循环免疫细胞中的体外 STAT3 磷酸化:早期癌症诊断和抗 PD-1 治疗反应的新型生物标记物。

IF 20.1 1区 医学 Q1 ONCOLOGY
Sung-Woo Lee, Young Ju Kim, Saei Jeong, Kyung Na Rho, Jeong Eun Noh, Hee-Ok Kim, Hyun-Ju Cho, Yoo Duk Choi, Deok Hwan Yang, Eu Chang Hwang, Woo Kyun Bae, Sook Jung Yun, Ju Sik Yun, Cheol-Kyu Park, In-Jae Oh, Jae-Ho Cho
{"title":"循环免疫细胞中的体外 STAT3 磷酸化:早期癌症诊断和抗 PD-1 治疗反应的新型生物标记物。","authors":"Sung-Woo Lee,&nbsp;Young Ju Kim,&nbsp;Saei Jeong,&nbsp;Kyung Na Rho,&nbsp;Jeong Eun Noh,&nbsp;Hee-Ok Kim,&nbsp;Hyun-Ju Cho,&nbsp;Yoo Duk Choi,&nbsp;Deok Hwan Yang,&nbsp;Eu Chang Hwang,&nbsp;Woo Kyun Bae,&nbsp;Sook Jung Yun,&nbsp;Ju Sik Yun,&nbsp;Cheol-Kyu Park,&nbsp;In-Jae Oh,&nbsp;Jae-Ho Cho","doi":"10.1002/cac2.12631","DOIUrl":null,"url":null,"abstract":"<p>Basal signal transducer and activator of transcription 3 (STAT3) activation is well-documented in the tumor microenvironment (TME) due to its association with cancer prognosis [<span>1</span>]. However, its presence and clinical relevance in the bloodstream remain unexplored. Given that STAT3-inducing cytokines, such as interleukin-6 (IL-6), are often elevated in the bloodstream of various cancer patients [<span>2, 3</span>], we aimed to investigate basal STAT3 activation in blood by developing a methodology to assess <i>ex vivo</i> phosphorylation of STAT3 (pSTAT3<i><sup>ex vivo</sup></i>) in circulating immune cells.</p><p>Since phosphorylation is a transient process prone to dephosphorylation, we sought to minimize the time between blood collection and the experiment. Specifically, 1) we limited the use of peripheral blood mononuclear cell (PBMC) samples to those processed within 1 hour of blood collection, and 2) immediately fixed the samples after thawing (Figure 1A). Notably, 135 non-small cell lung cancer (NSCLC) patient samples processed in this way exhibited significantly higher levels of pSTAT3<i><sup>ex vivo</sup></i>-positive cells compared to healthy controls (Figure 1B and Supplementary Table S1). Prolonged handling and extended experimental steps significantly decreased pSTAT3<i><sup>ex vivo</sup></i> expression (Supplementary Figure S1), underscoring the importance of our novel approach in controlling the time between blood collection and the experiment.</p><p>We next investigated the cell types within PBMCs that express pSTAT3<i><sup>ex vivo</sup></i>. CD4<sup>+</sup> T cells exhibited the highest pSTAT3<i><sup>ex vivo</sup></i> expression, followed by CD8<sup>+</sup> T cells, whereas monocytes, B cells, and natural killer (NK) cells showed minimal pSTAT3<i><sup>ex vivo</sup></i> expression (Figure 1C). Within both CD4<sup>+</sup> and CD8<sup>+</sup> T cells, pSTAT3<i><sup>ex vivo</sup></i> expression was highest in the least differentiated CD27<sup>+</sup> CD45RA<sup>+</sup> naïve subset (Figure 1C) [<span>4</span>]. A similar pattern was observed across multiple other cancer types (Figure 1D and Supplementary Figure S2).</p><p>Focusing on CD4<sup>+</sup> naïve T cells, pSTAT3<i><sup>ex vivo</sup></i> expression showed a stark contrast between NSCLC patients and healthy donors, even at stage I (Figure 1E). The area under the receiver operating characteristic curve for distinguishing stage I NSCLC patients from healthy donors was 0.9851, with a sensitivity of 0.92 at 95% specificity (Figure 1F). No tumor-specific or patient-specific clinical variables correlated with pSTAT3<i><sup>ex vivo</sup></i> expression in NSCLC patients (Supplementary Figure S3), while surgical removal of the tumor decreased pSTAT3<i><sup>ex vivo</sup></i> expression (Figure 1G), supporting a direct association between pSTAT3<i><sup>ex vivo</sup></i> and tumor burden. These findings underscore the potential of pSTAT3<i><sup>ex vivo</sup></i> as a blood-based diagnostic biomarker for early cancer detection, particularly useful in screening for cancer before proceeding to more invasive standard confirmation methods.</p><p>Next, we investigated the inducer of pSTAT3<i><sup>ex vivo</sup></i>. Among the various cytokines tested, in vitro IL-6 stimulation of STAT3 best matched the pSTAT3<i><sup>ex vivo</sup></i> expression profiles in NSCLC patients (Supplementary Figure S4A-C). To confirm the involvement of IL-6 in pSTAT3<i><sup>ex vivo</sup></i> expression, we cultured healthy PBMCs with patient serum and assessed pSTAT3 expression. Serum-induced pSTAT3 expression tightly correlated with both the pSTAT3<i><sup>ex vivo</sup></i> levels in the serum donors and the IL-6 concentration in the corresponding serum (Figure 1H-I). Importantly, the addition of anti-IL-6Rα or anti-IL-6 antibodies to the serum completely abrogated pSTAT3 induction (Figure 1J and Supplementary Figure S4D). These findings strongly suggest that IL-6 is the predominant inducer of pSTAT3<i><sup>ex vivo</sup></i> expression.