The clinical synergistic antitumor efficacy of Lienal Polypeptide combined with EGFR-TKIs for advanced NSCLC

The clinical synergistic antitumor efficacy of Lienal Polypeptide combined with EGFR-TKIs for advanced NSCLC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The clinical synergistic antitumor efficacy of Lienal Polypeptide combined with EGFR-TKIs for advanced NSCLC Yun Chen, Xinyin Liu, Jiaqi Yao, Shidai Jin, Jun Li, Jiali Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1517195/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 Purpose : EGFR-TKIs are the first-line therapy for advanced NSCLC harboring EGFR-sensitive mutations. A robust immunity is an essential foundation for patients to tolerate continuous drug treatments. Lienal polypeptide (LP) is an immunomodulator widely applied to regulate immunity in clinical practice. Nevertheless, its potential impact on EGFR-TKIs therapy has not been illustrated. This study aimed to explore the immunomodulatory and antitumor efficacy of LP in combination with EGFR-TKIs threapy in advanced NSCLC. Patients and methods : Retrospective analysis on variation of lymphocytes in 106 NSCLC patients after EGFR-TKIs combined with LP treatment was performed. Proliferation experiment, transwell and wound healing assays were performed in PC9-GR cells to estimate influence of LP on tumor proliferation, invasion and migrati n in vitro . Flow cytometry was performed to detect cell apoptosis and cell cycle. The expression of p-EGFR and EGFR were detected by Western blot to investigate antitumor effect of LP. Results : The levels of CD3 + , CD4 + T cells and the CD4 + /CD8 + ratio were higher in NSCLC patients treated with Gefitinib in conjunction with LP. Gefitinib combined with LP inhibited tumor invasion and migration, triggered G0/G1 phase arrest to block cellular proliferation and promote cell apoptosis in vitro . Furthermore, the expression of p-EGFR was decreased after Gefitinib-combining-LP treatment. Conclusions : LP had a synergistic anticancer effect with EGFR-TKIs in NSCLC. LP in combination with EGFR-TKIs therapy has clinical curative effect in treatment of advanced NSCLC with EGFR driving mutations, can effectively enhance physical immunity and resensitize drug-resistant cells to EGFR-TKIs, which has a certain clinical application value. Lienal polypeptide EGFR-TKIs drug resistance combination therapy NSCLC Figures Figure 1 Figure 2 Figure 3 Introduction Lung cancer remains the leading cause of cancer related deaths worldwide 1 . Non-small cell lung cancer (NSCLC) is divided to different molecular subtypes, among which epidermal growth factor receptor (EGFR) mutation is the most common subtype 2 – 4 . EGFR-TKIs such as Gefitinib extraordinarily prolonged median overall survival (OS) of advanced NSCLC patients, as well as improved the quality of life 5 . EGFR-TKIs have been recommended as first-line treatment for patients with advanced NSCLC harboring EGFR mutaions 6 – 8 . Nevertheless, a considerable proportion of patients have to discontinue treatment due to adverse effects, which lead to disease progression and failure of therapy 9 , 10 . Long-term inflammatory responses due to treatment cause massive infiltration of inflammatory cells and increased cytokines levels. Such tumor microenvironment flooding with chronic inflammatory cells and inflammatory mediators may result in gene silencing or abnormal expression, epigenetic changes, mismatched repair enzyme inactivation, DNA damage or gene mutation in tumor cells, eventually contributing to malignant transformation and drug resistance of tumor cells 11 – 14 . Lienal polypeptide (LP) is extracted from the spleen of healthy calves, which functioning as an immune modulator with the ability of correcting immune dysfunction, activating non-specific immune function, as well as improving the immune function of lymphocytes, therefore enhancing the body's defensive capabilities to infection 15 . At present, LP is mainly applied to cellular immunodeficiency diseases and malignant tumors caused by chemo-radiotherapy, which can ameliorate cancer cachexia 16 , 17 . LP has also been widely used in the treatment of multiple malignancy tumors nowadays. Recently studies found that EGFR-TKIs can moderate T lymphocytes and natural killer cells to deregulate carcinogenesis 18 . However the chronic inflammatory reaction induced by EGFR-TKIs inversely results in treating termination or drug resistance. LP, an immunomodifier, could significantly improve immune function and correct immune disorders. Therefore, we hypothesized that LP treatment could reduce the persistent chronic inflammation and drug-related adverse effects caused by EGFR-TKIs in NSCLC. In this study, we aim to use Gefitinib combined with LP to treat NSCLC harboring EGFR-sensitive mutations, observe the efficacy of the combined therapy, and investigate the effects of drug combination on tumor biological behaviors and immune function, we further clarified the molecular mechanisms of combination therapy. In conclusion, our study provides new insights in LP combined with EGFR-TKIs in treatment of advanced NSCLC. Patients And Methods Patients A total of 106 patients diagnosed with III and IV stage NSCLC in Jiangsu Province Hospital from January 2019 to January 2021 were enrolled in this study. This study was approved by the ethical committee of the Jiangsu Province Hospital Medical Ethics Committee and was carried out in accordance with the approved guidelines. All participants had a good knowledge about the study and signed written informed consents. The inclusion criteria were as follows: pathologically diagnosed with lung adenocarcinoma, carrying EGFR mutation concluding in-frame deletions in exon 19 (19DEL) or a point mutation in exon 21 (L858R), accepting oral administration of EGFR-TKIs per day and/or combined with intravenous infusion of lienal polypeptide liquid injection once a month (a treatment cycle). Clinical characteristics were described in Table 1 . Table 1 Patient characteristics. Baseline characteristics and treatment information of 106 patients included in this study. The control group refers to patients who were orally administered EGFR-TKIs only. The treatment group included patients who received the treatment of EGFR-TKIs combined with lienal polypeptide injection. Characteristic Control Group Treatment Group Sex,n,(%) Female 26(49) 25(47) Male 27(51) 28(53) Age,years(%) <65 35(66) 29(55) ≥65 18(34) 24(45) Median age,years 61.9 ± 8.2 62.3 ± 8.4 Smoking history,(%) Yes 9(17) 8(15) No 44(83) 45(85) TNM Stage,n,(%) III 7(13) 11(20) IV 46(87) 42(80) EGFR mutation,n,(%) 19DEL 28(53) 23(43) 21L858R 25(47) 30(57) EGFR-TKIs,n,(%) Gefitinib 17(32) 21(40) Eerlotinib 1(2) 0(0) Icotinib 29(54) 27(50) Afatinib 2(4) 2(4) Osimertinib 4(8) 1(2) Almonertinib 0(0) 2(4) Total,n,(%) 53(100) 53(100) Cell culture and agents The human NSCLC cell line PC9 and the Gefitinib resistant cell line PC9-GR were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium which was supplemented with 10% fetal bovine serum (10% FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator at 37°C with 5% CO2. Lienal polypeptide injection (Batch No. 20130405) was given by Jilin Fengsheng Pharmaceutical Co., Ltd (Jilin, China). Gefitinib was purchased from AstraZeneca Biotechnology limited company (London, England, UK). Cell grouping and treatment PC9 and PC9-GR cells were cultured in different concentration gradients of LP and logarithmic phase growth cells were collected for further study. PC9-GR cells were respectively grouped into: Gefitinib group (treated with Gefitinib), LP group (treated with lienal popypeptide liquid), Gefitinib-LP group (treated with Gefitinib and lienal polypeptide liquid) and control group (treated with DMSO). Cell proliferation experiment Cell proliferation was measured by CCK8 assay (cell counting kit-8, Selleck, Shanghai, China). PC9 cells and PC9-GR cells under logarithmic phase were seeded in 96-well plates maintaining in media containing 10% FBS at a density of 3500 cells/well and incubated overnight. Subsequently, the cells were exposed to different concentrations of LP for 72 h. After that, 10 µL of CCK8 was added into each well and incubated at 37°C for 1 hour. The optical density was measured at 450 nm by an enzyme-labeled instrument. For the colony formation experiment, PC9-GR cells were placed into a six-well plate with the density of 500cells a well and cultured in the medium containing 10% FBS for 14 days, culturing medium was replaced every 5 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St.Louis, MO, USA) in PBS for 15 min. Colony formation was detected by counting the number of stained colonies. For each treatment group, wells were counted in triplicate. Wound healing assay On the back of the 6-well plate, a marker pen was used to draw uniform horizontal lines with the assistance of a straightedge. The lines were at intervals of 0.8 cm and crossing the wells, with at least five lines for each well. In each well, 5 × 10 5 cells were added, and the confluency reached 100%. 