Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence

Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence Yo Kawaguchi, Keigo Okamoto, Yoko Kataoka, Kohei Shibata, Hiroki Saitoh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3954307/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 7 You are reading this latest preprint version Abstract 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 TAMs (tumor associated macrophages). Furthermore, even after the blood MCP-1 and monocytes decreased enough 72 hours 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. Postoperative recurrence micro metastasis surgical stress monocytes MCP-1 TAM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction 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 ) 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. 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 Clinicopathological characteristics in lung cancer patients between high or low blood monocytes after surgery. Variables N Low monocytes N (%) High monocytes N (%) p-value Total 192 151(79) 41 (21) - Age (years) > 70 103 81 (79) 22 (21) 1.000 ≦ 70 89 70 (79) 19 (21) Sex Male 117 89 (76) 28 (24) 0.276 Female 75 62 (83) 13 (17) PS 0 151 119 (79) 32(21) 0.916 1 or 2 41 32 (78) 9 (22) Smoking states Never 55 44 (78) 11 (22) 0.772 Smoker 137 107 (47) 30 (39) COPD (-) 118 94 (80) 24 (20) 0.665 (+) 74 57 (77) 17 (23) p-TNM stage Ⅰ 136 109 (80) 27 (20) 0.429 Ⅱ 31 24 (77) 7 (23) 0.856 Ⅲ 25 18 (72) 7 (28) 0.385 Surgical procedure Lobectomy 144 114 (79) 30 (21) 0.760 Limited resection 48 37 (77) 11 (23) Histological subtype Adeno 133 101 (76) 32 (24) 0.170 Squamous 51 45 (88) 6 (12) 0.051 The others 12 9 (75) 3 (25) 0.750 Pleural invasion (+) 39 28 (72) 11 (28) 0.287 Lymph duct invasion (+) 49 36 (73) 13 (27) 0.328 Vascular invasion (+) 92 71 (77) 21 (23) 0.791 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% CO 2 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 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 × 10 5 cells), RERF-LC-MS (1 × 10 5 cells), and LLC (1 × 10 5 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 hours. 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 minutes). 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 minutes 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 minutes. 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 ware mixed with 2.0 mL of red blood cell lysis buffer for 3 minutes 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% CO 2 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. Isolated white blood cells, tumor-infiltrating cells, or bone marrow cells were stained with 2 µL FITC-conjugated Ly6c and APC-conjugated CD206 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 × 10 4 ), A549 (1 × 10 4 ), RERF-LC-MS (1 × 10 4 ) and LLC (1 × 10 4 ) 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 hours. 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 × 10 5 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 hours, the Ly6c monocytes migrating to the lower chamber were counted using flow cytometer. Macrophage differentiation assay Bone marrow cells (20 × 10 5 ) 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 hours. 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 recurrent mouse model LLC cells (1×10 5 ) were injected subcutaneously into the right flank of mice. Two weeks later, the tumor was resected. Three weeks after LLC injection, the mice were euthanized, and the lungs were harvested. In contrast, the control mice received only inhalational anesthesia, and the tumor was left in the mice until the lungs were harvested. Experimental lung metastasis mouse model LLC cells were suspended to a density of 5 × 10 5 cells/100 µL PBS and were injected into the tail vein. Six hours after LLC injection, Ly6c monocytes (5 × 10 5 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. Micrometastasis mouse model Main tumor: LLC cells (1 × 10 5 ) were injected subcutaneously into the left flank of mice. Micrometastasis: Ten days later, LLC cells (1 × 10 5 ) were subcutaneously injected into the right flank of the same mice. After more 10 days, a mouse model of micrometastasis 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 analysis. Post-operative spontaneous metastasis mouse model LLC cells (1 × 10 5 ) were injected subcutaneously into the right flank of mice. Two weeks after the LLC injection, the tumor was resected. Four weeks after LLC injection, the mice were euthanized, and the lungs were harvested. Nimesulide was administered three hours before tumor resection and the administration was continued daily until the day of the lung harvest. 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 × b 2 /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 χ 2 test. Survival curves were prepared using the Kaplan–Meier method and compared using the log-rank test. Statistical significance was set at P < 0.05. Results High blood monocytes increased the risk of postoperative recurrences in lung cancer patients. First, we investigated whether post-operative blood monocytes promoted lung cancer recurrence. We found that the blood monocyte count increased after lung cancer surgery (Fig. 1 a). In addition, post operative high blood monocytes were significantly associated with tumor recurrence (Fig. 1 b). Univariate analysis showed that high blood monocytes were not associated with p-TNM stage, lymph duct invasion, or vascular invasion, which are the common risk factors for tumor recurrence (Table 1 ). We believe that increased monocyte count after lung cancer surgery functions as an independent risk factor for recurrence. Blood monocytes increased after surgery in post-operative recurrent mouse model We established the post-operative recurrent mouse model ( Fig. 1 c). In this mouse model, the number of blood Ly6c monocytes increased 6 hours after surgery (Fig. 1 d). 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. 1 e, 1 f). 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. 2 a). We also demonstrated that, along with tumor growth, blood MCP-1 levels increased in a subcutaneous inoculation mouse model of LLC (Fig. 2 b). 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. 2 c). Transwell chamber assays demonstrated that MCP-1 recruited Ly6c monocytes from bone marrow cells (Fig. 2 d). Furthermore, MCP-1 differentiated bone marrow cells into Ly6c monocytes and partially differentiated them into CD206 TAMs (Fig. 2 e). In the subcutaneous inoculation mouse model, along with an increase in serum MCP-1 levels, the number of monocytes in the blood (Fig. 2 f) and TAMs in the tumor tissue increased (Fig. 2 g). 