Fluoxetine was purchased from Sigma (St. Louis, MO, United States). The ELISA kits of 5-HT, Trp, kynurenine, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were from Shanghai Jianglai biological technology Co., Ltd. (Shanghai, China). TDO, IDO1, IDO2 antibodies were obtained from Abcam (United States). Caspase-1, Caspase-3, Caspase-4, Caspase-5, Caspase-7, Caspase-12 antibodies were obtained from Immunoway Biotechnology Co., Ltd. (Beijing, China). AhR antibodies were obtained from Proteintech (United States). FITC-conjugated anti-mouse CD3, APC/Cyanine7-conjugated anti-mouse CD4, PE/Cyanine7-conjugated anti-mouse CD8a, APC-conjugated anti-mouse CD25, PE-conjugated anti-mouse Foxp3, APC-conjugated anti-mouse NK-1.1, and PE-conjugated anti-mouse TCR-β antibodies were obtained from Biolegent (California, United States).

Human NSCLC cell line A549 and mice NSCLC cell line LLC were obtained from Shanghai Institutes of Cell Biology (Shanghai, China). Cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 units/ml penicillin at 5% CO₂ at 37°C.

Female C57BL/6 mice weighing 18–20 g were bought from the Shanghai Jiesijie laboratory animal technology Co., Ltd. (Animal Quality Certificate: 20180004000898). Mice were housed in the laboratory animal center, East China Normal University, Shanghai (Animal experiment license: SYXK 2010-0094) in a specific pathogen-free (SPF) lab until they were acclimated to their surroundings for 7 days to habituate to the experimenter. The research related animal used has been compiled with all relevant national regulations and institutional policies for the care and use of animals and has been approved by the Animal Research Ethics Committee in Fengxian Hospital, Southern Medical University.

The open-field test was conducted in a quiet room. Briefly, the open-field consisted of an opaque plastic box (100 × 100 × 40 cm) divided equally into 20 × 20 cm² squares. Mice were placed in the center of this field, and the number of squares mice moved and standing times were monitored using a ZSZFT Video Analysis System (ZSZRDC science and technology Co., Ltd., China) as indexes of locomotion activity and exploratory behavior, respectively in 5 min. Mouse with behavior scores of >120 or <30 was considered as abnormal and ruled out of the experiment.

Mice were housed individually and exposed to sucrose solution (2% in tap water, Sigma, St Louis, Mo, United States) for 72 h, followed by 18 h of water deprivation and 2 h exposure to two identical bottles, one was filled with 2% sucrose solution and the other was filled with water. The weight of the sucrose solution and water exposed for 2 h was measured. Sucrose preference was defined as the ratio of sucrose weight to total weight (sucrose + water) during the 2 h test, normalized to the body weight of each animal.

The CUMS-induced depression-like behavior model was established in C57BL/6 mice. The CUMS-induced mice were individually housed in cages for 8 weeks and exposed to the stressors in random order (Table 1). The same stress was not allowed to appear in two consecutive days to avoid prediction. Control mice were fed normally without any stress during this period. 8 weeks later, the open-field test, the sucrose preference experiment and the level of 5-HT were used to verify the success of the depression model. Then, LLC lung cancer cells (1 × 10⁶ cells) suspended in 0.2 ml PBS was subcutaneously inoculated in the right flank of C57BL/6 mice. The entire experimental procedure is shown in Figure 1.

Mice were randomized into seven groups as follows (eight animals each): group A (blank control), group B (tumor-bearing control), group C (tumor-bearing + FLX), group D (CUMS control), group E (CUMS + FLX), group F (tumor-bearing + CUMS), and group G (tumor-bearing + CUMS + FLX). Healthy mice were set as a control without any treatment. After 3 days, fluoxetine (20 mg/kg/day) was administrated via gavage for 2 weeks. Four control groups (groups A, B, D, and F) were given vehicle for 2 weeks. At 24 h after the last administration, all mice were killed. Blood was collected from the inner canthus. Tumors were isolated and weighed.

The levels of 5-HT, Tryptophan, kynurenine, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α in serum were assessed using ELISA kits according to the operating guide.

