The expression of PD-1 ligands in the immune microenvironment was altered in TTF-1-negative lung adenocarcinoma

The expression of PD-1 ligands in the immune microenvironment was altered in TTF-1-negative lung adenocarcinoma | 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 expression of PD-1 ligands in the immune microenvironment was altered in TTF-1-negative lung adenocarcinoma Hiroyuki Yamada, Hiromu Yano, Eri Matsubara, Shukang Zhao, Yusuke Shinchi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6311106/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Aug, 2025 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Immunotherapies targeting the programmed cell death-1 (PD-1) pathway have been adopted in lung adenocarcinoma (LUAD) treatment. Expression of programmed cell death-ligand 1 (PD-L1) and PD-L2 is observed in both cancer cells and tumor-associated macrophages (TAMs). PD-L1/L2 expression in TAMs is thought to be induced by inflammatory cytokines, such as interferon (IFN)-g. Thyroid transcription factor-1 (TTF-1) is a common diagnostic marker for LUAD, with TTF-1 negativity associated with poorer survival outcomes and reduced response to chemotherapy and immunotherapy. The present study investigated the correlation between TTF-1 expression and the immune microenvironment. Methods: TTF-1 status was evaluated using immunohistochemistry on paraffin-embedded samples from 226 patients with LUAD, examining the correlation of TTF-1 status with immune-related markers and immune cell infiltration. For further investigation, in silico RNA-seq analysis was performed. Results: TTF-1-negative cases showed worse progression-free survival and cancer-specific survival compared to TTF-1-positive cases. Additionally, TTF-1-negative cases exhibited lower PD-L1/L2 expression in TAMs, although no association was found between TTF-1 status and PD-L1/L2 expression in cancer cells. Consistent findings were observed through in silico analysis. The CD8 + T-cell density was generally lower in TTF-1-negative cases, a significant difference when cases with mutant epidermal growth factor receptor (EGFR) were excluded. Conclusions: These findings suggest that TTF-1-negative LUAD represents an immunologically “COLD” tumor, characterized by reduced infiltration of CD8 + T cells and a low IFN-g signature. lung adenocarcinoma TTF-1 NKX2-1 macrophage PD-L1 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide, and associated rates of mortality and morbidity are increasing [ 1 , 2 ]. Despite the widespread adoption of computed tomography scans, which has contributed to early detection and mortality reduction [ 3 , 4 ], most lung cancers still are found at advanced stages. In recent years, immunotherapy has rapidly spread to become the standard therapeutic option for lung cancer. Immune checkpoint inhibitors, particularly those targeting programmed cell death-1 (PD-1) and programmed cell death-1 ligand 1 (PD-L1), have shown significant efficacy against certain types of lung cancer [ 5 , 6 ]. The interaction between PD-L1 (on tumor cells) and PD-1 (on CD8 + T cells) causes T cell exhaustion, fostering immune tolerance [ 7 ]. PD-L1 expression in LUAD cells serves as a biomarker for predicting the effectiveness of anti-PD-L1/PD-1 therapy [ 8 – 10 ]. On the other hand, macrophages are one of the most common immune cells in the tumor immune microenvironment (TIME), and those that infiltrate tumor tissues are called tumor-associated macrophages (TAMs). TAMs are recognized for their tumor-promoting activity [ 11 , 12 ]. Not only PD-L1 but also PD-L2 (another ligand of PD-1) are known to be expressed in TAMs, and both ligands have been shown to be involved in immunosuppression [ 13 – 15 ]. Moreover, our previous research on lung adenocarcinoma (LUAD) cases has highlighted the correlation between PD-L1/L2 expression in TAMs and clinical prognosis [ 16 , 17 ]. Thyroid transcription factor-1 (TTF-1, also known as NK2 homeobox 1 [NKX2-1]) is specifically expressed in normal distal lung cells, such as Clara cells and type II alveolar cells [ 18 ]. TTF-1 plays essential roles in the development and differentiation of the peripheral lung and in maintaining normal respiratory function through the regulation of the expression of pulmonary surfactants [ 19 , 20 ]. TTF-1 also is expressed in most LUADs derived from the distal lung, serving as a diagnostic marker for primary LUAD [ 21 , 22 ]. Studies have shown that patients with LUAD who are negative for TTF-1, comprising about 30% of all cases, tend to have poorer survival outcomes [ 23 – 25 ] and exhibit lower responsiveness to pemetrexed [ 26 ] or immune checkpoint inhibitors [ 27 ], compared to those who are positive for TTF-1. Although many studies have discussed both the tumor-suppressive and oncogenic roles of TTF-1 [ 28 ], with the former including the reduction of invasion and metastasis [ 29 – 31 ], there have been few reports on the relationship between TTF-1 and TIME. In particular, the involvement of TTF-1 with CD8 + T cells and TAMs remains unclear. In the present study, we observed that PD-L1 and PD-L2 expression in TAMs was decreased in cases of LUAD that were negative for TTF-1. 2. Materials and Methods 2.1. Samples Paraffin-embedded tissue samples were obtained from 226 patients diagnosed with LUAD between 2010 and 2013 at Kumamoto University Hospital. All tissue specimens were reviewed by two pathologists. We also employed tissue microarrays previously prepared by our group [ 32 ]. 2.2. Immunohistochemistry (IHC) analysis Paraffin-embedded sections were subjected to IHC using anti-TTF-1 antibody (clone 8G7G3/1; Agilent Technologies, Santa Clara, CA, USA) and anti-CD8 antibody (clone C8/144B; Nichirei, Tokyo, Japan). 3,3’-Diaminobenzidine (DAB; Nichirei) was used for the visualization of immunoreactions. Two investigators (Y.K., H.Y.), both of whom were blinded to the patients’ characteristics and outcomes, evaluated the immunostained sections. Data on PD-L1 and PD-L2 expression in cancer cells and TAMs from the same cohort were published in previous studies [ 16 , 17 ]. For representative cases with high and low expression of PD-L1 and PD-L2 in TAMs, multichannel pseudocolored images were created using HALO (version 4.0.5107.318; Indica Labs, Albuquerque, NM, USA). The slides were digitized with a NanoZoomer S20 (Hamamatsu Photonics, Hamamatsu, Japan) whole-slide scanner, and pseudocolored images showing staining intensities for both PD-L1 and PU.1 were generated using the color deconvolution function. 