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 lung cancer recurrent mouse model, the blood MCP-1 level rapidly increased 6 hours after surgery and decreased 24 hours after surgery (Fig. 3 a). As blood MCP-1 levels increased, the number of blood monocytes also temporarily increased (Fig. 3 b). 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. 1 ). To test whether monocytes were recruited early in the metastasis process, we administered monocytes 6 hours 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. 3 c). 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. 3 d). These results suggested that an increasing number of blood monocytes are recruited around micrometastases via MCP-1 and promote tumor growth. Ly6c monocytes were recruited to micrometastases and triggered the 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 micrometastasis mouse model (Fig. 4 a). This model did not have real micrometastases, but a main (large) tumor and a micrometastasis-like (small) tumor 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 micrometastases were much fewer than those in the main tumor (Fig. 4 b). In addition, the growth rate of the micrometastases was suppressed (Fig. 4 c). Next, the main tumor was resected, and the TAMs in the micrometastases were counted using flow cytometry at 0.5, 6, 24, and 72 hours after the main tumor resection. As a results, TAMs in micrometastases drastically increased immediately after the main tumor resection and remained abundant after 72 hours (Fig. 4 d). In response to the increasing numbers of monocytes, the growth rate of micrometastases recovered after the main tumor resection (Fig. 4 e). Based on these results, we considered the mechanism by which surgery triggers the outgrowth of micrometastases as follows. (Fig. 4 f ) : 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. 4 g): 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 hours after tumor resection. ( Fig. 4 h): Even after blood MCP-1 levels and blood monocytes decreased in 24–72 hours, 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. 4 e). MCP-1 inhibitor decreased blood MCP-1 and 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. 2 ). We also checked that administration of the MCP-1 inhibitor (40 mg/kg BW/day) to LLC bearing mice ( Supplemental Fig. 3a ) decreased TAMs ( Supplemental Fig. 3b ) and suppressed the growth of the tumor ( Supplemental Fig. 3c ). Perioperative administration of the MCP-1 inhibitor reduced serum MCP-1 levels in mice after tumor resection (Fig. 5 a). Furthermore, blood monocytes after tumor resection were also decreased by MCP-1 inhibitor treatment (Fig. 5 b) compared to the control (Fig. 3 b). In response to the decrease in serum MCP-1 and blood monocytes, the number of TAMs in the micrometastases also decreased (Fig. 5 c, 5 d). 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 spontaneous 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 spontaneous metastasis mouse model for lung cancer (Fig. 6 a). We evaluated the presence of lung metastases, both macroscopically and microscopically (Fig. 6 b). Lung metastases were observed in all mice when their lungs were harvested four weeks after LLC inoculation (Fig. 6 c). When the MCP-1 inhibitor treatment was initiated at the time of tumor resection, the incidence of lung metastases decreased (Fig. 6 c). In addition, we noted that the induction of the MCP-1 inhibitor treatment significantly decreased the number of lung metastases (Fig. 6 d). Discussion Systemic response to surgery facilitates cancer recurrence by reactivating the dormant micrometastasis[ 3 – 5 ]. However, the molecular mechanisms that trigger postoperative 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, 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) change the immune cells in the TME towards immunosuppression. TAMs secrete interferon (IFN)-γ to induce the expression of programmed cell death-ligand 1 (PD-L1) in lung cancer cells[ 17 ]. PD-L1 also exists in TAMs, where it inhibits the antitumor effects of cytotoxic T cells[ 18 , 19 ]. Additionally, macrophage-derived CCL-22 promotes immunosuppressive TME by recruiting Tregs. Furthermore, TAMs produce several cytokines; and to maintain immunosuppressive TME, CCL-22, CCL-17, transforming growth factor (TGF)-β, Arginase-1 and Galectin-3[ 20 ], and NK cell cytotoxicity is also suppressed[ 21 ]. TAMs promote tumor growth by secreting proangiogenic cytokines in the hypoxic TME, including vascular endothelial growth factor (VEGF) to facilitate tumor angiogenesis[ 18 ]. Based on these results, we concluded that depleting TAMs might be an effective treatment 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 , 22 ]. In addition, the overexpression of MCP-1 in patients with lung cancer is associated with poor survival[ 23 ]. 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[ 24 ]. We previously reported that the inhibition of 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[ 25 ]. Because nimesulide also inhibits the secretion of MCP-1 from injured cells[ 24 ], the elevation of blood 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 micrometastasis mouse model to observe changes in TAMs in the micrometastatic region after surgery. Finally, we succeeded to observe the dynamic increase of TAMs in micrometastasies after surgery, which indicated the immunological mechanism in outgrowth of metastases. As a limitation, it is unclear whether our micrometastatic mouse model can reflect real micrometastasis. We expect that the applicability of this model will be proven in future studies. 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. Abbreviations NSCLC; non-small cell lung cancer MCP-1; monocyte chemoattractant protein-1 TAM; tumor associated macrophage CTC; circulating tumor cell TME; tumor microenvironment NK; natural killer PD-L1; programmed cell death-ligand 1 Declarations Acknowledgements N/A Funding Information: This study was supported by the YOKOYAMA Foundation for Clinical Pharmacology (YRY‐2005). Competing Interest: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: Keigo Okamoto, Yoko Kataoka and Keiko Ueda supported parts of the experiments. Kohei Shibata, Hiroki Saitoh, Takuya shiratori supported patient’s data collection. Yoko Kataoka and Yasuhiko Ohshio supported making conceptualization. Jun Hanaoka supervised the work. Yo Kawaguchi 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. Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. 