</p><p>Given the role of IL-6 in inducing pSTAT3<i><sup>ex vivo</sup></i>, we further investigated their relationship. Notably, the two parameters closely followed a dose-response curve, with a half-maximal effective concentration of 11.63 pg/mL (Figure 1K) [<span>5</span>]. Interestingly, this value is 1000-fold lower than previous in vitro estimates [<span>6, 7</span>] providing strong evidence that serum IL-6 can actively induce the signaling cascade. Moreover, the dose-response curve suggests that biological activity correlates with the logarithmic form of IL-6 concentration. Accordingly, while patients with low (0%-20%), intermediate (21%-60%), and high (61%-100%) pSTAT3<i><sup>ex vivo</sup></i> (pSTAT3<i><sup>ex vivo</sup></i>-lo, pSTAT3<i><sup>ex vivo</sup></i>-int, and pSTAT3<i><sup>ex vivo</sup></i>-hi, respectively) showed a stepwise increase in pSTAT3<i><sup>ex vivo</sup></i> expression, IL-6 levels in the pSTAT3<i><sup>ex vivo</sup></i>-hi group were markedly higher than in the other two groups, rendering them relatively similar (Figure 1L). These results suggest that measuring pSTAT3<i><sup>ex vivo</sup></i> expression provides a more accurate representation of systemic IL-6 activity—its capacity to trigger intracellular signaling cascades—than direct IL-6 measurements.</p><p>Given the potential of pSTAT3<i><sup>ex vivo</sup></i> as a marker of systemic IL-6 activity, we investigated the relationship between pSTAT3<i><sup>ex vivo</sup></i> and immune cell populations. Initially, we compared pSTAT3<i><sup>ex vivo</sup></i> expression with the distribution of immune cell populations in peripheral blood. However, we did not observe any gross alterations (Supplementary Figure S5). We then explored immune cell populations within the TME. Notably, the frequency of M2-like CD206<sup>hi</sup> tumor-associated macrophages and CD39<sup>hi</sup> regulatory T cells correlated with pSTAT3<i><sup>ex vivo</sup></i> and were most abundant in the pSTAT3<i><sup>ex vivo</sup></i>-hi group (Supplementary Figure S6A-G), suggesting a link between systemic IL-6 activity and the formation of immunosuppressive TMEs.</p><p>Interestingly, the distribution of CD8<sup>+</sup> tumor-infiltrating lymphocytes (TILs) showed a unique relationship with pSTAT3<i><sup>ex vivo</sup></i> expression. We performed uniform manifold approximation and projection analysis on CD8<sup>+</sup> TILs by integrating flow cytometry data from 24 NSCLC patients (Figure 1M). Among the four CD8<sup>+</sup> TIL clusters, those with high CD103 expression (C3.CD103<sup>hi</sup>CD39<sup>lo</sup> and C4.CD103<sup>hi</sup>CD39<sup>hi</sup>) were specifically elevated in the pSTAT3<i><sup>ex vivo</sup></i>-int group, whereas the C2.CD27<sup>hi</sup>CD103<sup>lo</sup> cluster was reduced (Figure 1N-O and Supplementary Figure S6H). Given that CD103<sup>+</sup>CD8<sup>+</sup> TILs include tumor-reactive cells (Supplementary Figure S7) [<span>8, 9</span>], these results suggest that the accumulation of tumor-reactive CD8<sup>+</sup> T cells within tumors increases at a specific range of systemic IL-6 activity but diminishes at higher levels.</p><p>Given the strong correlation between pSTAT3<i><sup>ex vivo</sup></i> expression and key elements of anti-tumor immunity, we hypothesized that pSTAT3<i><sup>ex vivo</sup></i> expression could serve as a prognostic marker for cancer immunotherapy. We analyzed pSTAT3<i><sup>ex vivo</sup></i> expression in pre-therapy PBMCs from 49 NSCLC patients who received anti-PD-1 therapy (± chemotherapy) (Supplementary Table S2). The pSTAT3<i><sup>ex vivo</sup></i>-hi group showed the worst response (partial response (PR) 20%; Figure 1P), consistent with their highly immunosuppressive TMEs. Notably, the pSTAT3<i><sup>ex vivo</sup></i>-int group exhibited significantly better outcomes than pSTAT3<i><sup>ex vivo</sup></i>-lo (PR 66.7% versus 25%; Figure 1P), suggesting a potential connection with CD103<sup>+</sup>CD8<sup>+</sup> T cells. Moreover, the biomarker performance of pSTAT3<i><sup>ex vivo</sup></i> was significantly greater than tumor PD-L1 expression (Supplementary Figure S8). These findings suggest an unappreciated non-linear relationship between systemic IL-6 activity and anti-PD-1 responsiveness.