24 hours later, the pipette (10 µL) was used to scratch along the straightedge vertical to the horizontal lines on the back. After scratching, cells were rinsed by PBS for three times to remove the scratched cells. With culture medium, the plate was incubated in a 5% CO2 incubator at 37°C. The samples were collected at 0, 24, 48 hours and photographed under an inverted microscope. The healing area of scratches was calculated by National Instrument Vision Assistant 8.6 software: migration rate = healing area of scratch/initial area of scratch × 100%. Experiments were carried out three times and mean value was calculated. Transwell assay Cells were digested after culturing in different groups. Every 5 × 10 4 cells in serum-free RPMI 1640 were seeded in the upper chamber (8 mm; Millipore), and the lower chamber was added with RPMI 1640 containing 10% FBS. After 24 hours’ incubation, the cells migrated through the membrane were fixed by 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. The images were taken by an IX7 inverted microscope (Olympus, Tokyo, Japan), five fields of view were randomly selected for photographing and counting. The number of cells adhering to the Matrigel of the side in the lower chamber was considered as the number of invasive cells. All experiments were conducted in triplicate. Flow cytometric analysis of apoptosis and cell cycle The PC9-GR cells in different groups as described above were cultured for 48 hours. Then, the cells were harvested by trypsinization and double stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide using the FITC Annexin V apoptosis detection kit (BD Biosciences). Cell apoptosis ratio was determined by a flow cytometer (FACScan, BD Biosciences). The percentage of cells in G0/G1, S, or G2/M phases was estimated by the specific BD Cycle Test Plus DNA Reagent Kit (BD Biosciences, Shanghai, China) in compliance with the manufacturer’s protocol. Every experiment was performed three times independently. Western blotting assay and antibodies The total cellular protein lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with specific antibodies against EGFR, p-EGFR overnight at 4℃. GAPDH was used as an internal control. All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Statistical analysis SPSS 17.0 statistical software (Chicago, IL, USA) was used for the statistical analysis. The independent samples t-test was used to compare the changes of CD4 + cells, CD8 + cells, NK cell activity and CD4+/CD8 + of peripheral blood between two groups of patients before and after therapy. P < 0.05 was considered statistically significant. Results Patients’ characteristics A total of 106 patients carrying EGFR mutations (19DEL and 21L858R) and diagnosed with advanced NSCLC were enrolled in this study. 53 of them were only oral administered with EGFR-TKIs, named control group, and remaining 53 participants received combined treatment of LP and EGFR-TKIs, named treatment group. The control group consisted of 27 males and 26 females, with a median age of 61.9 ± 8.2 years, and accordingly 28 males and 25 females in treatment group, with a median age of 62.3 ± 8.4 years. III and IV stage patients were 7 and 46 in control group, while 11 and 42 in treatment group. Detailed information including smoking history, EGFR mutation subtypes and the type of EGFR-TKIs were shown in Table 1 . LP combined with Gefitinib enhances immunity in advanced NSCLC To investigate the immune regulating ability of LP in real world, we retrospectively analyzed the changes of lymphocyte populations involving CD3 + T cells, CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B lymphocytes and the ratio of CD4 + /CD8 + in peripheral blood before and after treatment (Fig. 1 and Table 2 ). Compared with control group, the levels of CD3 + , CD4 + T lymphcytes increased dramatically in the treatment group and had significant differences ( p <0.05). In particular, the ratio of CD4 + /CD8 + displayed an upward trend in the treatment group after the related treatment. On the contrary, all the above indicators showed a descending tendency in control group ( p <0.05). These results indicated that LP combined with Gefitinib presented a notable benefit in improving immune function in advanced NSCLC patients. Table 2 Changes in peripheral blood lymphocyte subtypes of 106 NSCLC patients after different treatments in two groups. Lymphcyte subtypes Control Group(n = 53) Treatment Group(n = 53) Before treatment After treatment Before treatment After treatment CD3 + (%) 70.19 ± 1.47 59.74 ± 13.02 a 65.52 ± 11.07 74.21 ± 9.12 a,b CD4 + (%) 40.84 ± 10.89 32.33 ± 11.71 a 36.85 ± 9.68 42.31 ± 10.50 a,b CD8 + (%) 24.34 ± 10.06 23.24 ± 9.37 25.71 ± 8.56 27.87 ± 10.43 a,b NK(%) 17.20 ± 10.98 27.73 ± 15.32 a 18.47 ± 11.34 13.58 ± 8.19 a,b B(%) 10.60 ± 4.71 10.03 ± 6.66 10.71 ± 4.82 10.08 ± 4.51 a The ratio of CD4 + /CD8 + 2.08 ± 1.31 1.83 ± 1.18 a 1.65 ± 0.89 1.81 ± 0.95 a,b a: p <0.05, compared with treatments. b: p <0.05, compared with control group. LP combined with Gefitinib represses tumor growth of PC9-GR cells As a previous study demonstrated that EGFR-TKIs resistant tumors possess an immunosupressive microenvironment with relative more immunosuppressive cells and fewer immune-activated cells 18 . We hypothesise exogenous supplementation of immunomodulator could regulate intrinsic characteristics of tumor microenviroment, so as to increase sensitivity to EGFR-TKIs. Therefore, we investigate the influence of LP on Gefitinib-resistant cells PC9-GR in vitro . As shown in Fig. 2 A and 2 B, LP exhibited an encouraging effect on inhibiting PC9-GR cell proliferation and this effect exhibited a dose-dependent manner. Compared with Gefitinib group, cell proliferation in Gefitnib-LP group was significantly inhibited ( p < 0.05), with IC50 values of 2.405mg/ml in Gefitinib group versus 1.653mg/ml in Gefitinib-LP group. Furthermore, cloning formation experiments (Fig. 2 C) showed the same result: cell viability was particularly diminished in Gefitinib-LP group. Collectively, these data indicated that LP could enhance sensitivity of PC9-GR to Gefitinib by repressing cell proliferation. LP combined with Gefitinib inhibits PC9-GR cell invasion and migration Distant organ metastasis can generally be observed when EGFR-TKIs therapy fails, subsequently resulting in recurrence or disease progression. To evaluate the function of LP on tumor metastasis, transwell experiment was used to observe PC9-GR cell invasion ability. Compared with the control group, Gefitinib group and LP group, the number of invasive PC9-GR cells was reduced in Gefitinib-LP group (Fig. 2 D). Wound healing experiment was used to estimate PC9-GR cell migration. Gefitinib-LP group showed a decrease in PC9-GR cell migration compared with other groups. The combination of Gefitinib with LP could markedly inhibit PC-9GR cell migration (Fig. 2 E). These results suggested that LP might restore the sensitivity of PC9-GR cells to Gefitinib, hence further inhibiting tumor invasion and migration. LP combined with Gefitinib promotes PC9-GR cell apoptosis in vitro To investigate the potential mechanism of LP on tumor cell growth, cell apoptosis was detected by flow cytometry. As showed in Fig. 3 A and 3 C, PC9-GR cells in Gefitinib-LP group had a higher apoptotic proportion than Gefitinib group (6.00% vs. 5.67%), namely, LP induced more cells to develop apoptosis, and the same phenomenon was observed in the Gefitinib-LP group versus Gefitinib group (15.25% versus 13.97%). These results demonstrated that LP combined with Gefitinib promoted PC9GR cell apoptosis. LP combined with Gefitinib can arrest PC9-GR cell cycle We consequently investigated the influence of LP on cell cycle. The results (Fig. 3 B and 3 D) indicated that treatment of Gefitinib combined with LP increased the proportion of G0/G1 phase cells and decreased proportion of S and G2/M phase cells. LP combined with Gefitinib could trigger G0/G1 phase arrest to block cellular proliferation, therefore played an enhancing antiproliferative effect and tumor-supressor role. Taken together, all above results demonstrated LP combined with Gefitinib owns spectacular property in inhibiting tumor cell growth, migration and invasion as well as resensitizing drug-resistant cells to Gefitinib. LP combined with Gefitinib inhibits the expression of p-EGFR in PC9-GR cells Hyperactivation of the EGFR ultimately results in resistance in EGFR-TKIs targeted therapy. Therefore Western blotting assay was used to determine whether the combining administration of LP can influence the activity of phosphorylated-EGFR. As expected, Fig. 3 E showed that LP combined with Gefitinib markedly diminished EGFR activation as measured by phosphorylation levels. The result above further validated that immunomodifier LP owns the ability of resensitizing resistant cells to EGFR-TKIs. Discussion EGFR-TKIs play a dedicated therapeutic effect on NSCLC carrying EGFR sensitive mutations, such as EGFR 19 exon in-frame deletion(19DEL) and substitutional mutation of arginine for leucine (L858R) in exon 21 2, 4, 19 . Although EGFR-TKIs exhibited spectacular therapeutic benefits in clinical practice, the occurrence of adverse effects can not be ignored at any time. Adverse events may impact the efficacy of anticancer therapies even result in treatment termination and disease progression. In addition to the reduction or stabilization of local lesions, systemic or local inflammatory reactions also occur in patients during the whole process of EGFR-TKIs therapy 18 . Common inflammatory reactions including rash, paronychia and chondriasis, hair disorders, mucitis, etc. Other inflammatory reactions include interstitial pneumonitis, chronic inflammation of intestinal tract and liver 20 – 22 . As an immune modulator, LP is generally prescribed in the treatment of numerous malignant tumors. Previous studies pointed out that LP can regulate the body’s immune function so as to improve physical condition of cancer patients and diminish cancer/treatment-related painfulness or adverse effects 15 . Additionally, LP administration could elevate the cellular level of CD3 + , CD4 + and NK cells in malignant cancer patients who underwent radiotherapies, which proved that LP could enhance the body's cellular immunity and reduce the toxicities of radiotherapy 16 . Similarly, our study showed higher levels