
==== Front
Cancer Immunol Immunother
Cancer Immunol Immunother
Cancer Immunology, Immunotherapy : CII
0340-7004
1432-0851
Springer Berlin Heidelberg Berlin/Heidelberg

39235612
3800
10.1007/s00262-024-03800-8
Research
Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence
Kawaguchi Yo kawaguchi1228@yahoo.co.jp

Okamoto Keigo
Kataoka Yoko
Shibata Kohei
Saito Hiroki
Shiratori Takuya
Ueda Keiko
Ohshio Yasuhiko
Hanaoka Jun
https://ror.org/00d8gp927 grid.410827.8 0000 0000 9747 6806 Division of General Thoracic Surgery, Department of Surgery, Shiga University of Medical Science, Tsukinowacho, Seta, Otsu City, Shiga 520-2192 Japan
5 9 2024
5 9 2024
11 2024
73 11 21213 2 2024
5 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Patients with lung cancer have a high incidence of tumor recurrence even after curative surgical resection. Some reports indicated that immunosuppressive cells induced by surgical stress could contribute to tumor recurrence after surgery; however, the underlying mechanisms are not fully understood. In this study, we found that increased postoperative blood monocytes served as a risk factor for tumor recurrence in 192 patients with non-small cell lung cancer (NSCLC). We established the lung cancer recurrent mouse model after tumor resection and showed that the surgical stress immediately increased the level of serum monocyte chemoattractant protein-1 (MCP-1), which subsequently increased blood monocytes. These blood monocytes were rapidly recruited into distant micrometastases and became tumor growth-promoting tumor associated macrophages (TAMs). Furthermore, even after the blood MCP-1 and monocytes decreased enough 72 h after tumor resection, TAMs in micrometastases remained rich because the MCP-1 secreted by micrometastases themselves continued to recruit monocytes around the tumor. Consequently, tumor resection triggered the outgrowth of distant metastases via the MCP-1–Monocyte–TAM axis. When we administered the MCP-1 inhibitor to the lung cancer recurrent model mice, blood monocytes decreased after tumor resection, and TAMs in micrometastases also dramatically decreased. Finally, peri- and postoperative treatment with the MCP-1 inhibitor suppressed distant metastases after surgery. Targeting the MCP-1–Monocyte–TAM axis may inhibit surgical stress-induced NSCLC recurrence by attenuating postoperative immunosuppressive monocytes in micrometastases.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-024-03800-8.

Keywords

Postoperative recurrence
Micro metastasis
Surgical stress
Monocytes
MCP-1
TAM
YOKOYAMA Foundation for Clinical PharmacologyYRY‐2005 Kawaguchi Yo issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2024
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pmcIntroduction

Lung cancer is the leading cause of cancer-related mortality worldwide [1]. One of the reasons for this is postoperative recurrence. Although curative surgical resection is the most effective treatment for non-small cell lung cancer (NSCLC), up to 60% of tumor recurrence is observed within five years, especially during stages 2–3A of NSCLC, even after the induction of adjuvant cisplatin-based chemotherapy [2]. therefore, a thorough investigation of the molecular mechanisms underlying postoperative metastasis and better perioperative management to reduce tumor recurrence are vital.

Surgical stress accelerates tumor recurrence after surgery [3–5]. Circulating tumor cells (CTCs) float in the blood and reach metastatic organs not only in advanced lung cancer, but also in early stage lung cancer that can be curatively resected [6]. Usually, most CTCs are eliminated by the host’s anti-tumor immune response. In contrast, only a few CTCs become dormant tumor cells, but their growth is suppressed by host immune cells. When dormant tumor cells recruit immunosuppressive cells around them, they begin to grow because immunosuppressive cells are released from the suppression of tumor growth by anti-tumor immune cells, resulting in growing metastasis [7]. Surgical resection of the primary tumor can activate dormant tumor cells in distant organs (micrometastases) and induce metastatic outgrowth by increasing the number of immunosuppressive cells [4, 5, 8].

Cancer-associated fibroblasts, neutrophils, and platelets are major immunosuppressive cells at metastatic sites [7]. In contrast, only a few reports have indicated that monocytes function as immunosuppressive cells in breast [9] and colorectal [10] cancer metastases. In our study, we showed that blood monocytes increased in patients after lung cancer surgery and the postoperative recurrence rate was higher in patients with high blood monocytes. Furthermore, our recent study demonstrated that blood monocytes are recruited around lung cancer cells and differentiated into tumor-associated macrophages (TAMs) by the tumor-secreted monocyte chemoattractant protein-1 (MCP-1). The MCP-1 blockade decreases TAM levels in tumor microenvironment (TME) and suppresses lung cancer growth [11]. Based on these results, we hypothesized that increasing monocytes after lung cancer resection are recruited to metastatic lesions and differentiated into TAMs, then dormant tumor cells at metastatic sites start to grow. However, this mechanism has not been clarified.

In this study, we established a lung cancer recurrent mouse model after surgery and showed that tumor resection triggered the outgrowth of distant metastases via the MCP-1–Monocyte–TAM axis. Moreover, the MCP-1 inhibitor suppressed this axis, consequently, recurrence dramatically decreased. Our findings indicated that peri- and postoperative treatment with the MCP-1 inhibitor attenuates the impact of surgery on recurrence, suggesting that this treatment may improve the prognosis of patients with lung cancer who received surgery.