</p><p>Collectively, these findings suggest that systemic IL-6, despite its extremely low concentration (median ∼10 pg/mL) [<span>3</span>], can actively induce the STAT3 signaling cascade in vivo and modulate anti-tumor immunity. Our refined methodology enabled quantification of systemic IL-6 activity as pSTAT3<i><sup>ex vivo</sup></i>, which could serve as a biomarker for cancer diagnosis and a predictor of responsiveness to anti-PD-1 therapy. Overall, our study offers new avenues for exploring systemic cytokines in various disease models, as demonstrated in our cancer patient cohort.</p><p>Sung-Woo Lee and Jae-Ho Cho administered this project; Sung-Woo Lee, Young Ju Kim, and Jae-Ho Cho conceptualized, designed methodologies, performed formal analysis, curated data, and wrote the original draft; Sung-Woo Lee, In-Jae Oh, and Jae-Ho Cho supervised, acquired funding, reviewed and edited the manuscript; Saei Jeong, Kyung Na Rho, Jeong Eun Noh, Hee-Ok Kim, Hyun-Ju Cho, Yoo Duk Choi, Deok Hwan Yang, Eu Chang Hwang, Woo Kyun Bae, Sook Jung Yun, Ju Sik Yun, Cheol-Kyu Park, and In-Jae Oh curated resources.</p><p>The authors declare no competing interests.</p><p>This work is funded by National Research Foundation of Korea (2020R1A5A2031185, 2020M3A9G3080281, 2022R1A2C2009385, 2020M3A9G3080330, and 2022R1A6A3A01086438).</p><p>This study was approved by the Institutional Review Boards of Chonnam National University Medical School and Hwasun Hospital (CNUHH-2022-021 and CNUHH-2024-034). All cancer patients provided written informed consent. Written informed consent from healthy donors from the Korean Red Cross was formally waived in accordance with the Korean Bioethics and Safety Act.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":"45 1","pages":"58-62"},"PeriodicalIF":20.1000,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758149/pdf/","citationCount":"0","resultStr":"{\"title\":\"Ex vivo STAT3 phosphorylation in circulating immune cells: a novel biomarker for early cancer diagnosis and response to anti-PD-1 therapy\",\"authors\":\"Sung-Woo Lee,&nbsp;Young Ju Kim,&nbsp;Saei Jeong,&nbsp;Kyung Na Rho,&nbsp;Jeong Eun Noh,&nbsp;Hee-Ok Kim,&nbsp;Hyun-Ju Cho,&nbsp;Yoo Duk Choi,&nbsp;Deok Hwan Yang,&nbsp;Eu Chang Hwang,&nbsp;Woo Kyun Bae,&nbsp;Sook Jung Yun,&nbsp;Ju Sik Yun,&nbsp;Cheol-Kyu Park,&nbsp;In-Jae Oh,&nbsp;Jae-Ho Cho\",\"doi\":\"10.1002/cac2.12631\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Basal signal transducer and activator of transcription 3 (STAT3) activation is well-documented in the tumor microenvironment (TME) due to its association with cancer prognosis [<span>1</span>]. However, its presence and clinical relevance in the bloodstream remain unexplored. Given that STAT3-inducing cytokines, such as interleukin-6 (IL-6), are often elevated in the bloodstream of various cancer patients [<span>2, 3</span>], we aimed to investigate basal STAT3 activation in blood by developing a methodology to assess <i>ex vivo</i> phosphorylation of STAT3 (pSTAT3<i><sup>ex vivo</sup></i>) in circulating immune cells.</p><p>Since phosphorylation is a transient process prone to dephosphorylation, we sought to minimize the time between blood collection and the experiment. Specifically, 1) we limited the use of peripheral blood mononuclear cell (PBMC) samples to those processed within 1 hour of blood collection, and 2) immediately fixed the samples after thawing (Figure 1A). Notably, 135 non-small cell lung cancer (NSCLC) patient samples processed in this way exhibited significantly higher levels of pSTAT3<i><sup>ex vivo</sup></i>-positive cells compared to healthy controls (Figure 1B and Supplementary Table S1). Prolonged handling and extended experimental steps significantly decreased pSTAT3<i><sup>ex vivo</sup></i> expression (Supplementary Figure S1), underscoring the importance of our novel approach in controlling the time between blood collection and the experiment.