of CD3 + , CD4 + and the ratio of CD4 + /CD8 + in advanced NSCLC patients treated by LP combined with Gefitinib. CD3 + T cell is representative of whole immune cells level, normally the ratio of CD4 + /CD8 + is larger than 1, the bigger the ratio is, the greater number of helper T cells are, and corresponding the less suppressor T cells are, which reflects a better immune status. Furthermore, increased CD4 + /CD8 + ratio can serve as an independent prognostic factor in NSCLC 23 – 26 . Our study innovatively found that LP showed a synergistic antitumor effect and could enhance the efficacy of EGFR-TKIs. LP combined with Gefitinib exhibited a more magnificent inhibitory capability on tumor cell biological behavior. More importantly, LP showed a synergistic effect in blocking G0/G1 phase transition to inhibit tumor cell proliferation in our study. A study focused on the combination therapy of cyclophosphamide (CTX) with LP based on murine lung carcinoma model pointed out that LP itself did not possess the property of direct antitumor effect, but it showed a synergistic enhancing phenomenon of antitumor effect when applied with CTX 27 . This probably attribute to LP relieving immunosuppressive tumor microenvironment by stimulating and activating lymphocytes in tumor microenvironment. Likewise, that might explain the tinny difference between the LP group and control group in our study. Taken together, our study demonstrated that LP in conjunction with Gefitinib could enhance body immune function so that empower patients tolerate longer treatment exposure and derive more benefit from EGFR-TKIs therapy, accordingly, arriving longer survival time. Furthermore, LP promotes the sensitivity of PC9-GR cells to EGFR-TKIs, arrests cell cycle, inhibits tumor proliferation, invasion and migration, as well as plays an important role in reducing the expression of phosphorylation of EGFR protein, and consequencely enhances anticancer effect of EGFR-TKIs therapy. In conclusion, LP in combination with Gefitinib was an effective treatment for patients of advanced NSCLC, which may improve the life quality of patients, and potentially improve prognosis. Further investigations are still required to explore the specific mechanisms to enhance the immunity of LP. Declarations Acknowledgements and founding The work of this study was supported by grants from the National Natural Science Foundation of China (grant no. 81972188) and the Wu Jie-ping Foundation (320.6799.15032). Author contributions Yun Chen, Xinyin Liu and Jiaqi Yao contributed to designing and organizing the experiments, carrying out data analysis, and writing the manuscript. Shidai Jin, Jun Li and Jiali Xu contributed to laboratory measurements and data analysis. Renhua Guo contributed to conceiving the ideas, supervising the study, and writing the manuscript. All authors read and approved the final manuscript. Conflicts of interest The authors have declared that no conflict of interest exists. References Sung, H.; Ferlay, J.; Siegel, R. L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021, 71 (3), 209-249. Sholl, L. M.; Aisner, D. L.; Varella-Garcia, M.; Berry, L. D.; Dias-Santagata, D.; Wistuba, I. I.; Chen, H.; Fujimoto, J.; Kugler, K.; Franklin, W. A.; Iafrate, A. J.; Ladanyi, M.; Kris, M. G.; Johnson, B. E.; Bunn, P. A.; Minna, J. D.; Kwiatkowski, D. J., Multi-institutional Oncogenic Driver Mutation Analysis in Lung Adenocarcinoma: The Lung Cancer Mutation Consortium Experience. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2015, 10 (5), 768-777. Chen, Z.; Fillmore, C. M.; Hammerman, P. S.; Kim, C. F.; Wong, K.-K., Non-small-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer 2014, 14 (8), 535-546. Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A., Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007, 7 (3), 169-181. Mok, T. S.; Wu, Y.-L.; Thongprasert, S.; Yang, C.-H.; Chu, D.-T.; Saijo, N.; Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y.; Nishiwaki, Y.; Ohe, Y.; Yang, J.-J.; Chewaskulyong, B.; Jiang, H.; Duffield, E. L.; Watkins, C. L.; Armour, A. A.; Fukuoka, M., Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. The New England journal of medicine 2009, 361 (10), 947-957. Ettinger, D. S.; Wood, D. E.; Aggarwal, C.; Aisner, D. L.; Akerley, W.; Bauman, J. R.; Bharat, A.; Bruno, D. S.; Chang, J. Y.; Chirieac, L. R.; D'Amico, T. A.; Dilling, T. J.; Dobelbower, M.; Gettinger, S.; Govindan, R.; Gubens, M. A.; Hennon, M.; Horn, L.; Lackner, R. P.; Lanuti, M.; Leal, T. A.; Lin, J.; Loo, B. W.; Martins, R. G.; Otterson, G. A.; Patel, S. P.; Reckamp, K. L.; Riely, G. J.; Schild, S. E.; Shapiro, T. A.; Stevenson, J.; Swanson, S. J.; Tauer, K. W.; Yang, S. C.; Gregory, K.; Hughes, M., NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 1.2020. J Natl Compr Canc Netw 2019, 17 (12), 1464-1472. Dingemans, A. M. C.; Früh, M.; Ardizzoni, A.; Besse, B.; Faivre-Finn, C.; Hendriks, L. E.; Lantuejoul, S.; Peters, S.; Reguart, N.; Rudin, C. M.; De Ruysscher, D.; Van Schil, P. E.; Vansteenkiste, J.; Reck, M., Small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology 2021, 32 (7), 839-853. Wu, Y. L.; Planchard, D.; Lu, S.; Sun, H.; Yamamoto, N.; Kim, D. W.; Tan, D. S. W.; Yang, J. C. H.; Azrif, M.; Mitsudomi, T.; Park, K.; Soo, R. A.; Chang, J. W. C.; Alip, A.; Peters, S.; Douillard, J. Y., Pan-Asian adapted Clinical Practice Guidelines for the management of patients with metastatic non-small-cell lung cancer: a CSCO-ESMO initiative endorsed by JSMO, KSMO, MOS, SSO and TOS. Annals of oncology : official journal of the European Society for Medical Oncology 2019, 30 (2), 171-210. Hosomi, Y.; Morita, S.; Sugawara, S.; Kato, T.; Fukuhara, T.; Gemma, A.; Takahashi, K.; Fujita, Y.; Harada, T.; Minato, K.; Takamura, K.; Hagiwara, K.; Kobayashi, K.; Nukiwa, T.; Inoue, A., Gefitinib Alone Versus Gefitinib Plus Chemotherapy for Non-Small-Cell Lung Cancer With Mutated Epidermal Growth Factor Receptor: NEJ009 Study. J Clin Oncol 2020, 38 (2), 115-123. Hsu, W. H.; Yang, J. C. H.; Mok, T. S.; Loong, H. H., Overview of current systemic management of EGFR-mutant NSCLC. Annals of oncology : official journal of the European Society for Medical Oncology 2018, 29 (suppl_1), i3-i9. Mayekar, M. K.; Bivona, T. G., Current Landscape of Targeted Therapy in Lung Cancer. Clin Pharmacol Ther 2017, 102 (5), 757-764. Altorki, N. K.; Markowitz, G. J.; Gao, D.; Port, J. L.; Saxena, A.; Stiles, B.; McGraw, T.; Mittal, V., The lung microenvironment: an important regulator of tumour growth and metastasis. Nat Rev Cancer 2019, 19 (1). Grivennikov, S. I.; Greten, F. R.; Karin, M., Immunity, inflammation, and cancer. Cell 2010, 140 (6), 883-899. Biswas, S. K.; Mantovani, A., Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol 2010, 11 (10), 889-896. Wang, J.; Zheng, M.; Min, Q.; Gao, Y.; Sun, W., The dual regulatory function of lienal peptide on immune system. Int Immunopharmacol 2018, 55 , 245-253. Chu, A.; Song, R.; Hou, G.; Yuan, J.; Wang, C.; Yang, Y.; Qin, N.; Liu, Y.; Liang, B.; Zhang, Y.; Liu, Z., Experimental Study and Clinical Observation on the Improvement Effect of Lienal Polypeptide on Blood Toxicity and Immune Injury Induced by Radiotherapy. Genet Test Mol Biomarkers 2020, 24 (2), 73-77. Zhou, J.; Niu, G.; Pei, Y.; Cao, C.; Ding, C.; Sun, G.; Guo, J.; Liu, Y.; Yu, Y., The effect and clinical efficacy of lienal polypeptide injection combined with FOLFOX chemotherapy regimen in colon cancer patients. Oncol Lett 2016, 12 (5), 3191-3194. Liu, L.; Wang, C.; Li, S.; Bai, H.; Wang, J., Tumor immune microenvironment in epidermal growth factor receptor-mutated non-small cell lung cancer before and after epidermal growth factor receptor tyrosine kinase inhibitor treatment: a narrative review. Transl Lung Cancer Res 2021, 10 (9), 3823-3839. Paez, J. G.; Jänne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B. E.; Meyerson, M., EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004, 304 (5676), 1497-1500. Park, K.; Tan, E.-H.; O'Byrne, K.; Zhang, L.; Boyer, M.; Mok, T.; Hirsh, V.; Yang, J. C.-H.; Lee, K. H.; Lu, S.; Shi, Y.; Kim, S.-W.; Laskin, J.; Kim, D.-W.; Arvis, C. D.; Kölbeck, K.; Laurie, S. A.; Tsai, C.-M.; Shahidi, M.; Kim, M.; Massey, D.; Zazulina, V.; Paz-Ares, L., Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial. The Lancet. Oncology 2016, 17 (5), 577-589. Oshima, Y.; Tanimoto, T.; Yuji, K.; Tojo, A., EGFR-TKI-Associated Interstitial Pneumonitis in Nivolumab-Treated Patients With Non-Small Cell Lung Cancer. JAMA oncology 2018, 4 (8), 1112-1115. Zhou, C.; Wu, Y.-L.; Chen, G.; Feng, J.; Liu, X.-Q.; Wang, C.; Zhang, S.; Wang, J.; Zhou, S.; Ren, S.; Lu, S.; Zhang, L.; Hu, C.; Hu, C.; Luo, Y.; Chen, L.; Ye, M.; Huang, J.; Zhi, X.; Zhang, Y.; Xiu, Q.; Ma, J.; Zhang, L.; You, C., Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. The Lancet. Oncology 2011, 12 (8), 735-742. Galon, J.; Costes, A.; Sanchez-Cabo, F.; Kirilovsky, A.; Mlecnik, B.; Lagorce-Pagès, C.; Tosolini, M.; Camus, M.; Berger, A.; Wind, P.; Zinzindohoué, F.; Bruneval, P.; Cugnenc, P.-H.; Trajanoski, Z.; Fridman, W.-H.; Pagès, F., Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006, 313 (5795), 1960-1964. deLeeuw, R. J.; Kost, S. E.; Kakal, J. A.; Nelson, B. H., The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature. Clinical cancer research : an official journal of the American Association for Cancer Research 2012, 18 (11), 3022-3029. Ruffini, E.; Asioli, S.; Filosso, P. L.; Lyberis, P.; Bruna, M. C.; Macrì, L.; Daniele, L.; Oliaro, A., Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg 2009, 87 (2). Zhang, J.; Huang, S.-H.; Li, H.; Li, Y.; Chen, X.-L.; Zhang, W.-Q.; Chen, H.-G.; Gu, L.