Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade.

While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

Functional brain network changes associated with maintenance of cognitive function in multiple sclerosis.

In multiple sclerosis (MS) functional changes in connectivity due to cortical reorganization could lead to cognitive impairment (CI), or reflect a re-adjustment to reduce the clinical effects of widespread tissue damage. Such alterations in connectivity could result in changes in neural activation as assayed by executive function tasks. We examined cognitive function in MS patients with mild to moderate CI and age-matched controls. We evaluated brain activity using functional magnetic resonance imaging (fMRI) during the successful performance of the Wisconsin card sorting (WCS) task by MS patients, showing compensatory maintenance of normal function, as measured by response latency and error rate. To assess changes in functional connectivity throughout the brain, we performed a global functional brain network analysis by computing voxel-by-voxel correlations on the fMRI time series data and carrying out a hierarchical cluster analysis. We found that during the WCS task there is a significant reduction in the number of smaller size brain functional networks, and a change in the brain areas representing the nodes of these networks in MS patients compared to age-matched controls. There is also a concomitant increase in the strength of functional connections between brain loci separated at intermediate-scale distances in these patients. These functional alterations might reflect compensatory neuroplastic reorganization underlying maintenance of relatively normal cognitive function in the face of white matter lesions and cortical atrophy produced by MS.

Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.

Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.

Context-specific sensitization of cocaine-induced locomotor activity and associated neuronal ensembles in rat nucleus accumbens.

Repeated cocaine administration to rats outside their home cage induces behavioral sensitization that is strongly modulated by the drug administration environment. We hypothesized that stimuli in the drug administration environment activate specific sets of striatal neurons, called neuronal ensembles, for further cocaine-enhanced activation, and that repeated activation of these neuronal ensembles underlies context-specific sensitization. In the present study, we repeatedly administered cocaine or saline to rats on alternate days in two distinct environments outside the home cage, one paired with cocaine and the other with saline. On test day, cocaine challenge injections in the cocaine-paired environment produced strongly enhanced levels of locomotor activity, while cocaine challenge injections in the saline-paired environment did not. The corresponding record of past neuronal activation in nucleus accumbens and caudate-putamen during repeated drug administration was assessed using FosB immunohistochemistry, while acute neuronal activation on test day was assessed using c-fos in situ hybridization. Although only 2% of striatal neurons were FosB labeled, 87% of these FosB-labeled neurons were co-labeled with c-fos when cocaine was injected in the cocaine-paired environment. The degree of co-labeling was significantly less following cocaine or saline challenge injections in the saline-paired environment. Furthermore, the total number of c-fos-labeled neurons was greater with either cocaine or saline challenge injections in the cocaine-paired environment than in the saline-paired environment. These findings demonstrate that the drug administration environment partly determines which striatal neuronal ensembles are activated, and to what extent, following context-specific sensitization to cocaine.

Repeated amphetamine administration outside the home cage enhances drug-induced Fos expression in rat nucleus accumbens.

Induction of the immediate early gene protein product Fos has been used extensively to assess neural activation in the striatum after repeated amphetamine administration to rats in their home cages. However, this technique has not been used to examine striatal activation after repeated administration outside the home cage, an environment where repeated drug administration produces more robust psychomotor sensitization. We determined the dose-response relationship for amphetamine-induced psychomotor activity and Fos expression in nucleus accumbens and caudate-putamen 1 week after repeated administration of amphetamine or saline in locomotor activity chambers. Repeated administration of amphetamine enhanced amphetamine-induced locomotor activity and stereotypy and Fos expression in nucleus accumbens, but not in caudate-putamen. In comparison, levels of Fos expression induced by 1mg/kg amphetamine were not altered in nucleus accumbens or caudate-putamen by repeated amphetamine administration in the home cage. Double-labeling of Fos protein and enkephalin mRNA indicates that Fos is expressed in approximately equal numbers of enkephalin-negative and enkephalin-positive neurons in nucleus accumbens and caudate-putamen following injections outside the home cage. Furthermore, repeated amphetamine administration increased drug-induced Fos expression in enkephalin-positive, but not enkephalin-negative, neurons in nucleus accumbens. We conclude that repeated amphetamine administration outside the home cage recruits the activation of enkephalin-containing nucleus accumbens neurons during sensitized amphetamine-induced psychomotor activity.

Cocaine-induced locomotor activity and Fos expression in nucleus accumbens are sensitized for 6 months after repeated cocaine administration outside the home cage.