To determine the percentages of Th cells, Tc cells, and Treg cells in the total T cell population, the pretreated cells were stained with FITC-conjugated anti-mouse CD3 and treated with fixation and permeabilization regents as described in the manufacturer’s instructions. Then, the cells were stained with antibodies. To determine the percentages of Th in the total CD3⁺ T cell population, the cells were stained with FITC-conjugated anti-mouse CD3, APC/Cyanine7-conjugated anti-mouse CD4, and PE/Cyanine7-conjugated anti-mouse CD8a antibodies. To determine the percentages of regulatory T cells in the total CD4⁺ T cell population, the cells were stained with APC/Cyanine7-conjugated anti-mouse CD4 and APC-conjugated anti-mouse CD25 antibodies and treated with fixation and permeabilization regents. Then, the cells were stained with PE-conjugated anti-mouse Foxp3 antibodies. All the cells were resuspended in 0.2 ml of the staining buffer before being detected by flow cytometry. The data were analyzed with FACS Diva™ software. All antibodies used in the experiment were purchased by Biolegent (California, United States).

Total RNA was extracted with Trizol according to the protocol (Sangon Biotech, SK1312/BS409, Shanghai, China) and RNA concentration were measured with NanoDrop ND-100 Spectrophotometer (Thermo Scientific, Wilmington, DE, United States). For qRT-PCR, TliRNaseH Plus was used according to the manufacturer’s protocol. The primers sequences are shown in Table 2.

The tumor tissues and cells were collected and sonicated in lysis buffer on ice. The samples were centrifuged and proteins was quantified by BCA protein assay kit. Total protein (20 μg) from each sample was run on SDS-PAGE and transferred to PVDF membranes. The blots were steeped with 5% fat-free milk in Tris-buffered saline-Tween 20 (TBST) for 4 h, followed by incubation with primary antibody overnight at 4°C. All antibodies were diluted with 5% fat-free milk in TBST buffer (dilution rate: TDO, IDO1, IDO2, and AhR were 1:1,000, cleaved-caspases and β-actin were 1:2,000). The blots were washed with TBST buffer three times (10 min each time), and then labeled with secondary antibody at room temperature for 2 h respectively. Finally, membranes were visualized using ECL detection reagents and analyzed by ImageJ software.

The vitro experiments were divided into two groups as follows: control group and fluoxetine group. A549 Cells (5,000 cells per well) were plated into 96-well plates, then treated with fluoxetine for 72 h. Cell viability was assayed using CCK-8 according to the manufacturer’s instructions. The OD value at 450 nm was detected and recorded with GloMaxMuiti+(Promega, E8032, United States).

Five hundred cells were seeded in DMEM with 10% FBS on 60 mm plates, and treated with fluoxetine for 12 h, then cultured for 7 days or 10 days. The number of the clones (P50 cells) was assessed by counting under a microscope.

Cells were seeded into 6-well plates and treated with fluoxetine for 72 h. The cells were collected and seeded at 0.1 ml (5×10⁴ cells/well) into transwell upper chambers with DMEM. The bottom chambers were filled with 0.6 ml DMEM with 10% FBS as a chemoattractant. After 8 h or 24 h, non-migratory cells were carefully removed with a cotton swab.

A549 Cells were seeded into 6-well plates, treated as above for 24 h/48 h, then collected and washed with PBS twice, stained with Annexin V-FITC and PI (556419, 556421, BD Biosciences). Samples were analyzed on Flow cytometry (BD, FACS Canto, United States). Annexin V-FITC positive and PI negative, Annexin V-FITC positive and PI positive were considered as apoptotic cells.

All analyses were performed using the SPSS 25.0. Data were expressed as mean ± SD. Statistical comparisons between the two groups were performed using the two independent-samples t-test and the inter group were performed using the One-way analysis of variance (ANOVA). (two-tailed) p < 0.05 was considered to be statistically significant.

An open-field behavioral test, sucrose preference test, and biochemical test revealed significant differences between two groups after 8 weeks of chronic stress treatment. At the end of 8 weeks of CUMS, the locomotion and exploratory scores, sucrose preference, and 5-HT levels are decreased in CUMS model group mice compared to the baseline. No significant change occurred over time in the normal control group mice (Figure 2). All results confirmed that CUMS successfully induce depression-like behavior in mice.