Materials and methods

Clinical samples

This study was approved by the Institutional Review Board of Shiga University of Medical Science (R2020-048).

We reviewed the clinicopathological data of 192 patients (Table 1, Supplemental Table 1) with NSCLC who underwent surgery at Shiga University of Medical Science Hospital between January 2015 and December 2016. The data of blood monocyte count were collected at post-operative day 3. Monocyte counts greater than 500/µL were defined as “high” and used as a cut-off value to indicate lung cancer recurrence after surgery. This definition showed a sensitivity of 63.2%, specificity of 51.1%, and area under the receiver operating characteristic curve of 0.554 (Supplemental Fig. 1a). Histological assessments were performed according to the World Health Organization histological classification. The seventh edition of the TNM classification for lung cancer was used for pathological staging.Table 1 Lung cancer recurrence rate after surgery in patients with several clinicopathological characteristics

Variables		N	Cancer recurrence	5-year recurrence rate (%)	p-value	
(−)	(+)	
Total		192	135	57	34.2	–	
Age (years)	 > 70	103	69	34	40.1	0.146	
	≦ 70	89	66	23	28.2		
Sex	Male	117	76	41	41.6	0.018	
	Female	75	59	16	23.6		
PS	0	151	107	44	32.8	0.370	
	1 or 2	41	28	13	39.8		
Smoking states	Never	55	43	12	23.8	0.083	
	Smoker	137	92	45	38.6		
COPD	(−)	118	88	30	28.7	0.052	
	(+)	74	47	27	43.7		
p-TNM stage	I	136	107	29	22.6	 < 0.001	
	II	31	18	13	48.2	0.036	
	III	25	10	15	73.7	 < 0.001	
Surgical procedure	Lobectomy	144	99	45	34.5	0.519	
	Limited resection	48	36	12	32.4		
Histological subtype	Adeno	133	98	35	29.7	0.037	
	Squamous	51	32	19	45.3	0.126	
	The others	12	7	5	52.0	0.031	
Pleural invasion	(+)	39	20	19	28.5	 < 0.001	
	(−)	111	37		60.1		
Lymph duct invasion	(+)	49	26	23	26.7	 < 0.001	
	(−)	141	108	33	57.1		
Vascular invasion	(+)	92	53	39	19.4	 < 0.001	
	(−)	99	82	17	50.5		
Blood monocytes	≧ 500/µL	105	68	37	40.1	0.043	
	 < 500/µL	87	67	20	27.5		

Cell lines and culture

The human lung adenocarcinoma cell line A549 and murine carcinoma cell line LLC were provided by the Cell Resource Center for Biomedical Research of Tohoku University. The human lung adenocarcinoma cell line RERF-LC-MS was provided by the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (Osaka, Japan). These cell lines were maintained in DMEM (Nacalai tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Biosera, South Africa origin) and 1% penicillin–streptomycin-amphotericin B (PSA; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C in 5% CO2 in a humidified incubator.

Reagents

The FITC-conjugated anti-mouse Ly6c antibody for flow cytometry was purchased from BioLegend (San Diego, CA, USA). The APC-conjugated anti-mouse CD206 antibody for flow cytometry and recombinant mouse MCP-1 were purchased from R&D Systems (Minneapolis, MN, USA). The PE-conjugated anti-mouse CD4 antibody, FITC-conjugated anti-mouse CD8, APC-conjugated anti-mouse Foxp3, APC-conjugated anti-mouse CD11b, and PE-conjugated anti-mouse Ly6G for flow cytometry were purchased from eBioscience (San Diego, CA, USA). The FITC-conjugated anti-mouse CD3 for flow cytometry was purchased from Invitrogen (Carlsbad, CA, USA). The PE-conjugated anti-mouse NK1.1 for flow cytometry was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The MCP-1 inhibitor (nimesulide) and isoflurane were purchased from Fujifilm Wako Pure Chemical Corporation. The MTT solution (cell titer) was purchased from Promega (Madison, WI, USA). The red blood cell lysis buffer was purchased from Invitrogen (Carlsbad, CA, USA). Collagenase was obtained from Roche (Basel, Switzerland). Paraformaldehyde phosphate (4%) was purchased from Nacalai Tesque.

ELISA

A549 (5 × 105 cells), RERF-LC-MS (1 × 105 cells), and LLC (1 × 105 cells) cells were seeded onto a 6-cm dish and cultured in 10% FBS- and 1% PSA-containing DMEM with nimesulide at the various concentrations for 48 h. Their supernatants were collected and the levels of secreted MCP-1 were determined using ELISA (MCP-1 kit; R&D Systems).

For mouse serum MCP-1 detection, mice were euthanized via isoflurane inhalation, and the blood was immediately collected via cardiac puncture. Blood plasma was isolated by incubation for two hours to promote clotting and centrifugation (2000×g, 20 min). The samples were analyzed using the same ELISA kits.

Preparation of cells

To collect circulating immune cells, mice were euthanized via isoflurane inhalation. The blood was immediately isolated via cardiac puncture and collected in EDTA tubes (Greiner Bio-One, Kremsmünster, Austria). To purify white blood cells, 300 µL of whole blood was added to 2.0 mL of red blood cell lysis buffer for 3 min at 37 °C to deplete erythrocytes. White cells were washed twice with PBS and analyzed using flow cytometry.

