Zhang et al. Cancer Cell Int (2020) 20:583 https://doi.org/10.1186/s12935-020-01671-4 Cancer Cell International PRIMARY RESEARCH Open Access CD44, a marker of cancer stem cells, is positively correlated with PD-L1 expression and immune cells infltration in lung adenocarcinoma Chenyue Zhang1†, Hui Wang2†, Xia Wang3, Chenglong Zhao4 and Haiyong Wang3* Abstract Background: PD-L1 inhibitors is widely applied in lung adenocarcinoma patients. Tumor cells with high PD-L1 expression could trigger immune evasion. Cancer stem cells (CSCs) can evade from immunesurveillance due to their immunomodulating efects. However, the correlation between CSC and PD-L1 and some immune-related markers is seldom reported in patients with lung adenocarcinoma. Therefore, we aimed to ascertain their association in lung adenocarcinoma patients. Methods: We assessed CD44 expression and its association with PD-L1 in lung adenocarcinoma, using Tumor Immune Estimation Resource (TIMER), which was further validated in our patient cohort. The immune cells infltration was depicted by CIBERSORT using GEO database. The correlation between CD44 and immune cells was also analyzed. We further evaluated the prognostic role of CD44 in patients with lung adenocarcinoma both using Kaplan–Meier plotter and validated in our patient cohort. Results: Positive association between CD44 and PD-L1 were found in lung adenocarcinoma patients. T cells CD4 memory resting cells and mast cells resting cells varied signifcantly between patients with CD44 high and those with CD44 low. Furthermore, positive association could be found between CD44 expression and immune cells. Arm-level depletion of CD44 was linked with B cell, CD4+ T cell, neutrophil and dendritic cell infltration. Patients with higher CD44 levels had worsened overall survival (OS). Conclusions: In summary, these results demonstrate that CD44 was associated with PD-L1 and infltration of immune cells, and was a negative prognostic factor for predicting worsened OS in lung adenocarcinoma. Keywords: Cancer stem cell, CD44, Programmed cell death ligand-1, Lung adenocarcinoma Background Lung cancer is the most lethal cancer worldwide with a 5-year survival rate of 15% [1]. Although adenocarci- noma (ADC) accounts for a large proportion of lung can- *Correspondence: [email protected] cer, most ADC patients developed metastasis upon initial †Chenyue Zhang and Hui Wang are frst authors and contributed equally to this work diagnosis, resulting in poor prognosis. Immunotherapy, 3 Department of Internal Medicine Oncology, Shandong Cancer Hospital aiming to enhance the host antitumor immune defense, and Institute, Shandong First Medical University and Shandong Academy has rendered prolonged survival in ADC [2]. of Medical Sciences, Number 440, Ji Yan Road, Jinan 250117, China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Cancer Cell Int (2020) 20:583 Page 2 of 8 Programmed cell death ligand-1 (PD-L1) is expressed Shandong Provincial Hospital afliated to Shandong in many solid tumors including lung cancer. Interac- University were included in this study. All these patients tion between programmed cell death-1 (PD-1) and its underwent lung cancer radical resection, and forma- ligands could attenuate immune response via evasion of lin-fxed parafn-embedded (FFPE) specimens were immune elimination. It has been reported that PD-L1 collected from patients. Detailed information on age, is overexpressed in lung adenocarcinoma patients and gender, smoking habits, tumor stage and lymph node is associated with dismal prognosis. Terefore, immune stage were all documented. Scoring of CD44 expression checkpoint inhibitors (ICIs) have been widely applied in in tumor from each lung adenocarcinoma patient was lung adenocarcinoma for its function in the blockade of confrmed by pathologists. Tumor staging was deter- PD-L1 [3–6]. mined according to the International Association for the Cancer stem cells (CSCs) represent an exclusive cohort Study of Lung Cancer (IASLC). Tis study was approved of long-lived cells with their capacity to generate cellu- by Shandong Cancer Hospital. All included patients in lar progeny throughout life and fuel tumor growth [7, 8]. this study ofered written informed consent. Overall sur- Teir existence may account for the inefective response vival (OS) was defned as the interval between diagnosis of many therapies including ICIs, leading to poor prog- and death or between diagnosis and the last observation nosis and recurrence among patients with diferent