2.3. Single-cell RNA-seq (scRNA-seq) analysis Two scRNA-seq datasets of LUAD (GSE162498 and GSE131907), consisting of data from 20 patients, were downloaded from the Gene Expression Omnibus [ 33 , 34 ]. Cells captured in scRNA-seq analysis were clustered and analyzed using the Seurat package (version 5.1.0) in R (version 4.4.0; R Foundation for Statistical Computing, Vienna, Austria) [ 35 , 36 ]. Cells with unique feature counts of 200 or under, or of 5,000 or over, or mitochondrial counts of 15% or over, were filtered out as part of the quality control process. After merging samples, a standard workflow using the LogNormalize method was implemented to normalize gene expression for each cell. We calculated and identified the top-2000 most-variably expressed genes among the cells, and principal component (PC) analysis was performed for linear dimensional reduction. To determine how many PCs to use for the cluster analysis, the ElbowPlot method was implemented; this approach suggested that using the first 15 PCs was reasonable for representing the true dimensionality. Based on the K-nearest-neighbors graph generated by the FindNeighbors function, we applied the Louvain algorithm using the FindClusters function with a resolution of 1.0, yielding 28 cell clusters. The differentially expressed genes (DEGs) in each cluster were identified using the FindAllMarkers function. The epithelial cell cluster was classified into tumor and non-tumor cells, allowed by inference of the copy number variations (CNV) as previously described [ 34 ]. The AddModuleScore function was utilized to quantify the activity of a specific gene set in each cell. For the scoring of TTF-1 target genes, the following genes, which are known to be upregulated by TTF-1 [ 28 ], were defined as the gene set: SFTPA1 (Surfactant Protein A1), SFTPA2 (Surfactant Protein A2), SFTPB (Surfactant Protein B), SFTPC (Surfactant Protein C), SCGB1A1 (Secretoglobin Family 1A Member 1), SCGB3A2 (Secretoglobin Family 3A Member 2), ABCA3 (ATP Binding Cassette Subfamily A Member 3), and PDPN (Podoplanin). 2.4. Statistical analysis Statistical analysis was conducted using Prism (version 9.5.1; GraphPad Software, San Diego, CA, USA) and R (version 4.4.0). Fisher’s exact test was used to compare proportions between two groups. The Mann-Whitney U test was used to compare interval-scale data between two groups that were not assumed to follow a normal distribution. The association between prognostic survival and TTF-1 status was explored by comparing survival across groups using Kaplan-Meier analysis with logrank and Wilcoxon tests. A p value of < 0.05 was considered statistically significant in all analyses. 3. Results 3.1. TTF-1-negative cases show a worse clinical course in LUAD. Following IHC analysis (Fig. 1 a), 196 cases (86.7%) were positive for TTF-1, while 30 cases (13.3%) were negative for this marker. The distribution of TTF-1 expression was summarized according to patient characteristics (Table 1 ). TTF-1-negative status was significantly more prevalent among cases with epidermal growth factor receptor (EGFR)-wild-type status, male patients, smokers, and those with advanced disease stages. Notably, within the subgroup of patients with EGFR-mutant status, an overwhelming majority (98.1%) exhibited TTF-1-positive status. Further analysis using log-rank tests revealed that progression-free survival (PFS) and cancer-specific survival (CSS) were significantly lower in TTF-1-negative cases than in TTF-1-positive cases (Fig. 1 b). Table 1 TTF-1 expression and clinicopathological factors. TTF-1 expression Negative Positive p EGFR gene status Wild type 26 (92.9) 82 (43.9) < 0.001 Mutant 2 (7.1) 105 (56.1) Age < 65 5 (16.7) 61 (31.1) 0.13 ≥ 65 25 (83.3) 135 (68.9) Gender Male 22 (73.3) 91 (46.4) 0.010 Female 8 (26.7) 105 (53.6) Smoking BI < 600 9 (30.0) 131 (66.8) < 0.001 BI ≥ 600 21 (70.0) 65 (33.2) pStage 0-I 16 (53.3) 158 (80.6) 0.002 II-VI 14 (46.7) 38 (19.4) Grade 1,2 23 (76.7) 190 (96.9) < 0.001 3 7 (23.3) 6 (3.1) PD-L1 expression on cancer cells Low: < 50% 27 (90.0) 185 (94.4) 0.41 High: ≧ 50% 3 (10.0) 11 (5.6) PD-L1 expression on TAMs Low: < 50% 22 (73.3) 88 (44.9) 0.005 High: ≧ 50% 8 (26.7) 108 (55.1) PD-L2 expression on cancer cells Low: < 50% 24 (80.0) 129 (65.8) 0.15 High: ≧ 50% 6 (20.0) 67 (34.2) PD-L2 expression on TAMs Low: < 50% 22 (73.3) 75 (38.3) < 0.001 High: ≧ 50% 8 (26.7) 121 (61.7) Note: Fisher’s exact test was performed. Statistically significant ( p value < 0.05) are indicated with an underline. Abbreviations: TTF-1, thyroid transcription factor-1; EGFR, epidermal growth factor; BI, Brinkman index; PD-L1, programmed cell death-1 ligand 1; PD-L2, programmed cell death-1 ligand 2; TAMs, tumor-associated macrophages. 3.2. TTF-1-negative cases show lower expression of PD-L1 and PD-L2 in TAMs. Subsequently, we examined the correlations between TTF-1 status and PD-L1/L2 expression using previously published data on PD-L1/L2 in the same tissue array sections as described in the Materials and Methods (Section 2.2 ). Although no significant correlation was found between TTF-1 status and PD-L1/L2 expression in cancer cells (Table 1 ), significant correlations were observed between TTF-1 status and PD-L1/L2 positivity rates in TAMs (referred as the macrophage proportion score, MPS) (Table 1 and Fig. 2 a, 2 b). PD-L1/L2 expression in TAMs was lower in TTF-1-negative cases (compared to expression in TTF-1-positive cases), and this significant correlation also was preserved in the EGFR-wild-type group. Representative double-IHC images of sections co-stained for PD-L1 and PU.1, as well as for PD-L2 and PU.1, are shown in Fig. 2 b. Here, PU.1, a transcription factor, was employed as a marker to identify TAMs in co-staining with PD-L1 and PD-L2. The expression of PD-L1/L2 in TAMs was more clearly visualized in the pseudocolored images based on immunostaining for PD-L1/L2 and PU.1. 3.3. scRNA-seq indicates down-regulation of PD-L1 in TAMs from TTF-1-negative cases. To confirm the significant correlation between TTF-1 status and PD-L1/L2 expression in TAMs, public scRNA-seq data from 20 LUAD patients were re-analyzed. The epithelial cell cluster was further classified into tumor cells and normal cells using CNV inference (Fig. 3 a). The mean expression levels of NKX2-1 (the gene encoding TTF-1) were calculated for tumor cells in each patient, and patients were stratified into low and high groups based on the median value. Next, using the same approach, patients were also stratified based on the expression levels of TTF-1 target genes. Here, the TTF-1 target genes refer to the gene set consisting of SFTPA1 , SFTPA2 , SFTPB , SFTPC , SCGB1A1 , SCGB3A2 , ABCA3 , and PDPN , and their overall expression levels were quantified as a score using the method described in the Materials and Methods (Section 2.3 ). The mean expression levels of CD274 (cluster of differentiation 274, the gene encoding PD-L1) in macrophages were significantly lower in the low-TTF-1 target gene groups than in the high-TTF-1 target gene groups, whereas no significant difference was observed between the low- NKX2-1 and high- NKX2-1 groups (Fig. 3 b). On the other hand, the mean expression levels of PDCD1LG2 (Programmed Cell Death 1 Ligand 2, the gene encoding PD-L2) in macrophages showed no significant difference in either classification based on NKX2-1 or TTF-1 target genes (Fig. 3 c). 