Sijunzi Tang and gefitinib cooperate to inhibit Lung cancer progression by modulating the pre-metastatic niche

Sijunzi Tang and gefitinib cooperate to inhibit Lung cancer progression by modulating the pre-metastatic niche | 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 Sijunzi Tang and gefitinib cooperate to inhibit Lung cancer progression by modulating the pre-metastatic niche Mingyu Zhang, Wei Chen, Ruisheng Li, Xingjie Li, Chunyu Li, Guohui Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-144090/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Although targeted therapies usually trigger great initial responses in patients, the efficacy is transient due to tumor metastasis. The formation of pre-metastatic niche was proposed as the main cause for metastasis, of which the blocking way may be a potential method for inhibiting metastasis. Sijunzi Tang (SJZ), as a complementary drug for targeted therapy, can reduce the recurrence and metastasis of tumors and prolong the survival time of patients. However, how SJZ regulates the formation of pre-metastatic niche to improve the efficacy of targeted therapy remains unclear. Methods: Here, we investigated the anti-tumor activity and immunological mechanism of SJZ plus gefitinib based on pre-metastatic niche in Lewis lung carcinoma (LLC) incubated spontaneous metastatic mouse model, using histopathology and immunological methods. Results: The results showed that SJZ can improve the effect of gefitinib by inhibiting tumor cell growth, promoting tumor cell apoptosis and preventing metastasis in the lung. Besides, SJZ plus gefitinib could inhibit tumor cell aggregation and the expression of characteristic proteins of Mmp2 and Mmp9 in the lung areas of mice. We also confirmed that SJZ could downregulate the expression levels of c-kit and VEGFR2 on DCs, c-kit on neutrophils, c-kit, VEGFR2 on B lymphocyte in the blood, and c-kit and CXCR1 on monocytes in the lung; Gefitinib could decrease the expression levels of c-kit and VEGFR2 on DCs in the blood and c-kit and CXCR1 on monocytes in the lung, but increase the amount of c-kit + monocytes in the blood. SJZ plus gefitinib decrease the proportion of c-kit + and CXCR1 + monocytes in the lung. SJZ could regulate pro-metastatic inflammatory responses represented by down-regulating the expression of IL-23，RANTES, GRO-α against that of gefitinib. Moreover, there were significant rises in the expression level of IL-12p70 and IL-15，while declines in the expression level of IL-1β、IL-18、GRO-α in co-treatment group. Conclusions: This work identified the immune cells and cytokines in pre-metastatic niche associated with lung cancer affected by gefitinib and SJZ, and further revealed the immunological mechanism of SJZ improving the efficacy of gefitinib. Cancer Biology Translational Medicine Lung cancer Pre-metastatic niche Gefitinib Sijunzi Tang Immune cells Cytokines immunosuppressive Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Worldwide, lung cancer continues to attract special attention because of its high cancer-related morbidity and mortality [ 1 ] . Clinical evidence indicates that lung cancer with distant metastases is usually accompanied with poor prognosis [ 2 ] . It was reported that 85% of lung cancer patients are diagnosed as non-small cell lung cancer (NSCLC), who may probably be in the terminal stage of cancer [ 1 ] . In recent years, gefitinib, as the first-line drug of Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) greatly improved survival rate of patients for multiple types of cancer including NSCLC compared with chemotherapy [ 3 ] . Although many EGFR-mutated NSCLC patients benefit from EGFR-TKI therapy, whether gefitinib or osimertinib, tumor recurrence and metastasis are inevitable due to the prevalence of loss of drug sensitivity [ 4 ] . Increasing evidences show that Traditional Chinese medicine(TCM) has the characteristics of overall systematicity and multiple targets, which can improve the sensitivity of anti-tumor drugs and inhibit tumor metastasis [ 5 – 7 ] . However, the underlying mechanism of how TCM improves the sensitivity of EGFR-TKI remains unclear. Lately, a concept was proposed that metastasis is related to the formation of pre-metastatic niche which is driven by cooperation between tumors and their distant metastatic organs [ 8 ] . In this process, the primary tumor can regulate distant organs by secreting substances including inflammatory cytokines, adhesion molecules, chemokines and exosomes which may cause inflammation and immunosuppressiv [ 9 , 10 ] . For instance, Human Granulocyte Colony Stimulating Factor (G-CSF) secreted by tumor tissue will mobilize peripheral CD11b + Gr1 + cells into the lungs, as a result, force the formation of pre-metastatic niche in the lung [ 11 ] . Tumor-derived factors represented by inflammatory cytokines factors play an important role of in the initiate step of the formation of pre-metastatic niches (PMNs) [ 9 ] . The conception of PMNs has shifted our attention from cancer cell killing strategy to niche component regulation in the prevention of metastasis. XIAOPI formula treatment remarkably could reduce the populations of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and Myeloid-derived suppressor cells(MDSCs) in the lung tissues and reduce CXCL1 expression in a dose-dependent manner [ 12 ] .Sijunzi Tang (SJZ) is a typical Chinese medicine formula derived from ‘Prescriptions People's Welfare Pharmacy’ which consists of four herbs including Panax ginseng C.A.Mey.,Atractylodes macrocephala Koidz., Poria cocos (Schw.)Wolf and Nardostachys jatamansi DC.. As reported, it has been used as the adjunct drug which can enhance the efficacy of anti-tumor drugs including of EGFR-TKI, inhibit tumor recurrence and metastasis and prolong the survival of patients. Our previous study showed that gefitinib can increase the expression of cytokines represented by IL-6 and IL-1α in serum and tumor tissue, which may aggravate the inflammation in pre-metastatic niche [ 13 ] . The results suggested that the decreased efficacy of gefitinib may associated with the up-regulation of some inflammatory cytokines. Jia and colleagues found that there was either no significant change in antitumor effector cells and an increasing secretion of IL-10 and CCL2 in serum after a period of EGFR-TKI treatment [ 14 ] , which can exactly approve our point of view. However, the mechanistic details of the signaling network and the relevance to pulmonary physiology are poorly understood. Herein, to further investigate the efficacy of gefitinib and SJZ on LLC model in vivo and its immunological mechanism, we evaluated the situation of tumor growth and apoptosis. Further, we set out to identify the influence of gefitinib and SJZ on pro-metastatic inflammatory responses and pre-metastatic niche formation. Materials And Methods Reagents and antibodies Rabbit monoclonal antibodies against MMP9, MMP2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Cell culture The mouse lewis lung cancer cells were obtained from Guang’ anmen Hospital China academy of Chinese medical sciences and then cultured in DMEM medium, which is supplemented with 10 % fetal bovine serum, 1×10 5 u·L − 1 penicillin, and 100 mg·L − 1 streptomycin (Gibco, USA). The cell line was maintained in a humidified atmosphere at 37 °C and 5 % CO2. Preparation and quality control of Sijunzi Tang SJZ was extracted from a mixture of 4 herbs including Panax ginseng C.A.Mey., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf and Nardostachys jatamansi DC. by refluxing extraction method. Its quality control was applied by detecting content, heavy metals, pesticide residues and aflatoxin according to Pharmacopoeia of the People's Republic of China. The detailed preparation and quality control method has been previously reported [ 15 ] . Animals and Tumor model Animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, with the approval of animal ethics committee which belongs to Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China). Age-and sex-matched C57BL/6J mice (male, 6–8 weeks old, 16–20 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rodents used in the study were housed under standardized light- and temperature-controlled conditions with free access to food and water. To establish spontaneous lung metastatic models, 1×10 6 LLC cells were injected subcutaneously in the shaved right lateral axilla of mice. Treatment regime 84 mice were randomly divided into four groups with 21 mice in each group at each time point. The four groups of mice were administered the following dosing regimen once a day by intragastric administration (a) saline solution; (b) gefitinib (50 mg·kg − 1 ); (c) SJZ (25.74g·kg − 1 ); (d) gefitinib + SJZ. Gefitinib was suspended in 0.5% CMC-NA solution. Mice were sacrificed separately on 7, 14, 21 days. Flow cytometry assay Mouse peripheral blood was collected in sodium Heparin blood collection tube. Erythrocyte cells were harvested, washed, and resuspended in 100 µL PBS solution at a density of 1×10 6 cells. Mouse lung were also collected, shredded and digested with collagenase. The density of lung cell suspension is also 1×10 6 cells. All the cells were labeled by BD Pharmingen™ APC-Cy™7 Rat Anti-Mouse CD45 antibody (557659, BD Biosciences, San Jose, CA, USA). For neutrophils and monocytes analysis, BD Horizon™ BV711 Rat Anti-CD11b (563168, BD Biosciences, San Jose, CA, USA), PE-Cy™7 Rat Anti-Mouse Ly-6G (560601, BD Biosciences, San Jose, CA, USA) and BV605 Rat Anti-Mouse Ly-6C (563011, BD Biosciences, San Jose, CA, USA) were used. For detection of dendritic cells, Ms CD11c BUV395 HL3(563011, BD Biosciences, San Jose, CA, USA) and BV650 rat anti-mouse I-A/I-E (743873, BD Biosciences, San Jose, CA, USA) were used. For identification of Lymphocytes, lung cells and peripheral blood cells were incubated with BUV496 Hamster Anti-Mouse CD3e (612955, BD Biosciences, San Jose, CA, USA) and BUV737 Rat Anti-Mouse CD19 (564296, BD Biosciences, San Jose, CA, USA). Besides, the surface receptors of c-Kit, CXCR1, CCR2 and VEGFR2 on immune cells were also identified by PE anti-mouse CD117 (c-Kit) Antibody (105807, BD Biosciences, San Jose, CA, USA), PE Rat Anti-Mouse CD181 (CXCR1) (566383, BD Biosciences, San Jose, CA, USA), Alexa Fluor® 647 Mouse anti-Human CD192 (CCR2) (561744, BD Biosciences, San Jose, CA, USA) and BV421 Rat Anti-Mouse FLK-1 Clone Avas 12α1 (RUO) (562941, BD