</p><p>We next investigated the cell types within PBMCs that express pSTAT3<i><sup>ex vivo</sup></i>. CD4<sup>+</sup> T cells exhibited the highest pSTAT3<i><sup>ex vivo</sup></i> expression, followed by CD8<sup>+</sup> T cells, whereas monocytes, B cells, and natural killer (NK) cells showed minimal pSTAT3<i><sup>ex vivo</sup></i> expression (Figure 1C). Within both CD4<sup>+</sup> and CD8<sup>+</sup> T cells, pSTAT3<i><sup>ex vivo</sup></i> expression was highest in the least differentiated CD27<sup>+</sup> CD45RA<sup>+</sup> naïve subset (Figure 1C) [<span>4</span>]. A similar pattern was observed across multiple other cancer types (Figure 1D and Supplementary Figure S2).</p><p>Focusing on CD4<sup>+</sup> naïve T cells, pSTAT3<i><sup>ex vivo</sup></i> expression showed a stark contrast between NSCLC patients and healthy donors, even at stage I (Figure 1E). The area under the receiver operating characteristic curve for distinguishing stage I NSCLC patients from healthy donors was 0.9851, with a sensitivity of 0.92 at 95% specificity (Figure 1F). No tumor-specific or patient-specific clinical variables correlated with pSTAT3<i><sup>ex vivo</sup></i> expression in NSCLC patients (Supplementary Figure S3), while surgical removal of the tumor decreased pSTAT3<i><sup>ex vivo</sup></i> expression (Figure 1G), supporting a direct association between pSTAT3<i><sup>ex vivo</sup></i> and tumor burden. These findings underscore the potential of pSTAT3<i><sup>ex vivo</sup></i> as a blood-based diagnostic biomarker for early cancer detection, particularly useful in screening for cancer before proceeding to more invasive standard confirmation methods.</p><p>Next, we investigated the inducer of pSTAT3<i><sup>ex vivo</sup></i>. Among the various cytokines tested, in vitro IL-6 stimulation of STAT3 best matched the pSTAT3<i><sup>ex vivo</sup></i> expression profiles in NSCLC patients (Supplementary Figure S4A-C). To confirm the involvement of IL-6 in pSTAT3<i><sup>ex vivo</sup></i> expression, we cultured healthy PBMCs with patient serum and assessed pSTAT3 expression. Serum-induced pSTAT3 expression tightly correlated with both the pSTAT3<i><sup>ex vivo</sup></i> levels in the serum donors and the IL-6 concentration in the corresponding serum (Figure 1H-I). Importantly, the addition of anti-IL-6Rα or anti-IL-6 antibodies to the serum completely abrogated pSTAT3 induction (Figure 1J and Supplementary Figure S4D). These findings strongly suggest that IL-6 is the predominant inducer of pSTAT3<i><sup>ex vivo</sup></i> expression.</p><p>Given the role of IL-6 in inducing pSTAT3<i><sup>ex vivo</sup></i>, we further investigated their relationship. Notably, the two parameters closely followed a dose-response curve, with a half-maximal effective concentration of 11.63 pg/mL (Figure 1K) [<span>5</span>]. Interestingly, this value is 1000-fold lower than previous in vitro estimates [<span>6, 7</span>] providing strong evidence that serum IL-6 can actively induce the signaling cascade. Moreover, the dose-response curve suggests that biological activity correlates with the logarithmic form of IL-6 concentration. Accordingly, while patients with low (0%-20%), intermediate (21%-60%), and high (61%-100%) pSTAT3<i><sup>ex vivo</sup></i> (pSTAT3<i><sup>ex vivo</sup></i>-lo, pSTAT3<i><sup>ex vivo</sup></i>-int, and pSTAT3<i><sup>ex vivo</sup></i>-hi, respectively) showed a stepwise increase in pSTAT3<i><sup>ex vivo</sup></i> expression, IL-6 levels in the pSTAT3<i><sup>ex vivo</sup></i>-hi group were markedly higher than in the other two groups, rendering them relatively similar (Figure 1L). These results suggest that measuring pSTAT3<i><sup>ex vivo</sup></i> expression provides a more accurate representation of systemic IL-6 activity—its capacity to trigger intracellular signaling cascades—than direct IL-6 measurements.</p><p>Given the potential of pSTAT3<i><sup>ex vivo</sup></i> as a marker of systemic IL-6 activity, we investigated the relationship between pSTAT3<i><sup>ex vivo</sup></i> and immune cell populations. Initially, we compared pSTAT3<i><sup>ex vivo</sup></i> expression with the distribution of immune cell populations in peripheral blood. However, we did not observe any gross alterations (Supplementary Figure S5). We then explored immune cell populations within the TME. Notably, the frequency of M2-like CD206<sup>hi</sup> tumor-associated macrophages and CD39<sup>hi</sup> regulatory T cells correlated with pSTAT3<i><sup>ex vivo</sup></i> and were most abundant in the pSTAT3<i><sup>ex vivo</sup></i>-hi group (Supplementary Figure S6A-G), suggesting a link between systemic IL-6 activity and the formation of immunosuppressive TMEs.