3.4. CD8 + T-cell infiltration is suppressed in TTF-1-negative cases within the EGFR-wild-type group. It is well known that PD-L1/L2 expression in macrophages is enhanced by interferon-gamma (IFN-γ). Therefore, overexpression of PD-L1/L2 in macrophages is thought to be induced by increased anti-cancer immune responses within the tumor microenvironment [ 37 ]. To investigate differences in anti-cancer immune responses between TTF-1-positive and TTF-1-negative cases, we evaluated CD8 expression by IHC. The results showed that CD8 + T-cell density tended to be lower in TTF-1-negative cases, although this difference was not statistically significant in the entire cohort. However, in EGFR wild-type cases, the density was significantly lower in TTF-1-negative cases compared to TTF-1-positive cases (Fig. 4 a). Representative CD8-stained IHC images are shown in Fig. 4 b. 4. Discussion In the present study, TTF-1-negative LUAD was shown to exhibit a significantly worse clinical course than TTF-1-positive LUAD, consistent with the results of several previous reports [ 23 – 25 ]. TTF-1 is recognized as a lineage-survival oncoprotein in TTF-1-positive LUAD [ 28 ], such that survival signals are mediated through the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) axis via the receptor tyrosine kinase-like orphan receptor 1 (ROR1) [ 38 ]. Additionally, a functional relationship with EGFR is strongly suggested, given that TTF-1 expression appears to be essential for the development of EGFR-mutant LUAD [ 38 ]. On the other hand, TTF-1 is known to exhibit tumor-suppressive roles. This factor transcriptionally activates genes such as myosin binding protein H ( MYBPH ), occludin ( OCLN ), claudin 1 ( CLDN1 ), and CLDN18 [ 29 – 31 ], which are involved in cytoskeleton regulation and cell-cell adhesion, thereby acting to suppress cancer cell invasion and metastasis. Furthermore, TTF-1 has been reported to inhibit the transforming growth factor (TGF)-β-mediated epithelial-mesenchymal transition [ 39 ] and to suppress the development of Kirsten rat sarcoma (KRAS)-mutant mucinous adenocarcinoma [ 40 ]. Thus, TTF-1 has dual-functional significance in cancer cell growth; however, only a few studies have explored this factor’s correlation with the TIME. Specifically, previous work has suggested that TTF-1-negative LUAD is associated with a more-immunosuppressive environment [ 41 , 42 ]. In the present study, we observed that TTF-1-negative LUAD was associated with reduced CD8 + T cell infiltration in the TIME, indicating that immune suppression in TTF-1-negative LUAD may contribute to this disease’s poorer clinical outcomes. Using a previous research database containing information on a tissue array of LUAD specimens, we found that PD-L1/L2 expression in TAMs was lower in TTF-1-negative cases, while no significant association was observed between TTF-1 status and PD-L1/L2 expression in cancer cells. It is well known that PD-L1/L2 expression in TAMs is induced predominantly by inflammatory mediators, such as IFN-γ, a factor that is secreted into the TIME by activated T cells [ 43 , 44 ]. Thus, the lower levels of inflammatory cytokines, due to the immunologically “COLD” status of TTF-1-negative cases, may be associated with decreased PD-L1/L2 expression in TAMs. Gene mutations, such as amplification of the gene encoding PD-L1, may directly affect PD-L1 overexpression in cancer cells; less is known about PD-L2 [ 45 , 46 ]. In the present study, differential expression of PD-L1/L2 was observed only in TAMs and not in cancer cells, suggesting that the immunosuppressive characteristics of TTF-1-negative LUAD more likely reflect the immunological status of the TIME, rather than underlying gene mutations. In scRNA-seq analysis, no correlation was observed between NKX2-1 expression in tumor cells and CD274 expression in macrophages. The functions of TTF-1 are known to be regulated by posttranslational modifications, including acetylation, phosphorylation, and physical inhibition [ 47 – 49 ]. To evaluate how effectively TTF-1 functions as a transcriptional activator in tumor cells, we quantified the expression levels of its target gene set, including surfactant-related genes. We found that the expression of these target genes positively correlated with CD274 expression in macrophages. The results suggest that signaling derived from either functional TTF-1 activity or lineage-specific characteristics influences PD-L1 expression in TAMs. In contrast, no correlation was observed between TTF-1-related gene expression and PDCD1LG2 expression in TAMs. It is generally known that mRNA and protein expression are not always correlated, which may account for the discrepancy observed between PD-L2 expression by IHC and PDCD1LG2 expression by scRNA-seq. We previously reported that granulocyte macrophage colony-stimulating factor (GM-CSF) derived from cancer cells contributes to PD-L1 expression in TAMs, although its correlation with PD-L2 remains unclear [ 16 ]. That study also demonstrated relatively higher GM-CSF production in the TTF-1-positive H358 and H1975 cell lines, and lower production in the TTF-1-negative A549 and PC-9 cell lines. Consistently, Wood et al. reported that TTF-1-transfected A549 cells showed increased levels of GM-CSF in CM compared to controls [ 50 ]. These findings led us to hypothesize that GM-CSF derived from TTF-1-positive cancer cells may influence PD-L1/L2 expression in TAMs. 5. Conclusions Lower infiltration of CD8 + T cells and reduced expression of PD-L1/L2 in TAMs were observed in TTF-1-negative LUAD. These findings suggest that immune responses are suppressed in TTF-1-negative LUAD, potentially contributing to its poorer clinical outcomes. Further studies are warranted to elucidate the immunosuppressive characteristics of the TIME in TTF-1-negative LUAD. Declarations • Funding This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20H03459). • Conflicts of interest/Competing interests The authors declare no conflict of interest. • Ethics approval The study design was approved by the Institutional Review Board of Kumamoto University (Approval No. 1174) and was conducted in compliance with the Declaration of Helsinki and subsequent amendments. • Informed consent Written informed consent was obtained from all patients prior to participation in the study. • Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements We thank Ms. Yuka Watanabe for the technical assistance. Author Contributions Conceptualization, H.Y., Y.F. and Y.K.; methodology, Y.F. and Y.K.; software, H.Y. and C.P.; validation, H.Y., C.P., Y.F. and Y.K.; formal analysis, H.Y.; investigation, H.Y., H.Y., E.M., S.Z., Y.S. and C.P.; resources, E.M., Y.S., K.F., K.I., and Y.K.; data curation, H.Y., E.M., S.Z. and Y.S.; writing – original draft preparation, H.Y. and Y.K.; writing – review and editing, T.K., K.F., K.I., Y.K. and M.S.; visualization, H.Y., E.M., Y.S. and C.P.; supervision, M.S.; project administration, Y.K.; funding acquisition, Y.K. All authors read and approved the final version of the manuscript to be submitted. References Sung H, Ferlay J, Siegel RL, 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 May;71(3):209-249. https://doi.org/10.3322/caac.21660. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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Maeda Y, Tsuchiya T, Hao H, Tompkins DH, Xu Y, Mucenski ML, Du L, Keiser AR, Fukazawa T, Naomoto Y, Nagayasu T, Whitsett JA. Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung. J Clin Invest. 2012 Dec;122(12):4388-400. https://doi.org/10.1172/JCI64048. Saito RA, Watabe T, Horiguchi K, Kohyama T, Saitoh M, Nagase T, Miyazono K. Thyroid transcription factor-1 inhibits transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells. Cancer Res. 2009 Apr 1;69(7):2783-91. https://doi.org/10.1158/0008-5472.CAN-08-3490. Mollaoglu G, Jones A, Wait SJ, Mukhopadhyay A, Jeong S, Arya R, Camolotto SA, Mosbruger TL, Stubben CJ, Conley CJ, Bhutkar A, Vahrenkamp JM, Berrett KC, Cessna MH, Lane TE, Witt BL, Salama ME, Gertz J, Jones KB, Snyder EL, Oliver TG. The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment. 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Br J Cancer. 2015 Apr 28;112(9):1501-9. https://doi.org/10.1038/bjc.2015.101. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, Shin T, Tsuchiya H, Pardoll DM, Okumura K, Azuma M, Yagita H. Expression of programmed death 1 ligands by murine T cells and APC. J Immunol. 2002 Nov 15;169(10):5538-45. https://doi.org/10.4049/jimmunol.169.10.5538. Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms Controlling PD-L1 Expression in Cancer. Mol Cell. 2019 Nov 7;76(3):359-370. https://doi.org/10.1016/j.molcel.2019.09.030. Fan Z, Wu C, Chen M, Jiang Y, Wu Y, Mao R, Fan Y. The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation. Acta Pharm Sin B. 2022 Mar;12(3):1041-1053. https://doi.org/10.1016/j.apsb.2021.09.010. Yang L, Yan D, Bruggeman M, Du H, Yan C. Mutation of a lysine residue in a homeodomain generates dominant negative thyroid transcription factor 1. Biochemistry. 2004 Oct 5;43(39):12489-97. https://doi.org/10.1021/bi049283o. Yan C, Whitsett JA. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1. J Biol Chem. 1997 Jul 11;272(28):17327-32. https://doi.org/10.1074/jbc.272.28.17327. Li C, Zhu NL, Tan RC, Ballard PL, Derynck R, Minoo P. Transforming growth factor-beta inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors. J Biol Chem. 2002 Oct 11;277(41):38399-408. https://doi.org/10.1074/jbc.M203188200. Wood LW, Cox NI, Phelps CA, Lai SC, Poddar A, Talbot C Jr, Mu D. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome. Sci Rep. 2016 Feb 25;6:19857. https://doi.org/10.1038/srep19857. Cite Share Download PDF Status: Published Journal Publication published 18 Aug, 2025 Read the published version in Human Cell → Version 1 posted Editorial decision: Major Revisions Needed 17 May, 2025 Reviewers agreed at journal 21 Apr, 2025 Reviewers invited by journal 28 Mar, 2025 Editor assigned by journal 28 Mar, 2025 First submitted to journal 26 Mar, 2025 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-6311106","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435561531,"identity":"5f09dd50-58e2-4bab-8736-769bac49b514","order_by":0,"name":"Hiroyuki 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Komohara","email":"","orcid":"https://orcid.org/0000-0001-9723-0846","institution":"Kumamoto University Faculty of Life Sciences School of Medicine: Kumamoto Daigaku Daigakuin Seimei Kagaku Kenkyubu Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Komohara","suffix":""},{"id":435561542,"identity":"824ddb13-aee2-4dbb-a493-8d2a007fb5a6","order_by":11,"name":"Makoto Suzuki","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Suzuki","suffix":""}],"badges":[],"createdAt":"2025-03-26 09:57:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6311106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6311106/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-025-01275-y","type":"published","date":"2025-08-18T16:29:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80813753,"identity":"045a55c3-f740-4cee-90c5-c19b3b5b6619","added_by":"auto","created_at":"2025-04-17 10:44:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6558741,"visible":true,"origin":"","legend":"\u003cp\u003eThyroid transcription factor-1 (TTF-1) expression and its clinical significance (a) Representative results of immunohistochemical analysis of TTF-1 expression (100x magnification, scale bar: 100 mm; 400x magnification, scale bar: 20 mm). (b) Kaplan-Meier analysis of progression-free survival (PFS) and cancer-specific survival (CSS) based on TTF-1 status. Logrank and Wilcoxon tests were performed, and a \u003cem\u003ep\u003c/em\u003e value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6311106/v1/6d660db5c9b25bd86a9851aa.png"},{"id":80813754,"identity":"f6302259-5b04-4052-abb9-995e77748077","added_by":"auto","created_at":"2025-04-17 10:44:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16968743,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in PD-L1/L2 expression in TAMs based on TTF-1 status (a) PD-L1/L2 positivity rates in TAMs (macrophage proportion score, MPS) are shown for both the whole group and the EGFR-wild-type subgroup. Fisher’s exact test was used. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt; 0.05 was considered statistically significant. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ns = not significant. (b) Representative images are provided for each group (Top row: 100x magnification, scale bar: 100 mm; bottom two rows: 400x magnification, scale bar: 20 mm). Pseudocolored images were generated from chromogenic signals using the color deconvolution function in HALO software, where PU.1 is shown in green, PD-L1 and PD-L2 in blue-magenta.\u003c/p\u003e\n\u003cp\u003eAbbreviations: PD-L1, programmed cell death-1 ligand 1; PD-L2, programmed cell death-1 ligand 2; TAMs, tumor-associated macrophages; EGFR, epidermal growth factor receptor.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6311106/v1/f9df9a05103b742fd9dc304d.png"},{"id":80813746,"identity":"0cdd549e-59bc-41eb-808d-2fee294e2327","added_by":"auto","created_at":"2025-04-17 10:44:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3392351,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-cell RNA sequencing analysis of public data (a) Data from 20 lung adenocarcinoma patients were analyzed, with clustering results displayed as a UMAP plot. Tumor cells were identified among epithelial cells using CNV inference. Cells above the 99.75th percentile on the x-axis were omitted from the epithelial cell plot for clarity, but were included in the analysis. (b, c) For each patient, tumor cells were evaluated for the expression of \u003cem\u003eNKX2-1\u003c/em\u003e (the gene encoding TTF-1) or the TTF-1 target gene set, and patients were stratified into low and high groups based on the median value. The mean expression levels of \u003cem\u003eCD274\u003c/em\u003e (the gene encoding PD-L1) and \u003cem\u003ePDCD1LG2\u003c/em\u003e (the gene encoding PD-L2) in macrophages were then compared between the two groups. The y-axis represents the log-transformed normalized UMI counts. The Mann-Whitney \u003cem\u003eU\u003c/em\u003e test was performed, and a \u003cem\u003ep\u003c/em\u003e value of \u0026lt; 0.05 was considered statistically significant. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ns = not significant.\u003c/p\u003e\n\u003cp\u003eAbbreviations: UMAP, Uniform Manifold Approximation \u0026amp; Projection; CNV, copy number variations; \u003cem\u003eNKX2-1\u003c/em\u003e, \u003cem\u003eNK2 homeobox 1\u003c/em\u003e; \u003cem\u003eCD274\u003c/em\u003e, \u003cem\u003ecluster of differentiation 274\u003c/em\u003e; \u003cem\u003ePDCD1LG2\u003c/em\u003e, \u003cem\u003eProgrammed Cell Death 1 Ligand 2\u003c/em\u003e; UMI, Unique Molecular Identifier.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6311106/v1/02fb77abc4002eb79f77d7ae.png"},{"id":80813752,"identity":"7dfb8dc8-19f0-41e7-a57e-4562bab81eed","added_by":"auto","created_at":"2025-04-17 10:44:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6847312,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T-cell density based on TTF-1\u0026nbsp;status (a) CD8\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;T-cell density\u0026nbsp;is\u0026nbsp;shown for both the whole group and the EGFR-wild-type subgroup.\u0026nbsp;The Mann-Whitney\u0026nbsp;\u003cem\u003eU\u003c/em\u003e\u0026nbsp;test was used. A\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026nbsp;value of\u0026nbsp;\u0026lt; 0.05 was considered statistically significant.