胃癌（Gastric cancer, GC）作为消化系统最为常见的恶性肿瘤之一，其有着早期诊断率低、病程隐匿、进展迅速的特征，是全球范围内第四大恶性肿瘤相关性死因。针对胃癌的大量临床和基础研究使得胃癌的诊疗取得一定的发展，但进展期胃癌患者的5年生存率仍不足40%，对化疗药物的耐受是患者预后欠佳的主要因素之一。 恶性肿瘤的发生和进展不仅取决于肿瘤细胞本身的恶性生物学行为，同时还受肿瘤微环境（Tumour microenvironment, TME）的调节和支持。肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）是肿瘤微环境中最为丰富的间质细胞群，其在TME中的作用主要由经典活化型（Classic activation of macrophage, M1）或替代活化型（Alternative activation of macrophage, M2）的极化表型特征决定。多项研究发现M2型TAMs参与恶性肿瘤的进展，其浸润的密度与多种实体瘤的化疗耐药和不良预后密切相关。一方面TAMs的M2型极化受包括肿瘤细胞在内的TME所诱导，另一方面M2型极化的TAMs转而可影响肿瘤的恶性生物学行为，进而影响肿瘤的治疗效果。目前针对胃癌的肿瘤微环境的研究仅关注于其中的TAMs单向促肿瘤机制，而对胃癌细胞与TAMs之间交互作用对胃癌化疗耐药和恶性进展影响的研究较少，并且有关新辅助化疗胃癌患者的肿瘤组织中TAMs的浸润程度与患者预后之间的关系研究报道甚少。 母体胚胎亮氨酸拉链激酶 (Maternal embryonic leucine zipper kinase, MELK)属于Ser/Thr蛋白激酶，多项研究证实MELK是肿瘤发生发展的关键驱动基因，参与子宫平滑肌肉瘤、肺癌、骨髓瘤、肝癌和胆管癌等肿瘤细胞的增殖、迁移、侵袭和耐药，但其在胃癌进展尤其是胃癌耐药中的作用尚未明确。 因此，探究TAMs与胃癌细胞的交互作用和MELK在胃癌进展中的作用可为临床治疗胃癌提供新的靶点和思路，同时有助于发现胃癌新辅助化疗的预后因素。本课题具体研究将从以下四个部分进行阐述：

第一部分 肿瘤相关巨噬细胞与胃癌细胞的交互作用增强胃癌耐药和迁移侵袭能力 研究目的： 本部分研究主旨是应用胃癌细胞亲本/耐药株与巨噬细胞构建非接触共培养模型，检测细胞间的交互作用对巨噬细胞的极化和胃癌细胞的耐药和迁移侵袭能力的影响，并探讨其相关机制。研究方法： 1.药物持续接触浓度递增法建立对氟尿嘧啶（5-fluorouracil, 5-FU）耐药的人胃癌癌细胞株MKN45-R 和 HGC27-R，采用细胞计数试剂盒（Cell counting kit-8, CCK-8）、蛋白免疫印迹法（Western blot, WB）、划痕实验和Transwell对胃癌细胞耐药性和迁移侵袭能力进行检测。 2.胃癌细胞亲本株MKN45-S, HGC27-S和耐药株MKN45-R, HGC27-R分别与THP-1来源的M0巨噬细胞非接触式共培养，得到MS和MR巨噬细胞，采用实时荧光定量聚合酶链反应法（realtime quantitative polymerase chain reaction, RT-qPCR）和流式细胞术（Flow cytometry, FCM）比较胃癌细胞亲本株和耐药株对巨噬细胞极化的影响。 3.MS和MR巨噬细胞分别与胃癌细胞亲本株共培养，采用CCK-8、平板克隆、FCM和WB实验比较MS和MR巨噬细胞对胃癌细胞耐药性和迁移侵袭能力的影响。 4.RT-qPCR和酶联免疫吸附试验（Enzyme linked immunosorbent assay, ELISA）对介导作用的细胞因子进行筛选。 5.FCM、Transwell和WB实验对该细胞因子的介导作用进行鉴定，并对相关通路进行验证。 研究结果： 1.MKN45-R和HGC27-R细胞的耐药性和迁移侵袭能力显著高于MKN45-S和HGC27-S细胞。 2.胃癌细胞可诱导巨噬细胞M0向M2型极化，MR巨噬细胞相较于MS其向M2型极化程度更高。 3.与MS巨噬细胞相比，MR巨噬细胞与胃癌亲本细胞共培养后显著增强胃癌细胞的耐药性和迁移侵袭能力。 4.巨噬细胞来源的细胞因子CXCL5在巨噬细胞增强胃癌细胞耐药和侵袭转移能力过程中通过激活PI3K/AKT/mTOR信号通路发挥介导作用，并能招募单核细胞进而诱导形成更多M2型TAMs。 研究结论： 胃癌细胞耐药后，其迁移侵袭能力同时增强，并且能有效诱导巨噬细胞向M2型极化，M2型巨噬细胞转而继续增强胃癌细胞耐药性和迁移侵袭能力，形成正反馈回路。巨噬细胞来源的CXCL5通过激活PI3K/AKT/mTOR通路发挥促瘤作用，并招募单核细胞诱导极化形成更多的M2型TAMs。 

第二部分 MELK高表达促进胃癌耐药并诱导肿瘤相关巨噬细胞向M2型极化 研究目的： 本部分研究主旨是探讨MELK与胃癌耐药的关系，并进一步探讨其在肿瘤相关巨噬细胞向M2型极化过程中的作用及相关机制。 研究方法： 1.RT-qPCR和WB实验比较人胃粘膜上皮细胞GES-1和人胃癌细胞亲本株MKN45-S、HGC27-S及耐药株MKN45-R、HGC27-R的MELK表达水平。 2.RT-qPCR和免疫组织化学（Immunohistochemistry, IHC）对比正常胃粘膜组织、化疗敏感和化疗耐药患者胃癌组织中MELK的水平。 3.采用慢病毒感染的方法，在胃癌细胞亲本株构建MELK过表达稳转株（LV-MELK），在胃癌细胞耐药株构建MELK敲低稳转株（sh-MELK），CCK-8、平板克隆、FCM凋亡实验和动物实验检测MELK对肿瘤细胞耐药性的影响。 4.将MELK过表达稳转细胞株和敲低稳转细胞株分别与M0巨噬细胞非接触式共培养，RT-qPCR和FCM分析MELK对巨噬细胞M2型极化的影响。 5.RT-qPCR和ELISA对介导作用的细胞因子进行筛选。FCM和WB实验对细胞因子CSF-1的介导作用进行鉴定并对相关通路进行检测。 6.IHC技术对新辅助化疗患者的胃癌组织中的MELK、CSF-1和CD206的表达水平及其相关性进行检测，评价MELK、CSF-1和CD206的表达与预后的关系。 研究结果： 1.MELK与胃癌耐药相关。相比于GES-1，胃癌细胞高表达MELK，并且胃癌细胞耐药株的表达水平要显著高于胃癌细胞亲本株。MELK 在化疗耐药的胃癌组织中的表达显著高于化疗敏感的胃癌组织和正常胃粘膜组织。 2.MELK高表达可增强胃癌细胞的耐药性。过表达MELK可显著增强胃癌细胞亲本株对5-FU的耐受性及抗凋亡能力，相反，敲低MELK可显著降低胃癌细胞耐药株对5-FU的耐受性及抗凋亡能力。 3.MELK高表达可诱导巨噬细胞向M2型极化。胃癌细胞与巨噬细胞共培养后，通过RT-qPCR和FCM检测发现胃癌细胞亲本株过表达MELK后，其诱导巨噬细胞向M2型极化的能力显著增强，相反，耐药株敲低MELK后，其诱导巨噬细胞向M2型极化的能力显著减弱。 4.RT-qPCR结合ELISA筛选出胃癌细胞来源的细胞因子CSF-1在胃癌细胞诱导巨噬细胞M2型极化过程中发挥介导作用，FCM和WB实验进一步验证了CSF-1通过JAK2/STAT3通路发挥介导作用。 5.新辅助化疗患者的胃癌组织中MELK表达与CSF-1和M2型巨噬细胞标志物CD206表达呈现正相关关系，MELK、CSF-1和CD206高表达组患者的OS和DFS均显著短于低表达组患者。 研究结论： MELK的高表达可促进胃癌发生耐药，并通过细胞因子CSF-1介导激活肿瘤相关巨噬细胞的JAK2/STAT3通路诱导其向M2型巨噬细胞极化，MELK、CSF-1和CD206的高表达均预示着新辅助治疗胃癌患者的不良预后。