</p><p>Interestingly, the distribution of CD8<sup>+</sup> tumor-infiltrating lymphocytes (TILs) showed a unique relationship with pSTAT3<i><sup>ex vivo</sup></i> expression. We performed uniform manifold approximation and projection analysis on CD8<sup>+</sup> TILs by integrating flow cytometry data from 24 NSCLC patients (Figure 1M). Among the four CD8<sup>+</sup> TIL clusters, those with high CD103 expression (C3.CD103<sup>hi</sup>CD39<sup>lo</sup> and C4.CD103<sup>hi</sup>CD39<sup>hi</sup>) were specifically elevated in the pSTAT3<i><sup>ex vivo</sup></i>-int group, whereas the C2.CD27<sup>hi</sup>CD103<sup>lo</sup> cluster was reduced (Figure 1N-O and Supplementary Figure S6H). Given that CD103<sup>+</sup>CD8<sup>+</sup> TILs include tumor-reactive cells (Supplementary Figure S7) [<span>8, 9</span>], these results suggest that the accumulation of tumor-reactive CD8<sup>+</sup> T cells within tumors increases at a specific range of systemic IL-6 activity but diminishes at higher levels.</p><p>Given the strong correlation between pSTAT3<i><sup>ex vivo</sup></i> expression and key elements of anti-tumor immunity, we hypothesized that pSTAT3<i><sup>ex vivo</sup></i> expression could serve as a prognostic marker for cancer immunotherapy. We analyzed pSTAT3<i><sup>ex vivo</sup></i> expression in pre-therapy PBMCs from 49 NSCLC patients who received anti-PD-1 therapy (± chemotherapy) (Supplementary Table S2). The pSTAT3<i><sup>ex vivo</sup></i>-hi group showed the worst response (partial response (PR) 20%; Figure 1P), consistent with their highly immunosuppressive TMEs. Notably, the pSTAT3<i><sup>ex vivo</sup></i>-int group exhibited significantly better outcomes than pSTAT3<i><sup>ex vivo</sup></i>-lo (PR 66.7% versus 25%; Figure 1P), suggesting a potential connection with CD103<sup>+</sup>CD8<sup>+</sup> T cells. Moreover, the biomarker performance of pSTAT3<i><sup>ex vivo</sup></i> was significantly greater than tumor PD-L1 expression (Supplementary Figure S8). These findings suggest an unappreciated non-linear relationship between systemic IL-6 activity and anti-PD-1 responsiveness.</p><p>Collectively, these findings suggest that systemic IL-6, despite its extremely low concentration (median ∼10 pg/mL) [<span>3</span>], can actively induce the STAT3 signaling cascade in vivo and modulate anti-tumor immunity. Our refined methodology enabled quantification of systemic IL-6 activity as pSTAT3<i><sup>ex vivo</sup></i>, which could serve as a biomarker for cancer diagnosis and a predictor of responsiveness to anti-PD-1 therapy. Overall, our study offers new avenues for exploring systemic cytokines in various disease models, as demonstrated in our cancer patient cohort.</p><p>Sung-Woo Lee and Jae-Ho Cho administered this project; Sung-Woo Lee, Young Ju Kim, and Jae-Ho Cho conceptualized, designed methodologies, performed formal analysis, curated data, and wrote the original draft; Sung-Woo Lee, In-Jae Oh, and Jae-Ho Cho supervised, acquired funding, reviewed and edited the manuscript; Saei Jeong, Kyung Na Rho, Jeong Eun Noh, Hee-Ok Kim, Hyun-Ju Cho, Yoo Duk Choi, Deok Hwan Yang, Eu Chang Hwang, Woo Kyun Bae, Sook Jung Yun, Ju Sik Yun, Cheol-Kyu Park, and In-Jae Oh curated resources.</p><p>The authors declare no competing interests.</p><p>This work is funded by National Research Foundation of Korea (2020R1A5A2031185, 2020M3A9G3080281, 2022R1A2C2009385, 2020M3A9G3080330, and 2022R1A6A3A01086438).</p><p>This study was approved by the Institutional Review Boards of Chonnam National University Medical School and Hwasun Hospital (CNUHH-2022-021 and CNUHH-2024-034). All cancer patients provided written informed consent. Written informed consent from healthy donors from the Korean Red Cross was formally waived in accordance with the Korean Bioethics and Safety Act.</p>\",\"PeriodicalId\":9495,\"journal\":{\"name\":\"Cancer Communications\",\"volume\":\"45 1\",\"pages\":\"58-62\"},\"PeriodicalIF\":20.1000,\"publicationDate\":\"2024-11-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758149/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer Communications\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12631\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12631","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
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摘要