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J Leukoc Biol 42:682–688. https://doi.org/10.1002/jlb.42.6.682 Porrello A, Leslie PL, Harrison EB, Gorentla BK, Kattula S, Ghosh SK, Azam SH, Holtzhausen A, Chao YL, Hayward MC, Waugh TA, Bae S, Godfrey V, Randell SH, Oderup C, Makowski L, Weiss J, Wilkerson MD, Hayes DN, Earp HS, Baldwin AS, Wolberg AS, Pecot CV (2018) Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking. Nat Commun 9:1988. https://doi.org/10.1038/s41467-018-04355-w Li L, Liu YD, Zhan YT, Zhu YH, Li Y, Xie D, Guan XY (2018) High levels of CCL2 or CCL4 in the tumor microenvironment predict unfavorable survival in lung adenocarcinoma. Thorac Cancer 9:775–784. https://doi.org/10.1111/1759-7714.12643 Genç S, Attar E, Gürdöl F, Kendigelen S, Bilir A, Serdaroğlu H (2007) The effect of COX-2 inhibitor, nimesulide, on angiogenetic factors in primary endometrial carcinoma cell culture. Clin Exp Med 7:6–10. https://doi.org/10.1007/s10238-007-0119-x Kimball A, Schaller M, Joshi A, Davis FM, denDekker A, Boniakowski A, Bermick J, Obi A, Moore B, Henke PK, Kunkel SL, Gallagher KA (2018) Ly6C. Arterioscler Thromb Vasc Biol 38:1102–1114. https://doi.org/10.1161/ATVBAHA.118.310703 Additional Declarations No competing interests reported. Supplementary Files SupplementalFigure1.tif Supplemental Figure 1: Ly6c monocytes isolated from mouse bone marrow cells using a cell sorter. SupplementalFigure2.tif Supplemental Figure 2: 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 SupplementalFigure3.tif Supplemental Figure 3: 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). Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2024 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 15 Mar, 2024 Reviews received at journal 25 Feb, 2024 Reviewers agreed at journal 24 Feb, 2024 Reviewers invited by journal 23 Feb, 2024 Submission checks completed at journal 14 Feb, 2024 Editor assigned by journal 14 Feb, 2024 First submitted to journal 13 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3954307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272768539,"identity":"a2f4b3de-d044-41b4-810f-5fc40049af16","order_by":0,"name":"Yo Kawaguchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYBACCSidwMbMfPDBhwoGBgOoCDNhLexsyYYzzpCihYGfx0yatw2hBSeQbD9j+PFrm10eHzODgeTMeYflzdmbDzD8qGBgN8ehRZonx1hati25mI2ZIcHg47bDhjt7jiUw9pxhYLZswK5FjiF3g7Rk24HENmaGA4kztx1m3HAjx4CZsY2B2eAADi38bzf/hmhhbDjMO+ewPUEt0hK52yQ/grUwMzbzNhxOJKhFcsb7b9YM55KBWtiYGWccS0/ecOZYwsGeMxI4/SJxPi355o8yu8T5/ee///hQY2274XjzwQc/KmyScYUYCDDzINjNYBLoJIlkfBHE+APBroOz7AjG6SgYBaNgFIwUAABmC13Xg9rSeAAAAABJRU5ErkJggg==","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":true,"prefix":"","firstName":"Yo","middleName":"","lastName":"Kawaguchi","suffix":""},{"id":272768540,"identity":"03820429-6304-48c2-a226-5c575fc91ed9","order_by":1,"name":"Keigo Okamoto","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Keigo","middleName":"","lastName":"Okamoto","suffix":""},{"id":272768541,"identity":"2da4cc31-3e7d-44f7-9841-00cf3c00f1bc","order_by":2,"name":"Yoko Kataoka","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Yoko","middleName":"","lastName":"Kataoka","suffix":""},{"id":272768542,"identity":"535301e5-1665-4285-a13d-4d0eb20d6d48","order_by":3,"name":"Kohei Shibata","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Shibata","suffix":""},{"id":272768543,"identity":"d659b44c-47b4-4323-a03f-675f15b08cc2","order_by":4,"name":"Hiroki Saitoh","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Saitoh","suffix":""},{"id":272768544,"identity":"9829e362-0095-4e34-86de-54b5e6a0b5a9","order_by":5,"name":"Takuya Shiratori","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Shiratori","suffix":""},{"id":272768545,"identity":"3b0d860d-e900-4731-b068-00d143a6f529","order_by":6,"name":"Keiko Ueda","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Keiko","middleName":"","lastName":"Ueda","suffix":""},{"id":272768546,"identity":"6a3daa7e-de4b-4cf6-ac37-9528d095c13b","order_by":7,"name":"Yasuhiko Ohshio","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Yasuhiko","middleName":"","lastName":"Ohshio","suffix":""},{"id":272768547,"identity":"eb6adf82-1ba2-4650-af58-c0f4283f9a8d","order_by":8,"name":"Jun Hanaoka","email":"","orcid":"","institution":"Shiga University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Hanaoka","suffix":""}],"badges":[],"createdAt":"2024-02-13 19:46:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3954307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3954307/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-024-03800-8","type":"published","date":"2024-09-05T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51190554,"identity":"c920488c-eb5e-4c14-bc25-d8723162d1ca","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203935,"visible":true,"origin":"","legend":"\u003cp\u003eHigh 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) Postoperative high blood monocytes count (≧500 µL) was significantly associated with tumor recurrence after surgery. (C) Schemeof establishing post-operative recurrent mouse model. (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.\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05, and ***P \u0026lt; 0.001 (Student’s t test).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/599d85c65de151aada1e29ee.png"},{"id":51190557,"identity":"e11f8f44-2d27-44e4-b178-0e2ecbf7bace","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145989,"visible":true,"origin":"","legend":"\u003cp\u003eLung 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) Blood 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. (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.\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05, and **P \u0026lt; 0.01 (Student’s t test).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/a18424888274ef4787b82c47.png"},{"id":51190559,"identity":"47d1aa48-8a70-4644-a32f-819311433512","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":341985,"visible":true,"origin":"","legend":"\u003cp\u003eLy6c monocytes promote lung cancer metastases via MCP-1. (A) Blood MCP-1 level rapidly increased in 6 h and decreased 24 h after tumor resection. (B) Blood monocytes also increased in 6 hours and decreased 24 hours 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).\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05 (Student’s t test).