To isolate tumor-infiltrating cells, the tumors were removed from mice, dissected, and then digested with 40 µL of collagenase at 37 °C for 30 min. The cells were washed twice with PBS, passed through 95-µm mesh, and analyzed using flow cytometry.

Bone marrow cells were isolated from the femur of 8-week-old C57BL/6J mice (Japan SLC, Hamamatsu, Japan). The cells were mixed with 2.0 mL of red blood cell lysis buffer for 3 min at 37 °C. Monocytes were isolated from the bone marrow cells using a FACSAria Fusion cell sorter (BD Biosciences, San Jose, CA, USA) with an anti-mouse FITC-conjugated Ly6c antibody. Isolated monocytes were maintained in 20% FBS- and 1% PSA-supplemented DMEM at 37 °C in 5% CO2 in a humidified incubator.

Flow cytometry

Ly6c antibodies were used as monocyte markers in mouse blood and pan-TAM markers in tumor tissue. CD206 antibodies were used as tumor-promoting TAM markers. The CD8 antibody was used as a marker of cytotoxic T lymphocytes (CTLs). CD4/Foxp3 antibodies were used as regulatory T cell (Treg) markers. CD11b/Ly6G antibodies were used as myeloid-derived suppressor cell (MDSC) markers. CD3/NK1.1 antibodies were used as natural killer (NK) cells markers. Isolated white blood cells, tumor-infiltrating cells, or bone marrow cells were stained with 2 µL antibodies in 20 µL of FACS buffer (PBS containing 2% FBS and 0.1% sodium azide). After 30 min, the cells were washed twice with PBS and resuspended in 500 µL FACS buffer for flow cytometric analysis. Data were analyzed using a FACSCanto flow cytometer (BD Biosciences).

MTT assay

Bone marrow (4 × 104) cells were seeded into 96-well plates and incubated with 0–10 ng/mL recombinant MCP-1 in the RPMI containing 2% FBS and 1% PSA for 48 h. A549 (1 × 104), RERF-LC-MS (1 × 104) and LLC (1 × 104) cells were seeded into 96-well plates and incubated with 0–200 µM nimesulide in the DMEM containing 2% FBS and 1% PSA for 48 h. Subsequently, 10 µL of MTT solution was added. After 4 h of incubation, the absorbance was measured at 492 nm using a microplate reader (TECAN, Infinite M200, Mannedorf, Switzerland).

Cell migration assay

Cell migration assays were performed using a 24-well transwell system. Five-micrometer pore transwell inserts (Chemotaxicell; Kurabo, Osaka, Japan) and 24-well plates (Corning, PA, USA) were used as the upper and lower chambers, respectively. Bone marrow cells (2 × 105 cells) were placed in the upper chamber containing 200 µL RPMI with 2% FBS and 1% PSA. In the lower chamber, 500 µL RPMI with 2% FBS and 1% PSA containing 0–10 ng/mL recombinant MCP-1 were placed. After incubation at 37 °C for 6 h, the Ly6c monocytes migrating to the lower chamber were counted using flow cytometer.

Macrophage differentiation assay

Bone marrow cells (20 × 105) were seeded on a 5-cm dish and cultured with recombinant MCP-1 (0 or 10 ng/mL) in FBS-free RPMI with 1% PSA for 48 h. Differentiation of bone marrow cells into Ly6c monocytes/CD206 TAMs was quantified using a flow cytometer.

Animals

Eight-week-old female C57BL/6J mice (Japan SLC) were maintained under specific pathogen-free conditions. All animal experiments were performed in compliance with the Guidelines for Animal Experimentation of Shiga University of Medical Science (approval number: 2022-6-23). During tumor resection, a 3-cm skin incision was made, the tumor was resected, and the skin was sutured under isoflurane inhalational anesthesia. The mice were administered 40 mg/kg BW/day of MCP-1 inhibitor (nimesulide) suspended in 100 µL PBS via oral gavage.

Post-operative metastasis mouse model

LLC cells (1 × 105) were injected subcutaneously into the right flank of mice. Two weeks later, the tumor was resected.

To compare lung metastases between mice with or without tumor resection, the control mice received only inhalational anesthesia, and the tumor was left in the mice until the lungs were harvested at three weeks.

To compare lung metastases between mice with or without MCP-1 inhibitor treatment, the mice were euthanized at the four weeks, and the lungs were harvested. MSP-1 inhibitor (nimesulide) was administered three hours before tumor resection and the administration was continued daily until the day of the lung harvest.

Experimental lung metastasis mouse model

LLC cells were suspended to a density of 5 × 105 cells/100 µL PBS and were injected into the tail vein. Six hours after LLC injection, Ly6c monocytes (5 × 105 cells/100 µL PBS) were additionally injected into the tail vein. The mice were euthanized ten days after the injection of LLC cells and the lungs were harvested. Nimesulide was administered one hour before the LLC injection and the administration was continued daily until nine days after the LLC injection.

Lung cancer mouse model with micrometastasis-like lesion

Main tumor: LLC cells (1 × 105) were injected subcutaneously into the left flank of mice.

Micrometastasis-like lesion: Ten days later, LLC cells (1 × 105) were subcutaneously injected into the right flank of the same mice. After more 10 days, a lung cancer mouse model with micrometastasis-like lesion was established. Nimesulide was administered three hours before the main tumor resection in the established mouse model, and administration was continued daily until the day of micrometastasis-like lesion analysis.