基础信号换能器和转录激活因子3 (STAT3)的激活在肿瘤微环境(TME)中被充分证实与癌症预后相关[1]。然而,它在血液中的存在和临床相关性仍未被探索。鉴于诱导STAT3的细胞因子,如白细胞介素-6 (IL-6),在各种癌症患者的血液中经常升高[2,3],我们旨在通过开发一种方法来评估循环免疫细胞中STAT3的体外磷酸化(pSTAT3ex vivo),研究血液中STAT3的基础活化。由于磷酸化是一个短暂的过程,容易发生去磷酸化,我们试图减少血液采集和实验之间的时间。具体来说,1)我们将外周血单个核细胞(PBMC)样本的使用限制在采集血液1小时内处理的样本,2)解冻后立即固定样本(图1A)。值得注意的是,以这种方式处理的135例非小细胞肺癌(NSCLC)患者样本显示,与健康对照组相比,pSTAT3ex体内阳性细胞水平明显更高(图1B和补充表S1)。长时间的处理和延长的实验步骤显著降低了pSTAT3ex - vivo表达(补充图S1),强调了我们的新方法在控制采血和实验之间时间的重要性。接下来,我们研究了pmcs体内表达pstat3的细胞类型。CD4+ T细胞的pstat3体外表达最高,其次是CD8+ T细胞,而单核细胞、B细胞和自然杀伤(NK)细胞的pstat3体外表达最低(图1C)。在CD4+和CD8+ T细胞中,pstat3体外表达在分化程度最低的CD27+ CD45RA+ naïve亚群中最高(图1C)[4]。在其他多种癌症类型中也观察到类似的模式(图1D和补充图S2)。关注CD4+ naïve T细胞,即使在I期,NSCLC患者和健康供体的pstat3体外表达也显示出明显的差异(图1E)。区分I期NSCLC患者与健康供体的受者工作特征曲线下面积为0.9851,在95%特异性下敏感性为0.92(图1F)。在NSCLC患者中,没有肿瘤特异性或患者特异性临床变量与pSTAT3ex - vivo表达相关(补充图S3),而手术切除肿瘤可降低pSTAT3ex - vivo表达(图1G),支持pSTAT3ex - vivo与肿瘤负荷之间的直接关联。这些发现强调了pSTAT3ex体内作为早期癌症检测的血液诊断生物标志物的潜力,特别是在进行更具侵入性的标准确认方法之前进行癌症筛查。接下来,我们在体外研究了pstat3诱导因子。在测试的各种细胞因子中,体外IL-6刺激STAT3最符合非小细胞肺癌患者体内pstat3的表达谱(补充图S4A-C)。为了证实IL-6参与pSTAT3的体外表达,我们用患者血清培养了健康的pbmc,并评估了pSTAT3的表达。血清诱导的pSTAT3表达与供体血清中pSTAT3体外水平及相应血清中IL-6浓度密切相关(图1h - 1)。重要的是,在血清中加入抗il - 6r α或抗il -6抗体完全消除了pSTAT3的诱导作用(图1J和补充图S4D)。这些发现强烈提示IL-6是pstat3体外表达的主要诱导剂。鉴于IL-6在体内诱导pstat3的作用,我们进一步研究了它们之间的关系。值得注意的是,这两个参数密切遵循剂量-反应曲线,半最大有效浓度为11.63 pg/mL(图1K)[5]。有趣的是,这个数值比之前的体外估计值低1000倍[6,7],这有力地证明了血清IL-6可以积极诱导信号级联。此外,剂量-反应曲线表明,生物活性与IL-6浓度的对数形式相关。因此,体内低(0%-20%)、中(21%-60%)和高(61%-100%)pSTAT3ex(分别为pSTAT3ex vivo-lo、pSTAT3ex vivo-int和pSTAT3ex vivo-hi)患者的体内pSTAT3ex表达水平逐步升高,但pSTAT3ex vivo-hi组的IL-6水平明显高于其他两组,两者相对相似(图1L)。这些结果表明,与直接测量IL-6相比,测量pSTAT3ex - vivo表达可以更准确地表示系统IL-6活性(其触发细胞内信号级联的能力)。鉴于体内pSTAT3ex作为全身IL-6活性标记物的潜力,我们研究了体内pSTAT3ex与免疫细胞群之间的关系。首先,我们比较了pstat3在体内的表达与外周血中免疫细胞群的分布。 然而,我们没有观察到任何明显的变化(补充图S5)。然后,我们探索了TME内的免疫细胞群。值得注意的是,m2样CD206hi肿瘤相关巨噬细胞和CD39hi调节性T细胞的频率与pSTAT3ex体内相关,并且在pSTAT3ex体内-hi组中最为丰富(补充图S6A-G),这表明全身IL-6活性与免疫抑制性TMEs的形成之间存在联系。有趣的是,CD8+肿瘤浸润淋巴细胞(TILs)的分布与pstat3体外表达有独特的关系。通过整合24例NSCLC患者的流式细胞术数据,我们对CD8+ TILs进行了均匀流形近似和投影分析(图1M)。在4个CD8+ TIL簇中,CD103高表达的(C3;CD103hiCD39lo和C4.CD103hiCD39hi)在pSTAT3ex活体int组中特异性升高,而C2. CD103hiCD39lo和C4.CD103hiCD39hi在pSTAT3ex活体int组中特异性升高。CD27hiCD103lo集群减少(图1N-O和补充图S6H)。考虑到CD103+CD8+ til包括肿瘤反应性细胞(Supplementary Figure S7)[8,9],这些结果表明肿瘤内肿瘤反应性CD8+ T细胞的积累在特定的全身IL-6活性范围内增加,但在更高水平时减少。鉴于pSTAT3ex体内表达与抗肿瘤免疫的关键要素之间存在很强的相关性,我们假设pSTAT3ex体内表达可以作为癌症免疫治疗的预后标志物。我们分析了49例接受抗pd -1治疗(±化疗)的NSCLC患者治疗前PBMCs中pstat3体外表达(补充表S2)。pSTAT3ex在体组反应最差(部分缓解(PR) 20%;图1P),与它们高度免疫抑制的TMEs一致。值得注意的是,pSTAT3ex活体-int组的预后明显优于pSTAT3ex活体-lo组(PR = 66.7% vs . 25%;图1P),提示可能与CD103+CD8+ T细胞有关。此外,pstat3在体内的生物标志物表现显著高于肿瘤中PD-L1的表达(Supplementary Figure S8)。这些发现表明,系统IL-6活性与抗pd -1反应性之间存在未被认识到的非线性关系。总的来说,这些发现表明,尽管全身IL-6的浓度极低(中位数~ 10 pg/mL),但它可以在体内积极诱导STAT3信号级联并调节抗肿瘤免疫。我们的精细化方法可以量化全身IL-6在体内的pstat3活性,这可以作为癌症诊断的生物标志物和抗pd -1治疗反应性的预测因子。总的来说,我们的研究为探索各种疾病模型中的系统细胞因子提供了新的途径,正如我们的癌症患者队列所证明的那样。李成宇、赵在浩负责该项目;李成宇、金永柱、赵在浩负责构思、设计方法、进行形式分析、整理资料、撰写初稿;李成宇、吴仁宰、赵在浩负责监督、筹集资金、审查和编辑稿件;Jeong Saei, Kyung Na Rho, Jeong Eun Noh, Kim Hee-Ok, Cho Hyun-Ju, Yoo Duk Choi, Yang Deok Hwan, Hwang euchang, Woo Kyun, Sook Jung Yun, Ju Sik Yun, Cheol-Kyu Park, In-Jae Oh担任资源总监。作者声明没有利益冲突。本课题由韩国国家研究基金资助(2020R1A5A2031185、2020M3A9G3080281、2022R1A2C2009385、2020M3A9G3080330、2022R1A6A3A01086438)。本研究由全南国立大学医学院和华顺医院机构审查委员会(CNUHH-2022-021和CNUHH-2024-034)批准。所有癌症患者均提供书面知情同意书。根据《生命伦理安全法》,正式放弃了大韩红十字会健康捐赠者的知情同意书。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Ex vivo STAT3 phosphorylation in circulating immune cells: a novel biomarker for early cancer diagnosis and response to anti-PD-1 therapy

