Visceral Obesity Promotes Lung Cancer Progression – Towards Resolution of the Obesity Paradox in Lung Cancer
内脏性肥胖促进肺癌进展 - 走向解决肺癌肥胖悖论

Image analysis 图像分析

CT scans obtained as part of a pre-operative PET-CT were analyzed. A single image at L3 level was identified and exported as a DICOM image. In the unusual cases where a pre-operative PET-CT scan was unavailable in the clinical record, images at L2 or L1 obtained from chest CT scans were used for analysis. Images at L3, L2 and L1 levels were obtained for a cohort of 46 patients to assess the validity of using images at these levels in lieu of L3. Image acquisition was performed by CR, EK, XW, SS, RG and SY, and measurements were performed using NIH ImageJ (v1.52 Java 1.8.0_112), as described in supplementary data (text S1). The entire cross-section of the patient was selected and total fat area (TFA) was calculated. The visceral fat area (VFA) was then selected and measured. Central adiposity was estimated as the ratio between the visceral fat area (VFA) to the total fat area (TFA) and was labelled the viscera fat index (VFI).
CT 扫描作为术前 PET-CT 的一部分进行分析。在 L3 水平识别并导出一幅图像作为 DICOM 图像。在临床记录中术前 PET-CT 扫描不可用的情况下，使用从胸部 CT 扫描中获得的 L2 或 L1 水平的图像进行分析。为了评估在缺少 L3 图像的情况下使用这些水平的图像的有效性，对 46 名患者进行了 L3、L2 和 L1 水平的图像获取。图像采集由 CR、EK、XW、SS、RG 和 SY 执行，使用 NIH ImageJ（v1.52 Java 1.8.0_112）进行测量，如补充数据（文本 S1）中所述。选择患者的整个横截面，并计算总脂肪面积（TFA）。然后选择并测量内脏脂肪面积（VFA）。将中心性脂肪沉积估计为内脏脂肪面积（VFA）与总脂肪面积（TFA）之间的比率，并标记为内脏脂肪指数（VFI）。

Survival analyses 生存分析

As established categories of VFI as a measure of central obesity do not exist, VFI was first analyzed as a continuous variable. Age, sex, grade, race, histology, diffusion capacity for carbon monoxide (DLCO), American Society of Anesthesiology Score (ASA) and smoking status were used as covariates in model generation. Univariate analyses (Kaplan Meier) were performed to evaluate the association of individual variables with OS and RFS. Only variables associated with OS and RFS on univariate analysis were included in model generation by multivariable analyses (Cox proportional hazards). Given the association of VFI with age and sex, interaction variables were included in model generation. Variables were excluded at a significance > 0.1 at each step of model generation. In a separate analysis, patients were classified into tertiles (‘Top’, ‘Middle’ and ‘Bottom’) based on VFI. Univariate (Kaplan-Meier) and multivariable (Cox proportional hazards) survival analyses were performed to compare the OS and RFS of the ‘Top’ and ‘Bottom’ tertile of patients. Similar analyses were performed with disease-specific survival (DSS) as well. All analyses were performed in SPSS ver. 25.
由于不存在作为中心性肥胖度量的 VFI 的已建立类别，因此首先将 VFI 作为连续变量进行分析。年龄、性别、年级、种族、组织学、一氧化碳弥散能力（DLCO）、美国麻醉学会评分（ASA）和吸烟状况被用作模型生成的协变量。单变量分析（Kaplan Meier）用于评估各个变量与 OS 和 RFS 的关联。只有在单变量分析中与 OS 和 RFS 相关的变量才会通过多变量分析（Cox 比例危险）纳入模型生成。鉴于 VFI 与年龄和性别的关联，交互变量被纳入模型生成。在模型生成的每个步骤中，显著性排除变量> 0.1。在单独的分析中，患者根据 VFI 被分类为三分位数（“顶部”、“中部”和“底部”）。进行了单变量（Kaplan-Meier）和多变量（Cox 比例危险）生存分析，以比较“顶部”和“底部”三分位数患者的 OS 和 RFS。类似的分析也适用于疾病特异性生存（DSS）。所有分析均在 SPSS ver. 25 中进行。