\u0026nbsp;*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ns = not significant. (b) Representative images are provided for each group\u0026nbsp;(100x magnification, scale bar: 100\u0026nbsp;mm; 400x magnification, scale bar: 20\u0026nbsp;mm).\u003c/p\u003e\n\u003cp\u003eAbbreviation:\u0026nbsp;CD8, cluster of differentiation 8.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6311106/v1/1c79a8a601fbd7ed73e5ba51.png"},{"id":89847209,"identity":"87ee9dd0-2c28-4cc6-bbdf-53715b420b86","added_by":"auto","created_at":"2025-08-25 16:42:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31424068,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6311106/v1/ecde2718-4995-43a6-8045-59fe9b3e48d8.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe expression of PD-1 ligands in the immune microenvironment was altered in TTF-1-negative lung adenocarcinoma\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is the leading cause of cancer-related deaths worldwide, and associated rates of mortality and morbidity are increasing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the widespread adoption of computed tomography scans, which has contributed to early detection and mortality reduction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], most lung cancers still are found at advanced stages. In recent years, immunotherapy has rapidly spread to become the standard therapeutic option for lung cancer. Immune checkpoint inhibitors, particularly those targeting programmed cell death-1 (PD-1) and programmed cell death-1 ligand 1 (PD-L1), have shown significant efficacy against certain types of lung cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe interaction between PD-L1 (on tumor cells) and PD-1 (on CD8\u003csup\u003e+\u003c/sup\u003e T cells) causes T cell exhaustion, fostering immune tolerance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. PD-L1 expression in LUAD cells serves as a biomarker for predicting the effectiveness of anti-PD-L1/PD-1 therapy [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the other hand, macrophages are one of the most common immune cells in the tumor immune microenvironment (TIME), and those that infiltrate tumor tissues are called tumor-associated macrophages (TAMs). TAMs are recognized for their tumor-promoting activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Not only PD-L1 but also PD-L2 (another ligand of PD-1) are known to be expressed in TAMs, and both ligands have been shown to be involved in immunosuppression [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, our previous research on lung adenocarcinoma (LUAD) cases has highlighted the correlation between PD-L1/L2 expression in TAMs and clinical prognosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThyroid transcription factor-1 (TTF-1, also known as NK2 homeobox 1 [NKX2-1]) is specifically expressed in normal distal lung cells, such as Clara cells and type II alveolar cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. TTF-1 plays essential roles in the development and differentiation of the peripheral lung and in maintaining normal respiratory function through the regulation of the expression of pulmonary surfactants [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. TTF-1 also is expressed in most LUADs derived from the distal lung, serving as a diagnostic marker for primary LUAD [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Studies have shown that patients with LUAD who are negative for TTF-1, comprising about 30% of all cases, tend to have poorer survival outcomes [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and exhibit lower responsiveness to pemetrexed [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] or immune checkpoint inhibitors [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], compared to those who are positive for TTF-1. Although many studies have discussed both the tumor-suppressive and oncogenic roles of TTF-1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with the former including the reduction of invasion and metastasis [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], there have been few reports on the relationship between TTF-1 and TIME. In particular, the involvement of TTF-1 with CD8\u003csup\u003e+\u003c/sup\u003e T cells and TAMs remains unclear. In the present study, we observed that PD-L1 and PD-L2 expression in TAMs was decreased in cases of LUAD that were negative for TTF-1.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Samples\u003c/h2\u003e \u003cp\u003eParaffin-embedded tissue samples were obtained from 226 patients diagnosed with LUAD between 2010 and 2013 at Kumamoto University Hospital. All tissue specimens were reviewed by two pathologists. We also employed tissue microarrays previously prepared by our group [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Immunohistochemistry (IHC) analysis\u003c/h2\u003e \u003cp\u003eParaffin-embedded sections were subjected to IHC using anti-TTF-1 antibody (clone 8G7G3/1; Agilent Technologies, Santa Clara, CA, USA) and anti-CD8 antibody (clone C8/144B; Nichirei, Tokyo, Japan). 3,3\u0026rsquo;-Diaminobenzidine (DAB; Nichirei) was used for the visualization of immunoreactions. Two investigators (Y.K., H.Y.), both of whom were blinded to the patients\u0026rsquo; characteristics and outcomes, evaluated the immunostained sections. Data on PD-L1 and PD-L2 expression in cancer cells and TAMs from the same cohort were published in previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For representative cases with high and low expression of PD-L1 and PD-L2 in TAMs, multichannel pseudocolored images were created using HALO (version 4.0.5107.318; Indica Labs, Albuquerque, NM, USA). The slides were digitized with a NanoZoomer S20 (Hamamatsu Photonics, Hamamatsu, Japan) whole-slide scanner, and pseudocolored images showing staining intensities for both PD-L1 and PU.1 were generated using the color deconvolution function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Single-cell RNA-seq (scRNA-seq) analysis\u003c/h2\u003e \u003cp\u003eTwo scRNA-seq datasets of LUAD (GSE162498 and GSE131907), consisting of data from 20 patients, were downloaded from the Gene Expression Omnibus [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Cells captured in scRNA-seq analysis were clustered and analyzed using the Seurat package (version 5.1.0) in R (version 4.4.0; R Foundation for Statistical Computing, Vienna, Austria) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Cells with unique feature counts of 200 or under, or of 5,000 or over, or mitochondrial counts of 15% or over, were filtered out as part of the quality control process. After merging samples, a standard workflow using the LogNormalize method was implemented to normalize gene expression for each cell. We calculated and identified the top-2000 most-variably expressed genes among the cells, and principal component (PC) analysis was performed for linear dimensional reduction. To determine how many PCs to use for the cluster analysis, the ElbowPlot method was implemented; this approach suggested that using the first 15 PCs was reasonable for representing the true dimensionality. Based on the K-nearest-neighbors graph generated by the FindNeighbors function, we applied the Louvain algorithm using the FindClusters function with a resolution of 1.0, yielding 28 cell clusters. The differentially expressed genes (DEGs) in each cluster were identified using the FindAllMarkers function. The epithelial cell cluster was classified into tumor and non-tumor cells, allowed by inference of the copy number variations (CNV) as previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The AddModuleScore function was utilized to quantify the activity of a specific gene set in each cell. For the scoring of TTF-1 target genes, the following genes, which are known to be upregulated by TTF-1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], were defined as the gene set: \u003cem\u003eSFTPA1\u003c/em\u003e (Surfactant Protein A1), \u003cem\u003eSFTPA2\u003c/em\u003e (Surfactant Protein A2), \u003cem\u003eSFTPB\u003c/em\u003e (Surfactant Protein B), \u003cem\u003eSFTPC\u003c/em\u003e (Surfactant Protein C), \u003cem\u003eSCGB1A1\u003c/em\u003e (Secretoglobin Family 1A Member 1), \u003cem\u003eSCGB3A2\u003c/em\u003e (Secretoglobin Family 3A Member 2), \u003cem\u003eABCA3\u003c/em\u003e (ATP Binding Cassette Subfamily A Member 3), and \u003cem\u003ePDPN\u003c/em\u003e (Podoplanin).