Biosciences, San Jose, CA, USA). After incubation, cells were washed with PBS and subjected into the FACS Canto™ II flow cytometer (BD Biosciences, San Jose, CA, USA). HE staining H&E staining was performed as described [ 16 ] . Lungs were inflated with 10% formalin and embedded in paraffin. Subsequently, 5-µm sections were cut for hematoxylin/eosin staining. TUNEL staining Cell apoptosis in tumor tissues were detected using the TUNEL method. The assay was carried out with Roche TUNEL assay kit according to the manufacturer's protocols. The tumor tissues of mice were first immersed in 10% neutral formalin. After dehydration with graded ethanol (70%-100%) and degrease disposing with xylene, the paraffin embedded tumor tissues were made for further use. Sections were deparaffinized, re-hydrated and incubated with 20 mg/ml proteinase K for 20 min at room temperature. The TUNEL reaction mixture was prepared as follows: 50 µL TdT enzyme, 450µL Biotin-dUTP. The slides were incubated in the mixture for 60 min at 37 °C and then incubated in the 50 µl converter-POD for 30 min at 37 °C. And DAB solution was applied to the sections for 10 min at 15–25℃. Images of the cells were captured using a microscope. Immunohistochemistry 5 µm sections of the paraffin embedded femur were incubated at 60℃ for 24 h and then followed by defatting in xylene and hydrating with graded ethanol (100%-70%). After successively incubating with antigen retrieval solution and 3% H 2 O 2 for 30 min, the slides were rinsed and incubated with the primary antibody (MMP-2, MMP9) overnight at 4℃. For the negative controls, the primary antibodies were replaced by non-immunized goat serum. The next day, the slides were rinsed and incubated with the second antibody for a period of time followed by DAB and haematoxylin staining, respectively. Quantification of positive cells was examined in at least five random fields from each section. Cytokine measurement Mouse testing samples were obtained by centrifugation from tumor tissue homogenate at 10000 rpm for 15 min. After protein quantification to 10 pg·mL − 1 , the samples were stored in -80℃ environment. Cytokines (ENA-78, Eotaxin, G-CSF, GM-CSF, GROα, IFN-γ, IFNα, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-22, IL-23, IL-27, IL-28, IL-31, IP-10, LIF, MCP-1, M-CSF, MCP-3, MIP-1α, MIP-1β, MIP-2, RANTAS, TNF-α) in tumor issue were tested by ProcartaPlex™ Mouse Cytokine&Chemokine Panel. Statistical analysis Numerical data are presented as mean ± SD. All data analyses were performed by Graph Pad Prism (version8.0, Graph Pad Software, Inc.). Between-group analyses were conducted using one-way ANOVA and unpaired t-Test. Results SJZ plus gefitinib enhance the anti -tumor activity The LLC spontaneous model was generated to investigate the anti-tumor activity after treatment by gefitinib and SJZ. The mice were sacrificed separately on 7, 14, 21 days. Based on the previous study, gefitinib and SJZ was administered via intragastric administration (i.g.) route at the dose of 50 mg·kg − 1 and 25.74 g·kg − 1 , respectively [ 15 ] . As shown in Fig. 1 , the tumor growth curve indicated that gefitinib combined with SJZ can significantly reduce the tumor volume compared with the model group. The average volume of tumors in co-treatment group increased at a significantly slower rate than the average volume of tumors in the model group at 14,21days( P < 0.001). All of three treatment groups significantly decreased the tumor weight compared with that of the model group, and the co-treatment group behave more obvious at 14༌21days( P < 0.01 or P < 0.001) (Fig. 1 D, E). Furthermore, the co-treatment group did not show obvious side effects on weight loss in body weight and internal organs (lung, liver, kidney, and heart) during the experiment (Fig. 1 B, C). These results demonstrated that SJZ could enhance the anti-tumor activity of gefitinib without obvious toxic side effects on internal organs. In addition, the TUNEL assay was conducted to evaluated the cell death following treatment with gefitinib, SJZ or both. The result showed that the amounts of TUNEL-positive cells in the gefitinib or SJZ-treated cells were significantly higher compared with that in the model group. By contrast, the addition of SJZ further promoted the apoptotic effect of gefitinib, leading to a markedly higher amounts of apoptotic cells in the gefitinib plus SJZ group (Fig. 1 F). Thus, it can be concluded that SJZ was able to enhance the apoptotic effect of gefitinib on the LLC cells. SJZ and gefitinib treatment prevents lung pre-metastatic niches formation and metastasis The pre-metastatic niche plays a critical role in the metastatic process as it prepares the ‘fertile soil’ in distinct organs for tumor metastasis [ 8 ] . In order to confirm the anti-metastasis effects of SJZ and gefitinib in the spontaneous metastatic mouse model, the formation of micro-metastases was examined by HE staining. The results showed that a large number of tumor cells aggregated and surrounded the small bronchioles, consequently forming the micro-metastases in the lung in model group. There was lower aggregation in lung metastasis in the gefitinib or SJZ group than in the model. However, there was no tumor cell aggregation in the lung areas of mice after co-treatment with gefitinib and SJZ (Fig. 2 A). Based on the result of H&E staining, we found that a more severe level of micro-metastasis in the model group. Furthermore, the expression of characteristic proteins of Mmp2 and Mmp9, which are reported to promote tumor cell invasion, migration and colonization in the metastatic niche, were also assessed by IHC analysis. We found that the treated group by gefitinib or SJZ can effectively downregulate the expression of Mmp2, Mmp9 after administration than model group. Besides,the gefitinib plus SJZ group showed a significantly lower expression of Mmp2, Mmp9 after administration than single treatment. Thus, SJZ may prevent the formation of the pre-metastatic niche and improve the therapeutic efficacy of gefitinib. Therefore, the combination of SJZ and gefitinib can effectively inhibit the lung pre-metastatic niche formation. The effect of SJZ and gefitinib on immune cells in the pre-metastatic niche Several lines of evidence have shown that BMDCs are recruited to metastatic organ sites to form a metastatic niche. It has been shown that neutrophils may facilitate cancer lung metastasis. According to the recent published researches, CD11b + myeloid cells were reported to remarkably expanded in the pre-metastatic niche, among which CD45 + CD11b + Ly6G + Ly6C int neutrophils were the most dominant [ 17 ] . Therefore, we analyzed the cellular composition of the lung and blood in mice after tumor inoculation and drug administration. The fluorescence-activated cell sorting (FACS) analysis revealed that CD45 + CD11b + Ly6G + Ly6C int neutrophils and CD11c + Ia/e + dendritic cells were remarkably decreased in the treatment group in the pre-metastatic niche,while Ly6G − Ly6C + monocytes, lymphocytes and F4/80 + macrophages showed no difference (Fig. 3 B). These data indicate that neutrophils and DCs may be key biomarkers in pre-metastatic niche that gefitinib and SJZ focus on, although other myeloid cells may be also involved. The above findings indicated that gefitinib and SJZ could inhibit myeloid cells accumulation especially for neutrophils and DCs in target organs, since myeloid cells were reported to modulate by chemokines or cytokines relying on the chemokine receptors or proto-oncogene on their surface, it is necessary to explore the changes of receptors expression in response to gefitinib and SJZ treatment. c-kit + , CCR2 + , CXCR1 + and VEGFR1 + myeloid cells are the main BMDCs gathered in the pre-metastatic niche [ 17 , 18 ] . As showed in Fig. 4 , there is a significantly change in some receptor positive myeloid cell following the treatment. Obviously, compared with the model group, gefitinib or SJZ treatment can downregulate the expression of c-kit and VEGFR2 on DCs in the blood (Fig. 4 A). Besides, the c-kit + neutrophils and c-kit + , VEGFR2 + B lymphocytes in the blood was significantly reduced after SJZ treatment compared with the model group (Fig. 4 B and 4 D). Surprisingly, we found that gefitinib can elevated the c-kit + monocytes in the blood (Fig. 4 C). We also tested the receptors expression on the immune cells in the lung of mice which is considered as the metastatic niche (Fig. 4 E- 4 H). The result showed that gefitinib or SJZ treatment apparently can’t affect the receptors expression of neutrophils and dendritic cells. However, gefitinib and co-treatment can effectively decrease the proportion of c-kit + and CXCR1 + monocytes (Fig. 4 G). In addition to this, the expression of CXCR1 of B lymphocytes significantly reduced after gefitinib treatment. Therefore, gefitinib and SJZ treatment have different effects on immune cells in the pre-metastatic. The regulation on immune cells in the pre-metastatic by SJZ might be the major reason for improving the efficacy of gefitinib. The effect of SJZ and gefitinib on cytokines secretion in the pre-metastatic niche The tumor microenvironment plays a vital role in the initiation and progression of many cancers [ 19 ] . Primary tumor-secreted soluble molecules contribute to preparing distant sites for the pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism [ 9 ] . Therefore,these soluble molecules is also considered as niche-promoting molecules. In our study, the secretion of cytokine levels in tumor issues were measured using Luminex xMAP technology. The expression differences of the cytokines levels in each treatment group were analyzed during the pre-metastatic niche formation. We identified the 9 main inflammatory cytokines (IL-12p70, IL-1β, TNF-α, IL-18, IL-23, GRO-α, IP -10, RANTES, IL-15) as the mediator of primary tumor after drug treatment. As shown in the Fig. 5 , compared with model group, the expression levels of IL-23 and RANTES increased after gefitinib treatment ( P < 0.05), while the expression level of TNF-α decreased( P < 0.05). Besides, compared with the model group, the expression levels of TNF-α, IL-18, IL-23, GRO-α, IP -10, RANTES showed a downward trend in the SJZ group, while the expression levels of GRO-α and IP-10 decreased remarkably( P < 0.05). Compared with the model group, co-treatment with gefitinib and SJZ could increase the activity of IL-12p70 and IL-15 and