-J., Preoperative lymphocyte count is a favorable prognostic factor of disease-free survival in non-small-cell lung cancer. Med Oncol 2013, 30 (1), 352. Wu, Y.-P.; Deng, J.; Ouyang, S.-H.; Mao, Z.-F.; Wang, G.-E.; Kurihara, H.; He, R.-R.; Li, Y.-F., Immune regulation effect of lienal polypeptides extract in Lewis lung carcinoma-bearing mice treated with cyclophosphamide. Experimental biology and medicine (Maywood, N.J.) 2018, 243 (1), 66-77. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-1517195","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":96012344,"identity":"1bebc9e3-daad-4e84-830c-e7bf437831b1","order_by":0,"name":"Yun Chen","email":"","orcid":"","institution":"Jiangsu Province People's Hospital and Nanjing Medical University First Affiliated Hospital: Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Chen","suffix":""},{"id":96012345,"identity":"50761b11-ea36-427f-ac2c-7a3dd1701c0e","order_by":1,"name":"Xinyin Liu","email":"","orcid":"","institution":"Jiangsu Province People's Hospital and Nanjing Medical University First Affiliated Hospital: Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinyin","middleName":"","lastName":"Liu","suffix":""},{"id":96012346,"identity":"bc64b5ab-611c-4a70-857d-5eeed122f2dc","order_by":2,"name":"Jiaqi Yao","email":"","orcid":"","institution":"Wuxi Ninth People's Hospital: Wuxi Hand Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Yao","suffix":""},{"id":96012347,"identity":"aa039645-8eb9-410e-9093-a6592ef4978f","order_by":3,"name":"Shidai Jin","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shidai","middleName":"","lastName":"Jin","suffix":""},{"id":96012348,"identity":"91d761b2-8d43-444b-856c-e96e67b72fa6","order_by":4,"name":"Jun Li","email":"","orcid":"","institution":"Jiangsu Province People's Hospital and Nanjing Medical University First Affiliated Hospital: Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":96012349,"identity":"04bb9fec-e871-4506-a33c-7e55213af61a","order_by":5,"name":"Jiali Xu","email":"","orcid":"","institution":"Jiangsu Province People's Hospital and Nanjing Medical University First Affiliated Hospital: Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Xu","suffix":""},{"id":96012350,"identity":"62f59bac-01c6-4ed9-be31-48b1db4265b3","order_by":6,"name":"Renhua Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCRBRwcAM5vAQr+UMyVoY26AcorSYz26/JvFx3h123RkJjA/etjHImxPSInPnTJnkzG3PmM1uJDAbzm1jMNzZQMhdEjlp0rzbDoO0sEnztjEkGBwgSsscsBb230RqST8mzdsAsYWZWFuYLWccA2o587BZcs45CcMNRNjy8MaHmsPJZseTD354U2YjT9AWYFwYgMhkYOw0MEBjlhBgfwAi7YhROgpGwSgYBSMUAAAxWjw32lbxgQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4475-8617","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":true,"prefix":"","firstName":"Renhua","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2022-04-02 16:58:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1517195/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1517195/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20032435,"identity":"a1dfe619-71be-4cb4-95e8-ae1ac53e66a0","added_by":"auto","created_at":"2022-04-06 16:43:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3562987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLP combined with Gefitinib can enhance immune function\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA, C\u003c/strong\u003e, The variation of lymphocyte subtypes between pre-and post-treatment in two groups. \u003cstrong\u003eB, D,\u003c/strong\u003e The ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+ \u003c/sup\u003ebetween pre-and post-treatment in two groups. \u003cstrong\u003eE, \u003c/strong\u003eThe absoute difference of lymphocyte subtypes between two groups after treatment. \u003cstrong\u003eF\u003c/strong\u003e, The absoute difference of the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e bettween two groups after treatment (the difference less than zero refers to the ratio going down after treatments, and the difference greater than zero refer to the ratio rising up after treatment) . *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05，**\u003cem\u003ep\u003c/em\u003e\u0026lt;0.005，***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001，ns, have no statistical significance.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1517195/v1/affeecb7d563805296952d05.jpg"},{"id":20032433,"identity":"acec574c-fa5d-4da5-9478-12e7d88cff51","added_by":"auto","created_at":"2022-04-06 16:43:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLP combined with Gefitinib inhibited PC9-GR cell proliferation, migration and invasion. A\u003c/strong\u003e, CCK8 assay was used to detect PC9-GR cell viability after treating with gradient concentration of LP (0, 0.25, 0.50, 0.75, 1.00, 1.25mg/ml) for 72h. \u003cstrong\u003eB\u003c/strong\u003e, The IC50 values of PC9-GR cells treated with Gefitinib or LP combined with Gefitinib. \u003cstrong\u003eC\u003c/strong\u003e, Colony-formation experiments assessed the proliferating ability of PC9-GR cells treated with 1µmol/L Gefitinib and/or 1mg/ml LP. \u003cstrong\u003eD\u003c/strong\u003e, Wound healing assay to estimate migrating ability of PC9-GR cells treated with 1µmol/L Gefitinib and/or 1mg/ml LP for 24h.\u003cstrong\u003e E\u003c/strong\u003e, Transwell experiment were performed to observe PC9-GR cells invading condition after treated with 1µmol/L Gefitinib and/or 1mg/ml LP for 24h.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1517195/v1/8af275c807553556d34296f4.jpg"},{"id":20032434,"identity":"1c563678-20ce-4678-9658-05122912bd48","added_by":"auto","created_at":"2022-04-06 16:43:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":545841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLP combined with Gefitinib induced G0/G1 phase arrest and depressed the expression of phosphorylated-EGFR in PC9-GR cells. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA, B\u003c/strong\u003e, Flow cytometry was carried out to detect apoptosis and cell cycle of PC9-GR cells after treated with 1µmol/L Gefitinib and/or 1mg/ml LP for 48 h. \u003cstrong\u003eC\u003c/strong\u003e, Quantitative analysis of G0/G1, S, G2/M stage changes of PC9-GR cells in different groups. \u003cstrong\u003eD,\u003c/strong\u003e Quantitative analysis of apoptotic rates of PC9-GR cells treated with 1µmol/L Gefitinib and/or 1mg/ml LP for 48 h. \u003cstrong\u003eE\u003c/strong\u003e, expression of EGFR, p-EGFR in PC9-GR cells with 1µmol/L Gefitinib and/or 1mg/ml LP treatment for 72 h by Western blot.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1517195/v1/a40e2c2a2885644ee502c16c.jpg"},{"id":20506199,"identity":"8654b2e2-c44f-43ab-9924-1fa76de8cd5f","added_by":"auto","created_at":"2022-04-19 14:46:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":810274,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1517195/v1/79520845-e694-4ca0-b28f-ba95a1141a69.pdf"}],"financialInterests":"","formattedTitle":"The clinical synergistic antitumor efficacy of Lienal Polypeptide combined with EGFR-TKIs for advanced NSCLC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer remains the leading cause of cancer related deaths worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Non-small cell lung cancer (NSCLC) is divided to different molecular subtypes, among which epidermal growth factor receptor (EGFR) mutation is the most common subtype\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. EGFR-TKIs such as Gefitinib extraordinarily prolonged median overall survival (OS) of advanced NSCLC patients, as well as improved the quality of life\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. EGFR-TKIs have been recommended as first-line treatment for patients with advanced NSCLC harboring EGFR mutaions\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Nevertheless, a considerable proportion of patients have to discontinue treatment due to adverse effects, which lead to disease progression and failure of therapy\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Long-term inflammatory responses due to treatment cause massive infiltration of inflammatory cells and increased cytokines levels. Such tumor microenvironment flooding with chronic inflammatory cells and inflammatory mediators may result in gene silencing or abnormal expression, epigenetic changes, mismatched repair enzyme inactivation, DNA damage or gene mutation in tumor cells, eventually contributing to malignant transformation and drug resistance of tumor cells\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLienal polypeptide (LP) is extracted from the spleen of healthy calves, which functioning as an immune modulator with the ability of correcting immune dysfunction, activating non-specific immune function, as well as improving the immune function of lymphocytes, therefore enhancing the body's defensive capabilities to infection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. At present, LP is mainly applied to cellular immunodeficiency diseases and malignant tumors caused by chemo-radiotherapy, which can ameliorate cancer cachexia\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. LP has also been widely used in the treatment of multiple malignancy tumors nowadays.\u003c/p\u003e \u003cp\u003eRecently studies found that EGFR-TKIs can moderate T lymphocytes and natural killer cells to deregulate carcinogenesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However the chronic inflammatory reaction induced by EGFR-TKIs inversely results in treating termination or drug resistance. LP, an immunomodifier, could significantly improve immune function and correct immune disorders. Therefore, we hypothesized that LP treatment could reduce the persistent chronic inflammation and drug-related adverse effects caused by EGFR-TKIs in NSCLC. In this study, we aim to use Gefitinib combined with LP to treat NSCLC harboring EGFR-sensitive mutations, observe the efficacy of the combined therapy, and investigate the effects of drug combination on tumor biological behaviors and immune function, we further clarified the molecular mechanisms of combination therapy. In conclusion, our study provides new insights in LP combined with EGFR-TKIs in treatment of advanced NSCLC.