Animals

C57BL/6J mice (Tokyo Laboratory Animals Science, Tokyo, Japan) and CRH-ires-Cre mice [B6(Cg)-Crhtm1(cre)Zjh/J; Stock #012704, Jackson Laboratory, Bar Harbor, ME, USA] were used for this study. All mice were housed individually and maintained under a room temperature of 24 ± 1 °C with a 12-h light–dark cycle (lights on 8:00 a.m. to 8:00 p.m.). Food and water were available ad libitum during every experimental period. All experiments were conducted in accordance with the ethical guidelines for the care and use of laboratory animals of Hoshi University School of Pharmacy and Pharmaceutical Sciences.

Stereotaxic AAV injection

Mice were anesthetized with isoflurane (3%, inhalation) before surgery. At the start of the surgical procedures, mice were fixed in a stereotaxic apparatus (RWD Life Science, CA, USA). For manipulation of CRH neurons in the PVN, AAV10-hSyn-Flex-hM3Dq-mCherry was delivered bilaterally to the PVN of male CRH-ires-Cre mice (A/P, − 0.69 mm; M/L, ± 1.06 mm from the bregma; D/V, − 4.66 mm from the dura; with a 10° angle toward the midline in the coronal plane) through an internal cannula (Eicom Co., Kyoto, Japan) at a rate of 0.25 μL/min for 4 min (1 μL total volume per side) with a glass micropipette and an air pressure injector system (Micro-syringe Pump-Model ESP-32; Eicom Co.). After each injection, the cannula was left in the brain for 2 min. After the surgical procedures, mice were allowed to recover in their home cages for at least 2 weeks until AAV vector-derived transgene expressed enough Gq-coupled human muscarinic M3 (hM3Dq). The virus used in this study was a kind gift from Dr. Akihiro Yamanaka (Nagoya University, Nagoya, Japan).

Drug treatment

Hydrocortisone (10 mg/kg, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), suspended in physiological saline (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan) containing 5% (v/v) Tween-80 (Sigma-Aldrich, Inc., MO, USA), was injected intraperitoneally once a day for 20 days. The dose (10 mg/kg) of hydrocortisone used in the present study was chosen based on the results of a previous study using rodents [10]. Clozapine N-oxide (CNO; 3 mg/kg, Abcam plc., Cambridge, UK) to activate hM3Dq was dissolved in saline.

Electrophysiological evaluation of hM3Dq receptor activation by CNO

Whole-cell recordings from PVN neurons were performed as previously described [9]. Briefly, coronal slices (250 µm thick) were cut on a vibratome (VT-1200S, Leica Biosystems, Wetzlar, Germany) in oxygenated ice-cold sucrose-based solution. Slices were transferred to an oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 128 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 24 NaHCO3, 1.25 NaH2PO4, and 10 glucose. The slices were kept in ACSF to recover for at least 1 h before recording. Patch recording electrodes (4–6 MΩ) were filled with K-gluconate solution containing biocytin. CRH neurons expressing hM3Dq-mCherry in the PVN were visually identified with a Nikon FN-1 fluorescence microscope (Nikon, Tokyo, Japan). Data were acquired using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) and analyzed using pCLAMP 10 software (Molecular Devices). In the current-clamp mode, after a stable membrane potential was recorded, CNO was bath-applied to determine the effects of hM3Dq receptor activation. After recording, slices were fixed in 4% paraformaldehyde, and biocytin-filled neurons were visualized using streptoavidin-conjugated Alexa Fluor-350 (1:2000; S11249, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Cancer cell implantation

Lewis lung carcinoma (LLC) cells and LLC-luc cells were suspended with extracellular matrix gel (Sigma-Aldrich, Inc.) and Hank’s Balanced Salt solution (Thermo Fisher Scientific Inc.) at a concentration of 1 × 106 cells/mL. The cell suspension (1 × 106 cells/mL; 0.5 mL) was transplanted subcutaneously into the right lower back of WT/hM3Dq mice, CRH-Cre/hM3Dq mice and C57BL/6J mice that had been injected with vehicle or hydrocortisone.