\u003c/p\u003e\n\u003cp\u003en.s.; not significant\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/9a076f7b51faf61d36fa39b3.png"},{"id":51190561,"identity":"43d92cea-813a-4609-aa79-9821628fb36e","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148983,"visible":true,"origin":"","legend":"\u003cp\u003eLy6c monocytes were recruited to micrometastases after main tumor resection, and then triggered the outgrowth. (A) The picture of micrometastasis mouse model. This mouse model had both main (large) tumor and micrometastatic-like (small) tumor. (B) Ly6c TAMs in micrometastasis were much fewer than that in main tumor. (C) Growth of micrometastasis was suppressed when coexisting with main tumor (n=4, in each group). (D) Immediately after the main tumor resection, TAMs in micrometastases were drastically increased. (E) In response to increasing monocytes, the growth speed of micrometastases was recovered after main tumor resection (n=4, in each group). (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 hours after tumor resection. (H) Even after the blood MCP-1 level and blood monocytes decrease in 24–72 hours, TAMs persist in micrometastases because micrometastases could easily recruit the blood monocytes via their MCP-1.\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05 (Student’s t test).\u003c/p\u003e\n\u003cp\u003en.s.; not significant\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/c18fecdef4e252ff9d685480.png"},{"id":51191233,"identity":"0d60cf44-dfc5-4494-860f-a1f9b5db8b09","added_by":"auto","created_at":"2024-02-15 17:02:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111271,"visible":true,"origin":"","legend":"\u003cp\u003eMCP-1 inhibitor decreased serum MCP-1 and monocyte levels after tumor resection, resulting in suppression of TAMs in micrometastasis.\u003cstrong\u003e \u003c/strong\u003e(A)The perioperative administration of MCP-1 inhibitor reduced the serum MCP-1 level, (B) blood monocytes, and (C) TAMs in the micrometastasis after tumor resection. (D) Postoperative TAMs in the micrometastases with MCP-1 inhibitor treatment significantly lower than that without treatment (n=4, in each group).\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05, **P \u0026lt; 0.01 and ***P \u0026lt; 0.001 (Student’s t test).\u003c/p\u003e\n\u003cp\u003en.s.; not significant\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/f01f00265c58f339690e68fe.png"},{"id":51190560,"identity":"9a8c9679-e872-4a89-a1b9-b2d5c0d569ee","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":165242,"visible":true,"origin":"","legend":"\u003cp\u003eMCP-1 inhibitor suppressed lung metastasis after surgery.\u003cstrong\u003e \u003c/strong\u003e(A) Scheme of establishing the post-operative spontaneous metastasis mouse model. (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 spontaneous 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).\u003c/p\u003e\n\u003cp\u003eData are shown as mean ±SD. *P ≤ 0.05 (Student’s t test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/c5ee54c4f6ef7999daafc599.png"},{"id":64185699,"identity":"8884bd15-44e4-4487-a4b7-3fe5610355b3","added_by":"auto","created_at":"2024-09-09 16:21:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1974779,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/7e46808a-0061-41de-8da7-574784282f65.pdf"},{"id":51190555,"identity":"76b05dba-9cb4-48ca-ab56-884c362ef88d","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":73590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e: Ly6c monocytes isolated from mouse bone marrow cells using a cell sorter.\u003c/p\u003e","description":"","filename":"SupplementalFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/48b935087c4ad4b45a24ccaa.tif"},{"id":51190558,"identity":"ce8c54ab-2e29-47d5-9501-0905aedded62","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":75396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2\u003c/strong\u003e: 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.\u003c/p\u003e\n\u003cp\u003eData are shown as mean + SD. *P ≤ 0.05 and **P \u0026lt; 0.01 (Student’s t test).\u003c/p\u003e\n\u003cp\u003en.s.; not significant\u003c/p\u003e","description":"","filename":"SupplementalFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/9e4cd468bea3d6aff30ddd2d.tif"},{"id":51190562,"identity":"cfe50ef9-11e9-4921-94f6-11603c0db7bf","added_by":"auto","created_at":"2024-02-15 16:54:57","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":93690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 3\u003c/strong\u003e: 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).\u003c/p\u003e\n\u003cp\u003eData are shown as mean +SD. *P ≤ 0.05 (Student’s t test).\u003c/p\u003e","description":"","filename":"SupplementalFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3954307/v1/2e2f3cf768e017ae4b1d17bf.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related mortality worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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\u0026ndash;3A of NSCLC, even after the induction of adjuvant cisplatin-based chemotherapy[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. therefore, a thorough investigation of the molecular mechanisms underlying postoperative metastasis and better perioperative management to reduce tumor recurrence are vital.\u003c/p\u003e \u003cp\u003eSurgical stress accelerates tumor recurrence after surgery[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCancer-associated fibroblasts, neutrophils, and platelets are major immunosuppressive cells at metastatic sites[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, only a few reports have indicated that monocytes function as immunosuppressive cells in breast[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and colorectal[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] 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[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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\u0026ndash;Monocyte\u0026ndash;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.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003e This study was approved by the Institutional Review Board of Shiga University of Medical Science (R2020-048).\u003c/p\u003e \u003cp\u003eWe reviewed the clinicopathological data of 192 patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) 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. 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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinicopathological characteristics in lung cancer patients between high or low blood monocytes after surgery.