第三部分 肿瘤相关巨噬细胞与胃癌新辅助化疗效果和预后的相关性研究 研究目的： 本部分研究主旨是评估胃癌肿瘤组织中巨噬细胞的浸润与新辅助化疗化疗效果、临床病理指标及患者预后之间的关系，并分析M2型TAMs与CXCL5表达的相关性。 研究方法： 1.应用IHC检测新辅助化疗敏感组和耐药组胃癌组织中TAMs总标志物CD68的表达，比较两组之间的差异。 2.回顾性收集患者的临床病理指标，统计分析TAMs浸润的情况与各项临床病理指标之间的关系；IHC检测胃癌组织中CD163、CD206和CXCL5表达量，斯皮尔曼等级相关分析TAMs浸润与CXCL5表达量之间的相关性。 3.COX回归分析新辅助化疗胃癌患者的预后因素，Kaplan-Meier法进行生存分析，log-rank检验用于比较生存差异。 研究结果： 1.新辅助化疗耐药的胃癌组织TAMs浸润数目显著高于化疗敏感的胃癌组织。 2.以胃癌组织中TAMs的浸润数目中位值将胃癌患者分为TAMs高浸润组和TAMs低浸润组，统计分析TAMs高浸润组胃癌患者的新辅助化疗耐药率、淋巴结转移率较TAMs低浸润组高，且术后T分期和TNM分期较晚，而患者年龄、性别、BMI、肿瘤大小、临床T分期、肿瘤部位、分化程度、新辅助化疗方案、脉管侵犯均与TAMs浸润无显著相关性。 3.斯皮尔曼等级相关分析发现CD163、CD206与CXCL5表达量之间呈正相关性。 4.单因素COX回归分析分析发现胃癌患者预后与TAMs浸润程度及CXCL5表达量相关，Kaplan-Meier生存分析发现TAMs高浸润组、CXCL5高表达组的总生存期分别短于TAMs低浸润组、CXCL5低表达组，多因素COX回归分析结果显示TAMs高浸润及CXCL5高表达均为胃癌患者预后的独立危险因素。 研究结论： 胃癌组织中TAMs的浸润与新辅助化疗效果、病理T分期、病理TNM分期及淋巴结转移相关，M2型TAMs与CXCL5表达量呈正相关性。TAMs高浸润与CXCL5高表达均是胃癌新辅助化疗预后的独立危险因素。 

第四部分 胃癌新辅助化疗后的肿瘤病理反应与淋巴结状态在评估胃癌患者预后中的价值对比和应用 研究目的： 本部分研究主旨是比较和探讨胃癌患者新辅助化疗后的肿瘤病理反应和淋巴结状态在对患者预后评估中的价值。 研究方法： 根据新辅助化疗后的肿瘤病理反应和淋巴结转移状态将纳入的160位胃癌患者分别分为肿瘤病理反应（Pathological response, PR）组、无肿瘤病理反应（No pathological response, nPR）组和淋巴结转移组 (Nodal status+, NS+)、淋巴结无转移组 (Nodal status-, NS-)，评估肿瘤病理反应和淋巴结转移与临床病理指标之间的关系。Kaplan-Meier法进行生存分析，log-rank检验用于生存差异的比较，COX回归分析新辅助化疗胃癌患者的预后相关因素。 研究结果： 1.PR组在肿瘤体积大小和印戒细胞癌特征水平与nPR组比较存在有统计学意义的差异 (P<0.05)。NS+组在肿瘤体积大小、术后病理T分期、分化程度、印戒细胞癌特征水平、脉管癌栓和术后TNM分期与NS-组比较存在有统计学意义的差异 (P<0.05)。 2.Kaplan-Meier提示PR组胃癌患者的生存时间与nPR组相比差异无统计学意义 (P>0.05)，NS-组胃癌患者的DFS显著高于NS+组 (95% CI 0.194–0.468, P=0.002)。 3.在肿瘤病理反应的患者中，无淋巴结转移相较有淋巴结转移具有更好的OS和DFS (OS: HR 0.346, 95% CI 0.173-0.693; P=0.004; DFS: HR 0.312, 95% CI 0.148-0.659, P=0.001)。同样地，对于无肿瘤病理反应的患者，其淋巴结状态对OS和DFS有同样显著的预后意义(OS: HR 0.439, 95% CI 0.192-1.001, P= 0.019; DFS: HR 0.320, 95% CI 0.177-0.579, P<0.001)。然而，根据肿瘤病理反应进行分层分析时，患者的淋巴结是否转移，生存曲线显示肿瘤病理反应均与OS和DFS无关(均P>0.05)。 4.单因素COX回归分析结果显示胃癌患者的临床T分期、淋巴结状态、术后T分期和术后TNM分期均与预后相关，多因素COX回归分析发现淋巴结转移是胃癌患者生存的独立危险因素 (OS: HR 1.756, 95% CI 1.114–3.278, P=0.029; DFS: HR 1.901, 95% CI 1.331–3.093, P=0.012)。 研究结论： 对于新辅助化疗的胃癌患者，新辅助化疗后的淋巴结状态是患者的独立预后因素，其可能比肿瘤病理反应更有预后价值，淋巴节状态结合肿瘤病理反应能更好地评估胃癌患者新辅助化疗效果及其预后生存。 