Graft tumor growth assay

After tumor implantation, tumor size was measured using calipers, and tumor volume was calculated as (L × W2)/2, (L = length and W = width). In CRH-Cre/hM3Dq mice, WT/hM3Dq mice and C57BL/6J mice that had been injected with vehicle or hydrocortisone, tumor size was measured every other day for 18–21 days after implantation.

Corticosterone ELISA

Blood was collected from the abdominal aorta of mice by a 26 gauge needle (TERUMO Corp., Osaka, Japan), and transferred to a collection tube that contained an anticoagulant such as EDTA under isoflurane anesthesia (3%, inhalation). To separate the plasma fraction, the blood sample was centrifuged at 5000 rpm for 5 min at room temperature, and the upper layer was then gently collected. The assay was performed using the DetectX® Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA) according to the instructions from the manufacturer.

Flow cytometry

Under isoflurane anesthesia (3%, inhalation), spleen was harvested from the mice for the analysis of spleen-derived immune cells, and then homogenized using a syringe plunger with PBS (Thermo Fisher Scientific Inc.). After the cell suspension was filtered through a nylon mesh and 100 μm cell strainer to remove cell aggregates, it was then treated with ammonium chloride hemolysis.

For the analysis of tumor-infiltrating lymphocytes (TILs) and LLC-luc cells, tumor tissue (< 1.0 g) was harvested from the mice under isoflurane anesthesia (3%, inhalation) and minced into small pieces. 2× Tumor & Tissue Dissociation Reagent (TTDR; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) diluted with Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific Inc.) was added to the tissue suspension, and the mixture was then incubated for 30 min at 37 °C. After the incubation period, 1% Albumin, from Bovine Serum, Fraction V pH7.0 (BSA; FUJIFILM Wako Pure Chemical Corp.)/2 mM ethylenediaminetetraacetic acid (EDTA; NIPPON GENE Co., Ltd., Tokyo, Japan) in PBS (25 mL) was added to the suspension. The homogenized suspension was segregated using a 70 μm cell strainer to remove cell aggregates. Subsequently, a hemolytic agent ammonium chloride was added to a cell suspension.

Before specific extracellular staining to classify immune cells, cells were incubated with an anti-CD16/32 antibody (Becton, Dickinson and Company) to avoid non-specific binding. Next, cells were stained with the following antibodies; APC/Cy-conjugated anti-CD45.2 (104), FITC or PE-conjugated anti-CD4 (GK1.5), PE-conjugated anti-CD8a (53–6.7), PE or APC-conjugated anti-CD3ε (145-2C11), APC-conjugated anti-CD49b (DX5), PE/Cy7-conjugated anti-Ly6G (1A8), APC-conjugated anti-CD11b (M1/70), PE-conjugated anti-Ly6C (HK1.4), and PE-conjugated anti-F4/80 (BM8). All antibodies were purchased from BioLegend (San Diego, CA, USA). Dead cells were segregated by staining with propidium iodide (PI; Sigma-Aldrich, Inc.) or 7-aminoactinomycin D (7-AAD; Becton, Dickinson and Company). Immune cells were sorted and analyzed using a BD FACS Aria™ II Cell Sorter (Becton, Dickinson and Company) and Flowjo™ software (Becton, Dickinson and Company).

Quantitative real-time reverse-transcription PCR (qRT-PCR) for mRNA

Total RNA obtained from immune cells of the mice described above was extracted using a mirVana miRNA Isolation Kit (Thermo Fisher Scientific Inc.), and purified total RNA was then reverse-transcribed into cDNA using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. qPCR was performed using a StepOne Plus™ System (Thermo Fisher Scientific Inc.) with a Fast SYBR® Green Master Mix (Thermo Fisher Scientific Inc.) and synthesized primers (Additional file 2: Table S1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control.

Statistical analysis

The results are presented as the mean ± S.E.M. The statistical significance of differences between groups was determined using an unpaired t-test or two-way ANOVA followed by Bonferroni’s multiple comparisons test. Statistical analyses and graph generations were performed with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).