FFPE tumor gene expression
FFPE 肿瘤基因表达

RNA was extracted from each FFPE sample and processed for targeted RNA-seq, as previously described³⁴. Gene expression was evaluated by amplicon sequencing of 394 immune transcripts on samples that met validated quality control (QC) thresholds (text S1).
从每个 FFPE 样本中提取 RNA，并按照先前描述的方法进行目标 RNA 测序 ³⁴ 。通过对符合经过验证的质量控制（QC）阈值的样本进行 394 个免疫转录本的引物测序来评估基因表达（文本 S1）。

Murine lung cancer models
小鼠肺癌模型

Age-matched cohorts of obese and non-obese male C57BL/6 mice were purchased from the Jackson Laboratory, Bar Harbor, ME (stock numbers 380050 and 380056, respectively). These mice were generated by feeding them a high-fat diet (5.2 kcal/gram, 60% fat calories) beginning at 6 weeks of age and continuing for approximately 14 weeks to induce obesity. Mice fed a conventional chow diet (3.8 kcal/gram, 5–10% fat calories) were used as normal-weight controls. Mice were weighed before and periodically after initiation of experiments to confirm obese/normal status. All mice were housed in a specific pathogen free facility and all procedures were approved by the Institutional Animal Care and Use Committee. The Lewis Lung Carcinoma (LLC) cell line and its EF1 promoter-driven firefly luciferase-expressing variant (LLC-luc) were purchased from ATCC, Manassas, VA (product identifiers CRL-1642 and CRL-1642-LUC2, respectively). Both cell lines were maintained as adherent cultures in DMEM medium (Gibco) supplemented with 10% v/v fetal bovine serum (FBS; Gemini Bioproducts). For tumor challenge experiments, cells were grown to near confluency, detached with trypsin/EDTA, washed and resuspended in sterile PBS. For subcutaneous (s.c.) tumor challenge, 1×10⁵ tumor cells were implanted into the shaved flanks of obese and normal weight mice (n = 5–7/group). Tumor volumes were measured every 2–3 days using a digital caliper. About 21 days post-implantation mice were euthanized. Tumor sections were harvested and either snap frozen for RNA sequencing analysis or mechanical dissociation to generate single-cell suspensions. Tumor-infiltrating leukocytes (TILs) were recovered from the latter after washing and Percoll gradient centrifugation. These as well as the cells of both tumor-draining and tumor-distal lymph nodes and spleen were analyzed by flow cytometry. For studies of pulmonary metastases, 0.25×10⁶ LLC-luc cells were injected intravenously (i.v.) by the tail vein into obese and non-obese mice (n = 5/group), and the development of secondary tumors primarily in the lung was quantified by in vivo bioluminescence imagery (BLI) using Xenogen IVIS Spectrum technology (Perkin-Elmer, Waltham, MA). Just prior to imaging session, mice were injected intraperitoneally (i.p.) with D-luciferin (150 mg/kg body weight; Gold Biotechnology) and anesthetized by isoflurane inhalation. Photonic flux (photon per second) in representative images was quantified with Living Image software (version 4.3.1.0.15880; Perkin-Elmer).
年龄相匹配的肥胖和非肥胖 C57BL/6 雄性小鼠队列分别从缅因州巴港的杰克逊实验室购买（库存编号分别为 380050 和 380056）。这些小鼠通过从 6 周龄开始饲喂高脂饮食（5.2 千卡/克，60%脂肪热量）并持续约 14 周诱导肥胖。饲喂传统饲料（3.8 千卡/克，5-10%脂肪热量）的小鼠被用作正常体重对照。在实验开始前和定期称重以确认肥胖/正常状态。所有小鼠都被安置在特定无病原体设施中，所有程序均获得机构动物护理和使用委员会批准。刘易斯肺癌（LLC）细胞系及其 EF1 启动子驱动的萤火虫荧光素表达变体（LLC-luc）分别从弗吉尼亚州马纳萨斯的 ATCC 购买（产品标识符分别为 CRL-1642 和 CRL-1642-LUC2）。这两个细胞系均在含有 10%体积/体积胎牛血清（FBS；Gemini Bioproducts）的 DMEM 培养基（Gibco）中作为贴壁培养维持。 对于肿瘤挑战实验，细胞被培养至接近充实状态，用胰酶/EDTA 脱离，洗涤并悬浮在无菌 PBS 中。对于皮下（s.c.）肿瘤挑战，将 1×10 ⁵ 肿瘤细胞植入肥胖和正常体重小鼠的剃光侧腹部（每组 n = 5-7）。使用数字卡尺每 2-3 天测量肿瘤体积。植入后约 21 天小鼠被安乐死。收集肿瘤切片，要么立即冷冻用于 RNA 测序分析，要么机械分离以生成单细胞悬浮液。从后者中洗涤和 Percoll 梯度离心分离出肿瘤浸润性白细胞（TILs）。这些以及肿瘤引流和肿瘤远端淋巴结和脾脏的细胞通过流式细胞术进行分析。对于肺转移的研究，将 0.25×10 ⁶ LLC-luc 细胞通过尾静脉注射（i.v.）到肥胖和非肥胖小鼠中（每组 n = 5），并使用 Xenogen IVIS Spectrum 技术（Perkin-Elmer，Waltham，MA）进行体内生物发光成像（BLI）来定量主要在肺部的继发肿瘤的发展。 在成像会话之前，小鼠被腹腔注射 D-琥珀酸酯（150 毫克/千克体重；Gold Biotechnology），并通过异氟醚吸入麻醉。代表性图像中的光子通量（每秒光子）由 Living Image 软件（版本 4.3.1.0.15880；Perkin-Elmer）进行定量分析。