As one of the most common malignant tumors in the digestive system, gastric cancer has the characteristics of low rate of early diagnosis, insidious symptoms and rapid progress, accounting for the fourth leading cause of cancer-related mortality worldwide. A large number of clinical and basic researches have achieved great progress in the diagnosis and treatment of gastric cancer, however, the 5-year survival rate of patients is still less than 50%, chemoresistance is one of the major contributing factors leading to poor prognosis of gastric cancer patients. The occurrence and progression of malignant tumor not only depend on the malignant biology of tumor cells, but also the regulation and support of tumour microenvironment (TME). Tumor-associated macrophages (TAMs) is the most abundant interstitial cell population, which role in TME depends on the phenotype of classic activation (M1 macrophages) or alternative activation (M2 macrophages) of macrophages. Several studies have found that M2 macrophages are related to the progression of malignant tumors, whose invasion is closely associated with the chemoresistance of several solid tumors. M2-type polarization of TAMs is induced by TME, including tumor cells. On the other hand, M2 macrophages effect the malignant biological properties of tumor in turn. Study on TME of gastric cancer only concentrated on the mechanism for the promotion of tumor rather than the crosstalk between gastric cancer cells and TAMs, especially the correlation between TAMs infiltrating in gastric cancer and prognosis in patients who recieved neoadjuvant chemotherapy. Maternal embryonic leucine zipper kinase (MELK) is a member of Ser/Thr protein kinase, which has been confirmed as the key driving gene for the occurrence and development of malignant tumor, involving in the proliferation, migration, invasion and chemoresistance of leiomyosarcoma of uterus, lung cancer, myeloma, liver cancer and cholangiocarcinoma. However, few study has reported its effect on the chemoresistance of gastric cancer. Therefore, exploring the crosstalk between gastric cancer cells and TAMs and the role of MELK in the progression of gastric cancer can put forth the novel targets and methods for the treatment of gastric cancer as well as the prognostic factors for neoadjuvant chemotherpay. Our study will be elaborated from the following four parts:

Part I Crosstalk between tumor-associated macrophages and tumor cells promotes chemoresistance and enhences migration and invasion ability of gastric cancer cells Objective: The purpose of this part is to establish chemoresistant gastric cancer cell line, constructing indirect co-culture model using parental or chemoresistant gastric cancer cell and TAMs to detect the effect of interaction between cells on the polarization of macrophages and the chemoresistance, the ability of migration and invasion of gastric cancer cells, and explore the mechanism involved. Methods: 1.5-FU-resistent cell lines MKN45-R and HGC27-R were generated by repetitively exposing gastric cancer cells to increasing concentrations of 5-FU. Cell counting kit-8, western blot, wound-healing and transwell assay were used to evaluate the ability of chemoresistance, migration and invasion. 