\u003c/p\u003e"},{"header":"Patients And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eA total of 106 patients diagnosed with III and IV stage NSCLC in Jiangsu Province Hospital from January 2019 to January 2021 were enrolled in this study. This study was approved by the ethical committee of the Jiangsu Province Hospital Medical Ethics Committee and was carried out in accordance with the approved guidelines. All participants had a good knowledge about the study and signed written informed consents. The inclusion criteria were as follows: pathologically diagnosed with lung adenocarcinoma, carrying EGFR mutation concluding in-frame deletions in exon 19 (19DEL) or a point mutation in exon 21 (L858R), accepting oral administration of EGFR-TKIs per day and/or combined with intravenous infusion of lienal polypeptide liquid injection once a month (a treatment cycle). Clinical characteristics were described in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePatient characteristics. Baseline characteristics and treatment information of 106 patients included in this study. The control group refers to patients who were orally administered EGFR-TKIs only. The treatment group included patients who received the treatment of EGFR-TKIs combined with lienal polypeptide injection.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl Group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment Group\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex,n,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26(49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25(47)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27(51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28(53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge,years(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35(66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29(55)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18(34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24(45)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian age,years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSmoking history,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9(17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8(15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44(83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45(85)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNM Stage,n,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7(13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11(20)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46(87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42(80)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eEGFR mutation,n,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19DEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28(53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23(43)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21L858R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25(47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30(57)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEGFR-TKIs,n,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGefitinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17(32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21(40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEerlotinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIcotinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29(54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27(50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAfatinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOsimertinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4(8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlmonertinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal,n,(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eCell culture and agents\u003c/h2\u003e\n \u003cp\u003eThe human NSCLC cell line PC9 and the Gefitinib resistant cell line PC9-GR were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium which was supplemented with 10% fetal bovine serum (10% FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator at 37\u0026deg;C with 5% CO2. Lienal polypeptide injection (Batch No. 20130405) was given by Jilin Fengsheng Pharmaceutical Co., Ltd (Jilin, China). Gefitinib was purchased from AstraZeneca Biotechnology limited company (London, England, UK).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eCell grouping and treatment\u003c/h2\u003e\n \u003cp\u003ePC9 and PC9-GR cells were cultured in different concentration gradients of LP and logarithmic phase growth cells were collected for further study. PC9-GR cells were respectively grouped into: Gefitinib group (treated with Gefitinib), LP group (treated with lienal popypeptide liquid), Gefitinib-LP group (treated with Gefitinib and lienal polypeptide liquid) and control group (treated with DMSO).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eCell proliferation experiment\u003c/h2\u003e\n \u003cp\u003eCell proliferation was measured by CCK8 assay (cell counting kit-8, Selleck, Shanghai, China). PC9 cells and PC9-GR cells under logarithmic phase were seeded in 96-well plates maintaining in media containing 10% FBS at a density of 3500 cells/well and incubated overnight. Subsequently, the cells were exposed to different concentrations of LP for 72 h. After that, 10 \u0026micro;L of CCK8 was added into each well and incubated at 37\u0026deg;C for 1 hour. The optical density was measured at 450 nm by an enzyme-labeled instrument. For the colony formation experiment, PC9-GR cells were placed into a six-well plate with the density of 500cells a well and cultured in the medium containing 10% FBS for 14 days, culturing medium was replaced every 5 days. Colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St.Louis, MO, USA) in PBS for 15 min. Colony formation was detected by counting the number of stained colonies. For each treatment group, wells were counted in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eWound healing assay\u003c/h2\u003e\n \u003cp\u003eOn the back of the 6-well plate, a marker pen was used to draw uniform horizontal lines with the assistance of a straightedge. The lines were at intervals of 0.8 cm and crossing the wells, with at least five lines for each well. In each well, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were added, and the confluency reached 100%. 24 hours later, the pipette (10 \u0026micro;L) was used to scratch along the straightedge vertical to the horizontal lines on the back. After scratching, cells were rinsed by PBS for three times to remove the scratched cells. With culture medium, the plate was incubated in a 5% CO2 incubator at 37\u0026deg;C. The samples were collected at 0, 24, 48 hours and photographed under an inverted microscope. The healing area of scratches was calculated by National Instrument Vision Assistant 8.6 software: migration rate\u0026thinsp;=\u0026thinsp;healing area of scratch/initial area of scratch \u0026times; 100%. Experiments were carried out three times and mean value was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eTranswell assay\u003c/h2\u003e\n \u003cp\u003eCells were digested after culturing in different groups. Every 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in serum-free RPMI 1640 were seeded in the upper chamber (8 mm; Millipore), and the lower chamber was added with RPMI 1640 containing 10% FBS. After 24 hours\u0026rsquo; incubation, the cells migrated through the membrane were fixed by 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. The images were taken by an IX7 inverted microscope (Olympus, Tokyo, Japan), five fields of view were randomly selected for photographing and counting. The number of cells adhering to the Matrigel of the side in the lower chamber was considered as the number of invasive cells. All experiments were conducted in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eFlow cytometric analysis of apoptosis and cell cycle\u003c/h2\u003e\n \u003cp\u003eThe PC9-GR cells in different groups as described above were cultured for 48 hours. Then, the cells were harvested by trypsinization and double stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide using the FITC Annexin V apoptosis detection kit (BD Biosciences). Cell apoptosis ratio was determined by a flow cytometer (FACScan, BD Biosciences). The percentage of cells in G0/G1, S, or G2/M phases was estimated by the specific BD Cycle Test Plus DNA Reagent Kit (BD Biosciences, Shanghai, China) in compliance with the manufacturer\u0026rsquo;s protocol. Every experiment was performed three times independently.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eWestern blotting assay and antibodies\u003c/h2\u003e\n \u003cp\u003eThe total cellular protein lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with specific antibodies against EGFR, p-EGFR overnight at 4℃. GAPDH was used as an internal control. All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eSPSS 17.0 statistical software (Chicago, IL, USA) was used for the statistical analysis. The independent samples t-test was used to compare the changes of CD4\u0026thinsp;+\u0026thinsp;cells, CD8\u0026thinsp;+\u0026thinsp;cells, NK cell activity and CD4+/CD8\u0026thinsp;+\u0026thinsp;of peripheral blood between two groups of patients before and after therapy. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003ePatients\u0026rsquo; characteristics\u003c/h2\u003e\n \u003cp\u003eA total of 106 patients carrying EGFR mutations (19DEL and 21L858R) and diagnosed with advanced NSCLC were enrolled in this study. 53 of them were only oral administered with EGFR-TKIs, named control group, and remaining 53 participants received combined treatment of LP and EGFR-TKIs, named treatment group. The control group consisted of 27 males and 26 females, with a median age of 61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2 years, and accordingly 28 males and 25 females in treatment group, with a median age of 62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4 years. III and IV stage patients were 7 and 46 in control group, while 11 and 42 in treatment group. Detailed information including smoking history, EGFR mutation subtypes and the type of EGFR-TKIs were shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eLP combined with Gefitinib enhances immunity in advanced NSCLC\u003c/h2\u003e\n \u003cp\u003eTo investigate the immune regulating ability of LP in real world, we retrospectively analyzed the changes of lymphocyte populations involving CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, natural killer (NK) cells, B lymphocytes and the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e in peripheral blood before and after treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with control group, the levels of CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e T lymphcytes increased dramatically in the treatment group and had significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). In particular, the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e displayed an upward trend in the treatment group after the related treatment. On the contrary, all the above indicators showed a descending tendency in control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). These results indicated that LP combined with Gefitinib presented a notable benefit in improving immune function in advanced NSCLC patients.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eChanges in peripheral blood lymphocyte subtypes of 106 NSCLC patients after different treatments in two groups.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eLymphcyte subtypes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eControl Group(n\u0026thinsp;=\u0026thinsp;53)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTreatment Group(n\u0026thinsp;=\u0026thinsp;53)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBefore treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAfter treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.74\u0026thinsp;\u0026plusmn;\u0026thinsp;13.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.52\u0026thinsp;\u0026plusmn;\u0026thinsp;11.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.12 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.84\u0026thinsp;\u0026plusmn;\u0026thinsp;10.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.33\u0026thinsp;\u0026plusmn;\u0026thinsp;11.71 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.85\u0026thinsp;\u0026plusmn;\u0026thinsp;9.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.31\u0026thinsp;\u0026plusmn;\u0026thinsp;10.50 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.34\u0026thinsp;\u0026plusmn;\u0026thinsp;10.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.24\u0026thinsp;\u0026plusmn;\u0026thinsp;9.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.71\u0026thinsp;\u0026plusmn;\u0026thinsp;8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.87\u0026thinsp;\u0026plusmn;\u0026thinsp;10.43 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNK(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.20\u0026thinsp;\u0026plusmn;\u0026thinsp;10.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.73\u0026thinsp;\u0026plusmn;\u0026thinsp;15.32 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.47\u0026thinsp;\u0026plusmn;\u0026thinsp;11.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.19 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;4.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.03\u0026thinsp;\u0026plusmn;\u0026thinsp;6.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ea: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, compared with treatments. b: \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, compared with control group.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eLP combined with Gefitinib represses tumor growth of PC9-GR cells\u003c/h2\u003e\n \u003cp\u003eAs a previous study demonstrated that EGFR-TKIs resistant tumors possess an immunosupressive microenvironment with relative more immunosuppressive cells and fewer immune-activated cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We hypothesise exogenous supplementation of immunomodulator could regulate intrinsic characteristics of tumor microenviroment, so as to increase sensitivity to EGFR-TKIs. Therefore, we investigate the influence of LP on Gefitinib-resistant cells PC9-GR \u003cem\u003ein vitro\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, LP exhibited an encouraging effect on inhibiting PC9-GR cell proliferation and this effect exhibited a dose-dependent manner. Compared with Gefitinib group, cell proliferation in Gefitnib-LP group was significantly inhibited (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with IC50 values of 2.405mg/ml in Gefitinib group versus 1.653mg/ml in Gefitinib-LP group. Furthermore, cloning formation experiments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) showed the same result: cell viability was particularly diminished in Gefitinib-LP group. Collectively, these data indicated that LP could enhance sensitivity of PC9-GR to Gefitinib by repressing cell proliferation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eLP combined with Gefitinib inhibits PC9-GR cell invasion and migration\u003c/h2\u003e\n \u003cp\u003eDistant organ metastasis can generally be observed when EGFR-TKIs therapy fails, subsequently resulting in recurrence or disease progression. To evaluate the function of LP on tumor metastasis, transwell experiment was used to observe PC9-GR cell invasion ability. Compared with the control group, Gefitinib group and LP group, the number of invasive PC9-GR cells was reduced in Gefitinib-LP group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Wound healing experiment was used to estimate PC9-GR cell migration. Gefitinib-LP group showed a decrease in PC9-GR cell migration compared with other groups. The combination of Gefitinib with LP could markedly inhibit PC-9GR cell migration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results suggested that LP might restore the sensitivity of PC9-GR cells to Gefitinib, hence further inhibiting tumor invasion and migration.\u003c/p\u003e\n \u003ch2\u003eLP combined with Gefitinib promotes PC9-GR cell apoptosis\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eTo investigate the potential mechanism of LP on tumor cell growth, cell apoptosis was detected by flow cytometry. As showed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, PC9-GR cells in Gefitinib-LP group had a higher apoptotic proportion than Gefitinib group (6.00% vs. 5.67%), namely, LP induced more cells to develop apoptosis, and the same phenomenon was observed in the Gefitinib-LP group versus Gefitinib group (15.25% versus 13.97%). These results demonstrated that LP combined with Gefitinib promoted PC9GR cell apoptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003eLP combined with Gefitinib can arrest PC9-GR cell cycle\u003c/h2\u003e\n \u003cp\u003eWe consequently investigated the influence of LP on cell cycle. The results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD) indicated that treatment of Gefitinib combined with LP increased the proportion of G0/G1 phase cells and decreased proportion of S and G2/M phase cells. LP combined with Gefitinib could trigger G0/G1 phase arrest to block cellular proliferation, therefore played an enhancing antiproliferative effect and tumor-supressor role. Taken together, all above results demonstrated LP combined with Gefitinib owns spectacular property in inhibiting tumor cell growth, migration and invasion as well as resensitizing drug-resistant cells to Gefitinib.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003eLP combined with Gefitinib inhibits the expression of p-EGFR in PC9-GR cells\u003c/h2\u003e\n \u003cp\u003eHyperactivation of the EGFR ultimately results in resistance in EGFR-TKIs targeted therapy. Therefore Western blotting assay was used to determine whether the combining administration of LP can influence the activity of phosphorylated-EGFR. As expected, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE showed that LP combined with Gefitinib markedly diminished EGFR activation as measured by phosphorylation levels. The result above further validated that immunomodifier LP owns the ability of resensitizing resistant cells to EGFR-TKIs.