a fraction of β-actin densitometry.

Trigeminal ganglia were harvested from MOC2-7 tumor-bearing or non-tumor animals and enzyme digested in papain, and then collagenase II/dispase at 37°C for 15 min. After washing and trituration, cells were plated onto a thin layer of Matrigel in glass bottom dishes and cultured with HamsF12 supplemented with 10% FBS. The cells were maintained in an incubator (5% CO₂, 37 °C) for 24 hr before they were used for calcium imaging experiments.

TGM neurons from non-tumor and tumor-bearing animals (n=4–6 mice/condition) were imaged on the same day. Neurons were incubated with the calcium indicator, Fluo-4AM, at 37 °C for 20 min. After dye loading, the cells were washed, and Live Cell Imaging Solution (Thermo-Fisher) with 20 mM glucose was added. Calcium imaging was conducted at room temperature. Changes in intracellular Ca²⁺ were measured using a Nikon scanning confocal microscope with a 10 x objective. Fluo-4AM was excited at 488 nm using an argon laser with intensity attenuated to 1%. The fluorescence images were acquired in the confocal frame (1024 × 1024 pixels) scan mode. After 1 min of baseline measurement, capsaicin (300 nM final concentration) was added. Ca²⁺ images were recorded before, during and after capsaicin application. Image acquisition and analysis were achieved using NIS-Elements imaging software. Fluo-4AM responses were standardized and shown as percent change from the initial frame. Data are presented as the relative change in fluorescence (ΔF/F₀), where F₀ is the basal fluorescence and ΔF=F F₀ with F being the measured intensity recorded during the experiment. Calcium responses were analyzed only for neurons responding to ionomycin (10 µM, positive control) to ensure neuronal health. Treatment with the cell permeable Ca²⁺ chelator, BAPTA (200 µM), served as a negative control.

Male mice were anesthetized with 2% isoflurane and placed in a stereotaxic head frame on a heating pad. A midline incision was made down the scalp and a craniotomy was made using a micro drill. A 10 μl Hamilton syringe was used to infuse 1 μl of AAV1/Syn-GCaMP6f-WPRESV40 (titer 4.65×10¹³ GC per ml, via Addgene) into the parabrachial nucleus (−5.3 mm anteroposterior, –1.3 mm mediolateral, −3 mm dorsoventral) via a microsyringe pump. After infusion, the needle was kept at the injection site for 5 min and then slowly withdrawn. Two weeks following stereotaxic surgeries, tumors were introduced by implanting MOC2-7 cells orthotopically into the oral cavity as described above.

GraphPad Prism (version 10.0.3, 2023) was used for all statistical analyses.