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using Prism (version 9.5.1; GraphPad Software, San Diego, CA, USA) and R (version 4.4.0). Fisher\u0026rsquo;s exact test was used to compare proportions between two groups. The Mann-Whitney \u003cem\u003eU\u003c/em\u003e test was used to compare interval-scale data between two groups that were not assumed to follow a normal distribution. The association between prognostic survival and TTF-1 status was explored by comparing survival across groups using Kaplan-Meier analysis with logrank and Wilcoxon tests. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant in all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. TTF-1-negative cases show a worse clinical course in LUAD.\u003c/h2\u003e \u003cp\u003eFollowing IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), 196 cases (86.7%) were positive for TTF-1, while 30 cases (13.3%) were negative for this marker. The distribution of TTF-1 expression was summarized according to patient characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). TTF-1-negative status was significantly more prevalent among cases with epidermal growth factor receptor (EGFR)-wild-type status, male patients, smokers, and those with advanced disease stages. Notably, within the subgroup of patients with EGFR-mutant status, an overwhelming majority (98.1%) exhibited TTF-1-positive status. Further analysis using log-rank tests revealed that progression-free survival (PFS) and cancer-specific survival (CSS) were significantly lower in TTF-1-negative cases than in TTF-1-positive cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTTF-1 expression and clinicopathological factors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eTTF-1 expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGFR gene status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (92.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82 (43.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMutant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (7.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105 (56.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61 (31.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (83.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e135 (68.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (73.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91 (46.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.010\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105 (53.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBI\u0026thinsp;\u0026lt;\u0026thinsp;600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e131 (66.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBI\u0026thinsp;\u0026ge;\u0026thinsp;600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (70.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65 (33.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epStage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0-I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (53.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e158 (80.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.002\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII-VI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (46.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38 (19.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 (76.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190 (96.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L1 expression on cancer cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow: \u0026lt; 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (90.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e185 (94.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh: ≧ 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (10.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (5.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L1 expression on TAMs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow: \u0026lt; 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (73.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88 (44.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.005\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh: ≧ 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108 (55.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L2 expression on cancer cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow: \u0026lt; 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (80.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e129 (65.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh: ≧ 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (20.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67 (34.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L2 expression on TAMs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow: \u0026lt; 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (73.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75 (38.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh: ≧ 50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e121 (61.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Fisher\u0026rsquo;s exact test was performed. Statistically significant (\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) are indicated with an underline.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eAbbreviations: TTF-1, thyroid transcription factor-1; EGFR, epidermal growth factor; BI, Brinkman index; PD-L1, programmed cell death-1 ligand 1; PD-L2, programmed cell death-1 ligand 2; TAMs, tumor-associated macrophages.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. TTF-1-negative cases show lower expression of PD-L1 and PD-L2 in TAMs.