decrease the activity of IL-1β、IL-18、GRO-α. Specially, compared with the model group, gefitinib can increase the levels of IL-23 and RANTES, while SJZ could decrease them and the co-treatment group couldn’t change them. However, compared with the gefitinib group, the levels of IL-23 and RANTES in the co-treatment group indicated a downward trend. Thus, in the pre-metastatic niche, the increased levels of inflammatory cytokines caused by gefitinib might be one of the important reasons for metastasis in the later stage of the tumor. Additionally, SJZ improved the efficacy of gefitinib by regulate the expression of immune cytokines. Discussion In this study, the inhibition rate and growth of tumors and the weight changes of mice were observed to evaluate the anti-tumor effects of gefitinib and SJZ. Compared with gefitinib or SJZ administration alone, the co-treatment has a better control of tumor growth and promoting tumor apoptosis. It has been proposed that the presence of apoptotic tumors enhances antigen-presenting ability in dendritic cells as well as T-cell response [ 20 ] . These results indicated that SJZ can significantly improve the effect of gefitinib in tumor-bearing mice. In 2005, Kaplan et al. put forward the concept of ‘pre-metastatic niche’ [ 8 ] . It refers to the adaptive transition in the metastatic organ before tumor metastasis, so as to create a ‘fertile soil’ suitable for tumor cell colonization [ 9 ] . In other words, the primary tumor can secrete tumor-derived factors such as chemokines, inflammatory factors or exosomes which can mobilize immune cells from bone marrow [ 21 ] . As for lung metastasis, tumor cells can move along blood vessels or lymphatics to the lung after the matrix prepared [ 22 ] . Of note, we found that there were no apparent lung metastatic lesions after LLC inoculation in 2th weeks in all groups, but HE staining showed that gefitinib and SJZ could inhibit the process of transferring of tumor cells to the lung, which means the co-treatment may block the formation of pre-metastatic niche in the lung. The formation of PMNs can be basically divided into three stages: 1) Tumor tissues secrete soluble cytokines or components into the circulatory system; 2) Immunosuppressive cells in bone marrow are activated and transferred in located site. 3) Cell matrix were captured in distant organ. In other words, the PMNs development is an overall process involving multiple components, cells and organs, which means there would be more possibilities improving clinical outcomes of patients by attempting more effective targeting strategies. Recognition of primary tumor-derived signals to initiate subsequent immune responses in distinct organs is a critical but poorly defined process in pre-metastatic niche formation. It was proved that chemokine receptors, which are mainly expressed on the surface of immune cells such as neutrophils and monocytes, can promote tumor metastasis, mediate inflammation and play an important role in regulating the recruitment of immune cells [ 23 ] . CXCR1 and CCR2 have been receiving particular interest on their roles in cancer metastasis [ 24 , 25 ] . The VEGF signaling pathway is associated with cellular proliferation and migration and endothelial cell permeability [ 26 ] . Vascular endothelial growth factor receptor 1 positive (VEGFR1 + ) BMDC clusters are recruited to the pre-metastatic sites before the arrival of LLC cells in the spontaneous metastasis model [ 27 ] .In our study, we found that gefitinib and SJZ single treatment can significantly downregulate the VEGFR2 + DC in blood of mouse, but they have no effect on the other proteins on immune cells. So, VEGFR2 protein may not be the dominant targets in PMNs modulated by gefitinib and SJZ. In the metastasis model, c-kit + neutrophils also expanded systemically, as tumors grew larger [ 18 ] . We surprisingly discovered that gefitinib can elevated the c-kit + monocytes in the blood. Besides, the gefitinib and SJZ single treatment can downregulate the expression of c-kit on DCs in blood of mice Besides, the c-kit + neutrophils and c-kit + B lymphocytes of was significantly reduced after SJZ single treatment compared with the model group in blood of mice. Notably, co-treatment can effectively decrease the proportion of c-kit + and CXCR1 + monocytes. Generally, c-kit and CXCR1 seem the potential targets that gefitinib and SJZ can do the joint regulation. As the initiate step, tumor-secreted cytokines play an important role in lung metastasis. In a word,PMNs formation is a complex procedure that involves multiple cytokines [ 12 ] . Our results showed that increased levels of RANTES and IL-23 were observed after gefitinib treatment. While SJZ could down-regulate the expression levels of RANTES and IL-23. RANTES, known as chemokine C-C motif ligand 5 (CCL5)༌has also been detected in ovarian cancer [ 28 ] , prostate cancer [ 29 ] ,pancreatic cancer [ 30 ] , and melanoma [ 31 ] and breast cancer [ 32 ] . RANTES produced by cells in the tumor microenvironment has been considered as an important contributor to metastatic disease [ 32 ] . It was proved that CCL5-deficiency inhibited tumor growth and metastasis of colorectal cancer cells by increasing the infiltration of CD8 + T cells into central tumor area. Besides, CCL5 can also modulate the differentiation of MDSCs to promote tumor progression in luminal and triple-negative breast cancer [ 33 ] . Interleukin (IL)-23 is a pro-inflammatory cytokine which consists of IL-12 p40 and IL-23 p19 subunits secreted by macrophages and dendritic cells, inducing autoimmunity by T-cell-mediated inflammation through impacting T helper 17 (Th17) cell response [ 34 ] . IL-23-mediated responses were indicated to be crucial in promoting tumor progression [ 35 ] . SHENG et al. found that blocking the function of IL-23 inhibited the proliferative activity and induced the apoptotic activity of tumor cells in MCF-7 cells [ 36 ] . IL-23 is associated with the metastasis of cancer, as IL-23 promoted the metastasis of hepatocellular carcinoma via matrix metalloproteinase 9 (Mmp9) [ 37 , 38 ] . It was found that in the transgenic mice with EGFR L858R driven mutation, gefitinib and osimertinib treatment can significantly improve the level of expression of IL-10 in mice serum and CCL-2 in tumor tissue [ 14 ] .The over secretion of IL-6 by tumor cells may be responsible for the decreased efficacy of tyrosine inhibitors such as icotinib and erlotinib, further resulting in the drug resistance [ 39 ] . Previously, we confirmed that gefitinib treatment induced higher level of inflammatory cytokines such as IL-1α which may cause inflammation in the pre-metastatic niche [ 13 ] . Therefore, the increased expression levels of IL-23 and RANTES might be associated with the formation of pre-metastatic niche and SJZ could inhibit it. IL-12p70, as well as IL-15, was a common immune promoting factors [ 40 , 41 ] ༌while IL-1α, IL-1β༌IL-18 and GRO-α were important cytokines involved in inflammatory processes which are considered as the facilitators in cancer metastasis [ 42 – 45 ] . We found that there were significant rises in the expression level of IL-12p70 and IL-15༌while declines in the expression level of IL-1β、IL-18、GRO-α in co-treatment group. It seems that SJZ treatment can maintain a relative balance in the tumor tissues, which means there were increased expression in immune promoting factors and decreased expression in immunosuppressive factors. Generally, we found that although SJZ treatment and co-treatment may cause different cytokines changes. Moreover, only the expression level of GRO-α showed a collaborative downward trend significantly. Studies have shown that GRO-α can mediate the activation of apoptosis signaling pathways through interacting with CXCR2 and increase the ability of tumor cell invasion and migration [ 46 ] . So if GRO-α could be the key target of controlling the initiate step for the formation of PMNs in tumor tissues that gefitinib and SJZ focus on has not been defined. Anyway, this study enlightened us that SJZ can modulate the tumor cell secretion to a state of balance which is favorable for improving anti-tumor effect of gefitinib. It is recognized that the process of tumor metastasis not only depends on the behavior of tumor cells themselves, but also depends on the adaptive change of extracellular matrix in the distant organ [ 47 ] . To rule out if gefitinib and SJZ can interfere the pre-metastatic niche with extracellular matrix, we performed IHC analysis of MMP2 and MM9 on lung tissues which are involved in the degradation of extracellular matrix (ECM). The results showed that gefitinib and SJZ could reduce the expression of these two proteins, and down-regulation of them were more obvious in co-treatment group. Recent studies reveal that the expression of matrix metalloproteinases (MMPs) is extremely high in lung tumors compared with non-malignant lung tissue. Recekamp et al. proved that the expression level of MMP-9 in serum of non-small cell lung cancer patients was closely related to the drug sensitivity of when receiving erlotinib treatment [ 48 ] . Conclusions In conclusion, these results implied that SJZ could improve the effect of gefitinib by targeting the tumor-derived cytokines as well as regulating matrix remodeling related proteins, therefore, resulting in the suppression of PMNs formation. Targeting the pre-metastatic niche is a potential approach to prevent metastasis. Abbreviations SJZ: Sijunzi Tang LLC: Lewis lung carcinoma NSCLC: non-small cell lung cancer EGFR-TKI: Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor TCM: Traditional Chinese medicine G-CSF: Granulocyte Colony Stimulating Factor PMNs: pre-metastatic niches MDSCs: Myeloid-derived suppressor cells VEGFR1+: Vascular endothelial growth factor receptor 1 positive Th17: T helper 17 MMPs: matrix metalloproteinases ECM: extracellular matrix Declarations Acknowledgements None. Funding This research was supported by grants from the CAMS Innovation Fund for Medical Sciences (CIFMS) (grant no. 2016-I2M-1-001). Author information Guohui Li designed the research. Mingyu Zhang carried out the experiments and performed data analysis. Chunyu Li and Chen wei participated part of the experiments. Mingyu Zhang and Chunyu Li wrote the manuscript. Ruisheng Li and Xingjie Li revised the manuscript. All of the authors have read and approved the final manuscript. Affiliations National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 10002，China Zhang-ming Yu, Chen Wei, Li-chun Yu & Li-guo Hui Research Center for Clinical and Translational Medicine, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China Li-rui Sheng & Li-xing Jie Contributions Guohui Li designed the research. Mingyu Zhang carried out the experiments and performed data analysis. Chunyu Li and Chen wei participated part of the experiments. Mingyu Zhang and Chunyu Li wrote the manuscript. Ruisheng Li and Xingjie Li revised the manuscript. All of the authors have read and approved the final manuscript. Corresponding authors Correspondence to Li-chun Yu ( [email protected] ) or Li-guo Hui ( [email protected] ). Ethics declarations Consent for publication Not applicable. 