The study protocol was approved by the Institutional Experimental Animal Ethics Committee of Fengxian Hospital, Southern Medical University (No. 2022-0334). Female C57BL/6 mice (15–18 g, aged 5 weeks) were obtained from Guangdong DK Pharmaceutical Co. Ltd. (License No: SCXK 2022-0060). The mice were housed in cages collectively for the control group and individually for the CUMS group at controlled temperature. A week-long acclimatization was provided, during which the mice had ad libitum access to food and water.

The scheme of the animal experimental design is shown in Figure 1A. At the endpoint, all mice were euthanized, and then blood serum, tumors, and spleens were collected. Tumors were photographed and weighed, with a portion of the tumor fixed in formalin for histopathology staining, and the remaining tumor tissue was used for Western blot and qRT-PCR experiments. The spleens were processed into single-cell suspensions for flow cytometry analysis.

The CUMS procedure was performed according to the protocol (Nollet, 2021), with a slight modification. Mice in the CUMS group were exposed to two different stressors daily. The detail of the specific interventions performed is shown in Supplementary Table S1.

Behavioral assessments were conducted consistently in 6 days, including the sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open field test (OFT). Each behavioral evaluation was conducted with a 24-h interval between tests. The detail of behavioral testing is shown in our previous research (Li et al., 2023).

The ID8 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in the laboratory. Cells were cultured in DMEM high glucose medium (Gibco, California, United States) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). Mice were subcutaneously injected with 1 × 10⁶/mL ID8 cells in 100 µL of phosphate-buffered saline (PBS) to establish a subcutaneous heterotopic transplantation tumor model. Tumor volume was measured every week.

Tumor tissues of mice were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, cut into 4-μm sections, and used for immunohistochemistry (IHC) staining with antibodies against Ki67 (1:1,000, Abcam, United Kingdom) and HDC polyclonal antibody (Immunoway Biotechnology, United States). The representative images were acquired under a light microscope (Olympus IX73, Tokyo, Japan). Ki67 index was calculated as the number of immunopositive cells × 100% divided by the total number of cells in seven random fields at ×400 magnification.

HIS (Elabscience, China), NE (Fine Test, China), COR (Fine Test, China), IL-6 (Fine Test, China), IL-17A (Elabscience, China), and 5-HT (Fine Test, China) in serum and in cell supernatant was analyzed using ELISA kits following the manufacturer’s instructions.

The tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sliced sections were dewaxed using solvents, stained with hematoxylin and eosin, and photographed under a microscope.

The expressions of HDC, STAT3, p-STAT3, S100A9, and GAPDH proteins were analyzed by Western blotting. The primary antibodies used include HDC polyclonal antibody (Immunoway Biotechnology, United States), Stat3 (124H6) antibody (Cell Signaling, Massachusetts, United States), Phospho-Stat3 (Tyr705) (D3A7) XP^® antibody (Cell Signaling, Massachusetts, United States), S100A9 antibody (Invitrogen California, Texas, United States), and GAPDH (ABclonal, China). The band density was analyzed using a gel imaging system and compared with an internal control.

Total RNA was extracted with TRIzol (Invitrogen™, Carlsbad, United States). The extracted RNA was reverse-transcribed to complementary DNA (cDNA) using an Evo M-MLV RT Mix Kit (ACCURATE BIOTECHNOLOGY CO., LTD., Changsha, China). For qRT-PCR, a SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY CO., LTD., Changsha, China) was utilized following the manufacturer’s protocol. Relative gene expression was calculated using the 2^−ΔΔCT method. The list of primers is shown in Supplementary Table S2.

Mouse spleen single-cell suspensions were stained with specific antibody for 20 min, including PE anti-mouse CD4 (Biolegend, California, United States), FITC anti-mouse CD8a (Biolegend, California, United States), and PerCP/Cyanine5.5 anti-mouse CD3 (Biolegend, California, United States) and then were analyzed using a flow cytometer (Canto; BD Biosciences, Shanghai, China) equipped with Cell-Quest software (BD Biosciences).