Induction of the immediate early gene protein product Fos has been used extensively to assess neural activation in the striatum after repeated cocaine administration to rats in their home cages but rarely after repeated administration outside the home cage, which produces more robust locomotor sensitization. In the present study, we found cocaine-induced Fos expression in nucleus accumbens, but not caudate-putamen, was enhanced 1 and 6 months after repeated drug administration in locomotor activity chambers. Double-labelling of Fos protein and enkephalin mRNA indicated that Fos expression in nucleus accumbens was enhanced in enkephalin-positive, but not enkephalin-negative, medium spiny neurons. In contrast, cocaine-induced Fos expression was absent altogether in nucleus accumbens and unaltered in caudate-putamen 1 month after repeated cocaine administration in the home cage. As cocaine-induced locomotor activity was also enhanced 1 and 6 months after repeated cocaine administration in locomotor activity chambers, we wanted to confirm that neuronal activity in nucleus accumbens mediates cocaine-induced locomotor activity using our particular treatment regimen. Bilateral infusions of the GABA agonists baclofen and muscimol (1 microg/side) into nucleus accumbens of sensitized rats blocked cocaine-induced Fos expression and locomotor activity. Thus, while neuronal activity in both D1- and D2-type neurons in nucleus accumbens can mediate acute cocaine-induced locomotor activity, the enhanced activation of enkephalinergic D2-type neurons suggests that these latter neurons mediate the enhancement of cocaine-induced locomotor activity for up to 6 months after repeated drug administration outside the home cage.

Cocaine-induced CREB phosphorylation in nucleus accumbens of cocaine-sensitized rats is enabled by enhanced activation of extracellular signal-related kinase, but not protein kinase A.

Repeated cocaine administration to rats outside their home cages sensitizes the behavioral effects of the drug, and enhances induction of the immediate early gene product Fos in nucleus accumbens. We hypothesized that the same treatment regimen would also enhance cocaine-induced activation of intracellular signaling kinases that phosphorylate cyclic AMP-regulated element-binding protein (CREB), an important mediator of c-fos transcription. Phosphorylation levels of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), calcium/calmodulin kinases (CaMKs) II and IV, and CREB were used to assess endogenous functional activity of these signaling molecules in rats behaviorally sensitized outside their home cages. Protein kinase A (PKA)-specific phosphorylation of Ser845 in the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 was used to assess endogenous functional activity of PKA. Using western blots and immunohistochemistry, we detected cocaine-induced CREB phosphorylation after repeated cocaine administration, but not after repeated saline administration. Using western blots and MAPK activity assays, we found that cocaine-induced phosphorylation and activation of ERK, but not of CaMKs II or IV or GluR1, was augmented in nucleus accumbens of cocaine-sensitized rats. Unilateral infusions of the MAPK kinase inhibitor U0126 into nucleus accumbens attenuated cocaine-induced ERK and CREB phosphorylation in cocaine-sensitized rats. In contrast, unilateral infusions of the PKA inhibitor Rp-isomer of adenosine-3',5'-cyclicmonophosphorothioate (Rp-cAMPs) did not affect cocaine-induced CREB phosphorylation. Therefore, enhanced activation of ERK, but not PKA, enables and mediates cocaine-induced CREB phosphorylation in nucleus accumbens of rats that are sensitized by repeated cocaine administration outside their home cages.

Preferences for cocaine- or pup-associated chambers differentiates otherwise behaviorally identical postpartum maternal rats.

RATIONALE AND OBJECTIVES: Our previous work uncovered a differential preference of maternal female rats for cues associated with pups versus cues associated with cocaine at three different postpartum time points. Our current study examines the preference for these cues in conjunction with an assessment of the capacity to express the maternal behavior at one of these time points. We examined dams at day 10 postpartum using a procedure that included two additional controls, and a complete assessment of the expression of maternal behavior and locomotor activity. METHODS: A conditioned place-preference procedure was used to determine the preference for cocaine- or pup-associated cues. The two controls were (1) a preconditioning test to verify no initial chamber preference and (2) a separate control group of postpartum day-10 dams exposed to chambers and cues but not to unconditioned stimuli. The expression of maternal behavior was determined by measurement of maternal nest building, retrieval of pups to the nest, grooming, crouching over pups, nursing, and maternal aggression. Locomotor activity was measured with an automated apparatus. RESULTS: Dams conditioned with cocaine or pups showed a preference for either the cocaine-associated chamber or the pup-associated chamber, confirming the existence of two similar-sized preference groups at this time point. Regardless of preference, dams had equal and robust expression of maternal behavior and similar locomotor capacity. The pre-conditioning test showed no initial chamber preferences and did not alter the conditioned preference response. The use of unconditioned stimuli in the place-preference conditioning procedure was effective and necessary for the preference response. CONCLUSION: Our current study has revealed that differences in the motivational state of the maternal dam emerge even while the expression of maternal behavior is constant and substantial. The data suggest that the difference in preference is a very specific appetitive response that is not linked to expression of maternal behavior or locomotor capacity.