Ex vivo STAT3 phosphorylation in circulating immune cells: a novel biomarker for early cancer diagnosis and response to anti-PD-1 therapy

Basal signal transducer and activator of transcription 3 (STAT3) activation is well-documented in the tumor microenvironment (TME) due to its association with cancer prognosis [1]. However, its presence and clinical relevance in the bloodstream remain unexplored. Given that STAT3-inducing cytokines, such as interleukin-6 (IL-6), are often elevated in the bloodstream of various cancer patients [2, 3], we aimed to investigate basal STAT3 activation in blood by developing a methodology to assess ex vivo phosphorylation of STAT3 (pSTAT3ex vivo) in circulating immune cells.

Since phosphorylation is a transient process prone to dephosphorylation, we sought to minimize the time between blood collection and the experiment. Specifically, 1) we limited the use of peripheral blood mononuclear cell (PBMC) samples to those processed within 1 hour of blood collection, and 2) immediately fixed the samples after thawing (Figure 1A). Notably, 135 non-small cell lung cancer (NSCLC) patient samples processed in this way exhibited significantly higher levels of pSTAT3ex vivo-positive cells compared to healthy controls (Figure 1B and Supplementary Table S1). Prolonged handling and extended experimental steps significantly decreased pSTAT3ex vivo expression (Supplementary Figure S1), underscoring the importance of our novel approach in controlling the time between blood collection and the experiment.

We next investigated the cell types within PBMCs that express pSTAT3ex vivo. CD4+ T cells exhibited the highest pSTAT3ex vivo expression, followed by CD8+ T cells, whereas monocytes, B cells, and natural killer (NK) cells showed minimal pSTAT3ex vivo expression (Figure 1C). Within both CD4+ and CD8+ T cells, pSTAT3ex vivo expression was highest in the least differentiated CD27+ CD45RA+ naïve subset (Figure 1C) [4]. A similar pattern was observed across multiple other cancer types (Figure 1D and Supplementary Figure S2).

Focusing on CD4+ naïve T cells, pSTAT3ex vivo expression showed a stark contrast between NSCLC patients and healthy donors, even at stage I (Figure 1E). The area under the receiver operating characteristic curve for distinguishing stage I NSCLC patients from healthy donors was 0.9851, with a sensitivity of 0.92 at 95% specificity (Figure 1F). No tumor-specific or patient-specific clinical variables correlated with pSTAT3ex vivo expression in NSCLC patients (Supplementary Figure S3), while surgical removal of the tumor decreased pSTAT3ex vivo expression (Figure 1G), supporting a direct association between pSTAT3ex vivo and tumor burden. These findings underscore the potential of pSTAT3ex vivo as a blood-based diagnostic biomarker for early cancer detection, particularly useful in screening for cancer before proceeding to more invasive standard confirmation methods.

Next, we investigated the inducer of pSTAT3ex vivo. Among the various cytokines tested, in vitro IL-6 stimulation of STAT3 best matched the pSTAT3ex vivo expression profiles in NSCLC patients (Supplementary Figure S4A-C). To confirm the involvement of IL-6 in pSTAT3ex vivo expression, we cultured healthy PBMCs with patient serum and assessed pSTAT3 expression. Serum-induced pSTAT3 expression tightly correlated with both the pSTAT3ex vivo levels in the serum donors and the IL-6 concentration in the corresponding serum (Figure 1H-I). Importantly, the addition of anti-IL-6Rα or anti-IL-6 antibodies to the serum completely abrogated pSTAT3 induction (Figure 1J and Supplementary Figure S4D). These findings strongly suggest that IL-6 is the predominant inducer of pSTAT3ex vivo expression.