A2780 and ES-2 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in the laboratory. A2780 cells were cultured in DMEM high glucose medium (Gibco, California, United States) supplemented with 10% FBS (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). ES-2 cells were cultured in RPMI 1640 medium (Gibco, California, United States) supplemented with 10% FBS (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). All cell lines were cultured in a humidified atmosphere of 5% CO₂ at 37°C.

For the in vitro cell test, four groups were set up with different treatment types: the normal control (NC) group was cultured in de-hormone medium; NE + COR group with 10 μM NE and 1.5 μM COR (Sigma-Aldrich, Saint Louis, United States) in the medium; NE + COR + HIS group with 10 μM NE, 1.5 μM COR, and 10 μM HIS (APExBIO, Houston, United States) in the medium; and BHOA group with 1 mM BHOA (APExBIO, Houston, United States) in the medium.

HDC knockdown or overexpression cell lines were generated with transiently transfected cells with HDC siRNA or HDC overexpression plasmid, respectively (Hanbio Tech, Shanghai, China). The vectors of OE-HDC were pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro. Specific HDC siRNA and HDC overexpression plasmid were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Massachusetts, United States) for 48 h. The transfection efficiency was observed through a fluorescence microscope, and HDC expression was determined through Western blotting analysis. Cells transfected with non-targeting siRNA (siNC group) and empty plasmid vectors (vector group) were used as controls. The transfected cells were then utilized to establish the siHDC group (HDC knockdown) and the OE-HDC group (HDC overexpression). Details of the siRNA and overexpression plasmid sequences used are listed in Supplementary Table S3.

Cells (3,000 per well) were seeded into 96-well plates. Ten microliters of Cell Counting Kit-8 solution (NCM Biotech, Suzhou, China) was added to each well after the cells were incubated at 37°C for 24–120 h. The optical density (OD) was then measured at 450 nm. Cell viability (%) = [(OD value of experimental group - OD value of blank control group)/(OD value of control group - OD value of blank control group)] × 100%.

Cells seeded in 6-well plates (1 × 10²/well) were serum-starved and cultured in hormone-depleted medium with various treatments for 10–14 days (medium changed every three days). Cells were fixed as the number of cells in a single clone exceeded 50, stained with crystal violet, dried, and photographed.

Cells were seeded in 6-well plates (1 × 10⁶/well) and serum-starved for 12 h. Wounds were scratched using sterile tips, followed by washing in PBS. Cell migration was observed and photographed at 0, 24, and 48 h, and the mobility was calculated.

Cells were resuspended in serum-free medium at 2.5 × 10⁵ cells/mL. The transwell chamber was placed in a 24-well plate, with 600 µL of 10% FBS and treatment solution (10 µM NE + 1.5 µM COR or 10 µM NE + 1.5 µM COR + 10 µM HIS) in the lower chamber and 200 µL of the cell suspension in the upper chamber. After 12–24 h of incubation at 37°C, cells on the underside of the membrane were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. Cells residing at the bottom of the chamber were gently removed, and the chamber was washed. After drying, six random fields were photographed under a microscope (100×) per chamber, and the number of migrated cells was counted and recorded.

Cells were coated with Matrigel (Corning, New York, United States) 4 h in advance, and the other steps were the same as for the cell migration assay.

All the data are expressed as mean ± standard deviation (SD). The GraphPad Prism 9.5 software package was used for statistical analysis and visualization. Three independent experiments were carried out with similar results in qRT-PCR, Western blotting, ELISA, clone formation assay, scratch healing assay, cell migration assay, and cell invasion assay. Data were analyzed with an unpaired Student’s t-test and one-way analysis of variance. p-value <0.05 was considered statistically significant.

To explore the effects of CUMS on depression-like behaviors in mice, the SPT, TST, FST, and OFT were performed before and after CUMS to verify the depression-like behaviors (Supplementary Figure S1). Specifically, the sucrose preference was significantly lower in the CUMS group in the SPT compared with the normal control (NC) group (p < 0.01). The immobility time in the CUMS group was increased significantly in the TST and FST compared with that in the NC group (p < 0.01). Mice exposed to CUMS tended to move less in the OFT, with significant reductions in total distance, distance in central area, time in central area, and number of entries to the central area compared with the NC mice (p < 0.01, respectively). These results confirm that the depression model was successfully built.