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Tumor Exosomal RNAs Promote Lung Pre-metastatic Niche Formation by Activating Alveolar Epithelial TLR3 to Recruit Neutrophils.Cancer Cell.2016; 30: 243-256. Coffelt SB, Kersten K, Doornebal CW, et al. IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis.Nature, 2015; 522: 345-348. Liotta LA,Kohn EC. The microenvironment of the tumour-host interface.Nature.2001; 411: 375-9. Liotta LA, Kohn EC.The microenvironment of the tumour-host interface.Nature.2001; 411: 375-9. Hiratsuka S, Watanabe A, Sakurai Y, et al. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nature Cell Biology. 2008; 10(11):1349-1355. Srivastava K, Hu JH, Korn C, et al. Postsurgical adjuvant tumor therapy by combining anti-angiopoietin-2 and metronomic chemotherapy limits metastatic growth.Cancer Cell. 2014; 26: 880-895. Varney ML, Singh S, Li A, et al. Small molecule antagonists for CXCR2 and CXCR1 inhibit human colon cancer liver metastases. Cancer Letters. 2011; 300(2): 0-188. Greene S, Robbins Y, Mydlarz WK, et al. Inhibition of MDSC Trafficking with SX-682, a CXCR1/2 Inhibitor, Enhances NK-Cell Immunotherapy in Head and Neck Cancer Models. Clin. Cancer Res..2020; 26: 1420-1431. Hao QY,Vadgama JV,Wang PW. CCL2/CCR2 signaling in cancer pathogenesis.Cell Commun. Signal.2020; 18: 82. Lowery CD,Blosser W, Dowless M, et al. Anti-VEGFR2 therapy delays growth of preclinical pediatric tumor models and enhances anti-tumor activity of chemotherapy.Oncotarget. 2019; 10(53): 5523-5533. Wang YH, Sun YP, Li Ding et al. Platelet P2Y12 is involved in murine pulmonary metastasis.PLoS ONE.2013; 8(11): e80780. Wertel I . Relationship between RANTES and dendritic cells in ovarian cancer patients. Frontiers in Bioence. 2011; 3(1):227-232. Vaday GG , Peehl DM , Kadam PA, et al. Expression of CCL5 (RANTES) and CCR5 in prostate cancer. Prostate. 2010; 66(2):124-134. Monti P , Marchesi F , Reni M , et al. A comprehensive in vitro characterization of pancreatic ductal carcinoma cell line biological behavior and its correlation with the structural and genetic profile. Virchows Archiv. 2004; 445(3):236-247. Mrowietz U,Schwenk U,Maune S, et al. The chemokine RANTES is secreted by human melanoma cells and is associated with enhanced tumour formation in nude mice. Br. J. Cancer. 1999; 79: 1025-31. Khalid A,Wolfram J,Ferrari I, et al. Recent Advances in Discovering the Role of CCL5 in Metastatic Breast Cancer. Mini Rev Med Chem. 2015; 15: 1063-72. Zhang SB, Zhong M,Wang C, et al. CCL5-deficiency enhances intratumoral infiltration of CD8 T cells in colorectal cancer.Cell Death Dis. 2018; 9: 766. Chen LL, He ZX, Slinger E, et al. IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology. Mucosal Immunol. 2015; 8: 390-402. Ahern P P , Izcue A , Maloy K J , et al. The interleukin-23 axis in intestinal inflammation. Immunological Reviews. 2010; 226(1):147-159. Langowski J , Zhang X , Wu L , et al. IL-23 promotes tumour incidence and growth. Nature. 2006; 442(7101):461. Sheng S , Zhang J , Ai J , et al. Aberrant expression of IL-23/IL-23R in patients with breast cancer and its clinical significance[J]. Molecular Medicine Reports. 2018; 17: 4639-4644. Li J, Lau G, Chen LL, et al. Interleukin 23 promotes hepatocellular carcinoma metastasis via NF-kappa B induced matrix metalloproteinase 9 expression.PLoS ONE. 2012;7: e46264. Zhang L, Li J, Li L,et al. IL-23 selectively promotes the metastasis of colorectal carcinoma cells with impaired Socs3 expression via the STAT5 pathway.Carcinogenesis.2014; 35: 1330-40. Wang JY,Wang YZ, Zheng CL, et al. Tyrosine kinase inhibitor-induced IL-6/STAT3 activation decreases sensitivity of EGFR-mutant non-small cell lung cancer to icotinib.Cell Biol. Int..2018; 42: 1292-1299. Sköld AE,Mathan T S M,van BJ J P,et al. Naturally produced type I IFNs enhance human myeloid dendritic cell maturation and IL-12p70 production and mediate elevated effector functions in innate and adaptive immune cells.Cancer Immunol. Immunother..2018; 67: 1425-1436. Knudson KM,Hodge JW,Schlom J, et al. Rationale for IL-15 superagonists in cancer immunotherapy. Expert Opin Biol Ther. 2020; 20: 705-709. Kuan EL,Ziegler SF. A tumor-myeloid cell axis, mediated via the cytokines IL-1α and TSLP, promotes the progression of breast cancer.Nat. Immunol..2018; 19: 366-374. Kaplanov I,Carmi Y,Kornetsky R, et al. Blocking IL-1β reverses the immunosuppression in mouse breast cancer and synergizes with anti-PD-1 for tumor abrogation. Proc. Natl. Acad. Sci. U.S.A..2019; 116: 1361-1369. Kaplanov I, Carmi Y, Kornetsky R, et al. Blocking IL-1β reverses the immunosuppression in mouse breast cancer and synergizes with anti-PD-1 for tumor abrogation.Proc. Natl. Acad. Sci. U.S.A..2019; 116: 1361-1369. Esmailbeig M,Ghaderi A. Interleukin-18: a regulator of cancer and autoimmune diseases. Eur. Cytokine Netw.. 2017; 28: 127-140. Yung MM,Tang HW,Cai PC, et al. GRO-α and IL-8 enhance ovarian cancer metastatic potential via the CXCR2-mediated TAK1/NFκB signaling cascade. Theranostics. 2018; 8: 1270-1285. Sharma B, Nannuru KC,Varney ML, et al. Host Cxcr2-dependent regulation of mammary tumor growth and metastasis.Clin. Exp. Metastasis. 2015; 32: 65-72. Merchant N, Nagaraju GP, Rajitha B, et al. Matrix metalloproteinases: their functional role in lung cancer. Carcinogenesis. 2017; 38: 766-780. Reckamp KL, Gardner BK , Figlin RA, et al. Tumor Response to Combination Celecoxib and Erlotinib Therapy in Non-small Cell Lung Cancer Is Associated with a Low Baseline Matrix Metalloproteinase-9 and a Decline in Serum-Soluble E-Cadherin. Journal of Thoracic Oncology. 2008; 3( 2):117-124. 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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-144090","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":7874875,"identity":"3ad1b65e-8360-4f2f-9e9b-2a05fe540338","order_by":0,"name":"Mingyu Zhang","email":"","orcid":"","institution":"Cancer Hospital Chinese Academy of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingyu","middleName":"","lastName":"Zhang","suffix":""},{"id":7874876,"identity":"b2d9f9e4-561d-49af-9823-55e31cf42968","order_by":1,"name":"Wei Chen","email":"","orcid":"","institution":"Cancer Hospital Chinese Academy of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Chen","suffix":""},{"id":7874877,"identity":"e9bfa8f1-8768-44d0-bd6f-9f905f509ec5","order_by":2,"name":"Ruisheng Li","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruisheng","middleName":"","lastName":"Li","suffix":""},{"id":7874878,"identity":"e3c2297b-0738-4b30-959a-1dee4ef9bd2d","order_by":3,"name":"Xingjie Li","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xingjie","middleName":"","lastName":"Li","suffix":""},{"id":7874879,"identity":"6173616b-beb6-4e1a-a3a9-490d87aeda95","order_by":4,"name":"Chunyu Li","email":"","orcid":"","institution":"Cancer Hospital Chinese Academy of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunyu","middleName":"","lastName":"Li","suffix":""},{"id":7874880,"identity":"fa7332a8-47a3-4f82-bc59-97897042a1fa","order_by":5,"name":"Guohui Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACPgYehgMfDGrkGJiJ1cLGwMP4cEbFMWOStDAbc5xhTmwg2mFs/GuPSTO2saX3tzM//HTjD4M8fwPzswd4tUi8S5MubJPJnXGYzVg6t43BcMYBNnMD/FrOmEnPbGPL3cDMwyCd28DAuIGBh02CoBbeNuZ0A2Ye5t85fxjsCWvh7zE25jnDnADUwiadw8aQSIQtPIagQDYE+sXMOrdNIhnEwKuFn/+MASgq5fn7Dz++nfPHxra/vfkZXi0MEgmoXAbCccp/gJCKUTAKRsEoGPEAAIzVPX/f6Kx+AAAAAElFTkSuQmCC","orcid":"","institution":"Cancer Hospital Chinese Academy of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Guohui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2021-01-09 15:36:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-144090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-144090/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5001292,"identity":"4267dafd-107d-4443-b660-fea79c872f34","added_by":"auto","created_at":"2021-01-15 17:58:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":478735,"visible":true,"origin":"","legend":"Anti-tumor activity of gefitinib and SJZ Tang in LLC mouse model. (A) Schematic illustration for the spontaneous metastatic models in this study. (B) The changes in body weight of mice (C)Tumor weight and (D) volume of mice was measured on 7, 12 and 21 days after gefitinib and SJZ treatment. The weight(E) and coefficient (F) of main organ (Heart, Liver, Spleen, Lung, Kidney). (G) Representative images of cell apoptosis in tumor tissue.Scale bar, 200μm.","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/0051911e5256d040c4f1bb9c.png"},{"id":5001293,"identity":"7393429f-50e1-47fa-b36d-7410224f5d53","added_by":"auto","created_at":"2021-01-15 17:58:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":852877,"visible":true,"origin":"","legend":"Gefitinib and Sijunzi Tang inhibit the formation of PMNs. (A) Representative images of lung sections stained of tumor-bearing mice after implantation. scale bars, 50μm. (B) Representative images of Mmp2 and Mmp9 staining in tumor tissues.","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/68e1031f7612c2fb615eb60e.png"},{"id":5001179,"identity":"31fa6436-8c56-4ad4-9d3e-b844b832ef77","added_by":"auto","created_at":"2021-01-15 17:55:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":290951,"visible":true,"origin":"","legend":"Different immune cells were detected by flow cytometry in C57 mice after LLC tumor inoculation after 14days. (A) Schematic diagram of sketch pattern of flow chart. (B) The proportions of macrophages, lymphocytes, dendritic cells, neutrophils and monocytes in the blood and lung.Results were presented as mean ± SEM. N = 2–3 per group. To compare differences between model group and treatment, unpaired student t test was used. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001. ","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/980f39a53c13e51d1ae4653a.png"},{"id":5001182,"identity":"9cdd261e-d2fd-46e2-8abc-2492b1eccae2","added_by":"auto","created_at":"2021-01-15 17:55:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146320,"visible":true,"origin":"","legend":"Fluorescence intensity of ckit, CCR2 , CXCR1 and CXCR4 expression on (A,E) dendritic cells, (B,F) neutrophils , (C,G) monocytes and (D,H) B lymphocytes in blood and lung detected by flow cytometry in C57 mice after LLC tumor inoculation after 14days. Results were presented as mean ± SEM. N = 2-3 per group. To compare differences between model group and treatment, unpaired student t test was used. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001. ","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/1bf0fa73419bad9382870da5.png"},{"id":5001183,"identity":"e352e219-d589-424e-829d-4f4320f3c849","added_by":"auto","created_at":"2021-01-15 17:55:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":253111,"visible":true,"origin":"","legend":"Gefitinib and Sijunzi Tang treatment changed tumor-derived inflammatory cytokines secretion. A.Luminex xMAP assay process. Briefly, 3mg of tumor tissue was dealt with PBS solution into tumor homogenate, then added up with beads, antibody and SAPE step by step. The complex was detected by Luminex test system. B.The levels of IL-12p70, IL-1β, TNF-α, IL-18, IL-23, GRO-α，IP-10, RANTES, IL-15, IL-1α and IL-6 concentration in tumor tissues from different treatment groups were measured by Luminex xMAP assay. Results were presented as mean ± SEM. N = 5–6 per group. To compare differences between model group and treatment, unpaired student t test was used. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001. ","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/5d5d7b40a37e9af353a8356c.png"},{"id":13646891,"identity":"aaf15463-0648-4ba5-8f73-382f6f6cb846","added_by":"auto","created_at":"2021-09-17 09:25:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2396389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-144090/v1/f224eae9-9d82-4fa1-9f8b-eb6418d3b4f8.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSijunzi Tang and gefitinib cooperate to inhibit Lung cancer progression by modulating the pre-metastatic niche\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eWorldwide, lung cancer continues to attract special attention because of its high cancer-related morbidity and mortality\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Clinical evidence indicates that lung cancer with distant metastases is usually accompanied with poor prognosis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. It was reported that 85% of lung cancer patients are diagnosed as non-small cell lung cancer (NSCLC), who may probably be in the terminal stage of cancer\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In recent years, gefitinib, as the first-line drug of Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) greatly improved survival rate of patients for multiple types of cancer including NSCLC compared with chemotherapy\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Although many EGFR-mutated NSCLC patients benefit from EGFR-TKI therapy, whether gefitinib or osimertinib, tumor recurrence and metastasis are inevitable due to the prevalence of loss of drug sensitivity\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Increasing evidences show that Traditional Chinese medicine(TCM) has the characteristics of overall systematicity and multiple targets, which can improve the sensitivity of anti-tumor drugs and inhibit tumor metastasis \u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanism of how TCM improves the sensitivity of EGFR-TKI remains unclear.\u003c/p\u003e \u003cp\u003eLately, a concept was proposed that metastasis is related to the formation of pre-metastatic niche which is driven by cooperation between tumors and their distant metastatic organs\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In this process, the primary tumor can regulate distant organs by secreting substances including inflammatory cytokines, adhesion molecules, chemokines and exosomes which may cause inflammation and immunosuppressiv\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. For instance, Human Granulocyte Colony Stimulating Factor (G-CSF) secreted by tumor tissue will mobilize peripheral CD11b\u003csup\u003e+\u003c/sup\u003eGr1\u003csup\u003e+\u003c/sup\u003ecells into the lungs, as a result, force the formation of pre-metastatic niche in the lung\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Tumor-derived factors represented by inflammatory cytokines factors play an important role of in the initiate step of the formation of pre-metastatic niches (PMNs)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The conception of PMNs has shifted our attention from cancer cell killing strategy to niche component regulation in the prevention of metastasis. XIAOPI formula treatment remarkably could reduce the populations of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and Myeloid-derived suppressor cells(MDSCs) in the lung tissues and reduce CXCL1 expression in a dose-dependent manner\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.Sijunzi Tang (SJZ) is a typical Chinese medicine formula derived from \u0026lsquo;Prescriptions People's Welfare Pharmacy\u0026rsquo; which consists of four herbs including Panax ginseng C.A.Mey.,Atractylodes macrocephala Koidz., Poria cocos (Schw.)Wolf and Nardostachys jatamansi DC.. As reported, it has been used as the adjunct drug which can enhance the efficacy of anti-tumor drugs including of EGFR-TKI, inhibit tumor recurrence and metastasis and prolong the survival of patients. Our previous study showed that gefitinib can increase the expression of cytokines represented by IL-6 and IL-1α in serum and tumor tissue, which may aggravate the inflammation in pre-metastatic niche\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The results suggested that the decreased efficacy of gefitinib may associated with the up-regulation of some inflammatory cytokines. Jia and colleagues found that there was either no significant change in antitumor effector cells and an increasing secretion of IL-10 and CCL2 in serum after a period of EGFR-TKI treatment\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, which can exactly approve our point of view. However, the mechanistic details of the signaling network and the relevance to pulmonary physiology are poorly understood. Herein, to further investigate the efficacy of gefitinib and SJZ on LLC model in vivo and its immunological mechanism, we evaluated the situation of tumor growth and apoptosis. Further, we set out to identify the influence of gefitinib and SJZ on pro-metastatic inflammatory responses and pre-metastatic niche formation.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eRabbit monoclonal antibodies against MMP9, MMP2 were purchased from Cell Signaling Technology (Danvers, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe mouse lewis lung cancer cells were obtained from Guang\u0026rsquo; anmen Hospital China academy of Chinese medical sciences and then cultured in DMEM medium, which is supplemented with 10 % fetal bovine serum, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e u\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin, and 100 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin (Gibco, USA). The cell line was maintained in a humidified atmosphere at 37\u0026nbsp;\u0026deg;C and 5 % CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and quality control of Sijunzi Tang\u003c/h2\u003e \u003cp\u003eSJZ was extracted from a mixture of 4 herbs including Panax ginseng C.A.Mey., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf and Nardostachys jatamansi DC. by refluxing extraction method. Its quality control was applied by detecting content, heavy metals, pesticide residues and aflatoxin according to Pharmacopoeia of the People's Republic of China. The detailed preparation and quality control method has been previously reported\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Tumor model\u003c/h2\u003e \u003cp\u003eAnimal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, with the approval of animal ethics committee which belongs to Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China). Age-and sex-matched C57BL/6J mice (male, 6\u0026ndash;8 weeks old, 16\u0026ndash;20\u0026nbsp;g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rodents used in the study were housed under standardized light- and temperature-controlled conditions with free access to food and water. To establish spontaneous lung metastatic models, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e LLC cells were injected subcutaneously in the shaved right lateral axilla of mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTreatment regime\u003c/h2\u003e \u003cp\u003e84 mice were randomly divided into four groups with 21 mice in each group at each time point. The four groups of mice were administered the following dosing regimen once a day by intragastric administration (a) saline solution; (b) gefitinib (50 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); (c) SJZ (25.74g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); (d) gefitinib\u0026thinsp;+\u0026thinsp;SJZ. Gefitinib was suspended in 0.5% CMC-NA solution. Mice were sacrificed separately on 7, 14, 21 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry assay\u003c/h2\u003e \u003cp\u003eMouse peripheral blood was collected in sodium Heparin blood collection tube. Erythrocyte cells were harvested, washed, and resuspended in 100 \u0026micro;L PBS solution at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells. Mouse lung were also collected, shredded and digested with collagenase. The density of lung cell suspension is also 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells. All the cells were labeled by BD Pharmingen\u0026trade; APC-Cy\u0026trade;7 Rat Anti-Mouse CD45 antibody (557659, BD Biosciences, San Jose, CA, USA). For neutrophils and monocytes analysis, BD Horizon\u0026trade; BV711 Rat Anti-CD11b (563168, BD Biosciences, San Jose, CA, USA), PE-Cy\u0026trade;7 Rat Anti-Mouse Ly-6G (560601, BD Biosciences, San Jose, CA, USA) and BV605 Rat Anti-Mouse Ly-6C (563011, BD Biosciences, San Jose, CA, USA) were used. For detection of dendritic cells, Ms CD11c BUV395 HL3(563011, BD Biosciences, San Jose, CA, USA) and BV650 rat anti-mouse I-A/I-E (743873, BD Biosciences, San Jose, CA, USA) were used. For identification of Lymphocytes, lung cells and peripheral blood cells were incubated with BUV496 Hamster Anti-Mouse CD3e (612955, BD Biosciences, San Jose, CA, USA) and BUV737 Rat Anti-Mouse CD19 (564296, BD Biosciences, San Jose, CA, USA). Besides, the surface receptors of c-Kit, CXCR1, CCR2 and VEGFR2 on immune cells were also identified by PE anti-mouse CD117 (c-Kit) Antibody (105807, BD Biosciences, San Jose, CA, USA), PE Rat Anti-Mouse CD181 (CXCR1) (566383, BD Biosciences, San Jose, CA, USA), Alexa Fluor\u0026reg; 647 Mouse anti-Human CD192 (CCR2) (561744, BD Biosciences, San Jose, CA, USA) and BV421 Rat Anti-Mouse FLK-1 Clone Avas 12α1 (RUO) (562941, BD Biosciences, San Jose, CA, USA). After incubation, cells were washed with PBS and subjected into the FACS Canto\u0026trade; II flow cytometer (BD Biosciences, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003eH\u0026amp;E staining was performed as described\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Lungs were inflated with 10% formalin and embedded in paraffin. Subsequently, 5-\u0026micro;m sections were cut for hematoxylin/eosin staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eCell apoptosis in tumor tissues were detected using the TUNEL method. The assay was carried out with Roche TUNEL assay kit according to the manufacturer's protocols. The tumor tissues of mice were first immersed in 10% neutral formalin. After dehydration with graded ethanol (70%-100%) and degrease disposing with xylene, the paraffin embedded tumor tissues were made for further use. Sections were deparaffinized, re-hydrated and incubated with 20\u0026nbsp;mg/ml proteinase K for 20\u0026nbsp;min at room temperature. The TUNEL reaction mixture was prepared as follows: 50 \u0026micro;L TdT enzyme, 450\u0026micro;L Biotin-dUTP. The slides were incubated in the mixture for 60\u0026nbsp;min at 37\u0026nbsp;\u0026deg;C and then incubated in the 50\u0026nbsp;\u0026micro;l converter-POD for 30\u0026nbsp;min at 37\u0026nbsp;\u0026deg;C. And DAB solution was applied to the sections for 10\u0026nbsp;min at 15\u0026ndash;25℃. Images of the cells were captured using a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003e5\u0026nbsp;\u0026micro;m sections of the paraffin embedded femur were incubated at 60℃ for 24\u0026nbsp;h and then followed by defatting in xylene and hydrating with graded ethanol (100%-70%). After successively incubating with antigen retrieval solution and 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 30\u0026nbsp;min, the slides were rinsed and incubated with the primary antibody (MMP-2, MMP9) overnight at 4℃. For the negative controls, the primary antibodies were replaced by non-immunized goat serum. The next day, the slides were rinsed and incubated with the second antibody for a period of time followed by DAB and haematoxylin staining, respectively. Quantification of positive cells was examined in at least five random fields from each section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCytokine measurement\u003c/h2\u003e \u003cp\u003eMouse testing samples were obtained by centrifugation from tumor tissue homogenate at 10000\u0026nbsp;rpm for 15\u0026nbsp;min. After protein quantification to 10 pg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the samples were stored in -80℃ environment. Cytokines (ENA-78, Eotaxin, G-CSF, GM-CSF, GROα, IFN-γ, IFNα, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-22, IL-23, IL-27, IL-28, IL-31, IP-10, LIF, MCP-1, M-CSF, MCP-3, MIP-1α, MIP-1β, MIP-2, RANTAS, TNF-α) in tumor issue were tested by ProcartaPlex\u0026trade; Mouse Cytokine\u0026amp;Chemokine Panel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNumerical data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. All data analyses were performed by Graph Pad Prism (version8.0, Graph Pad Software, Inc.). Between-group analyses were conducted using one-way ANOVA and unpaired t-Test.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSJZ plus gefitinib enhance the anti -tumor activity\u003c/h2\u003e \u003cp\u003eThe LLC spontaneous model was generated to investigate the anti-tumor activity after treatment by gefitinib and SJZ. The mice were sacrificed separately on 7, 14, 21 days. Based on the previous study, gefitinib and SJZ was administered via intragastric administration (i.g.) route at the dose of 50 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 25.74 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the tumor growth curve indicated that gefitinib combined with SJZ can significantly reduce the tumor volume compared with the model group. The average volume of tumors in co-treatment group increased at a significantly slower rate than the average volume of tumors in the model group at 14,21days(\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). All of three treatment groups significantly decreased the tumor weight compared with that of the model group, and the co-treatment group behave more obvious at 14༌21days(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Furthermore, the co-treatment group did not show obvious side effects on weight loss in body weight and internal organs (lung, liver, kidney, and heart) during the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). These results demonstrated that SJZ could enhance the anti-tumor activity of gefitinib without obvious toxic side effects on internal organs.\u003c/p\u003e \u003cp\u003eIn addition, the TUNEL assay was conducted to evaluated the cell death following treatment with gefitinib, SJZ or both. The result showed that the amounts of TUNEL-positive cells in the gefitinib or SJZ-treated cells were significantly higher compared with that in the model group. By contrast, the addition of SJZ further promoted the apoptotic effect of gefitinib, leading to a markedly higher amounts of apoptotic cells in the gefitinib plus SJZ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Thus, it can be concluded that SJZ was able to enhance the apoptotic effect of gefitinib on the LLC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSJZ and gefitinib treatment prevents lung pre-metastatic niches formation and metastasis\u003c/h2\u003e \u003cp\u003eThe pre-metastatic niche plays a critical role in the metastatic process as it prepares the \u0026lsquo;fertile soil\u0026rsquo; in distinct organs for tumor metastasis\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In order to confirm the anti-metastasis effects of SJZ and gefitinib in the spontaneous metastatic mouse model, the formation of micro-metastases was examined by HE staining. The results showed that a large number of tumor cells aggregated and surrounded the small bronchioles, consequently forming the micro-metastases in the lung in model group. There was lower aggregation in lung metastasis in the gefitinib or SJZ group than in the model. However, there was no tumor cell aggregation in the lung areas of mice after co-treatment with gefitinib and SJZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Based on the result of H\u0026amp;E staining, we found that a more severe level of micro-metastasis in the model group. Furthermore, the expression of characteristic proteins of Mmp2 and Mmp9, which are reported to promote tumor cell invasion, migration and colonization in the metastatic niche, were also assessed by IHC analysis. We found that the treated group by gefitinib or SJZ can effectively downregulate the expression of Mmp2, Mmp9 after administration than model group. Besides,the gefitinib plus SJZ group showed a significantly lower expression of Mmp2, Mmp9 after administration than single treatment. Thus, SJZ may prevent the formation of the pre-metastatic niche and improve the therapeutic efficacy of gefitinib. Therefore, the combination of SJZ and gefitinib can effectively inhibit the lung pre-metastatic niche formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of SJZ and gefitinib on immune cells in the pre-metastatic niche\u003c/h2\u003e \u003cp\u003eSeveral lines of evidence have shown that BMDCs are recruited to metastatic organ sites to form a metastatic niche. It has been shown that neutrophils may facilitate cancer lung metastasis. According to the recent published researches, CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells were reported to remarkably expanded in the pre-metastatic niche, among which CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003e+\u003c/sup\u003e Ly6C\u003csup\u003eint\u003c/sup\u003e neutrophils were the most dominant\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, we analyzed the cellular composition of the lung and blood in mice after tumor inoculation and drug administration. The fluorescence-activated cell sorting (FACS) analysis revealed that CD45\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003e+\u003c/sup\u003e Ly6C\u003csup\u003eint\u003c/sup\u003e neutrophils and CD11c\u003csup\u003e+\u003c/sup\u003e Ia/e\u003csup\u003e+\u003c/sup\u003e dendritic cells were remarkably decreased in the treatment group in the pre-metastatic niche,while Ly6G\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e monocytes, lymphocytes and F4/80\u003csup\u003e+\u003c/sup\u003e macrophages showed no difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These data indicate that neutrophils and DCs may be key biomarkers in pre-metastatic niche that gefitinib and SJZ focus on, although other myeloid cells may be also involved. The above findings indicated that gefitinib and SJZ could inhibit myeloid cells accumulation especially for neutrophils and DCs in target organs, since myeloid cells were reported to modulate by chemokines or cytokines relying on the chemokine receptors or proto-oncogene on their surface, it is necessary to explore the changes of receptors expression in response to gefitinib and SJZ treatment. c-kit\u003csup\u003e+\u003c/sup\u003e, CCR2\u003csup\u003e+\u003c/sup\u003e, CXCR1\u003csup\u003e+\u003c/sup\u003e and VEGFR1\u003csup\u003e+\u003c/sup\u003e myeloid cells are the main BMDCs gathered in the pre-metastatic niche \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. As showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, there is a significantly