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow monocytes\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh monocytes\u003c/p\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151(79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e≦\u0026thinsp;70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89 (76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28 (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.276\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62 (83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13 (17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32(21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.916\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 or 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSmoking states\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44 (78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.772\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmoker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e107 (47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 (39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCOPD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94 (80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.665\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17 (23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep-TNM stage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e109 (80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.429\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.856\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 (72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.385\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSurgical procedure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLobectomy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.760\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLimited resection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHistological subtype\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdeno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101 (76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32 (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSquamous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45 (88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThe others\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.750\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePleural invasion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 (72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.287\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLymph duct invasion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36 (73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13 (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.328\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVascular invasion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71 (77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21 (23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.791\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture\u003c/h2\u003e \u003cp\u003eThe 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\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe 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\u0026amp;D Systems (Minneapolis, MN, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eA549 (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells), RERF-LC-MS (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells), and LLC (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e 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 hours. Their supernatants were collected and the levels of secreted MCP-1 were determined using ELISA (MCP-1 kit; R\u0026amp;D Systems).\u003c/p\u003e \u003cp\u003eFor 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 \u0026times; g, 20 minutes). The samples were analyzed using the same ELISA kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of cells\u003c/h2\u003e \u003cp\u003eTo 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, Kremsm\u0026uuml;nster, Austria). To purify white blood cells, 300 \u0026micro;L of whole blood was added to 2.0 mL of red blood cell lysis buffer for 3 minutes at 37\u0026deg;C to deplete erythrocytes. White cells were washed twice with PBS and analyzed using flow cytometry.\u003c/p\u003e \u003cp\u003eTo isolate tumor-infiltrating cells, the tumors were removed from mice, dissected, and then digested with 40 \u0026micro;L of collagenase at 37\u0026deg;C for 30 minutes. The cells were washed twice with PBS, passed through 95-\u0026micro;m mesh, and analyzed using flow cytometry.\u003c/p\u003e \u003cp\u003eBone marrow cells were isolated from the femur of 8-week-old C57BL/6J mice (Japan SLC, Hamamatsu, Japan). The cells ware mixed with 2.0 mL of red blood cell lysis buffer for 3 minutes at 37\u0026deg;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\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eLy6c 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. Isolated white blood cells, tumor-infiltrating cells, or bone marrow cells were stained with 2 \u0026micro;L FITC-conjugated Ly6c and APC-conjugated CD206 antibodies in 20 \u0026micro;L 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 \u0026micro;L FACS buffer for flow cytometric analysis. Data were analyzed using a FACSCanto flow cytometer (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eBone marrow (4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e), A549 (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e), RERF-LC-MS (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) and LLC (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) cells were seeded into 96-well plates and incubated with 0\u0026ndash;10 ng/mL recombinant MCP-1 in the RPMI containing 2% FBS and 1% PSA for 48 hours. Subsequently, 10 \u0026micro;L 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell migration assay\u003c/h2\u003e \u003cp\u003eCell 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 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) were placed in the upper chamber containing 200 \u0026micro;L RPMI with 2% FBS and 1% PSA. In the lower chamber, 500 \u0026micro;L RPMI with 2% FBS and 1% PSA containing 0\u0026ndash;10 ng/mL recombinant MCP-1 were placed. After incubation at 37\u0026deg;C for 6 hours, the Ly6c monocytes migrating to the lower chamber were counted using flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMacrophage differentiation assay\u003c/h2\u003e \u003cp\u003eBone marrow cells (20 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) 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 hours. Differentiation of bone marrow cells into Ly6c monocytes/CD206 TAMs was quantified using a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eEight-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 \u0026micro;L PBS via oral gavage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePost-operative recurrent mouse model\u003c/h2\u003e \u003cp\u003eLLC cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were injected subcutaneously into the right flank of mice. Two weeks later, the tumor was resected. Three weeks after LLC injection, the mice were euthanized, and the lungs were harvested. In contrast, the control mice received only inhalational anesthesia, and the tumor was left in the mice until the lungs were harvested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExperimental lung metastasis mouse model\u003c/h2\u003e \u003cp\u003eLLC cells were suspended to a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/100 \u0026micro;L PBS and were injected into the tail vein. Six hours after LLC injection, Ly6c monocytes (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/100 \u0026micro;L 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicrometastasis mouse model\u003c/h2\u003e \u003cp\u003eMain tumor: LLC cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were injected subcutaneously into the left flank of mice.\u003c/p\u003e \u003cp\u003eMicrometastasis: Ten days later, LLC cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were subcutaneously injected into the right flank of the same mice. After more 10 days, a mouse model of micrometastasis 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 analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePost-operative spontaneous metastasis mouse model\u003c/h2\u003e \u003cp\u003eLLC cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were injected subcutaneously into the right flank of mice. Two weeks after the LLC injection, the tumor was resected. Four weeks after LLC injection, the mice were euthanized, and the lungs were harvested. Nimesulide was administered three hours before tumor resection and the administration was continued daily until the day of the lung harvest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTumor measurements and quantification of tumor nodules in the lungs\u003c/h2\u003e \u003cp\u003eTumor dimensions were measured twice a week using a caliper, and tumor volumes were calculated using the following formula: \u003cem\u003ea\u003c/em\u003e \u0026times; \u003cem\u003eb\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e/2, where \u003cem\u003ea\u003c/em\u003e is the largest diameter and \u003cem\u003eb\u003c/em\u003e is the smallest diameter.\u003c/p\u003e \u003cp\u003eTo 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-\u0026micro;m thick) were stained with hematoxylin\u0026ndash;eosin. The nodules were counted microscopically in the entire field of the section with a microscope of 400\u0026times; 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\u0026times;(mice with lung metastases/total mice).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS Statistics for Windows (version 25; IBM, Armonk, NY, USA). Significant differences between groups were determined using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. The association between variables were analyzed using the χ\u003csup\u003e2\u003c/sup\u003e test. Survival curves were prepared using the Kaplan\u0026ndash;Meier method and compared using the log-rank test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHigh blood monocytes increased the risk of postoperative recurrences in lung cancer patients.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, we investigated whether post-operative blood monocytes promoted lung cancer recurrence. We found that the blood monocyte count increased after lung cancer surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, post operative high blood monocytes were significantly associated with tumor recurrence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Univariate analysis showed that high blood monocytes were not associated with p-TNM stage, lymph duct invasion, or vascular invasion, which are the common risk factors for tumor recurrence (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We believe that increased monocyte count after lung cancer surgery functions as an independent risk factor for recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBlood monocytes increased after surgery in post-operative recurrent mouse model\u003c/h2\u003e \u003cp\u003eWe established the post-operative recurrent mouse model \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In this mouse model, the number of blood Ly6c monocytes increased 6 hours after surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMCP-1 recruited monocytes from bone marrow cells and differentiate them into TAMs\u003c/h2\u003e \u003cp\u003eTo 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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. First, we investigated MCP-1 secretion ability of LLC and showed that LLC secreted a considerable amount of MCP-1 into the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We also demonstrated that, along with tumor growth, blood MCP-1 levels increased in a subcutaneous inoculation mouse model of LLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Transwell chamber assays demonstrated that MCP-1 recruited Ly6c monocytes from bone marrow cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Furthermore, MCP-1 differentiated bone marrow cells into Ly6c monocytes and partially differentiated them into CD206 TAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In the subcutaneous inoculation mouse model, along with an increase in serum MCP-1 levels, the number of monocytes in the blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) and TAMs in the tumor tissue increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLy6c monocytes promote lung cancer metastases via MCP-1\u003c/h2\u003e \u003cp\u003eNext, we examined the role of Ly6c monocytes in promoting metastatic recurrence via MCP-1. In the lung cancer recurrent mouse model, the blood MCP-1 level rapidly increased 6 hours after surgery and decreased 24 hours after surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As blood MCP-1 levels increased, the number of blood monocytes also temporarily increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). 