2.TAMs from 5-FU-sensitive TME (MS) and 5-FU-resistant TME (MR) were obtained and harvested for experimental analysis. Realtime quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were used to compare the effect of parental and chemoresistant gastric cancer cell on the polarization of macrophages. 3.MS and MR cells co-cultured with parental gastric cancer cells, then Cell counting kit-8 (CCK-8), plate clone formation assay, FCM and Western blot (WB) were used to compare the effect of MS and MR cells on chemoresistance, migration and invasion of gastric cancer cells. 4.RT-qPCR and enzyme linked immunosorbent assay (ELISA) were adopted to screen out the cytokines exerting mediate effect. 5.FCM, transwell and WB were used to verify the mediate effect and the signal pathway involved. Results: 1.MKN45-R and HGC27-R cells were significantly stronger in the ability of chemoresistance, migration and invasion than MKN45-S and HGC27-S cells. 2.Co-culturing gastric cancer modulated macrophages to M2-like polarization, and chemoresistant cells were more effective in inducing M2 polarization. 3.Co-culturing MR cells significantly promoted chemoresistance, migration and invasion of MKN45-S and HGC27-S cells. 4.Macrophage-derived CXCL5 enhenced the ability of chemoresistance, migration and invasion via activating PI3K/AKT/mTOR signal pathway in gastric cancer cell, and recruited monocytes to be induce to be M2-like polarization TAMs. Conclusion: Interaction between TAMs and gastric cancer cells could induce macrophages polarization to M2 phenotype, which in turn further promoted the chemoresistance, migration and invasion of gastric cancer cells. TAMs-derived CXCL5 played a critical role in the cell-cell interaction and could recruit monocytes to form more M2-polarized macrophages in gastric cancer. 

Part II Upregulated MELK promotes chemoresistance and induces TAMs polarization to M2 phenotype in gastric cancer Objective: The purpose of this part is to investigate the effect of MELK on chemoresistance of gastric cancer, and further explore the its effect on M2 polarization of TAMs and the mechanism involved. Methods: 1.RT-qPCR and WB were used to detect and compare the expression of MELK in gastric epithelial cells GES-1, parental gastric cancer cells MKN45-S and HGC27-S, chemoresistant gastric cancer cells MKN45-R and HGC27-R. 2.Gastric cancer tissues from neoadjuvant chemotherapy patients were collected and divided into chemotherapy sensitive group and chemotherapy resistant group according to chemotherapeutic efficacy. RT-qPCR assay compared the expression of MELK in normal gastric tissue, gastric cancer tissue from chemotherapy sensitive group and chemotherapy resistant group in mRNA level. IHC was used to further compare the expression of MELK in chemotherapy sensitive group and chemotherapy resistant group in protein level. 