change in some receptor positive myeloid cell following the treatment. Obviously, compared with the model group, gefitinib or SJZ treatment can downregulate the expression of c-kit and VEGFR2 on DCs in the blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Besides, the c-kit\u003csup\u003e+\u003c/sup\u003e neutrophils and c-kit\u003csup\u003e+\u003c/sup\u003e, VEGFR2\u003csup\u003e+\u003c/sup\u003e B lymphocytes in the blood was significantly reduced after SJZ treatment compared with the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Surprisingly, we found that gefitinib can elevated the c-kit\u003csup\u003e+\u003c/sup\u003e monocytes in the blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also tested the receptors expression on the immune cells in the lung of mice which is considered as the metastatic niche (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The result showed that gefitinib or SJZ treatment apparently can\u0026rsquo;t affect the receptors expression of neutrophils and dendritic cells. However, gefitinib and co-treatment can effectively decrease the proportion of c-kit\u003csup\u003e+\u003c/sup\u003e and CXCR1\u003csup\u003e+\u003c/sup\u003e monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In addition to this, the expression of CXCR1 of B lymphocytes significantly reduced after gefitinib treatment. Therefore, gefitinib and SJZ treatment have different effects on immune cells in the pre-metastatic. The regulation on immune cells in the pre-metastatic by SJZ might be the major reason for improving the efficacy of gefitinib.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of SJZ and gefitinib on cytokines secretion in the pre-metastatic niche\u003c/h2\u003e \u003cp\u003eThe tumor microenvironment plays a vital role in the initiation and progression of many cancers\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Primary tumor-secreted soluble molecules contribute to preparing distant sites for the pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Therefore,these soluble molecules is also considered as niche-promoting molecules. In our study, the secretion of cytokine levels in tumor issues were measured using Luminex xMAP technology. The expression differences of the cytokines levels in each treatment group were analyzed during the pre-metastatic niche formation. We identified the 9 main inflammatory cytokines (IL-12p70, IL-1β, TNF-α, IL-18, IL-23, GRO-α, IP -10, RANTES, IL-15) as the mediator of primary tumor after drug treatment. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compared with model group, the expression levels of IL-23 and RANTES increased after gefitinib treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the expression level of TNF-α decreased(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Besides, compared with the model group, the expression levels of TNF-α, IL-18, IL-23, GRO-α, IP -10, RANTES showed a downward trend in the SJZ group, while the expression levels of GRO-α and IP-10 decreased remarkably(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the model group, co-treatment with gefitinib and SJZ could increase the activity of IL-12p70 and IL-15 and decrease the activity of IL-1β、IL-18、GRO-α. Specially, compared with the model group, gefitinib can increase the levels of IL-23 and RANTES, while SJZ could decrease them and the co-treatment group couldn\u0026rsquo;t change them. However, compared with the gefitinib group, the levels of IL-23 and RANTES in the co-treatment group indicated a downward trend. Thus, in the pre-metastatic niche, the increased levels of inflammatory cytokines caused by gefitinib might be one of the important reasons for metastasis in the later stage of the tumor. Additionally, SJZ improved the efficacy of gefitinib by regulate the expression of immune cytokines.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eIn this study, the inhibition rate and growth of tumors and the weight changes of mice were observed to evaluate the anti-tumor effects of gefitinib and SJZ. Compared with gefitinib or SJZ administration alone, the co-treatment has a better control of tumor growth and promoting tumor apoptosis. It has been proposed that the presence of apoptotic tumors enhances antigen-presenting ability in dendritic cells as well as T-cell response\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. These results indicated that SJZ can significantly improve the effect of gefitinib in tumor-bearing mice.\u003c/p\u003e \u003cp\u003eIn 2005, Kaplan \u003cem\u003eet al.\u003c/em\u003e put forward the concept of \u0026lsquo;pre-metastatic niche\u0026rsquo;\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. It refers to the adaptive transition in the metastatic organ before tumor metastasis, so as to create a \u0026lsquo;fertile soil\u0026rsquo; suitable for tumor cell colonization\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In other words, the primary tumor can secrete tumor-derived factors such as chemokines, inflammatory factors or exosomes which can mobilize immune cells from bone marrow\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. As for lung metastasis, tumor cells can move along blood vessels or lymphatics to the lung after the matrix prepared\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Of note, we found that there were no apparent lung metastatic lesions after LLC inoculation in 2th weeks in all groups, but HE staining showed that gefitinib and SJZ could inhibit the process of transferring of tumor cells to the lung, which means the co-treatment may block the formation of pre-metastatic niche in the lung. The formation of PMNs can be basically divided into three stages: 1) Tumor tissues secrete soluble cytokines or components into the circulatory system; 2) Immunosuppressive cells in bone marrow are activated and transferred in located site. 3) Cell matrix were captured in distant organ. In other words, the PMNs development is an overall process involving multiple components, cells and organs, which means there would be more possibilities improving clinical outcomes of patients by attempting more effective targeting strategies.\u003c/p\u003e \u003cp\u003eRecognition of primary tumor-derived signals to initiate subsequent immune responses in distinct organs is a critical but poorly defined process in pre-metastatic niche formation. It was proved that chemokine receptors, which are mainly expressed on the surface of immune cells such as neutrophils and monocytes, can promote tumor metastasis, mediate inflammation and play an important role in regulating the recruitment of immune cells\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. CXCR1 and CCR2 have been receiving particular interest on their roles in cancer metastasis\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The VEGF signaling pathway is associated with cellular proliferation and migration and endothelial cell permeability\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Vascular endothelial growth factor receptor 1 positive (VEGFR1\u003csup\u003e+\u003c/sup\u003e) BMDC clusters are recruited to the pre-metastatic sites before the arrival of LLC cells in the spontaneous metastasis model\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.In our study, we found that gefitinib and SJZ single treatment can significantly downregulate the VEGFR2\u003csup\u003e+\u003c/sup\u003e DC in blood of mouse, but they have no effect on the other proteins on immune cells. So, VEGFR2 protein may not be the dominant targets in PMNs modulated by gefitinib and SJZ. In the metastasis model, c-kit\u003csup\u003e+\u003c/sup\u003e neutrophils also expanded systemically, as tumors grew larger\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. We surprisingly discovered that gefitinib can elevated the c-kit\u003csup\u003e+\u003c/sup\u003e monocytes in the blood. Besides, the gefitinib and SJZ single treatment can downregulate the expression of c-kit on DCs in blood of mice Besides, the c-kit\u003csup\u003e+\u003c/sup\u003e neutrophils and c-kit\u003csup\u003e+\u003c/sup\u003e B lymphocytes of was significantly reduced after SJZ single treatment compared with the model group in blood of mice. Notably, co-treatment can effectively decrease the proportion of c-kit\u003csup\u003e+\u003c/sup\u003e and CXCR1\u003csup\u003e+\u003c/sup\u003e monocytes. Generally, c-kit and CXCR1 seem the potential targets that gefitinib and SJZ can do the joint regulation.\u003c/p\u003e \u003cp\u003eAs the initiate step, tumor-secreted cytokines play an important role in lung metastasis. In a word,PMNs formation is a complex procedure that involves multiple cytokines\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Our results showed that increased levels of RANTES and IL-23 were observed after gefitinib treatment. While SJZ could down-regulate the expression levels of RANTES and IL-23. RANTES, known as chemokine C-C motif ligand 5 (CCL5)༌has also been detected in ovarian cancer \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, prostate cancer\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e,pancreatic cancer\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, and melanoma\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e and breast cancer\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. RANTES produced by cells in the tumor microenvironment has been considered as an important contributor to metastatic disease\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. It was proved that CCL5-deficiency inhibited tumor growth and metastasis of colorectal cancer cells by increasing the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells into central tumor area. Besides, CCL5 can also modulate the differentiation of MDSCs to promote tumor progression in luminal and triple-negative breast cancer\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Interleukin (IL)-23 is a pro-inflammatory cytokine which consists of IL-12 p40 and IL-23 p19 subunits secreted by macrophages and dendritic cells, inducing autoimmunity by T-cell-mediated inflammation through impacting T helper 17 (Th17) cell response\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. IL-23-mediated responses were indicated to be crucial in promoting tumor progression\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. SHENG \u003cem\u003eet al.