Tumor weight and body weight of all tumor-bearing mice were examined. The tumor volume in group B (tumor-bearing control) was significantly smaller than that of group F (tumor-bearing + CUMS). The result indicated that CUMS promoted tumor growth. The tumor volume in group B (tumor-bearing control) was significantly larger than that of group C (tumor-bearing + FLX). Meanwhile the tumor volume in group F (tumor-bearing + CUMS) was significantly larger than that of group G (tumor-bearing + CUMS + FLX), suggesting fluoxetine could inhibit tumor growth in tumor both under stress and without stress (Figures 3A–C). However, there was no difference of the body weight in these four groups (Figure 3D).

As shown in Figure 4, tryptophan and 5-HT levels in group D (CUMS control), E (CUMS + FLX), F (tumor-bearing + CUMS), G (tumor-bearing + CUMS + FLX) were lower than those of group A (blank control). However, after fluoxetine administration, tryptophan and 5-HT levels in group C (tumor-bearing + FLX), E (CUMS + FLX) and G (tumor-bearing + CUMS + FLX) were higher than those of group B (tumor-bearing control), D (CUMS control) and F (tumor-bearing + CUMS), respectively (Figures 4A,B). Meanwhile, kynurenine levels in group C (tumor-bearing + FLX) and G (tumor-bearing + CUMS + FLX) were lower than those of group B (tumor-bearing control) and F (tumor-bearing + CUMS), respectively (Figure 4C). The ratio of tryptophan/kynurenine was increased after fluoxetine administration (Figure 4E). The data suggested that fluoxetine exerts antitumor effect via increasing the ratio of tryptophan/kynurenine.

The flow cytometry data showed that the numbers of CD4⁺ Th cells and CD8⁺ Tc cells were increased (Figures 5A,C), while the CD25⁺ FOXP3⁺ Treg cells were decreased (Figures 5B,D) after fluoxetine administration. Thus, the results revealed that fluoxetine enhanced Th and Tc cells responses, induced cellular immunity in vivo.

As shown in Figure 6, fluoxetine promoted differentiation of CD4⁺ T cells into Th1 cells that increased concentrations of IL-2, IFN-γ; inhibited differentiation of CD4⁺ T cells into Th2 and Th17 cells that decreased concentrations of IL-4, IL-6, IL-10, IL-17A. These data indicated that fluoxetine enhanced cellular immunity, weakened humoral immunity and inflammatory response.

We further investigated the related enzyme expression of kynurenine pathway. The western blot data revealed that CUMS obviously up-regulated IDO1, IDO2 expression, fluoxetine administration reversed this up-regulation (Figure 7). However, there was no significant change in TDO. Meanwhile the AhR expression showed a similar trend as well as IDO. This result indicated that fluoxetine down-regulated IDO1, IDO2, AhR to inhibit kynurenine pathway in vivo.

Further, we investigated the key immune related pro-apoptosis genes (cleaved-caspase 1, 4, 5, 12) and executioner caspase genes (cleaved-caspase 3, 7). As shown in Figure 8 , cleaved-caspase 4, 7, 12 showed a significant increase in group C (tumor-bearing + FLX) and group G (tumor-bearing + CUMS + FLX) compared with group B (tumor-bearing control) and group F (tumor-bearing + CUMS), respectively. Fluoxetine significantly potentiated the expression of cleaved-caspase 4, 7, and 12 in comparison with the vehicle group (Figure 8). However, there was no significant change in cleaved-caspase 1, 3, or 5. These results indicated the pro-apoptotic and regulated inflammation cytokines activities of fluoxetine administration in vivo.

We verified the antitumor effect of fluoxetine in A549 cells. The growth-inhibition curves showed that the 50% inhibition concentration (IC₅₀) of fluoxetine was 15 μmol/L (Figure 9A). Compared with the control group, fluoxetine induced apoptosis and reduced migration and clonal formation (Figures 9B–E). Moreover, fluoxetine down-regulated IDO1, IDO2, and AhR expression and up-regulated immune related pro-apoptosis protein cleaved-caspase 4, 5, and 7 (Figures 9F–I). But there was no significant change in cleaved-caspase 1, 3, or 5. The results of the in vitro experiment were consistent with those in vivo.