Tumor measurements and quantification of tumor nodules in the lungs

Tumor dimensions were measured twice a week using a caliper, and tumor volumes were calculated using the following formula: a × b2/2, where a is the largest diameter and b is the smallest diameter.

To assess lung metastasis, nodules on the lung surface were counted macroscopically immediately after the lungs were harvested. The lungs were then fixed with 4% paraformaldehyde phosphate for one day and embedded in paraffin. Maximum sections of paraffin-embedded lungs (4-µm thick) were stained with hematoxylin–eosin. The nodules were counted microscopically in the entire field of the section with a microscope of 400 × magnification. Lung metastases were calculated using macroscopic nodules on the lung surface and microscopic nodules in lung sections. The incidence of lung metastasis was calculated as: 100 × (mice with lung metastases/total mice).

Statistical analysis

Statistical analyses were performed using SPSS Statistics for Windows (version 25; IBM, Armonk, NY, USA). Significant differences between groups were determined using the Student’s t test. The association between variables were analyzed using the χ2 test. Survival curves were prepared using the Kaplan–Meier method and compared using the log-rank test. Cox’s proportional hazards model was used for multivariate analysis. Statistical significance was set at P < 0.05.

Results

High blood monocytes increased the risk of post-operative recurrences in lung cancer patients

First, we investigated whether post-operative blood monocytes promoted lung cancer recurrence. We found that the blood monocyte counts increased after lung cancer surgery (Fig. 1a). In addition, the 5-year recurrence rate was higher in patients with high post-operative blood monocytes (40.1%) than in patients with low post-operative blood monocytes (27.5%, p = 0.043) (Fig. 1b). Table 1 shows the 5-year recurrence rate in patients with several clinicopathological factors. Male sex, stage 2, stage 3, and histological type, along with pleural invasion, lymph duct invasion, vascular invasion, and high post-operative blood monocytes were significant risk factors for cancer recurrence according to univariate analysis. Post-operative high blood monocytes were not associated with these common risk factors for tumor recurrence according to the χ2 test (Supplemental Table 1). It was considered that increased monocytes after lung cancer surgery functions as one of the risk factors for recurrence. As shown in Table 2, factors of stage 2 or 3 disease (HR: 2.03) and pleural invasion (HR: 1.81) showed the significant risk for recurrence. A high post-operative blood monocyte level was not a significant risk factor according to this multivariate analysis, however, high post-operative blood monocytes tended to show higher risk for recurrence (hazard ratio; HR:1.49).Fig. 1 High levels of blood monocytes increased the risk of recurrences after lung cancer surgery. a Blood monocyte levels increased after lung cancer surgery in 192 patients with NSCLC. b Post-operative high blood monocytes count (≧ 500 µL) was significantly associated with tumor recurrence after surgery. c Scheme of establishing post-operative metastasis mouse model (n = 8, in each group). d The blood Ly6c monocytes increased 6 h after surgery. e Tumor resection mouse had more lung metastases (3.5 counts/mouse) than anesthesia mouse (0.8 counts/mouse) (n = 8, in each group). f Macroscopic lung surface (upper panels) and hematoxylin–eosin-stained microscopic lung section (lower panels) of tumor resection mouse with lung metastases (►) and only anesthesia mouse without lung metastases. Data are shown as mean + SD. *P ≤ 0.05, and ***P < 0.001 (Student’s t test)

Table 2 Cox proportional hazards analysis for recurrence

Variables		HR	95% CI	p-value	
Sex	Male	1.30	0.71–2.39	0.399	
p-TNM stage	II, III	2.03	1.10–3.75	0.023	
Histological subtype	The others	1.49	0.53–4.22	0.452	
Pleural invasion	(+)	1.81	1.02–3.21	0.042	
Lymph duct invasion	(+)	1.73	0.90–3.32	0.098	
Vascular invasion	(+)	0.59	0.31–1.15	0.123	
Blood monocytes	≧ 500/µL	1.49	0.83–2.67	0.185	

Blood monocytes increased after surgery in post-operative metastasis mouse model

We established the post-operative metastasis mouse model (Fig. 1c). In this mouse model, the number of blood Ly6c monocytes increased 6 h after surgery (Fig. 1d). Furthermore, we demonstrated that in tumor resection mouse, 3.5 lung metastases grew per mouse, whereas in the anesthetized mouse, only 0.8 lung metastases grew (Fig. 1e, 1f).