3.Lentivirus infection was used to construct MELK overexpressing stable cell line (LV-MELK) in parental gastric cancer cells, and MELK suppressing stable cell line (sh-MELK) was constructed in chemoresistant gastric cancer cells. CCK-8, plate clone formation assay and FCM were used to investigate the effect of MELK on chemoresistance of gastric cancer. 4.MELK overexpressing and suppressing stable cell lines co-culture with macrophages indirectly, and then the effect of MELK on M2 polarization of macrophages was explored. 5.RT-qPCR and ELISA assay screened out the cytokines exerting mediate effect. FCW and WB assay were used to verify the mediate effect of CSF-1 and explore the signal pathway involved. 6.The expression of MELK, CSF-1 and CD206 in gastric cancer tissue from neoadjuvant chemotherapy was detected by IHC, and the correlation with survival was further evaluated. Results: 1.MELK was associated with the resistance of gastric cancer to chemotherapy. Compared with CES-1, the expression of MELK was higher in gastric cancer cells, especially in chemoresistant gastric cancer cells. The results of IHC showes that the expression of MELK in chemotherapy resistant group was much higher than in chemotherapy sensitive group and normal gastric tissue. 2.Upregulated MELK could promote the chemoresistance of gastric cancer cells. Overexpressing MELK could significantly enhence the ability of parental gastric cancer cells in resistance to 5-FU and anti-apoptosis. On the contrary, suppressing MELK could reduce the ability of chemoresistant gastric cancer cells. 3.Upregulated MELK could induce macrophages polarization to M2 phenotype. RT-qPCR and FCM assay found that MELK overexpressing stable cell line was more efficient in inducing M2 macrophages polarization through co-culture. In contrast, suppressing MELK in chemoresistant gastric cancer cells could reduce the ability. 4.RT-qPCR and ELISA screened out that CSF-1 deriving from gastric cacer cells exerts mediate effect in gastric cancer cells inducing M2 macrophage polarization. FCM and WB assay further verified that CSF-1 exerts mediate effect via JAK2/STAT3 signal pathway. 5.The expression of MELK in gastric cancer tissue from neoadjuvant chemotherapy correlated positively with CSF-1 and CD206. The OS and DFS were significantly shorter in patients with high expression of MELK, CSF-1 and CD206. Conclusion: Upregulated MELK could promote the chemoresistance of gastric cancer cells and induce M2 macrophages polarization through CSF-1 deriving from gastric cancer cells via activating JAK2/STAT3 signal pathway. The high expression of MELK, CSF-1 and CD206 was associated poor prognosis of gastric cancer patients recieving neoadjuvant chemotherapy.

