Abstract
The prolonged interaction between the immune system and tumor antigens can result in T cell exhaustion. Extensive research has been conducted on strategies to reactivate exhausted T cells within the tumor microenvironment. However the exact contribution of the endocannabinoid system (ECS) and nociceptors in regulating CD8+ T cells within the framework of cancer-related inflammation has not been thoroughly studied. This study investigated the use of a TRPM8 antagonist (RQ-00203078), a selective cannabinoid receptor 1 (CB1) antagonist (AM251), and alpelisib (BYL-719) to control CD8+ T cell exhaustion. Our findings showed that administration of the CB1 antagonist AM251, either alone or in combination with alpelisib, significantly reduced the expression of PD-1 and Lag-3 on CD8+ T cells. Interestingly, treatment with the TRPM8 antagonist led to a notable increase in PD-1 expression on CD8+ T cells. These findings suggest that the decreased expression of inhibitory receptors on CD8+ T cells after treatment with the CB1 antagonist whether alone or with alpelisib and TRPM8 highlights the potential of ECS as a promising therapeutic target in cancer treatment.
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The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials, and can also be obtained from the corresponding author upon request.
Abbreviations
- CNS:
-
Central nervous system
- ECS:
-
Endocannabinoid system
- CB1:
-
Cannabinoid receptor 1
- CB1 ant:
-
Cannabinoid receptor 1 antagonist
- TRPM8:
-
Transient receptor potential cation channel subfamily M member 8
- TRPM8 ant:
-
Transient receptor potential cation channel subfamily M member 8 antagonist
- PI3K:
-
Phosphoinositide 3-kinase
- PD-1:
-
Programmed cell death protein 1
- CTLA-4:
-
Cytotoxic T-lymphocyte-associated antigen 4
- Lag-3:
-
Lymphocyte activation gene 3
- TIM-3:
-
T cell immunoglobulin domain and mucin domain
- BTLA:
-
B- and T-lymphocyte attenuator
- TIGIT:
-
T cell immunoreceptor with immunoglobulin and ITIM domain
- AEA:
-
Anandamide
- 2-AG:
-
2-Arachidonoylglycerol
- ICBs:
-
Immune checkpoint inhibitors
- IHC:
-
Immunohistochemistry
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A.M. conceptualized, designed, and conducted the majority of the experiments, performed statistical analyses, and authored the manuscript. S.D. made significant contributions to cell culture, cell processing, and animal handling tasks. A.T.M. assisted with software applications, analysis, visualization of data, and manuscript writing. A.A. provided consultation on drug preparations and partially drafted the manuscript related to the drug discussions and writing. R.M., a breast cancer surgeon, and A.A.P. offered valuable insights and critical revision feedback that enhanced the results and discussion sections. All authors participated in the editing process and approved the final manuscript.
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Ethics statement
To establish the 4T1-induced breast cancer model, it was confirmed that all animal care and experimental procedures conducted in this study adhered to the ethical guidelines approved by Urmia University of Medical Sciences, in alignment with ARRIVE guidelines (ARRIVE 2.0)61. All authors acknowledge the ethical considerations relevant to this manuscript. Ethical approval for this research was obtained from the Ethics Committee of Urmia University of Medical Sciences under approval code [IR.UMSU.AEC.1401.004]. Mice were maintained under standard housing conditions with a controlled temperature of 22°C, following a 12-h light/dark cycle. Food and water were available at all times, and appropriate air conditioning was implemented to minimize potential harm or distress. All necessary measures and precautions were employed to ensure the well-being of the animals. At the conclusion of the experiment, in accordance with IACUC guidelines, all euthanasia procedures for the mice were conducted using carbon dioxide (CO2) gas at a flow rate of 3 L/min until respiration ceased62. Prior to this procedure, the animal received anesthesia to minimize distress, which involved administering a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) via intraperitoneal injection. Euthanasia was subsequently performed using CO₂ asphyxiation, followed by the cervical dislocation method, which involved placing a pen directly behind the ears at the base of the skull and, while lifting the base of the tail, dislocating the cervical vertebrae. In this study, female BALB/c mice, weighing between 21 and 23 g and aged 6–8 weeks, were obtained from the Pasteur Institute in Tehran, Iran. Upon reaching the logarithmic growth phase, 4T1 cell lines were trypsinized (using Gibco), resuspended, and a suspension containing 1 × 105 4T1 cells in 100 μL of sterile phosphate-buffered saline (PBS) was injected into the mammary fat pad of the mice. Eight days post-implantation, the mice were evaluated for tumor growth and were randomly assigned to treatment and control groups. The experimental groups consisted of eight categories: Control or Untreated, Alp (Alpelisib treated), TRPM8 ant (TRPM8 antagonist treated), CB1 ant (CB1 antagonist treated), Alp/CB1 ant (Alpelisib & CB1 antagonist treated), TRPM8/CB1 ant (TRPM8 & CB1 antagonist treated), Alp/TRPM8 ant (Alpelisib & TRPM8 antagonist treated), and Alp/CB1/TRPM8 ant (Alpelisib & CB1 & TRPM8 antagonist treated), with n = 5 mice in each group. In total, n = 50 mice were utilized for tumor induction, resulting in n = 4–5 mice in each experimental group. Mice that did not develop a tumor mass or exhibited a tumor mass significantly larger than the others by day 8 of induction were excluded from the study. Additionally, 4–5 mice were randomly allocated to each cage (Large cage Size (W × L × H), 7.5″ × 11.75″ × 5″) for each experimental group.
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Mohammadzadeh, A., Moazzendizzaji, S., Mohammadi, A.T. et al. Targeting CB1 and TRPM8 receptors to counteract CD8+ T cell exhaustion. Sci Rep (2026). https://doi.org/10.1038/s41598-026-46794-2
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DOI: https://doi.org/10.1038/s41598-026-46794-2