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 (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e). To test whether monocytes were recruited early in the metastasis process, we administered monocytes 6 hours after injection of LLC cells, which is the time point at which almost all LLC cells adhere to the lungs[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The injection of Ly6c monocytes led to increase metastatic regions in the lungs compared to the injection of LLC cells alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These results suggested that an increasing number of blood monocytes are recruited around micrometastases via MCP-1 and promote tumor growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eLy6c monocytes were recruited to micrometastases and triggered the outgrowth after main tumor resection\u003c/h2\u003e \u003cp\u003eTo 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 micrometastasis mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This model did not have real micrometastases, but a main (large) tumor and a micrometastasis-like (small) tumor 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 micrometastases were much fewer than those in the main tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, the growth rate of the micrometastases was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Next, the main tumor was resected, and the TAMs in the micrometastases were counted using flow cytometry at 0.5, 6, 24, and 72 hours after the main tumor resection. As a results, TAMs in micrometastases drastically increased immediately after the main tumor resection and remained abundant after 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In response to the increasing numbers of monocytes, the growth rate of micrometastases recovered after the main tumor resection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Based on these results, we considered the mechanism by which surgery triggers the outgrowth of micrometastases as follows. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e: 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg): 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\u0026ndash;6 hours after tumor resection. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh): Even after blood MCP-1 levels and blood monocytes decreased in 24\u0026ndash;72 hours, 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMCP-1 inhibitor decreased blood MCP-1 and monocytes after tumor resection, resulting in suppression of TAMs in micrometastasis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe expected MCP-1 to be a therapeutic target for suppressing monocytes in micrometastasis and tumor outgrowth. We used nimesulide as MCP-1 inhibitor[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] 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 (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). We also checked that administration of the MCP-1 inhibitor (40 mg/kg BW/day) to LLC bearing mice (\u003cb\u003eSupplemental Fig.\u0026nbsp;3a\u003c/b\u003e) decreased TAMs (\u003cb\u003eSupplemental Fig.\u0026nbsp;3b\u003c/b\u003e) and suppressed the growth of the tumor (\u003cb\u003eSupplemental Fig.\u0026nbsp;3c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003ePerioperative administration of the MCP-1 inhibitor reduced serum MCP-1 levels in mice after tumor resection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Furthermore, blood monocytes after tumor resection were also decreased by MCP-1 inhibitor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In response to the decrease in serum MCP-1 and blood monocytes, the number of TAMs in the micrometastases also decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMCP-1 inhibitor suppressed lung metastasis after surgery\u003c/h2\u003e \u003cp\u003eFinally, we investigated whether treatment with the MCP-1 inhibitor decreased tumor recurrence after surgery. Because lung cancer recurrence mostly occurs in distant organs[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], a post-operative spontaneous metastasis mouse model is required to mimic the clinical scenario. The mouse model has already been established for breast cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but not for lung cancer. Therefore, we applied the method and established the post-operative spontaneous metastasis mouse model for lung cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We evaluated the presence of lung metastases, both macroscopically and microscopically (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Lung metastases were observed in all mice when their lungs were harvested four weeks after LLC inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). When the MCP-1 inhibitor treatment was initiated at the time of tumor resection, the incidence of lung metastases decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In addition, we noted that the induction of the MCP-1 inhibitor treatment significantly decreased the number of lung metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSystemic response to surgery facilitates cancer recurrence by reactivating the dormant micrometastasis[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the molecular mechanisms that trigger postoperative 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, 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.\u003c/p\u003e \u003cp\u003eA total of 99.99% of CTCs that adhere to distant organs die because of an attack by the host\u0026rsquo;s immune system[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. If they survive, most cells eventually enter dormancy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 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[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; therefore, depleting immunosuppressive cells around CTCs can be a therapeutic target to reduce metastatic recurrence.\u003c/p\u003e \u003cp\u003eDuring 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[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Ly6c monocytes function as immunosuppressive cells in breast[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and colorectal[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] cancer metastatic recurrence. However, whether monocytes function as immunosuppressive cells leading to lung cancer recurrence has not yet been proven[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our study demonstrated that MCP-1 recruited Ly6c monocytes for micrometastasis and promoted the growth of lung metastasis in a mouse model.