Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2^m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2^m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2^m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.

Neuropathic pain is one of the most intractable pain conditions associated with tumor growth compressing and damaging nerves. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, known as “tactile allodynia,” which is often refractory to currently available analgesics. Diurnal variations in pain hypersensitivity are common in patients with cancer, but the underlying mechanisms are enigmatic. Herein, we report that spinal expression of lipocalin-2 (LCN2) enhances pain sensitivity of NCTC2472 fibrosarcoma-implanted male mice during specific stages of the diurnal cycle. As the tumor grew, interleukin-6 (IL-6) levels increased in the spinal cord of the mice. Increased IL-6 levels stimulated LCN2 expression in spinal microglia, but this expression was periodically repressed by the circadian clock components REV-ERBα and REV-ERBβ. Notably, intraspinal dorsal horn injection of lentiviral vectors expressing REV-ERBα or REV-ERBβ in tumor-bearing mice alleviated tactile allodynia. Furthermore, intrathecal injection of SR9009, a synthetic agonist of REV-ERBs, also attenuated cancer-induced pain hypersensitivity, accompanied by suppressing spinal LCN2 expression. These results suggest that temporal elevation of LCN2 expression decreases the threshold of tactile pain hypersensitivity induced by tumor growth. We propose that the circadian clock component of REV-ERBs is an effective target for alleviation of cancer-induced tactile allodynia, identifying a new class of analgesic agents.

Immune checkpoint inhibitors (ICIs) are widely used to treat various types of cancer; however, their effectiveness varies, with some patients exhibiting resistance. Recent studies have shown that the efficacy of ICIs depends on the localization of immune cells, particularly T cells, within the tumor microenvironment (TME). Although the circadian clock is known to regulate immune cell migration into tumors, its role in orchestrating spatially precise intratumoral localization remains unclear. Here, we found that the distribution of CD8 ⁺ T cells within the TME varied according to the time of day, accompanied by diurnal expression of C‐X‐C chemokine receptor type 4 (CXCR4). The amplitude of the Cxcr4 expression rhythm was more pronounced in tumor‐infiltrated T cells than in those from the spleen and was associated with time‐dependent changes in their migration toward CXCL12‐expressing cancer‐associated fibroblasts (CAFs). Reanalysis of single‐cell RNA‐seq data from T cells of lung cancer patients also revealed that upregulation of CXCR4 expression in tumor‐infiltrated CD8 ⁺ T cells was linked to TGF‐β‐SMAD signaling. The TGF‐β‐SMAD signaling‐mediated transactivation of Cxcr4 was time‐dependently repressed by SMAD7, resulting in diurnal CXCR4 expression. Consequently, administration of a CXCR4 inhibitor during the circadian phase of elevated CXCR4 expression in tumor‐infiltrated CD8 ⁺ T cells promotes their dispersion throughout tumor tissues, thereby enhancing the efficacy of ICIs. Our findings highlight an unrecognized mechanism underlying diurnal changes in the aggregation and dispersion of CD8 ⁺ T cells within tumors, offering a novel approach to enhance the anti‐tumor immune effects of ICIs.