Given the role of IL-6 in inducing pSTAT3ex vivo, we further investigated their relationship. Notably, the two parameters closely followed a dose-response curve, with a half-maximal effective concentration of 11.63 pg/mL (Figure 1K) [5]. Interestingly, this value is 1000-fold lower than previous in vitro estimates [6, 7] providing strong evidence that serum IL-6 can actively induce the signaling cascade. Moreover, the dose-response curve suggests that biological activity correlates with the logarithmic form of IL-6 concentration. Accordingly, while patients with low (0%-20%), intermediate (21%-60%), and high (61%-100%) pSTAT3ex vivo (pSTAT3ex vivo-lo, pSTAT3ex vivo-int, and pSTAT3ex vivo-hi, respectively) showed a stepwise increase in pSTAT3ex vivo expression, IL-6 levels in the pSTAT3ex vivo-hi group were markedly higher than in the other two groups, rendering them relatively similar (Figure 1L). These results suggest that measuring pSTAT3ex vivo expression provides a more accurate representation of systemic IL-6 activity—its capacity to trigger intracellular signaling cascades—than direct IL-6 measurements.

Given the potential of pSTAT3ex vivo as a marker of systemic IL-6 activity, we investigated the relationship between pSTAT3ex vivo and immune cell populations. Initially, we compared pSTAT3ex vivo expression with the distribution of immune cell populations in peripheral blood. However, we did not observe any gross alterations (Supplementary Figure S5). We then explored immune cell populations within the TME. Notably, the frequency of M2-like CD206hi tumor-associated macrophages and CD39hi regulatory T cells correlated with pSTAT3ex vivo and were most abundant in the pSTAT3ex vivo-hi group (Supplementary Figure S6A-G), suggesting a link between systemic IL-6 activity and the formation of immunosuppressive TMEs.

Interestingly, the distribution of CD8+ tumor-infiltrating lymphocytes (TILs) showed a unique relationship with pSTAT3ex vivo expression. We performed uniform manifold approximation and projection analysis on CD8+ TILs by integrating flow cytometry data from 24 NSCLC patients (Figure 1M). Among the four CD8+ TIL clusters, those with high CD103 expression (C3.CD103hiCD39lo and C4.CD103hiCD39hi) were specifically elevated in the pSTAT3ex vivo-int group, whereas the C2.CD27hiCD103lo cluster was reduced (Figure 1N-O and Supplementary Figure S6H). Given that CD103+CD8+ TILs include tumor-reactive cells (Supplementary Figure S7) [8, 9], these results suggest that the accumulation of tumor-reactive CD8+ T cells within tumors increases at a specific range of systemic IL-6 activity but diminishes at higher levels.

Given the strong correlation between pSTAT3ex vivo expression and key elements of anti-tumor immunity, we hypothesized that pSTAT3ex vivo expression could serve as a prognostic marker for cancer immunotherapy. We analyzed pSTAT3ex vivo expression in pre-therapy PBMCs from 49 NSCLC patients who received anti-PD-1 therapy (± chemotherapy) (Supplementary Table S2). The pSTAT3ex vivo-hi group showed the worst response (partial response (PR) 20%; Figure 1P), consistent with their highly immunosuppressive TMEs. Notably, the pSTAT3ex vivo-int group exhibited significantly better outcomes than pSTAT3ex vivo-lo (PR 66.7% versus 25%; Figure 1P), suggesting a potential connection with CD103+CD8+ T cells. Moreover, the biomarker performance of pSTAT3ex vivo was significantly greater than tumor PD-L1 expression (Supplementary Figure S8). These findings suggest an unappreciated non-linear relationship between systemic IL-6 activity and anti-PD-1 responsiveness.

Collectively, these findings suggest that systemic IL-6, despite its extremely low concentration (median ∼10 pg/mL) [3], can actively induce the STAT3 signaling cascade in vivo and modulate anti-tumor immunity. Our refined methodology enabled quantification of systemic IL-6 activity as pSTAT3ex vivo, which could serve as a biomarker for cancer diagnosis and a predictor of responsiveness to anti-PD-1 therapy. Overall, our study offers new avenues for exploring systemic cytokines in various disease models, as demonstrated in our cancer patient cohort.

Sung-Woo Lee and Jae-Ho Cho administered this project; Sung-Woo Lee, Young Ju Kim, and Jae-Ho Cho conceptualized, designed methodologies, performed formal analysis, curated data, and wrote the original draft; Sung-Woo Lee, In-Jae Oh, and Jae-Ho Cho supervised, acquired funding, reviewed and edited the manuscript; Saei Jeong, Kyung Na Rho, Jeong Eun Noh, Hee-Ok Kim, Hyun-Ju Cho, Yoo Duk Choi, Deok Hwan Yang, Eu Chang Hwang, Woo Kyun Bae, Sook Jung Yun, Ju Sik Yun, Cheol-Kyu Park, and In-Jae Oh curated resources.

The authors declare no competing interests.

This work is funded by National Research Foundation of Korea (2020R1A5A2031185, 2020M3A9G3080281, 2022R1A2C2009385, 2020M3A9G3080330, and 2022R1A6A3A01086438).

This study was approved by the Institutional Review Boards of Chonnam National University Medical School and Hwasun Hospital (CNUHH-2022-021 and CNUHH-2024-034). All cancer patients provided written informed consent. Written informed consent from healthy donors from the Korean Red Cross was formally waived in accordance with the Korean Bioethics and Safety Act.

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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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