Mouse tumor gene expression
鼠肿瘤基因表达

Gene expression of tumors of three each of control and obese mice from each of two independent experiments was obtained by mRNA sequencing (text S1). Raw sequencing data was deposited in the European Nucleotide Archive under accession number PRJEB34297. Tumors of control and obese mice were compared for differential gene expression and gene set enrichment using DESeq2 and gsva Bioconductor software, with false discovery rate (FDR) cut-offs of 0.05 and 0.25 respectively used for significance testing (text S1).
两个独立实验中，每组三只对照组和肥胖小鼠的肿瘤基因表达通过 mRNA 测序获得（文本 S1）。原始测序数据已存储在欧洲核苷酸数据库，存取号为 PRJEB34297。使用 DESeq2 和 gsva Bioconductor 软件对对照组和肥胖小鼠的肿瘤进行差异基因表达和基因集富集比较，显著性检验分别使用了 0.05 和 0.25 的假发现率（FDR）截断值（文本 S1）。

Visceral obesity is associated with poor overall and recurrence free survival in early NSCLC patients undergoing surgical resection
内脏性肥胖与早期非小细胞肺癌患者在手术切除过程中的整体生存率和无复发生存率不良相关

We initially sought to establish the relationship between visceral obesity, as measured by VFI, and several clinically relevant metrics. We found that VFI was not associated with BMI (Pearson corr coeff = −0.06; P = 0.9; Supplementary Figure S2C). However, VFI is associated with sex as well as age. Specifically, the mean VFI of males is higher than females (0.56 vs. 0.37; P<0.01). VFI also tends to be elevated with increasing age (Pearson corr coeff 0.32; P<0.01).
我们最初试图建立内脏肥胖（由 VFI 测量）与几个临床相关指标之间的关系。我们发现 VFI 与 BMI 无关（Pearson 相关系数= -0.06；P = 0.9；附图 S2C）。然而，VFI 与性别以及年龄有关。具体而言，男性的平均 VFI 高于女性（0.56 vs. 0.37；P<0.01）。VFI 也倾向于随着年龄增长而升高（Pearson 相关系数 0.32；P<0.01）。

Univariate analyses demonstrated a statistically significant association of sex, ASA score, tumor grade, histology, age, DLCO and VFI with OS. Similarly, sex, tumor grade, tumor stage, ASA score and VFI were associated with RFS. Multivariable analysis resulted in a final model of overall survival that retained age, sex, DLCO, ASA score, VFI, tumor grade and the interaction term between VFI and age. Similar analysis resulted in a final model of recurrence free survival that retained only tumor grade, tumor stage and VFI as predictive variables. These results are summarized in Table 1.
单变量分析显示性别、ASA 评分、肿瘤分级、组织学、年龄、DLCO 和 VFI 与 OS 有显著关联。同样，性别、肿瘤分级、肿瘤分期、ASA 评分和 VFI 与 RFS 有关联。多变量分析得出了一个保留年龄、性别、DLCO、ASA 评分、VFI、肿瘤分级和 VFI 与年龄交互项的最终生存模型。类似的分析得出了一个保留只有肿瘤分级、肿瘤分期和 VFI 作为预测变量的最终复发无病生存模型。这些结果总结在表 1 中。