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEGFR-TKIs play a dedicated therapeutic effect on NSCLC carrying EGFR sensitive mutations, such as EGFR 19 exon in-frame deletion(19DEL) and substitutional mutation of arginine for leucine (L858R) in exon 21\u003csup\u003e2, 4, 19\u003c/sup\u003e. Although EGFR-TKIs exhibited spectacular therapeutic benefits in clinical practice, the occurrence of adverse effects can not be ignored at any time. Adverse events may impact the efficacy of anticancer therapies even result in treatment termination and disease progression. In addition to the reduction or stabilization of local lesions, systemic or local inflammatory reactions also occur in patients during the whole process of EGFR-TKIs therapy\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Common inflammatory reactions including rash, paronychia and chondriasis, hair disorders, mucitis, etc. Other inflammatory reactions include interstitial pneumonitis, chronic inflammation of intestinal tract and liver\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs an immune modulator, LP is generally prescribed in the treatment of numerous malignant tumors. Previous studies pointed out that LP can regulate the body\u0026rsquo;s immune function so as to improve physical condition of cancer patients and diminish cancer/treatment-related painfulness or adverse effects\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, LP administration could elevate the cellular level of CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e and NK cells in malignant cancer patients who underwent radiotherapies, which proved that LP could enhance the body's cellular immunity and reduce the toxicities of radiotherapy\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, our study showed higher levels of CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e and the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e in advanced NSCLC patients treated by LP combined with Gefitinib. CD3\u003csup\u003e+\u003c/sup\u003e T cell is representative of whole immune cells level, normally the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e is larger than 1, the bigger the ratio is, the greater number of helper T cells are, and corresponding the less suppressor T cells are, which reflects a better immune status. Furthermore, increased CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e ratio can serve as an independent prognostic factor in NSCLC\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study innovatively found that LP showed a synergistic antitumor effect and could enhance the efficacy of EGFR-TKIs. LP combined with Gefitinib exhibited a more magnificent inhibitory capability on tumor cell biological behavior. More importantly, LP showed a synergistic effect in blocking G0/G1 phase transition to inhibit tumor cell proliferation in our study. A study focused on the combination therapy of cyclophosphamide (CTX) with LP based on murine lung carcinoma model pointed out that LP itself did not possess the property of direct antitumor effect, but it showed a synergistic enhancing phenomenon of antitumor effect when applied with CTX\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This probably attribute to LP relieving immunosuppressive tumor microenvironment by stimulating and activating lymphocytes in tumor microenvironment. Likewise, that might explain the tinny difference between the LP group and control group in our study.\u003c/p\u003e \u003cp\u003eTaken together, our study demonstrated that LP in conjunction with Gefitinib could enhance body immune function so that empower patients tolerate longer treatment exposure and derive more benefit from EGFR-TKIs therapy, accordingly, arriving longer survival time. Furthermore, LP promotes the sensitivity of PC9-GR cells to EGFR-TKIs, arrests cell cycle, inhibits tumor proliferation, invasion and migration, as well as plays an important role in reducing the expression of phosphorylation of EGFR protein, and consequencely enhances anticancer effect of EGFR-TKIs therapy. In conclusion, LP in combination with Gefitinib was an effective treatment for patients of advanced NSCLC, which may improve the life quality of patients, and potentially improve prognosis. Further investigations are still required to explore the specific mechanisms to enhance the immunity of LP.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and founding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work of this study was supported by grants from the National Natural Science Foundation of China (grant no. 81972188) and the Wu Jie-ping Foundation (320.6799.15032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYun Chen, Xinyin Liu and Jiaqi Yao contributed to designing and organizing the experiments, carrying out data analysis, and writing the manuscript. Shidai Jin, Jun Li and Jiali Xu contributed to laboratory measurements and data analysis. Renhua Guo contributed to conceiving the ideas, supervising the study, and writing the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSung, H.; Ferlay, J.; Siegel, R. L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J Clin\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2021,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e71\u003c/em\u003e (3), 209-249.\u003c/li\u003e\n \u003cli\u003eSholl, L. M.; Aisner, D. L.; Varella-Garcia, M.; Berry, L. D.; Dias-Santagata, D.; Wistuba, I. I.; Chen, H.; Fujimoto, J.; Kugler, K.; Franklin, W. A.; Iafrate, A. J.; Ladanyi, M.; Kris, M. G.; Johnson, B. E.; Bunn, P. A.; Minna, J. D.; Kwiatkowski, D. J., Multi-institutional Oncogenic Driver Mutation Analysis in Lung Adenocarcinoma: The Lung Cancer Mutation Consortium Experience. \u003cem\u003eJournal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2015,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e10\u003c/em\u003e (5), 768-777.\u003c/li\u003e\n \u003cli\u003eChen, Z.; Fillmore, C. M.; Hammerman, P. S.; Kim, C. F.; Wong, K.-K., Non-small-cell lung cancers: a heterogeneous set of diseases. \u003cem\u003eNat Rev Cancer\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2014,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e14\u003c/em\u003e (8), 535-546.\u003c/li\u003e\n \u003cli\u003eSharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A., Epidermal growth factor receptor mutations in lung cancer. \u003cem\u003eNat Rev Cancer\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2007,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e7\u003c/em\u003e (3), 169-181.\u003c/li\u003e\n \u003cli\u003eMok, T. S.; Wu, Y.-L.; Thongprasert, S.; Yang, C.-H.; Chu, D.-T.; Saijo, N.; Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y.; Nishiwaki, Y.; Ohe, Y.; Yang, J.-J.; Chewaskulyong, B.; Jiang, H.; Duffield, E. L.; Watkins, C. L.; Armour, A. A.; Fukuoka, M., Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. \u003cem\u003eThe New England journal of medicine\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2009,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e361\u003c/em\u003e (10), 947-957.\u003c/li\u003e\n \u003cli\u003eEttinger, D. S.; Wood, D. E.; Aggarwal, C.; Aisner, D. L.; Akerley, W.; Bauman, J. R.; Bharat, A.; Bruno, D. S.; Chang, J. Y.; Chirieac, L. R.; D\u0026apos;Amico, T. A.; Dilling, T. J.; Dobelbower, M.; Gettinger, S.; Govindan, R.; Gubens, M. A.; Hennon, M.; Horn, L.; Lackner, R. P.; Lanuti, M.; Leal, T. A.; Lin, J.; Loo, B. W.; Martins, R. G.; Otterson, G. A.; Patel, S. P.; Reckamp, K. L.; Riely, G. J.; Schild, S. E.; Shapiro, T. A.; Stevenson, J.; Swanson, S. J.; Tauer, K. W.; Yang, S. C.; Gregory, K.; Hughes, M., NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 1.2020. \u003cem\u003eJ Natl Compr Canc Netw\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2019,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e17\u003c/em\u003e (12), 1464-1472.\u003c/li\u003e\n \u003cli\u003eDingemans, A. M. C.; Fr\u0026uuml;h, M.; Ardizzoni, A.; Besse, B.; Faivre-Finn, C.; Hendriks, L. E.; Lantuejoul, S.; Peters, S.; Reguart, N.; Rudin, C. M.; De Ruysscher, D.; Van Schil, P. E.; Vansteenkiste, J.; Reck, M., Small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. \u003cem\u003eAnnals of oncology : official journal of the European Society for Medical Oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2021,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e32\u003c/em\u003e (7), 839-853.\u003c/li\u003e\n \u003cli\u003eWu, Y. L.; Planchard, D.; Lu, S.; Sun, H.; Yamamoto, N.; Kim, D. W.; Tan, D. S. W.; Yang, J. C. H.; Azrif, M.; Mitsudomi, T.; Park, K.; Soo, R. A.; Chang, J. W. C.; Alip, A.; Peters, S.; Douillard, J. Y., Pan-Asian adapted Clinical Practice Guidelines for the management of patients with metastatic non-small-cell lung cancer: a CSCO-ESMO initiative endorsed by JSMO, KSMO, MOS, SSO and TOS. \u003cem\u003eAnnals of oncology : official journal of the European Society for Medical Oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2019,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e30\u003c/em\u003e (2), 171-210.\u003c/li\u003e\n \u003cli\u003eHosomi, Y.; Morita, S.; Sugawara, S.; Kato, T.; Fukuhara, T.; Gemma, A.; Takahashi, K.; Fujita, Y.; Harada, T.; Minato, K.; Takamura, K.; Hagiwara, K.; Kobayashi, K.; Nukiwa, T.; Inoue, A., Gefitinib Alone Versus Gefitinib Plus Chemotherapy for Non-Small-Cell Lung Cancer With Mutated Epidermal Growth Factor Receptor: NEJ009 Study. \u003cem\u003eJ Clin Oncol\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2020,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e38\u003c/em\u003e (2), 115-123.\u003c/li\u003e\n \u003cli\u003eHsu, W. H.; Yang, J. C. H.; Mok, T. S.; Loong, H. H., Overview of current systemic management of EGFR-mutant NSCLC. \u003cem\u003eAnnals of oncology : official journal of the European Society for Medical Oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2018,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e29\u003c/em\u003e (suppl_1), i3-i9.\u003c/li\u003e\n \u003cli\u003eMayekar, M. K.; Bivona, T. G., Current Landscape of Targeted Therapy in Lung Cancer. \u003cem\u003eClin Pharmacol Ther\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2017,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e102\u003c/em\u003e (5), 757-764.