Part III Infiltration of TAMs associated with chemotherapeutic efficacy and prognosis of gastric cancer after neoadjuvant chemotherapy Objective: The purpose of this part is to evaluate the correlation among the infiltration of TAMs with clinicopathologic characteristics and prognosis in gastric cancer after neoadjuvant chemotherapy and analyse correlation with the expression of CXCL5. Methods: 1.Gastric cancer tissues from neoadjuvant chemotherapy patients were collected and divided into chemotherapy sensitive group and chemotherapy resistant group according to chemotherapeutic efficacy. IHC was used to detect the expression of CD68 for TAMs infiltration analysis and the differences between the two groups were compared. 2.The clinicopathologic characteristics of patients were retrospectively collected. The correlation between the infiltration of TAMs and clinicopathologic characteristics was analyzed. IHC was used to detect the expression of CXCL5, the correlation between the infiltration of TAMs and the expression of CXCL5 was also analyzed. 3.Cox regression analysis was used to explore the prognostic factors of gastric cancer. Kaplan-Meier method was used to analyze the prognosis and the log-rank test was used to compare the survival difference. Results: 1.The number of TAMs infiltrated in chemotherapy resistant gastric cancer tissue was significantly higher than in chemotherapy sensitive gastric cancer tissue. 2.Patients were dedided into high infiltration group and low infiltration group by the median of TAMs. The chemoresistant rate and lymph node metastasis rate in high infiltration group were higher than in low infiltration group, Depth of tumor infiltration and TNM staging were later. Age, gender, tumor size, tumor location, differentiated degree, lymphatic and venous invasion had no correlation with the infiltration of TAMs. 3.The analysis of spearman rank correlation showed that the infiltration of TAMs had positive correaltion with the expression of CXCL5. 4.Univariate Cox regression analysis showed that the infiltration of TAMs and the expression of CXCL5 were associated with the prognosis of gastric cancer. Kaplan-Meier method found that the OS of patients with high infiltration of TAMs and high expression of CXCL5 was respectively shorter than patients with low infiltration of TAMs and low expression of CXCL5. Multivariate Cox regression analysis showed that high infiltration of TAMs and high expression of CXCL5 were both independent risk factors for the prognosis of gastric cancer. Conclusion: The infiltration of TAMs was associated with the chemotherapeutic efficacy, depth of tumor infiltration and lymph node metastasis rate, and had positive correaltion with the expression of CXCL5. The high infiltration of TAMs and high expression of CXCL5 were both independent risk factors for the prognosis of gastric cancer after neoadjuvant chemotherapy. 