can- point. For surviving patients, the data were censored at cer types. CSCs reside in a niche where immune cells, the last follow-up. microvesicles and cytokines could stimulate their self- renewal and promote metastasis [9]. Tese microvesicles Immunohistochemistry staining and cytokines might also exert immunosuppressive func- Immunohistochemistry protocols were performed as tions. Studies have found that CSCs can weaken T cell described previously [14]. Briefy, tumor sections were diferentiation, proliferation and anti-tumor efects. stained with rabbit anti-CD44 (abcam, ab216647), rabbit However, studies on CSC’s association with PD-L1 and anti-PD-L1 (ARIGO, ARG57681). Te Goat anti-rabbit the surrounding microenvironment in lung adenocarci- antibody was used as the secondary antibody. noma are rare documented. CD44 has been identifed as a crucial surface marker indicating CSC in lung cancer. Lung cancer cells expressing CD44 have been reported to Scoring of the staining be enriched for stem-like properties [10]. Immunostaining of CD44 and PD-L1 was analyzed under Terefore, in the present study, we explored the associ- light microscopy at 400× magnifcation by two independ- ation between CD44 and PD-L1 in lung adenocarcinoma. ent pathologists who were blinded to the clinical data of Moreover, we assessed some of the vital immune cells each patient. For evaluation of CD44, the staining was surrounding CSC in lung adenocarcinoma using Tumor graded as either high expression or low according to the Immune Estimation Resource (TIMER) and further vali- median value. X-tile software was used to determine the dated in our patient cohort. optimal cut-of value according to the user’s manual and previous studies. Methods Bioinformatics analysis of CD44 expression and its Statistical analysis association with PD‑L1 and immune cells Te chi-squared test was performed to assess the asso- For evaluation of CD44 expression among all cancer ciation between CD44, PD-L1 and clinical pathologic types, data collected from Tumor Immune Estimation features of patients with lung adenocarcinoma. Pear- Resource (TIMER) were collected [11]. For determina- son’s correlation test was used to analyze the correla- tion of CD44′s correlation with PD-L1 mRNA in lung tion between CSC marker CD44 and tumor infltrating adenocarcinoma, TIMER was also employed [11]. CD44 immune cells (B cell, CD8+ T cell, CD4+ T cell, mac- expression was measured in immunological subgroups rophage, neutrophil and dendritic cells). OS was defned of lung adenocarcinoma and drugs targeting CD44 were from the date of surgery to the date of death or last depicted as referenced by an integrated repository portal follow-up. To compare OS between patients in difer- for tumor-immune system interactions [12]. OS between ent groups, we have used Kaplan Meier analysis. And to lung adenocarcinoma patients with CD44 high and CD44 estimate the diference in survival, we have adopted log- low was calculated using Kaplan–Meier plotter [13]. rank test. Univariate and multivariate analysis was also conducted using the Cox proportional hazards regression Patients and samples model. All the analyses were performed using SPSS 17.0 A total of 76 patients diagnosed with lung adeno- software. A p value less than 0.05 was considered statisti- carcinoma from December, 2012 to October 2013 in cally signifcant. Zhang et al. Cancer Cell Int (2020) 20:583 Page 3 of 8 Results analyzed CD44 expression in lung adenocarcinoma Patients’ characteristics patients with stage I and stage II/III. Results have shown A total of 76 patients with lung adenocarcinoma were that no signifcant diference was found between patients included in the study. Among them, seventeen patients with stage I and stage II/III (Additional fle 1: Figure were ≥ 65 years at diagnosis. Most of the patients were S1). We further tested the correlation between CD44 non-smokers, accounting for 67.1%. Te majority of and PD-L1 (CD274) mRNA expression using TIMER patients were female (59.2%). again. Figure 1b have shown that CD44 level was in posi- Tere were 27 patients with at T1 stage, 41 patients tive proportion with PD-L1 mRNA expression (r = 0.46, with T2, 4 patients with T3 stage and 4 patients with T4 P = 2.86e−28). To validate the correlation between CD44 stage. A total of 48 patients were without lymph node and PD-L1 in lung adenocarcinoma patients with dif- metastasis (N0), 7 patients were at N1 stage and 21 ferent stages, we presented representative images using patients were at N2 stage.