To investigate the impact of HIS treatment on depression-like behaviors in mice, we compared the behavioral outcomes of mice before and after HIS treatment. We found that HIS treatment effectively alleviated depression-like behaviors induced by CUMS in the ovarian cancer comorbid depression mouse model. Specifically, HIS increased sucrose preference in the SPT (Figure 1B), decreased immobility time in the TST (Figure 1C) and FST (Figure 1D), and decreased total distance, distance in central area, time in central area, and number of entries to the central area in the OFT (Figures 1E, F) compared with the tumor + CUMS group.

To investigate the effects of chronic stress and histamine treatment on tumor progression in vivo, we measured diverse tumor features. The images of tumors in different groups are shown in Figure 2A. Compared with the tumor group, the tumor volume and weight of the tumor + CUMS group were increased significantly (p < 0.01 respectively), while the tumor volume and weight of the tumor + HIS group were decreased, with no significant difference. Compared with the tumor + CUMS group, the tumor volume (p < 0.05) and weight (p < 0.01) of the tumor + CUMS + HIS group decreased significantly (Figures 2B, C). Additionally, H&E staining of the tumor tissue showed that compared with the tumor group, the tumor + CUMS group exhibited an increase in the nucleus-to-cytoplasm ratio, distinct nucleoli, and pathological nuclear division. In contrast, compared with the tumor + CUMS group, the tumor + CUMS + HIS group showed a decrease in tumor cells and an increase in stroma (Figure 2D). Histopathological examination suggested that CUMS enhanced the Ki67 index, a marker of cell proliferation, while HIS treatment reduced the Ki67 index (p < 0.01, respectively, Figure 2E).

In exploring the interplay between chronic stress and HIS in tumor progression, we observed that CUMS profoundly altered key biochemical markers in mice serum. Compared with the tumor group, CUMS significantly decreased HDC and HIS levels and increased IL-6, IL-17A, NE, COR, and 5-HT levels. Exogenous HIS treatment significantly increased HIS levels and decreased IL-6, IL-17A, COR, and 5-HT levels, with no significant effect on NE. Our findings indicated that CUMS changed the levels of HDC, HIS, inflammatory factors, stress hormones, and 5-HT, and HIS treatment reversed these changes, except for HDC (Figure 3A).

Given that chronic stress promoted tumor growth while histamine treatment alleviated this effect, yet the regulatory mechanisms of HDC remain unclear, so we analyzed mouse tumor gene expression using immunohistochemistry, Western blot, and qRT-PCR. Immunohistochemical staining experiments specifically indicated that CUMS significantly reduced the expression of HDC in mouse tumors (Figure 3B).

Western blot analysis indicated that compared with the tumor group, the expression of HDC protein in the tumor + CUMS group was significantly decreased (p < 0.01). In contrast, the levels of p-STAT3 and S100A9 proteins were significantly increased (p < 0.01), while there was no significant difference in STAT3 protein expression. When comparing the tumor + CUMS group with the tumor + CUMS + HIS group, the expressions of p-STAT3 (p < 0.01) and S100A9 (p < 0.01) were significantly decreased in the tumor + CUMS + HIS group, with no significant differences in HDC and STAT3 proteins (Figures 3C, D). Similar results were observed for the changes in the mRNA expression levels (Figure 3E). The above results indicate that CUMS-induced downregulation of HDC leads to increased secretion of IL-6, phosphorylation of STAT3, and expression of S100A9, thereby activating the IL-6/STAT3/S100A9 pathway, whereas HIS effectively reversed the over-activation of this pathway induced by CUMS.