Part IV Comparison of the predictive value of pathological response at primary tumor and lymph node status after neoadjuvant chemotherapy in locally advanced gastric cancer Objective: This study aimed to investigate the prognostic value of pathological response at primary tumor and the lymph node status after receiving neoadjuvant chemotherapy. Methods: Data from 160 patients with loacally advanced gastric cancer treated with neoadjuvant chemotherapy followed by gastrectomy were retrospectively reviewed. Pathological evaluation after chemotherapy was based on the grade of pathological response of the primary tumor and the status of lymph node. Survival curves for overall survival (OS) and disease-free survival (DFS) were estimated via Kaplan-Meier method, and the log-rank test was used to compare survival difference. Univariate and multivariate analyses for prognostic factors were based on the Cox regression. Results: 1.Among 160 selected cases, 90 had pathological response (PR), while 70 had no pathological response (nPR) to chemotherapy. Smaller tumor size was presented in PR group, which also had lower level of signet ring cell features, compared to nPR group (all P < 0.05). Based on the status of lymph nodes, nodal status (-) group showed smaller tumor size, lower depth of tumor invasion, better differentiated degree, lower level of signet ring cell features, lower rate of lymphatic and venous invasion and less advanced postoperative pathological stage (all P < 0.05). 2.Survival was equivalent between PR and nPR group (all P > 0.05), while patients in NS- group had better DFS than in NS+group (HR 0.301, 95% CI 0.194-0.468, P = 0.002). 3.The stratified analysis by the status of lymph node was further performed. For patients with pathological response, OS and DFS were better in patients with no lymph node metastasis than that with lymph node metastasis (OS: HR 0.346, 95% CI 0.173-0.693, P = 0.004; DFS: HR 0.312, 95% CI 0.148-0.659, P = 0.001). Similarly, for patients with no pathological response, the lymph node status showed remarkable prognostic significance in OS and DFS (OS: HR 0.439, 95% CI 0.192-1.001, P = 0.019; DFS: HR 0.320, 95% CI 0.177-0.579, P < 0.001). However, when stratified analysis was performed by pathological response, whether patients had lymph node metastasis or not, survival curves showed that pathological response was not related to OS and DFS (all P > 0.05). 4.Univariable Cox regression analysis of prognostic factors identified several potential predictors of OS and DFS. Clinical T stage, pathological lymph node status and ypTNM stage were associated with OS and DFS. Stepwise selection of variables and multivariable Cox regression analysis identified that pathological lymph node status (HR 1.756, 95% CI 1.114-3.278, P = 0.029) as being independent prognostic factors associated with OS, similar results were obtained regarding DFS (HR 1.901, 95% CI 1.331-3.093, P = 0.012). Conclusion: Lymph node status after neoadjuvant chemotherapy can serve as a potential independent prognostic factor for gastricc cancer patients treated with neoadjuvant chemotherapy followed by surgery, and may be more efficient than pathological response in primary tumor to some extent. The combined application of nodal status and pathological response could contribute to the clinical evaluation of neoadjuvant chemotherapy, as well as the prognosis of gastric cancer.