MCP-1 recruited monocytes from bone marrow cells and differentiate them into TAMs

To identify the mediator that increases blood monocyte levels, we focused on MCP-1, which has been reported to be a key mediator of monocyte recruitment [11]. First, we investigated MCP-1 secretion ability of LLC and showed that LLC secreted a considerable amount of MCP-1 into the supernatant (Fig. 2a). We also demonstrated that, along with tumor growth, serum MCP-1 levels increased in a subcutaneous inoculation mouse model of LLC (Fig. 2b). Next, we analyzed the effects of MCP-1 on bone marrow cells. Using MTT assay, we demonstrated that MCP-1 promoted the proliferation of bone marrow cells (Fig. 2c). Transwell chamber assays demonstrated that MCP-1 recruited Ly6c monocytes from bone marrow cells. In addition, MCP-1 also recruited Ly6c negative immune cells. The percentages of migration cells were higher in Ly6c monocytes than that in Ly6c negative immune cells (Fig. 2d). These results indicated that although MCP-1 recruited several immune cells it was mainly Ly6c monocytes. Furthermore, MCP-1 differentiated bone marrow cells into Ly6c monocytes and partially differentiated them into CD206 TAMs (Fig. 2e). In the subcutaneous inoculation mouse model, along with an increase in serum MCP-1 levels, the number of monocytes in the blood (Fig. 2f) and TAMs in the tumor tissue increased (Fig. 2g).Fig. 2 Lung cancer secreting MCP-1 recruited monocytes from bone marrow and differentiate them into TAMs. a LLC secreted considerable amount of MCP-1 into the supernatant after a 48-h incubation. b Serum MCP-1 levels increased in subcutaneous LLC inoculation mouse along with the tumor growth (n = 3, in each group). c Recombinant MCP-1 promoted proliferation of bone marrow cells. d Recombinant MCP-1 actively recruited Ly6c monocytes from bone marrow cells. In addition, recombinant MCP-1 also recruited Ly6c negative immune cells. e Recombinant MCP-1 differentiated bone marrow cells into Ly6c monocytes and partially into CD206 TAMs. f In subcutaneous LLC inoculation mouse model, as the increasing of blood MCP-1 level, the blood monocytes increased and g TAMs in the tumor tissue also increased. Data are shown as mean + SD. *P ≤ 0.05, and **P < 0.01 (Student’s t test)

Ly6c monocytes promote lung cancer metastases via MCP-1

Next, we examined the role of Ly6c monocytes in promoting metastatic recurrence via MCP-1. In the post-operative metastasis mouse model, the serum MCP-1 level rapidly increased 6 h after surgery and decreased 24 h after surgery (Fig. 3a). As serum MCP-1 levels increased, the number of blood monocytes also temporarily increased (Fig. 3b). To determine whether this temporal increase in monocytes promotes the growth of micrometastases, we used an experimental lung metastasis mouse model. Ly6c monocytes were isolated from the bone marrow cells using a cell sorter (Supplemental Fig. 1b). To test whether monocytes were recruited early in the metastasis process, we administered monocytes 6 h after injection of LLC cells, which is the time point at which almost all LLC cells adhere to the lungs [9]. The injection of Ly6c monocytes led to increase metastatic regions in the lungs compared to the injection of LLC cells alone (Fig. 3c). We considered that metastatic tumors also secrete MCP-1 to recruit monocytes and promote the growth of metastases. To test this, we administered the MCP-1 inhibitor to mice and sequentially injected LLC cells through the tail vein. As a results, the MCP-1 inhibitor suppressed lung metastases (Fig. 3d). These results suggested that an increasing number of blood monocytes are recruited around micrometastases via MCP-1 and promote tumor growth.Fig. 3 Ly6c monocytes promote lung cancer metastases via MCP-1. a Serum MCP-1 level rapidly increased in 6 h and decreased 24 h after tumor resection. b Blood monocytes also increased in 6 h and decreased 24 h after tumor resection. c Additional injection of Ly6c monocytes to mouse increased lung metastases compared with injection of only LLC cells (n = 3, in each group). d MCP-1 inhibitor (nimesulide) suppressed the lung metastases (n = 3, in each group). Data are shown as mean + SD. *P ≤ 0.05 (Student’s t test). n.s.; not significant

Ly6c monocytes were recruited to micrometastasis-like lesions and triggered their outgrowth after main tumor resection