In order to observe the potential relationship between VFI and lung cancer survival outcomes, patients were stratified by VFI for Kaplan-Meier and Cox proportional hazards analyses. Comparison of the top and bottom VFI tertiles (VFItert) showed an association between high VFI and worse overall survival (OS) and recurrence free survival (RFS) (Figure 1A, ​,B).B). Multivariable modeling confirmed these results with high VFI group associated with RFS (HR = 1.79; 95% CI= 1.04 – 3.08; Figure 1C). The model of overall survival retained VFI tertile as well as the interaction terms between VFI and age as well as VFI and sex (Supplementary table S2). These results suggest that in contrast to high-BMI, visceral obesity as defined by elevated VFI is negatively associated with survival in early-stage lung cancer patients.
为了观察 VFI 与肺癌生存结果之间的潜在关系，患者根据 VFI 进行 Kaplan-Meier 和 Cox 比例危险分析分层。比较顶部和底部 VFI 三分位数（VFItert）显示高 VFI 与较差的总体生存率（OS）和无复发生存率（RFS）之间的关联（图 1A，B）。多变量建模证实了这些结果，高 VFI 组与 RFS 相关（HR = 1.79；95% CI= 1.04 – 3.08；图 1C）。总体生存模型保留了 VFI 三分位数以及 VFI 与年龄以及 VFI 与性别之间的交互项（附表 S2）。这些结果表明，与高 BMI 相反，由 VFI 升高定义的内脏肥胖与早期肺癌患者的生存率呈负相关。

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Figure 1. 图 1。

Overall and recurrence free survival of patients with High and Low VFI.
患有高和低 VFI 的患者的总体和无复发生存。

A) Overall survival curves generated for 513 patients separated categorized as having High (top tertile; N=171) and Low (bottom tertile; N=171) visceral adiposity as defined by VFI score demonstrate decreased survival with high visceral adiposity (HR=1.84; 95% CI = 1.21 – 2.81). B) Recurrence free survival curves demonstrate decreased survival with high visceral adiposity (HR = 1.82; 95%CI = 1.06 – 3.11). C) Expected recurrence free survival curves using the Cox proportional hazards model for demonstrate decreased survival with high visceral adiposity (HR = 1.79; 95% CI = 1.04 – 3.08).
A) 为 513 名患者生成的总体生存曲线，根据 VFI 评分将其分为高（顶部三分之一；N=171）和低（底部三分之一；N=171）内脏脂肪堆积两类，结果显示高内脏脂肪堆积患者的生存率降低（风险比=1.84；95%置信区间=1.21-2.81）。B) 复发无病生存曲线显示高内脏脂肪堆积患者的生存率降低（风险比=1.82；95%置信区间=1.06-3.11）。C) 利用 Cox 比例风险模型预期的复发无病生存曲线显示高内脏脂肪堆积患者的生存率降低（风险比=1.79；95%置信区间=1.04-3.08）。

Of the 513 patients, cause of death was unknown in 41 patients. Therefore, DSS was analyzed in 472 patients. Similar to OS and RFS, VFI tertile (Top tertile vs. bottom tertile) was associated with DSS on Kaplan Meier analysis (HR = 2.1; P=0.025). VFI as a continuous variable did not reach statistical significance however (P=0.1). On multivariate analysis, VFI tertile (Top tertile vs. bottom tertile) was associated with worse DSS (HR = 2.25; 95% 95% CI= 1.16 – 4.37; P=0.016; Supplementary figure S3) and was the only variable retained in the model. When VFI was analyzed as a continuous variable, age (0.006), VFI (P=0.001), tumor grade (P=0.003) and the interaction variable between VFI and age (P<0.001) were associated with DSS.
在 513 名患者中，41 名患者死因不明。因此，在 472 名患者中分析了 DSS。与 OS 和 RFS 类似，VFI 三分位数（顶部三分位数与底部三分位数）与 Kaplan Meier 分析中的 DSS 相关（HR = 2.1；P=0.025）。然而，VFI 作为连续变量并未达到统计显著性（P=0.1）。在多变量分析中，VFI 三分位数（顶部三分位数与底部三分位数）与更差的 DSS 相关（HR = 2.25；95% CI= 1.16 – 4.37；P=0.016；附图 S3），并且是模型中唯一保留的变量。当 VFI 作为连续变量进行分析时，年龄（0.006）、VFI（P=0.001）、肿瘤分级（P=0.003）以及 VFI 和年龄之间的交互变量（P<0.001）与 DSS 相关。