\u003c/em\u003e found that blocking the function of IL-23 inhibited the proliferative activity and induced the apoptotic activity of tumor cells in MCF-7 cells\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. IL-23 is associated with the metastasis of cancer, as IL-23 promoted the metastasis of hepatocellular carcinoma via matrix metalloproteinase 9 (Mmp9)\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. It was found that in the transgenic mice with EGFR\u003csup\u003eL858R\u003c/sup\u003e driven mutation, gefitinib and osimertinib treatment can significantly improve the level of expression of IL-10 in mice serum and CCL-2 in tumor tissue\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.The over secretion of IL-6 by tumor cells may be responsible for the decreased efficacy of tyrosine inhibitors such as icotinib and erlotinib, further resulting in the drug resistance\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Previously, we confirmed that gefitinib treatment induced higher level of inflammatory cytokines such as IL-1α which may cause inflammation in the pre-metastatic niche\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, the increased expression levels of IL-23 and RANTES might be associated with the formation of pre-metastatic niche and SJZ could inhibit it. IL-12p70, as well as IL-15, was a common immune promoting factors\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e༌while IL-1α, IL-1β༌IL-18 and GRO-α were important cytokines involved in inflammatory processes which are considered as the facilitators in cancer metastasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. We found that there were significant rises in the expression level of IL-12p70 and IL-15༌while declines in the expression level of IL-1β、IL-18、GRO-α in co-treatment group. It seems that SJZ treatment can maintain a relative balance in the tumor tissues, which means there were increased expression in immune promoting factors and decreased expression in immunosuppressive factors. Generally, we found that although SJZ treatment and co-treatment may cause different cytokines changes. Moreover, only the expression level of GRO-α showed a collaborative downward trend significantly. Studies have shown that GRO-α can mediate the activation of apoptosis signaling pathways through interacting with CXCR2 and increase the ability of tumor cell invasion and migration\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. So if GRO-α could be the key target of controlling the initiate step for the formation of PMNs in tumor tissues that gefitinib and SJZ focus on has not been defined. Anyway, this study enlightened us that SJZ can modulate the tumor cell secretion to a state of balance which is favorable for improving anti-tumor effect of gefitinib.\u003c/p\u003e \u003cp\u003eIt is recognized that the process of tumor metastasis not only depends on the behavior of tumor cells themselves, but also depends on the adaptive change of extracellular matrix in the distant organ\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. To rule out if gefitinib and SJZ can interfere the pre-metastatic niche with extracellular matrix, we performed IHC analysis of MMP2 and MM9 on lung tissues which are involved in the degradation of extracellular matrix (ECM). The results showed that gefitinib and SJZ could reduce the expression of these two proteins, and down-regulation of them were more obvious in co-treatment group. Recent studies reveal that the expression of matrix metalloproteinases (MMPs) is extremely high in lung tumors compared with non-malignant lung tissue. Recekamp \u003cem\u003eet al.\u003c/em\u003e proved that the expression level of MMP-9 in serum of non-small cell lung cancer patients was closely related to the drug sensitivity of when receiving erlotinib treatment \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, these results implied that SJZ could improve the effect of gefitinib by targeting the tumor-derived cytokines as well as regulating matrix remodeling related proteins, therefore, resulting in the suppression of PMNs formation. Targeting the pre-metastatic niche is a potential approach to prevent metastasis.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eSJZ: \u003c/strong\u003eSijunzi Tang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLLC: \u003c/strong\u003eLewis lung carcinoma\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNSCLC: \u003c/strong\u003enon-small cell lung cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEGFR-TKI:\u003c/strong\u003eEpidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCM: \u003c/strong\u003eTraditional Chinese medicine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG-CSF:\u003c/strong\u003eGranulocyte Colony Stimulating Factor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMNs: \u003c/strong\u003epre-metastatic niches\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMDSCs:\u003c/strong\u003eMyeloid-derived suppressor cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVEGFR1+:\u003c/strong\u003eVascular endothelial growth factor receptor 1 positive\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTh17:\u003c/strong\u003eT helper 17\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMMPs: \u003c/strong\u003ematrix metalloproteinases\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eECM:\u003c/strong\u003eextracellular matrix \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the CAMS Innovation Fund for Medical Sciences (CIFMS) (grant no. 2016-I2M-1-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuohui Li designed the research. Mingyu Zhang carried out the experiments and performed data analysis. Chunyu Li and Chen wei participated part of the experiments. Mingyu Zhang and Chunyu Li wrote the manuscript. Ruisheng Li and Xingjie Li revised the manuscript. All of the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 10002，China\u003c/p\u003e\n\u003cp\u003eZhang-ming Yu, Chen Wei, Li-chun Yu \u0026amp; Li-guo Hui\u003c/p\u003e\n\u003cp\u003eResearch Center for Clinical and Translational Medicine, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China\u003c/p\u003e\n\u003cp\u003eLi-rui Sheng \u0026amp; Li-xing Jie\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuohui Li designed the research. Mingyu Zhang carried out the experiments and performed data analysis. Chunyu Li and Chen wei participated part of the experiments. Mingyu Zhang and Chunyu Li wrote the manuscript. Ruisheng Li and Xingjie Li revised the manuscript. All of the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Li-chun Yu ([email protected]) or Li-guo Hui ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang XD, Adjei, AA. Lung cancer and metastasis: new opportunities and challenges. Cancer \u0026amp; Metastasis Reviews.2015; 34(2):169-171.\u003c/li\u003e\n\u003cli\u003eRosell R, Karachaliou N. 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Journal of Thoracic Oncology. 2008; 3( 2):117-124.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lung cancer, Pre-metastatic niche, Gefitinib, Sijunzi Tang, Immune cells, Cytokines, immunosuppressive ","lastPublishedDoi":"10.21203/rs.3.rs-144090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-144090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAlthough targeted therapies usually trigger great initial responses in patients, the efficacy is transient due to tumor metastasis. The formation of pre-metastatic niche was proposed as the main cause for metastasis, of which the blocking way may be a potential method for inhibiting metastasis. Sijunzi Tang (SJZ), as a complementary drug for targeted therapy, can reduce the recurrence and metastasis of tumors and prolong the survival time of patients. However, how SJZ regulates the formation of pre-metastatic niche to improve the efficacy of targeted therapy remains unclear.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eHere, we investigated the anti-tumor activity and immunological mechanism of SJZ plus gefitinib based on pre-metastatic niche in Lewis lung carcinoma (LLC) incubated spontaneous metastatic mouse model, using histopathology and immunological methods.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe results showed that SJZ can improve the effect of gefitinib by inhibiting tumor cell growth, promoting tumor cell apoptosis and preventing metastasis in the lung. Besides, SJZ plus gefitinib could inhibit tumor cell aggregation and the expression of characteristic proteins of Mmp2 and Mmp9 in the lung areas of mice. We also confirmed that SJZ could downregulate the expression levels of c-kit and VEGFR2 on DCs, c-kit on neutrophils, c-kit, VEGFR2 on B lymphocyte in the blood, and c-kit and CXCR1 on monocytes in the lung; Gefitinib could decrease the expression levels of c-kit and VEGFR2 on DCs in the blood and c-kit and CXCR1 on monocytes in the lung, but increase the amount of c-kit\u003csup\u003e+\u003c/sup\u003e monocytes in the blood. SJZ plus gefitinib decrease the proportion of c-kit\u003csup\u003e+\u003c/sup\u003e and CXCR1\u003csup\u003e+ \u003c/sup\u003emonocytes in the lung. SJZ could regulate pro-metastatic inflammatory responses represented by down-regulating the expression of IL-23，RANTES, GRO-α against that of gefitinib. Moreover, there were significant rises in the expression level of IL-12p70 and IL-15，while declines in the expression level of IL-1β、IL-18、GRO-α in co-treatment group.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThis work identified the immune cells and cytokines in pre-metastatic niche associated with lung cancer affected by gefitinib and SJZ, and further revealed the immunological mechanism of SJZ improving the efficacy of gefitinib.\u003c/p\u003e","manuscriptTitle":"Sijunzi Tang and gefitinib cooperate to inhibit Lung cancer progression by modulating the pre-metastatic niche","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-15 17:55:50","doi":"10.21203/rs.3.rs-144090/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"838efa2f-786c-4c2c-ac30-e00ebc8ff1b4","owner":[],"postedDate":"January 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3008380,"name":"Cancer Biology"},{"id":3008381,"name":"Translational Medicine"}],"tags":[],"updatedAt":"2021-01-15T17:58:52+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-15 17:55:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-144090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-144090","identity":"rs-144090","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}