To the best of our knowledge, whether increased blood monocytes after tumor resection are actually recruited to micrometastases remains unexplored. To confirm this hypothesis, we established a lung cancer mouse model with micrometastasis-like lesion (Fig. 4a). This model did not have real micrometastases, but a main (large) tumor and a micrometastasis-like (small) lesion coexisting in the same mouse. We expected that the dynamic change in immune cells in the micrometastatic lesion could be observed in this mouse model after the main tumor resection, which might imitate the change in real micrometastases. First, we showed that Ly6c TAMs in micrometastasis-like lesions were much fewer than those in the main tumor (Fig. 4b). In addition, the growth speed of the micrometastasis-like lesions was suppressed, compared to main tumor growth rate from 0 to 1.5 weeks after LLC inoculation (Fig. 4c). Next, the main tumor was resected, and the TAMs in the micrometastasis-like lesions were counted using flow cytometry at 0.5, 6, 24, and 72 h after the main tumor resection. As a results, TAMs in micrometastasis-like lesions drastically increased immediately after the main tumor resection and remained abundant after 72 h (Fig. 4d). In response to the increasing numbers of monocytes, the growth rate of micrometastasis-like lesions recovered after the main tumor resection at 1.5 weeks (Fig. 4e). Based on these results, we considered the mechanism by which surgery triggers the outgrowth of micrometastases as follows. (Fig. 4f): When the main tumor exists, it secretes a large amount of MCP-1, and the blood MCP-1 level becomes very high. Therefore, it is difficult for monocytes to move from the blood to the micrometastases. (Fig. 4g): When the main tumor was resected, the blood monocyte level increased and some of the blood monocytes start to move into the micrometastases 0.5–6 h after tumor resection. (Fig. 4h): Even after serum MCP-1 levels and blood monocytes decreased in 24–72 h, TAMs in micrometastases remained abundant. This is because after a sufficient decrease in blood MCP-1 level, micrometastases can easily recruit blood monocytes via their secretion of MCP-1. Based on the increase in the number of monocytes, micrometastases triggered their outgrowth (Fig. 4e).Fig. 4 Ly6c monocytes were recruited to micrometastasis like-lesion after main tumor resection, and then triggered the outgrowth. a Lung cancer mouse model with micrometastasis-like lesion. This mouse model had both main (large) tumor and micrometastasis-like lesion (small) tumor (n = 4, in each group in every experiment). b Ly6c TAMs in micrometastasis-like lesion were much fewer than that in main tumor. c Growth of micrometastasis was suppressed when coexisting with main tumor. Blue line shows main tumor growth curve from 0 to 1.5 weeks after LLC inoculation in mice. Orange line shows micrometastasis-like lesion from 0 to 1.5 weeks after inoculation in another mouse (n = 4, in each group). The tumor in each mouse group was resected at 1.5 weeks and weighed. d Immediately after the main tumor resection, TAMs in micrometastases were drastically increased. e In response to increasing monocytes, the growth rate of micrometastasis-like lesions was recovered after main tumor resection at 1.5 weeks (orange line). Blue line shows the main tumor growth rate in another mouse as a control (n = 4, in each group). The tumor in each mouse group was resected at 2.5 weeks and weighed. f–h The mechanism through which the surgery triggered the outgrowth of micrometastases. f Main tumor heavily secretes MCP-1 and blood MCP-1 levels become very high. Therefore, it is difficult for monocytes to move from the blood into micrometastases. g When the main tumor was resected, blood monocytes increase and some of them start to move into the micrometastases 0.5–6 h after tumor resection. h Even after the blood MCP-1 level and blood monocytes decrease in 24–72 h, TAMs persist in micrometastases because micrometastases could easily recruit the blood monocytes via their MCP-1. Data are shown as mean + SD. *P ≤ 0.05 (Student’s t test). n.s.; not significant

We also evaluated changes in other immune cells in micrometastasis-like lesions, as it has been reported that TAMs were increased at metastatic sites earlier than were other immunosuppressive cells, which altered the immunosuppressive environment. We found that Tregs (Supplemental Fig. 2a) and MDSCs (Supplemental Fig. 2b) were increased in micrometastasis -like lesions after main tumor resection, whereas CTLs (Supplemental Fig. 2c) and NK cells (Supplemental Fig. 2d) were not.

MCP-1 inhibitor decreased serum MCP-1 and blood monocytes after tumor resection, resulting in suppression of TAMs in micrometastasis

We expected MCP-1 to be a therapeutic target for suppressing monocytes in micrometastasis and tumor outgrowth. We used nimesulide as MCP-1 inhibitor [11] and checked that it suppressed the secretion of MCP-1 not only from the mouse lung cancer cell line (LLC), but also from human lung cancer cell lines (A549, RERF-LC–MS) without inhibition of cell growth (Supplemental Fig. 3). We also checked that administration of the MCP-1 inhibitor (40 mg/kg BW/day) to LLC bearing mice (Supplemental Fig. 4a) decreased TAMs (Supplemental Fig. 4b) and suppressed the growth of the tumor (Supplemental Fig. 4c).

Perioperative administration of the MCP-1 inhibitor reduced serum MCP-1 levels in mice after tumor resection (Fig. 5a). Furthermore, blood monocytes after tumor resection were also decreased by MCP-1 inhibitor treatment (Fig. 5b) compared to the control (Fig. 3b). In response to the decrease in serum MCP-1 and blood monocytes, the number of TAMs in the micrometastasis-like lesions also decreased (Fig. 5c, d).Fig. 5 MCP-1 inhibitor decreased serum MCP-1 and monocyte levels after tumor resection, resulting in suppression of TAMs in micrometastasis-like lesion. a The perioperative administration of MCP-1 inhibitor reduced the serum MCP-1 level, b blood monocytes, and c TAMs in the micrometastasis-like lesion after tumor resection. d Postoperative TAMs in the micrometastases-like lesion with MCP-1 inhibitor treatment significantly lower than that without treatment (n = 4, in each group). Data are shown as mean + SD. *P ≤ 0.05, **P < 0.01 and ***P < 0.001 (Student’s t test). n.s.; not significant

MCP-1 inhibitor suppressed lung metastasis after surgery

Finally, we investigated whether treatment with the MCP-1 inhibitor decreased tumor recurrence after surgery. Because lung cancer recurrence mostly occurs in distant organs [12], a post-operative metastasis mouse model is required to mimic the clinical scenario. The mouse model has already been established for breast cancer [9], but not for lung cancer. Therefore, we applied the method and established the post-operative metastasis mouse model for lung cancer (Fig. 6a). We evaluated the presence of lung metastases, both macroscopically and microscopically (Fig. 6b). Lung metastases were observed in all mice when their lungs were harvested four weeks after LLC inoculation (Fig. 6c). When the MCP-1 inhibitor treatment was initiated at the time of tumor resection, the incidence of lung metastases decreased (Fig. 6c). In addition, we noted that the induction of the MCP-1 inhibitor treatment significantly decreased the number of lung metastases (Fig. 6d).Fig. 6 MCP-1 inhibitor suppressed lung metastasis after surgery. a Scheme of establishing the post-operative metastasis mouse model (n = 8, in each group in every experiment). b Macroscopic metastases (►) on lung surface and hematoxylin–eosin-stained microscopic lung metastases (►) in lung section. c Lung metastases incident rate in the post-operative metastasis mouse model (n = 8, in each group). MCP-1 inhibitor treatment decreased the incident rate. d MCP-1 inhibitor treatment significantly decreased the number of lung metastases (n = 8, in each group). Data are shown as mean ± SD. *P ≤ 0.05 (Student’s t test)