\u003c/li\u003e\n \u003cli\u003eAltorki, N. K.; Markowitz, G. J.; Gao, D.; Port, J. L.; Saxena, A.; Stiles, B.; McGraw, T.; Mittal, V., The lung microenvironment: an important regulator of tumour growth and metastasis. \u003cem\u003eNat Rev Cancer\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2019,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e19\u003c/em\u003e (1).\u003c/li\u003e\n \u003cli\u003eGrivennikov, S. I.; Greten, F. R.; Karin, M., Immunity, inflammation, and cancer. \u003cem\u003eCell\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2010,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e140\u003c/em\u003e (6), 883-899.\u003c/li\u003e\n \u003cli\u003eBiswas, S. K.; Mantovani, A., Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. \u003cem\u003eNat Immunol\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2010,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e11\u003c/em\u003e (10), 889-896.\u003c/li\u003e\n \u003cli\u003eWang, J.; Zheng, M.; Min, Q.; Gao, Y.; Sun, W., The dual regulatory function of lienal peptide on immune system. \u003cem\u003eInt Immunopharmacol\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2018,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e55\u003c/em\u003e, 245-253.\u003c/li\u003e\n \u003cli\u003eChu, A.; Song, R.; Hou, G.; Yuan, J.; Wang, C.; Yang, Y.; Qin, N.; Liu, Y.; Liang, B.; Zhang, Y.; Liu, Z., Experimental Study and Clinical Observation on the Improvement Effect of Lienal Polypeptide on Blood Toxicity and Immune Injury Induced by Radiotherapy. \u003cem\u003eGenet Test Mol Biomarkers\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2020,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e24\u003c/em\u003e (2), 73-77.\u003c/li\u003e\n \u003cli\u003eZhou, J.; Niu, G.; Pei, Y.; Cao, C.; Ding, C.; Sun, G.; Guo, J.; Liu, Y.; Yu, Y., The effect and clinical efficacy of lienal polypeptide injection combined with FOLFOX chemotherapy regimen in colon cancer patients. \u003cem\u003eOncol Lett\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2016,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e12\u003c/em\u003e (5), 3191-3194.\u003c/li\u003e\n \u003cli\u003eLiu, L.; Wang, C.; Li, S.; Bai, H.; Wang, J., Tumor immune microenvironment in epidermal growth factor receptor-mutated non-small cell lung cancer before and after epidermal growth factor receptor tyrosine kinase inhibitor treatment: a narrative review. \u003cem\u003eTransl Lung Cancer Res\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2021,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e10\u003c/em\u003e (9), 3823-3839.\u003c/li\u003e\n \u003cli\u003ePaez, J. G.; J\u0026auml;nne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B. E.; Meyerson, M., EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. \u003cem\u003eScience\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2004,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e304\u003c/em\u003e (5676), 1497-1500.\u003c/li\u003e\n \u003cli\u003ePark, K.; Tan, E.-H.; O\u0026apos;Byrne, K.; Zhang, L.; Boyer, M.; Mok, T.; Hirsh, V.; Yang, J. C.-H.; Lee, K. H.; Lu, S.; Shi, Y.; Kim, S.-W.; Laskin, J.; Kim, D.-W.; Arvis, C. D.; K\u0026ouml;lbeck, K.; Laurie, S. A.; Tsai, C.-M.; Shahidi, M.; Kim, M.; Massey, D.; Zazulina, V.; Paz-Ares, L., Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial. \u003cem\u003eThe Lancet. Oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2016,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e17\u003c/em\u003e (5), 577-589.\u003c/li\u003e\n \u003cli\u003eOshima, Y.; Tanimoto, T.; Yuji, K.; Tojo, A., EGFR-TKI-Associated Interstitial Pneumonitis in Nivolumab-Treated Patients With Non-Small Cell Lung Cancer. \u003cem\u003eJAMA oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2018,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e4\u003c/em\u003e (8), 1112-1115.\u003c/li\u003e\n \u003cli\u003eZhou, C.; Wu, Y.-L.; Chen, G.; Feng, J.; Liu, X.-Q.; Wang, C.; Zhang, S.; Wang, J.; Zhou, S.; Ren, S.; Lu, S.; Zhang, L.; Hu, C.; Hu, C.; Luo, Y.; Chen, L.; Ye, M.; Huang, J.; Zhi, X.; Zhang, Y.; Xiu, Q.; Ma, J.; Zhang, L.; You, C., Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. \u003cem\u003eThe Lancet. Oncology\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2011,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e12\u003c/em\u003e (8), 735-742.\u003c/li\u003e\n \u003cli\u003eGalon, J.; Costes, A.; Sanchez-Cabo, F.; Kirilovsky, A.; Mlecnik, B.; Lagorce-Pag\u0026egrave;s, C.; Tosolini, M.; Camus, M.; Berger, A.; Wind, P.; Zinzindohou\u0026eacute;, F.; Bruneval, P.; Cugnenc, P.-H.; Trajanoski, Z.; Fridman, W.-H.; Pag\u0026egrave;s, F., Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. \u003cem\u003eScience\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2006,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e313\u003c/em\u003e (5795), 1960-1964.\u003c/li\u003e\n \u003cli\u003edeLeeuw, R. J.; Kost, S. E.; Kakal, J. A.; Nelson, B. H., The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature. \u003cem\u003eClinical cancer research : an official journal of the American Association for Cancer Research\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2012,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e18\u003c/em\u003e (11), 3022-3029.\u003c/li\u003e\n \u003cli\u003eRuffini, E.; Asioli, S.; Filosso, P. L.; Lyberis, P.; Bruna, M. C.; Macr\u0026igrave;, L.; Daniele, L.; Oliaro, A., Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. \u003cem\u003eAnn Thorac Surg\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2009,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e87\u003c/em\u003e (2).\u003c/li\u003e\n \u003cli\u003eZhang, J.; Huang, S.-H.; Li, H.; Li, Y.; Chen, X.-L.; Zhang, W.-Q.; Chen, H.-G.; Gu, L.-J., Preoperative lymphocyte count is a favorable prognostic factor of disease-free survival in non-small-cell lung cancer. \u003cem\u003eMed Oncol\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2013,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e30\u003c/em\u003e (1), 352.\u003c/li\u003e\n \u003cli\u003eWu, Y.-P.; Deng, J.; Ouyang, S.-H.; Mao, Z.-F.; Wang, G.-E.; Kurihara, H.; He, R.-R.; Li, Y.-F., Immune regulation effect of lienal polypeptides extract in Lewis lung carcinoma-bearing mice treated with cyclophosphamide. \u003cem\u003eExperimental biology and medicine (Maywood, N.J.)\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2018,\u0026nbsp;\u003c/strong\u003e\u003cem\u003e243\u003c/em\u003e (1), 66-77.\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":"Lienal polypeptide, EGFR-TKIs, drug resistance, combination therapy, NSCLC","lastPublishedDoi":"10.21203/rs.3.rs-1517195/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1517195/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: EGFR-TKIs are the first-line therapy for advanced NSCLC harboring EGFR-sensitive mutations. A robust immunity is an essential foundation for patients to tolerate continuous drug treatments. Lienal polypeptide (LP) is an immunomodulator widely applied to regulate immunity in clinical practice. Nevertheless, its potential impact on EGFR-TKIs therapy has not been illustrated. This study aimed to explore the immunomodulatory and antitumor efficacy of LP in combination with EGFR-TKIs threapy in advanced NSCLC.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePatients and methods\u003c/strong\u003e: Retrospective analysis on variation of lymphocytes in 106 NSCLC patients after\u0026nbsp;EGFR-TKIs combined with LP treatment was performed. Proliferation experiment, transwell and wound healing assays were performed in PC9-GR cells to estimate influence of LP on tumor proliferation, invasion and migrati n\u0026nbsp;\u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e. Flow cytometry was performed to detect cell apoptosis and cell cycle. The expression of p-EGFR and EGFR were detected by Western blot to investigate antitumor effect of LP.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The levels of CD3\u0026nbsp;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e, CD4\u0026nbsp;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells and the CD4\u0026nbsp;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e/CD8\u0026nbsp;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eratio were higher in NSCLC patients treated with Gefitinib in conjunction with LP. Gefitinib combined with LP inhibited tumor invasion and migration, triggered G0/G1 phase arrest to block cellular proliferation and promote cell apoptosis\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e. Furthermore, the expression of p-EGFR was decreased after Gefitinib-combining-LP treatment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: LP had a synergistic anticancer effect with EGFR-TKIs in NSCLC. LP in combination with EGFR-TKIs therapy has clinical curative effect in treatment of advanced NSCLC with EGFR driving mutations, can effectively enhance physical immunity and resensitize drug-resistant cells to EGFR-TKIs, which has a certain clinical application value.\u003c/p\u003e","manuscriptTitle":"The clinical synergistic antitumor efficacy of Lienal Polypeptide combined with EGFR-TKIs for advanced NSCLC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-06 16:43:38","doi":"10.21203/rs.3.rs-1517195/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":"407688bd-815e-43ca-a16f-bda91f7d72b3","owner":[],"postedDate":"April 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-19T14:46:24+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-06 16:43:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1517195","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1517195","identity":"rs-1517195","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}