\u003c/p\u003e \u003cp\u003eMonocytes that enter the tumor tissue are called TAMs. TAMs (known as tumor-promoting macrophages) change the immune cells in the TME towards immunosuppression. TAMs secrete interferon (IFN)-γ to induce the expression of programmed cell death-ligand 1 (PD-L1) in lung cancer cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. PD-L1 also exists in TAMs, where it inhibits the antitumor effects of cytotoxic T cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, macrophage-derived CCL-22 promotes immunosuppressive TME by recruiting Tregs. Furthermore, TAMs produce several cytokines; and to maintain immunosuppressive TME, CCL-22, CCL-17, transforming growth factor (TGF)-β, Arginase-1 and Galectin-3[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and NK cell cytotoxicity is also suppressed[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. TAMs promote tumor growth by secreting proangiogenic cytokines in the hypoxic TME, including vascular endothelial growth factor (VEGF) to facilitate tumor angiogenesis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Based on these results, we concluded that depleting TAMs might be an effective treatment by recovering anti-tumor immunity to suppress the growth of micrometastasis.\u003c/p\u003e \u003cp\u003eMCP-1 is the main mediator of macrophage recruitment to the TME and differentiation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The MCP-1 inhibitor may represent a suitable strategy to deplete TAMs in the TME to suppress the lung cancer growth[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, the overexpression of MCP-1 in patients with lung cancer is associated with poor survival[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We previously reported that the inhibition of 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[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. MCP-1 is secreted not only from cancer cells but also injured cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Because nimesulide also inhibits the secretion of MCP-1 from injured cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the elevation of blood 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.\u003c/p\u003e \u003cp\u003eIn this study, we generated the micrometastasis mouse model to observe changes in TAMs in the micrometastatic region after surgery. Finally, we succeeded to observe the dynamic increase of TAMs in micrometastasies after surgery, which indicated the immunological mechanism in outgrowth of metastases. As a limitation, it is unclear whether our micrometastatic mouse model can reflect real micrometastasis. We expect that the applicability of this model will be proven in future studies.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first study to explore the immunological mechanism and therapeutic potential of the MCP-1\u0026ndash;Monocyte\u0026ndash;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\u0026ndash;Monocyte\u0026ndash;TAM axis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNSCLC; non-small cell lung cancer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCP-1;\u0026nbsp;monocyte chemoattractant protein-1\u003c/p\u003e\n\u003cp\u003eTAM; tumor associated macrophage\u003c/p\u003e\n\u003cp\u003eCTC; circulating tumor cell\u003c/p\u003e\n\u003cp\u003eTME; tumor microenvironment\u003c/p\u003e\n\u003cp\u003eNK; natural killer\u003c/p\u003e\n\u003cp\u003ePD-L1; programmed cell death-ligand 1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information:\u003c/strong\u003e This study was supported by the YOKOYAMA Foundation for Clinical Pharmacology (YRY‐2005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u003c/strong\u003e \u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Keigo Okamoto, Yoko Kataoka and Keiko Ueda supported parts of the experiments. Kohei Shibata, Hiroki Saitoh, Takuya shiratori supported patient\u0026rsquo;s data collection. Yoko Kataoka and Yasuhiko Ohshio supported making conceptualization. Jun Hanaoka supervised the work. Yo Kawaguchi performed patient\u0026rsquo;s data collection, making the conceptualization, all the experiments, analyzing the data and writing \u003cem\u003efirst draft\u003c/em\u003e. \u003cem\u003eAll authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the\u0026nbsp;\u003c/em\u003eInstitutional Review Board of Shiga University of Medical Science (R2020-048)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Studies:\u0026nbsp;\u003c/strong\u003eAll mouse experiments were performed in compliance with the Guidelines for Animal Experimentation from Shiga University of Medical Science (Approval number:\u0026nbsp;2022-6-23).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e N/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e N/A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Arterioscler Thromb Vasc Biol 38:1102\u0026ndash;1114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/ATVBAHA.118.310703\u003c/span\u003e\u003cspan address=\"10.1161/ATVBAHA.118.310703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Postoperative recurrence, micro metastasis, surgical stress, monocytes, MCP-1, TAM","lastPublishedDoi":"10.21203/rs.3.rs-3954307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3954307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatients 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 TAMs (tumor associated macrophages). Furthermore, even after the blood MCP-1 and monocytes decreased enough 72 hours 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\u0026ndash;Monocyte\u0026ndash;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\u0026ndash;Monocyte\u0026ndash;TAM axis may inhibit surgical stress-induced NSCLC recurrence by attenuating postoperative immunosuppressive monocytes in micrometastases.\u003c/p\u003e","manuscriptTitle":"Increasing monocytes after lung cancer surgery triggers the outgrowth of distant metastases, causing recurrence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 16:54:52","doi":"10.21203/rs.3.rs-3954307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-15T18:32:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-26T03:46:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22c7dbe1-dccc-4e50-8ed7-9381ea43333a","date":"2024-02-25T02:11:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-23T16:22:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-14T05:31:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-14T05:31:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-02-13T19:30:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ce3e6b7f-65de-4a28-b3cb-1ce37e24edca","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T16:10:19+00:00","versionOfRecord":{"articleIdentity":"rs-3954307","link":"https://doi.org/10.1007/s00262-024-03800-8","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2024-09-05 15:57:00","publishedOnDateReadable":"September 5th, 2024"},"versionCreatedAt":"2024-02-15 16:54:52","video":"","vorDoi":"10.1007/s00262-024-03800-8","vorDoiUrl":"https://doi.org/10.1007/s00262-024-03800-8","workflowStages":[]},"version":"v1","identity":"rs-3954307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3954307","identity":"rs-3954307","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}