Stimulation of hypothalamic oxytocin neurons suppresses colorectal cancer progression in mice

Emerging evidence suggests that the nervous system is involved in tumor development in the periphery, however, the role of the central nervous system remains largely unknown. Here, by combining genetic, chemogenetic, pharmacological, and electrophysiological approaches, we show that hypothalamic oxytocin (Oxt)-producing neurons modulate colitis-associated cancer (CAC) progression in mice. Depletion or activation of Oxt neurons could augment or suppress CAC progression. Importantly, brain treatment with celastrol, a pentacyclic triterpenoid, excites Oxt neurons and inhibits CAC progression, and this anti-tumor effect was significantly attenuated in Oxt neuron-lesioned mice. Furthermore, brain treatment with celastrol suppresses sympathetic neuronal activity in the celiac-superior mesenteric ganglion (CG-SMG), and activation of β2 adrenergic receptor abolishes the anti-tumor effect of Oxt neuron activation or centrally administered celastrol. Taken together, these findings demonstrate that hypothalamic Oxt neurons regulate CAC progression by modulating the neuronal activity in the CG-SMG. Stimulation of Oxt neurons using chemicals, for example, celastrol, might be a novel strategy for colorectal cancer treatment.


Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed malignant tumor and the second leading cause of cancer death globally. There were 1.8 million new cases, and 900,000 patients fiber of CG-SMG (Figure 3-figure supplement 1A-C). Subsequently, the 6-min control (1% 193 DMSO in aCSF) spiking activity was acquired before CNO (1 µg per mouse) application 194 through the pre-implanted cannula. Single unit spikes from 30 (Sham) and 34 (Transection)  SMG neurons were isolated, and the firing rates were compared before and after CNO infusion 196 (Figure 3-figure supplement 1D). Group data showed that i.c.v. administration of CNO  The CG-SMG is required for lesion of Oxt neurons to promote CAC development 208 Next, we assessed the Oxt PVN neuron -> TH CG-SMG neuron connection using the CAC mouse 209 model. To this end, CAC was induced in the adult Oxt Cre and Oxt Cre ;DTA mice using AOM and 210 DSS. After the first cycle of DSS treatment, CG-SMG resection and sham surgeries were 211 performed in mice (Figure 3C,D). These manipulations did not significantly impact body weight 212 or food intake in mice (Figure 3-figure supplement 2A,B). While depletion of Oxt neurons 213 led to the increasing of CAC number and diameter, CG-SMG resection markedly attenuated 214 these effects (Figure 3E-G). We noted that colorectal length was not affected in these mice 215 (Figure 3-figure supplement 2C). In agreement with the data of tumor number and size, the 216 effects on cell proliferation and cell apoptosis were both attenuated when the CG-SMG were 217 removed from these mice (Figure 3H-K). Taken together, the promotion of CAC development 218 owing to Oxt neuron deficiency is mediated by the sympathetic CG-SMG.

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Celastrol enhances Oxt PVN neuron excitability by increasing their input resistance 220 Celastrol is a pentacyclic triterpenoid initially extracted from the root of thunder god vine. A 221 recent study showed that treatment with celastrol decreased the body weight in obese mice, but  Thereafter, adult male Oxt Cre mice were bilaterally injected with control and hM3Dq AAV into 278 the PVN, and then CAC was induced. These mice were i.p. administered with CNO every other 279 day, and were also i.p. injected with saline or isoprenaline on a daily basis. These treatments 280 were continued for 3 weeks (Figure 6-figure supplement 1C). Subsequently, these mice were 281 perfused with 4% PFA, and then brain tissues were sectioned. Immunofluorescent staining 282 showed that treatment with CNO elicited a robust c-Fos expression in the Oxt PVN neurons of 283 hM3Dq AAV-injected mice compared with the controls (Figure 6A,B), suggesting the activation 284 of these neurons.

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Treatment with CNO and/or isoprenaline did not impact the body weight or food intake in  Brain OTR is crucial for centrally administered celastrol to suppress CG-SMG neuronal 294 activity 295 Our data indicated that Oxt neurons are important for celastrol to restrict CAC development in 296 mice ( Figure 5). Next, we asked whether i.c.v. administered celastrol could similarly regulate CG-SMG neuronal activity. To address this question, adult male C57 BL/6 mice were implanted 298 with a guide cannula, and then were allowed to recover from surgeries. Subsequently, the 299 preganglionic fiber of CG-SMG was transected, or left intact (sham). These mice were i.c.v.