Discussion

Systemic response to surgery facilitates cancer recurrence by reactivating the dormant micrometastasis [3–5]. However, the molecular mechanisms that trigger post-operative metastasis in lung cancer remain unclear. In this study, we demonstrated for the first time that postoperative high blood monocytes were the risk factor for recurrence in patients with lung cancer using univariate analysis, suggesting that monocytes are essential effector cells for the induction of recurrence. In addition, we demonstrated that tumor cells in distant organ recruited an increasing number of blood monocytes after surgery and differentiated them into TAMs via MCP-1, resulting in metastasis in a lung cancer mouse model. Furthermore, we showed that inhibition of MCP-1 decreased monocytes and TAM in micrometastasis and reduced tumor recurrence.

A total of 99.99% of CTCs that adhere to distant organs die because of an attack by the host’s immune system [13]. If they survive, most cells eventually enter dormancy [14]. To avoid immune system attacks, CTCs adherent to distant organs interact with cancer-associated fibroblasts, neutrophils, and platelets. They protect CTCs as immunosuppressive cells from being attacked by anti-tumor immune cells such as natural killer (NK) cells. Increasing the number of immunosuppressive cells in micrometastases plays a critical role in promoting the growth switch of dormant tumor cells [7]. As the tumor die-dormancy-growth status has plasticity, changing the tumor status towards dormancy or death may be an effective treatment. Dormant cancer cells sometimes survive for several years and are resistant to adjuvant chemoradiotherapy [15]; therefore, depleting immunosuppressive cells around CTCs can be a therapeutic target to reduce metastatic recurrence.

During lung cancer recurrence, we suspect that monocytes mainly function as immunosuppressive cells. Monocytes are recruited to metastatic sites earlier than other immunosuppressive cells to alter the immunosuppressive environment [9]. Ly6c monocytes function as immunosuppressive cells in breast [9] and colorectal [10] cancer metastatic recurrence. However, whether monocytes function as immunosuppressive cells leading to lung cancer recurrence has not yet been proven [16]. Our study demonstrated that MCP-1 recruited Ly6c monocytes for micrometastasis and promoted the growth of lung metastasis in a mouse model.

Monocytes that enter the tumor tissue are called TAMs. TAMs (known as tumor-promoting macrophages) reportedly change the immune cells in the TME towards immunosuppression. TAM-derived CCL-22 promotes immunosuppressive TME by recruiting Tregs. Furthermore, TAMs secreting CXCL1 enhanced the proliferation, migration, and anti-CD8+ T cell functions of MDSCs [17]. Our study showed that the number of Tregs and MDSCs increased in response to an increase in the number of TAMs in the TME. In addition, our results showed that MCP-1 also recruited immune cells other than monocytes. This has indicated that MCP-1 also recruited immunosuppressive cells like Tregs [18] and MDSCs [19], resulting in early migration of these cells into micrometastasis-like lesions after the main tumor resection. TAMs also promote tumor growth by secreting proangiogenic cytokines in the hypoxic TME, including vascular endothelial growth factor (VEGF) to facilitate tumor angiogenesis [20]. Based on these results, we concluded that MCP-1/TAMs might be an effective therapeutic target by recovering anti-tumor immunity to suppress the growth of micrometastasis.

MCP-1 is the main mediator of macrophage recruitment to the TME and differentiation [11]. The MCP-1 inhibitor may represent a suitable strategy to deplete TAMs in the TME to suppress the lung cancer growth [11, 21]. In addition, the overexpression of MCP-1 in patients with lung cancer is associated with poor survival [22]. In the present study, we used nimesulide as an MCP-1 inhibitor. The MCP-1 is produced via the nuclear factor (NF)-κB pathway and nimesulide down-regulates this pathway to inhibit the production and secretion of MCP-1 [23]. We previously reported that the inhibition of human and mouse cancer cell-secreted MCP-1 by nimesulide suppressed the recruitment of monocytes into the TME and their differentiation into the tumor-promoting phenotype. We also examined the effects of nimesulide on other mediators, such as prostaglandin E2 (PGE2), colony stimulating factor-1 (CSF-1), and cellular communication network factor-3 (CCN3), which were previously reported as mediators associated with the recruitment and differentiation of TAM, and concluded that nimesulide suppressed TAMs only via MCP-1 inhibition [11]. MCP-1 is secreted not only from cancer cells but also injured cells. Injured muscle cells, injured skin cells, and neutrophils at the wound site secrete MCP-1 to recruit macrophages in mice [24, 25]. In addition, some studies demonstrated that the serum MCP-1 level is elevated by 2–threefold at 24 h after laparoscopic surgery in humans [26, 27]. Because nimesulide also inhibits the secretion of MCP-1 from these cells [23], the elevation of serum MCP-1 levels after surgery might be much suppressed. As a result, the number of blood monocytes decreased, consequently, the number of TAMs in the TME decreased.