Visceral obesity is associated with alterations in the tumor immune microenvironment (TME) in late-stage NSCLC patients
腹部肥胖与晚期非小细胞肺癌患者肿瘤免疫微环境（TME）的改变相关

Despite the reported survival advantage seen in high-BMI lung cancer patients^7–9 a preponderance of data, including that obtained from a number of preclinical mouse tumor models, have demonstrated a profoundly negative effect of obesity on the cells of the anti-tumor immune response^{14, 35–37}. To examine the potential association of VFI with tumor immune gene expression, we analyzed existing data from patients with advanced stage NSCLC that underwent molecular testing to guide their therapy. In these patients, targeted transcriptome analysis (an “immune report card”) was thus generated, as described in Methods. Thus, the expression of 395 immune genes was assessed for 159 patient lung tumors (see Supplementary Table S3 for a summary of patient characteristics).
尽管高 BMI 肺癌患者中报道了存活优势，但大量数据，包括从多个临床前小鼠肿瘤模型获得的数据，已经证明肥胖对抗肿瘤免疫反应细胞有着深刻的负面影响。为了检查 VFI 与肿瘤免疫基因表达的潜在关联，我们分析了接受分子检测指导治疗的晚期 NSCLC 患者的现有数据。在这些患者中，通过靶向转录组分析（一种“免疫报告卡”）生成了数据，具体方法如下所述。因此，对 159 例患者肺部肿瘤评估了 395 个免疫基因的表达（有关患者特征的摘要，请参见附表 S3）。

Unsupervised clustering analysis led to the discovery of three major inflammation clusters, namely, inflamed, borderline and non-inflamed tumors (Figure 2A). The inflammatory status of the tumors was significantly associated with both VFI as a continuous variable (Figure 2B) and VFI tertile as well (Figure 2C). Specifically, cases in the Top VFI tertile were significantly over-represented in the non-inflamed tumor cluster (p=0.04), whereas those in the bottom VFI tertile were over-represented among the inflamed tumors to a considerably significant degree (p= 0.0085) (Figure 2C). Within non-inflamed tumors (n=38), a significantly higher proportion of moderately proliferative tumor microenvironments were found in bottom tertile patients (p=0.015) compared to top tertile, where a higher proportion of highly proliferative tumor microenvironments were found, indicating that low visceral obesity may confer improved overall survival in NSCLC (Supplementary Figure S4)³⁸. Further gene-wise differential gene expression analysis confirmed the significant down regulation of inflammation-associated genes in the top VFI tertile. Notably, tumors from High-VFI patients displayed enhanced expression of CDKN3 (a driver of cell proliferation linked to poor prognosis when overexpressed in lung cancer³⁹), as well as BCL6, a promoter of tumor growth and survival in NSCLC⁴⁰ and CD44, a marker of cancer stemness and poor prognoses in multiple cancers. Among the transcripts significantly under-represented in the tumors of High-VFI patients were those encoding chemokines or their receptors (including those associated with Thelper 1 (Th1)-type anti-tumor immune responses), the Th1 transcription factor Tbet, T cell lineage markers (e.g., CD4 and CD8), components of the T cell receptor and co-stimulatory signaling cascades (e.g., CD3, CD86), and the machinery of antigen presentation (Supplementary Table S4). These results strongly link central obesity (defined by high VFI) to a diminished immune activity in the lung tumor microenvironment (TME) that may stem from impaired T cell recruitment or expansion in this niche - conditions likely permissive to the growth and progression of lung cancers.
无监督聚类分析发现了三个主要的炎症聚类，即发炎、边缘和非发炎肿瘤（图 2A）。肿瘤的炎症状态与 VFI 作为连续变量（图 2B）和 VFI 三分位数（图 2C）显著相关。具体来说，处于顶部 VFI 三分位数的病例在非发炎肿瘤聚类中显着过度表示（p=0.04），而处于底部 VFI 三分位数的病例在发炎肿瘤中显着过度表示（p=0.0085）（图 2C）。在非发炎肿瘤（n=38）中，与顶部三分位数相比，底部三分位数患者中发现了显着更高比例的中度增殖肿瘤微环境（p=0.015），在顶部三分位数中发现了更高比例的高度增殖肿瘤微环境，表明低内脏肥胖可能有助于改善 NSCLC 的总体生存率（附图 S4）。进一步的基因差异基因表达分析证实了顶部 VFI 三分位数中炎症相关基因的显著下调。 值得注意的是，高 VFI 患者的肿瘤表现出增强的 CDKN3 表达（这是与肺癌预后不良相关的细胞增殖驱动因子 ³⁹ ），以及 BCL6，一种 NSCLC 中促进肿瘤生长和存活的物质，以及 CD44，一种多种癌症中的癌干细胞标记和不良预后。在高 VFI 患者肿瘤中显着低表达的转录本中，包括编码趋化因子或其受体（包括与 Thelper 1（Th1）型抗肿瘤免疫反应相关的因子）、Th1 转录因子 Tbet、T 细胞系列标记物（例如 CD4 和 CD8）、T 细胞受体和共刺激信号级联的组分（例如 CD3、CD86）以及抗原呈递机制（附录表 S4）。这些结果强烈地将中心性肥胖（由高 VFI 定义）与肺肿瘤微环境（TME）中的免疫活性减弱联系起来，这可能源自于在这个环境中 T 细胞招募或扩增受阻的条件-这些条件可能有利于肺癌的生长和进展。