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Thereafter, we asked whether brain OTR is crucial for centrally administered celastrol to 306 suppress the CG-SMG neuronal activity. To this end, adult male C57 BL/6 mice were implanted 307 with a guide cannula directed to the third ventricle. After surgical recovery, these mice were i.c.v. 308 administered with vehicle control or L-368,899, the OTR antagonist, an hour before in vivo 309 single unit recordings. Subsequently, the 6-min control spiking activity was acquired before 310 celastrol application through the guide cannula ( Figure 7A,B). Single unit spikes from 68 CG-311 SMG neurons (vehicle) and 44 CG-SMG neurons (OTR antagonist) were isolated, and the firing 312 rates were compared before and after celastrol infusion (Figure 7C,D). Group data showed that 313 treatment with celastrol significantly reduced the firing frequency of CG-SMG neurons, 314 however, blockade of OTR abrogated this effect ( Figure 7E). Scatterplot of mean firing 315 frequency of individual CG-SMG neuron revealed a mixed modulation by celastrol ( Figure 7F). 316 The majority of CG-SMG neurons (63%) displayed a decreased firing frequency after celastrol 317 infusion. Only a small proportion of neurons (18%) showed an increased firing frequency. The remainder (19%) maintained their activity level during celastrol infusion. However, when L-319 368,899 was applied, the majority of CG-SMG neurons (57%) maintained their activity level 320 during celastrol infusion (Figure 7G), suggesting that blockade of OTR could attenuate the 321 inhibitory effect of celastrol on neuronal firing rate in CG-SMG. Together, these data suggest 322 that brain OTR is crucial for centrally administered celastrol to suppress the neuronal activity in 323 the CG-SMG. Negative mood is associated with the occurrences of cancers, however, the underlying 344 mechanisms remain less well understood. In this study, we show that excitation of Oxt PVN 345 neurons remarkably ameliorated CAC progression in mice, and that this effect was mediated by 346 inhibiting the neuronal activities in the CG-SMG. Also, brain treatment with celastrol suppressed 347 the progression of CAC, and this effect required hypothalamic Oxt neurons. Moreover, we show 348 that β2AR was involved in these processes. Together, our current work demonstrates that 349 modulating hypothalamic Oxt neurons can impact the CAC progression in mice.

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Negative moods, such as anxiety, depression and stress, are implicated in tumor progression. As   infusion cannula directed to third ventricle, and then were singly housed to allow recovery from 496 surgeries. On the recording day, aCSF and L-368,899 were applied through the pre-implanted cannula 1 hr before recordings. The 6-min control (5% DMSO in aCSF) spiking activity was 498 acquired before celastrol (0.5 µg per mouse) application through the infusion cannula.

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In the CG-SMG preganglionic nerve fiber transection experiment, adult Oxt Cre mice were 500 injected with control or hM3Dq AAV into the PVN. These mice were also implanted with an 501 infusion cannula directed to third ventricle. After recovery, the preganglionic nerve fiber of CG-502 SMG was transected before recording. In the control group, sham operations were carried out 503 before recording. Subsequently, the 6-min control (1% DMSO in aCSF) spiking activity was 504 acquired before CNO (1 µg per mouse) application through the infusion cannula.

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In vivo single-unit recordings data analysis 506 The single unit spike sorting was performed with Offline Sorter V4.0 (Plexon, Dallas, TX).

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Spikes were detected when a minimum waveform reached an amplitude threshold of -4.50 508 standard deviation greater than the noise amplitude. Principal component analysis (PCA) and 509 automatic scan were employed to separate single unit waveforms into individual clusters.

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Manual checking was then performed to ensure that the cluster boundaries were clearly 511 separated. All isolated single units exhibited recognizable refractory periods (> 1 ms) in the inter-512 spike interval (ISI) histograms. Only well-isolated units (L ratio < 0.2, isolation distance > 15) 513 were included in the data analysis.

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The response of single unit was analyzed with Neuroexplorer V5.0 (Plexon). Well separated units were used to analyze the responses before (baseline) and after celastrol or CNO infusion.

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Firing rates of neurons during baseline, 10-min and 20-min after celastrol or CNO infusion were 517 compared to determine the significance of difference in firing rates (paired Student's t-test, 95% 518 confidence interval). For heatmap analysis, z-score of each bin (10 sec) were calculated by the 519 following equation: z = (x-μ)/σ, in which x is the raw firing rate, and μ is the mean firing rate 520 during the baseline period, and σ is the corresponding standard deviation. Further normalization 521 was utilized for better presentation. All of the single unit z-scores were plotted using Matlab with 75%-100% ethanol, and then were embedded in paraffin and sectioned (thickness: 3 μm).

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The tissue sections were deparaffinized, and rehydrated using graded ethanol. the light one was exposed to room light. The exploratory activity was monitored for 5 min using      . Two hours later, the mice were anesthetized, and then were perfused with 4% PFA.

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Immunofluorescent staining for c-Fos (red) of brain tissue sections was carried out.