\u003c/h2\u003e \u003cp\u003eSubsequently, we examined the correlations between TTF-1 status and PD-L1/L2 expression using previously published data on PD-L1/L2 in the same tissue array sections as described in the Materials and Methods (Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e). Although no significant correlation was found between TTF-1 status and PD-L1/L2 expression in cancer cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), significant correlations were observed between TTF-1 status and PD-L1/L2 positivity rates in TAMs (referred as the macrophage proportion score, MPS) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). PD-L1/L2 expression in TAMs was lower in TTF-1-negative cases (compared to expression in TTF-1-positive cases), and this significant correlation also was preserved in the EGFR-wild-type group. Representative double-IHC images of sections co-stained for PD-L1 and PU.1, as well as for PD-L2 and PU.1, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. Here, PU.1, a transcription factor, was employed as a marker to identify TAMs in co-staining with PD-L1 and PD-L2. The expression of PD-L1/L2 in TAMs was more clearly visualized in the pseudocolored images based on immunostaining for PD-L1/L2 and PU.1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. scRNA-seq indicates down-regulation of PD-L1 in TAMs from TTF-1-negative cases.\u003c/h2\u003e \u003cp\u003eTo confirm the significant correlation between TTF-1 status and PD-L1/L2 expression in TAMs, public scRNA-seq data from 20 LUAD patients were re-analyzed. The epithelial cell cluster was further classified into tumor cells and normal cells using CNV inference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The mean expression levels of \u003cem\u003eNKX2-1\u003c/em\u003e (the gene encoding TTF-1) were calculated for tumor cells in each patient, and patients were stratified into low and high groups based on the median value. Next, using the same approach, patients were also stratified based on the expression levels of TTF-1 target genes. Here, the TTF-1 target genes refer to the gene set consisting of \u003cem\u003eSFTPA1\u003c/em\u003e, \u003cem\u003eSFTPA2\u003c/em\u003e, \u003cem\u003eSFTPB\u003c/em\u003e, \u003cem\u003eSFTPC\u003c/em\u003e, \u003cem\u003eSCGB1A1\u003c/em\u003e, \u003cem\u003eSCGB3A2\u003c/em\u003e, \u003cem\u003eABCA3\u003c/em\u003e, and \u003cem\u003ePDPN\u003c/em\u003e, and their overall expression levels were quantified as a score using the method described in the Materials and Methods (Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e). The mean expression levels of \u003cem\u003eCD274\u003c/em\u003e (cluster of differentiation 274, the gene encoding PD-L1) in macrophages were significantly lower in the low-TTF-1 target gene groups than in the high-TTF-1 target gene groups, whereas no significant difference was observed between the low-\u003cem\u003eNKX2-1\u003c/em\u003e and high-\u003cem\u003eNKX2-1\u003c/em\u003e groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). On the other hand, the mean expression levels of \u003cem\u003ePDCD1LG2\u003c/em\u003e (Programmed Cell Death 1 Ligand 2, the gene encoding PD-L2) in macrophages showed no significant difference in either classification based on \u003cem\u003eNKX2-1\u003c/em\u003e or TTF-1 target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration is suppressed in TTF-1-negative cases within the EGFR-wild-type group.\u003c/h2\u003e \u003cp\u003eIt is well known that PD-L1/L2 expression in macrophages is enhanced by interferon-gamma (IFN-γ). Therefore, overexpression of PD-L1/L2 in macrophages is thought to be induced by increased anti-cancer immune responses within the tumor microenvironment [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To investigate differences in anti-cancer immune responses between TTF-1-positive and TTF-1-negative cases, we evaluated CD8 expression by IHC. The results showed that CD8\u003csup\u003e+\u003c/sup\u003e T-cell density tended to be lower in TTF-1-negative cases, although this difference was not statistically significant in the entire cohort. However, in EGFR wild-type cases, the density was significantly lower in TTF-1-negative cases compared to TTF-1-positive cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Representative CD8-stained IHC images are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, TTF-1-negative LUAD was shown to exhibit a significantly worse clinical course than TTF-1-positive LUAD, consistent with the results of several previous reports [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. TTF-1 is recognized as a lineage-survival oncoprotein in TTF-1-positive LUAD [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], such that survival signals are mediated through the phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) axis via the receptor tyrosine kinase-like orphan receptor 1 (ROR1) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, a functional relationship with EGFR is strongly suggested, given that TTF-1 expression appears to be essential for the development of EGFR-mutant LUAD [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. On the other hand, TTF-1 is known to exhibit tumor-suppressive roles. This factor transcriptionally activates genes such as \u003cem\u003emyosin binding protein H\u003c/em\u003e (\u003cem\u003eMYBPH\u003c/em\u003e), \u003cem\u003eoccludin\u003c/em\u003e (\u003cem\u003eOCLN\u003c/em\u003e), \u003cem\u003eclaudin 1\u003c/em\u003e (\u003cem\u003eCLDN1\u003c/em\u003e), and \u003cem\u003eCLDN18\u003c/em\u003e [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which are involved in cytoskeleton regulation and cell-cell adhesion, thereby acting to suppress cancer cell invasion and metastasis. Furthermore, TTF-1 has been reported to inhibit the transforming growth factor (TGF)-β-mediated epithelial-mesenchymal transition [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and to suppress the development of Kirsten rat sarcoma (KRAS)-mutant mucinous adenocarcinoma [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, TTF-1 has dual-functional significance in cancer cell growth; however, only a few studies have explored this factor\u0026rsquo;s correlation with the TIME. Specifically, previous work has suggested that TTF-1-negative LUAD is associated with a more-immunosuppressive environment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the present study, we observed that TTF-1-negative LUAD was associated with reduced CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration in the TIME, indicating that immune suppression in TTF-1-negative LUAD may contribute to this disease\u0026rsquo;s poorer clinical outcomes.\u003c/p\u003e \u003cp\u003eUsing a previous research database containing information on a tissue array of LUAD specimens, we found that PD-L1/L2 expression in TAMs was lower in TTF-1-negative cases, while no significant association was observed between TTF-1 status and PD-L1/L2 expression in cancer cells. It is well known that PD-L1/L2 expression in TAMs is induced predominantly by inflammatory mediators, such as IFN-γ, a factor that is secreted into the TIME by activated T cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, the lower levels of inflammatory cytokines, due to the immunologically \u0026ldquo;COLD\u0026rdquo; status of TTF-1-negative cases, may be associated with decreased PD-L1/L2 expression in TAMs. Gene mutations, such as amplification of the gene encoding PD-L1, may directly affect PD-L1 overexpression in cancer cells; less is known about PD-L2 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the present study, differential expression of PD-L1/L2 was observed only in TAMs and not in cancer cells, suggesting that the immunosuppressive characteristics of TTF-1-negative LUAD more likely reflect the immunological status of the TIME, rather than underlying gene mutations.