To explore the effects of CUMS and HIS treatment on mouse immunity, we performed flow cytometry to measure the proportions of various immune cells (Figure 4A). Specifically, the proportion of CD3⁺ T cells (Figure 4B) was significantly reduced (p < 0.01) in the tumor group than in the NC group. HIS treatment significantly increased the levels of CD3⁺CD8⁺ Tc cells (Figure 4C) and CD3⁺ T cells compared with the tumor group (p < 0.05). Additionally, the proportion of CD3⁺ T cells in the tumor + CUMS + HIS group was significantly higher than that in the tumor + CUMS group (p < 0.05). The proportion of CD3⁺CD4⁺ Th (Figure 4D) cells remained stable. Furthermore, compared with the NC group, the spleen weight of mice in the tumor group was higher (p < 0.05), while HIS treatment reduced the spleen weight of mice in the tumor + HIS group compared with the tumor group (Figure 4E).

To further validate the impact of chronic stress on ovarian cancer progression, we employed an in vitro approach by treating A2780 and ES-2 cell lines with stress hormones and HIS. CCK-8 analysis showed that 100 μM and 500 µM HIS suppressed A2780 and ES-2 cell proliferation at 12 h, 24 h, and 48 h (p < 0.01, respectively), while 10 µM did not. Thus, 10 µM was chosen as a non-toxic concentration to study the effects of HIS on the IL-6/STAT3/S100A9 pathway, excluding direct tumor growth interference (Figure 5A).

To further explore the function of HIS in modulating the effects of stress hormones, A2780 and ES-2 cells were stimulated with the stress hormones NE and COR and then subsequently treated with HIS. We found that HIS treatment inhibited cell proliferation, migration, and invasion induced by stress hormones. Specifically, NE and COR treatment increased the number of colonies formed and promoted wound healing, cell migration, and invasion (p < 0.01, respectively). However, 10 µM HIS treatment decreased the number of colonies formed and promoted scratch healing, cell migration, and invasion (p < 0.01, respectively, Figures 5B–D).

Based on our previous results that chronic stress induced downregulation of HDC expression, we further explored the impact of HDC on tumor progression by constructing knockdown and overexpression of HDC in ovarian cell lines. Functionally, knockdown of HDC promoted cell proliferation, migration, and invasion in A2780 and ES-2 cells with siHDC compared with the NC groups (Figures 6A–C), while overexpression of HDC inhibited cell proliferation, migration, and invasion (Figures 6D–F).

In our preliminary experiments, we found that cells exposed to NE and/or COR downregulated the level of HDC protein and increased the phosphorylation of STAT3 and the expression of S100A9 (Supplementary Figure S2). The IL-6/STAT3/S100A9 pathway is a potential downstream target of HDC (Zhou et al., 2021). To elucidate the regulatory role of HDC in this signaling pathway, we conducted Western blot assay and ELISA to quantify protein expression and cytokine secretion in A2780 and ES-2 cell lines following various drug treatments, HDC knockdown, and HDC overexpression.

Following various drug treatments, the IL-6/STAT3/S100A9 pathway expressions in A2780 and ES-2 cell lines were detected by Western blot (Figure 7A) and ELISA (Figures 7B, C). NE + COR decreased HDC but increased IL-6, p-STAT3 (Tyr 705), and S100A9 levels (p < 0.01, respectively), while HIS treatment induced the opposite changes, except for HDC. The HDC inhibitor BHOA decreased HDC but increased IL-6, p-STAT3 (Tyr 705), and S100A9 levels (p < 0.01, respectively).

Knockdown of HDC in A2780 and ES-2 cells reduced HIS but increased IL-6, p-STAT3, and S100A9 (p < 0.01, respectively), activating the IL-6/STAT3/S100A9 pathway (Figures 7D–F). Conversely, HDC overexpression increased HIS and reduced IL-6, p-STAT3, and S100A9 (p < 0.01, respectively), inhibiting the IL-6/STAT3/S100A9 pathway (Figures 7G–I).

Overall, these results indicated that HDC downregulation induced by stress hormones promoted the proliferation, migration, and invasion of ovarian cancer cells through activation of the IL-6/STAT3/S100A9 signaling pathway.