In this study, we generated the lung cancer mouse model with micrometastasis-like lesions, as this model has not been reported previously. We needed to observe changes in immune cells in micrometastasis after primary tumor resection. To prepare the lung cancer mouse model with micrometastasis-like lesions, we first (day0) inoculated LLC tumor cells (primary-like large tumor at day 20) and then (day10) inoculated LLC tumor cells (micrometastasis-like very small tumor at day 20) in the same mouse. Finally, we succeeded to observe the dynamic increase of TAMs inmicrometastasis-like region after surgery, which indicated the immunological mechanism in outgrowth of metastases. As a limitation, it is unclear whether our micrometastasis-like lesion can reflect real micrometastasis. We expect that the applicability of this mouse model will be proven in future studies.

There were some limitations in this study. First, we could not demonstrate the significant correlation between high post-operative blood monocyte levels and tumor recurrence by multivariate analysis, because of insufficient power and confounding factors. The small number of patients in this study may not demonstrate significance, and confounding factors of recurrence like male and vascular invasion might affect this non-significant result. Second, to prepare lung cancer bearing mice, we inoculated LLC subcutaneously rather than in the lung. Thus, surgical stress differed from that experienced after actual lung cancer surgery. Third, we used the mouse lung cancer cell line LLC. Therefore, it is unclear whether the same results would be observed in human lung cancer. Finally, whether nimesulide clinically suppresses the serum MCP-1 level is unknown, as there are no data on nimesulide as an MCP-1 inhibitor in humans. However, a phase 1 study demonstrated the safety and tolerability of MCP-1 inhibitor treatment for patients with solid cancers [28]. We expect that if patients with lung cancer are treated with the MCP-1 inhibitor in perioperative period, metastatic recurrence after surgery can be reduced.

To our knowledge, this is the first study to explore the immunological mechanism and therapeutic potential of the MCP-1–Monocyte–TAM axis in lung cancer recurrence. The findings from the current study provide the following insights. First, surgery causes dynamic changes in immune status not only in systemic but also in local metastatic regions. CTCs that adhere to metastatic organs recruit increasing numbers of blood monocytes after surgery and differentiate them into TAMs, resulting in outgrowth of metastasis. Second, the MCP-1 inhibitor decreases the number of monocytes and TAMs in the micrometastases, leading to reduced metastatic recurrence after surgery. Our results suggest that the risk of metastatic recurrence after surgery can be reduced by developing a therapeutic method targeting the MCP-1–Monocyte–TAM axis.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file 1 Supplemental Figure 1: (a) Receiver operating characteristic curve for predicting lung cancer recurrence based on post-operative blood monocytes. (b) Ly6c monocytes isolated from mouse bone marrow cells using a cell sorter.

Supplementary file 2 Supplemental Figure 2: Change in the number of regulatory T cells (Tregs) (Supplemental Fig. 2a), myeloid-derived suppressor cells (MDSCs) (Supplemental Fig. 2b), cytotoxic T lymphocytes (CTLs) (Supplemental Fig. 2c), and natural killer (NK) cells (Supplemental Fig. 2d) in the micrometastasis like-lesions after main tumor resection.

Supplementary file 3 Supplemental Figure 3: MCP-1 inhibitor inhibited the secretion of MCP-1 from mouse lung cancer cell line LLC and human lung cancer cell lines A549, RERF-LC-MS. No significant inhibition of cell growth was observed. Data are shown as mean + SD. *P ≤ 0.05 and **P < 0.01 (Student’s t test). n.s.; not significant.

Supplementary file 4 Supplemental Figure 4: Administration of MCP-1 inhibitor decreased TAMs in tumor microenvironment and suppressed the tumor growth. (a) Scheme of administration of MCP-1 inhibitor to LLC bearing mouse. (b) When we administered the MCP-1 inhibitor (40 mg/kg BW/day) to mice in which LLC were subcutaneously injected, TAMs in the tumor decreased and (c) LLC tumor growth was suppressed (n=5, in each group). Data are shown as mean +SD. *P ≤ 0.05 (Student’s t test).

Supplementary file 5 (DOCX 19 kb)

Abbreviations

CTC Circulating tumor cell

CTL Cytotoxic T lymphocyte

MCP-1 Monocyte chemoattractant protein-1

MDSC Myeloid-derived suppressor cells

NK Natural killer

NSCLC Non-small cell lung cancer

TAM Tumor associated macrophage

TME Tumor microenvironment

Treg Regulatory T cell

Acknowledgements

N/A

Author contributions

K.O., Y.K. and K.U. supported parts of the experiments. K.S., H.S. and T.S. supported patient’s data collection. Y.K. and Y.O. supported making conceptualization. J.H. supervised the work. Y.K. performed patient’s data collection, making the conceptualization, all the experiments, analyzing the data and writing first draft. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the YOKOYAMA Foundation for Clinical Pharmacology (YRY‐2005).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Review Board of Shiga University of Medical Science (R2020-048).

Animal studies

All mouse experiments were performed in compliance with the Guidelines for Animal Experimentation from Shiga University of Medical Science (Approval number: 2022-6-23).

Consent to participate

N/A.

Consent to publish

N/A.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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