\u003c/p\u003e \u003cp\u003eIn scRNA-seq analysis, no correlation was observed between \u003cem\u003eNKX2-1\u003c/em\u003e expression in tumor cells and \u003cem\u003eCD274\u003c/em\u003e expression in macrophages. The functions of TTF-1 are known to be regulated by posttranslational modifications, including acetylation, phosphorylation, and physical inhibition [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. To evaluate how effectively TTF-1 functions as a transcriptional activator in tumor cells, we quantified the expression levels of its target gene set, including surfactant-related genes. We found that the expression of these target genes positively correlated with \u003cem\u003eCD274\u003c/em\u003e expression in macrophages. The results suggest that signaling derived from either functional TTF-1 activity or lineage-specific characteristics influences PD-L1 expression in TAMs. In contrast, no correlation was observed between TTF-1-related gene expression and \u003cem\u003ePDCD1LG2\u003c/em\u003e expression in TAMs. It is generally known that mRNA and protein expression are not always correlated, which may account for the discrepancy observed between PD-L2 expression by IHC and \u003cem\u003ePDCD1LG2\u003c/em\u003e expression by scRNA-seq.\u003c/p\u003e \u003cp\u003eWe previously reported that granulocyte macrophage colony-stimulating factor (GM-CSF) derived from cancer cells contributes to PD-L1 expression in TAMs, although its correlation with PD-L2 remains unclear [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. That study also demonstrated relatively higher GM-CSF production in the TTF-1-positive H358 and H1975 cell lines, and lower production in the TTF-1-negative A549 and PC-9 cell lines. Consistently, Wood et al. reported that TTF-1-transfected A549 cells showed increased levels of GM-CSF in CM compared to controls [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These findings led us to hypothesize that GM-CSF derived from TTF-1-positive cancer cells may influence PD-L1/L2 expression in TAMs.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eLower infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells and reduced expression of PD-L1/L2 in TAMs were observed in TTF-1-negative LUAD. These findings suggest that immune responses are suppressed in TTF-1-negative LUAD, potentially contributing to its poorer clinical outcomes. Further studies are warranted to elucidate the immunosuppressive characteristics of the TIME in TTF-1-negative LUAD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026bull; Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20H03459).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026bull; Conflicts of interest/Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026bull; Ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study design was approved by the Institutional Review Board of Kumamoto University (Approval No. 1174) and was conducted in compliance with the Declaration of Helsinki and subsequent amendments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026bull; Informed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients prior to participation in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026bull; Data availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. Yuka Watanabe for the technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, H.Y., Y.F. and Y.K.; methodology, Y.F. and Y.K.; software, H.Y. and C.P.; validation, H.Y., C.P., Y.F. and Y.K.; formal analysis, H.Y.; investigation, H.Y., H.Y., E.M., S.Z., Y.S. and C.P.; resources, E.M., Y.S., K.F., K.I., and Y.K.; data curation, H.Y., E.M., S.Z. and Y.S.; writing \u0026ndash; original draft preparation, H.Y. and Y.K.; writing \u0026ndash; review and editing, T.K., K.F., K.I., Y.K. and M.S.; visualization, H.Y., E.M., Y.S. and C.P.; supervision, M.S.; project administration, Y.K.;\u0026nbsp;funding acquisition, Y.K. \u003cstrong\u003eAll authors read and approved the final version of the manuscript to be submitted.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Sci Rep. 2016 Feb 25;6:19857. https://doi.org/10.1038/srep19857.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"lung adenocarcinoma, TTF-1, NKX2-1, macrophage, PD-L1","lastPublishedDoi":"10.21203/rs.3.rs-6311106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6311106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eImmunotherapies targeting the programmed cell death-1 (PD-1) pathway have been adopted in lung adenocarcinoma (LUAD) treatment. Expression of programmed cell death-ligand 1 (PD-L1) and PD-L2 is observed in both cancer cells and tumor-associated macrophages (TAMs). PD-L1/L2 expression in TAMs is thought to be induced by inflammatory cytokines, such as interferon (IFN)-g. Thyroid transcription factor-1 (TTF-1) is a common diagnostic marker for LUAD, with TTF-1 negativity associated with poorer survival outcomes and reduced response to chemotherapy and immunotherapy. The present study investigated the correlation between TTF-1 expression and the immune microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eTTF-1 status was evaluated using immunohistochemistry on paraffin-embedded samples from 226 patients with LUAD, examining the correlation of TTF-1 status with immune-related markers and immune cell infiltration. For further investigation, in silico RNA-seq analysis was performed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eTTF-1-negative cases showed worse progression-free survival and cancer-specific survival compared to TTF-1-positive cases. Additionally, TTF-1-negative cases exhibited lower PD-L1/L2 expression in TAMs, although no association was found between TTF-1 status and PD-L1/L2 expression in cancer cells. Consistent findings were observed through in silico analysis. The CD8\u003csup\u003e+\u003c/sup\u003e T-cell density was generally lower in TTF-1-negative cases, a significant difference when cases with mutant epidermal growth factor receptor (EGFR) were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eThese findings suggest that TTF-1-negative LUAD represents an immunologically “COLD” tumor, characterized by reduced infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells and a low IFN-g signature.\u003c/p\u003e","manuscriptTitle":"The expression of PD-1 ligands in the immune microenvironment was altered in TTF-1-negative lung adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:43:59","doi":"10.21203/rs.3.rs-6311106/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-05-17T18:29:15+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-22T00:26:41+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T00:05:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-28T19:40:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-03-26T05:57:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"daaf3259-54ed-4c60-8c28-1e7a5ffd8e71","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:32:30+00:00","versionOfRecord":{"articleIdentity":"rs-6311106","link":"https://doi.org/10.1007/s13577-025-01275-y","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2025-08-18 16:29:03","publishedOnDateReadable":"August 18th, 2025"},"versionCreatedAt":"2025-04-17 10:43:59","video":"","vorDoi":"10.1007/s13577-025-01275-y","vorDoiUrl":"https://doi.org/10.1007/s13577-025-01275-y","workflowStages":[]},"version":"v1","identity":"rs-6311106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6311106","identity":"rs-6311106","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}