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Figure 2. 图 2。

Changes in the tumor immune microenvironment with visceral obesity in advanced stage NSCLC.
晚期非小细胞肺癌中腹部肥胖与肿瘤免疫微环境的变化。

A) Heatmap of unsupervised clustering of immune response genes expression ranks (GEX Rank: columns) and 159 samples (rows) annotated by visceral fat index (VFI) tertiles, body mass index (BMI) thresholds and cell proliferation groups. B) Boxplot of inflammation cluster groups for VFI as a continuous variable with Wilcoxon test p values shown for all pairwise comparisons. C) Bar chart showing distribution of Inflammation clusters within top and bottom VFI tertiles with chi-square proportion test p values shown.
A) 免疫应答基因表达等级的无监督聚类热图（GEX Rank：列），159 个样本（行）按内脏脂肪指数（VFI）分为三等分，按体重指数（BMI）阈值和细胞增殖组进行注释。B) 炎症聚类组的箱线图，将 VFI 作为连续变量，显示所有成对比较的 Wilcoxon 检验 p 值。C) 柱状图显示炎症聚类在顶部和底部 VFI 三等分中的分布，显示卡方比例检验 p 值。

We further explored the potential association of VFI with co-variates such as BMI, race, gender, primary histology and staging. As expected, BMI category was inversely associated with VFI (p=0.00652), and pathology stage was significantly associated with VFI tertile (p=0.0024). Additionally, male gender was highly associated with high VFI status (p=1.54E-11) (Supplementary Table S5). These findings are in line with the generally accepted notion that males are more prone to visceral adiposity than females⁴¹. They also merit further investigation into the role of gender in visceral versus subcutaneous fat distribution with the two genders and the potential implications for long-term lung cancer patient outcomes and the anti-tumor immune response.
我们进一步探讨了 VFI 与诸如 BMI、种族、性别、原发性组织学和分期等共变量的潜在关联。如预期，BMI 类别与 VFI 呈负相关（p=0.00652），病理分期与 VFI 三分位显著相关（p=0.0024）。此外，男性性别与高 VFI 状态高度相关（p=1.54E-11）（附表 S5）。这些发现符合普遍接受的观念，即男性比女性更容易患内脏脂肪过多 ⁴¹ 。它们还值得进一步研究性别在内脏与皮下脂肪分布中的作用，以及两性之间的潜在影响对长期肺癌患者预后和抗肿瘤免疫反应的影响。

Interestingly, examining the correlation between traditional obesity status (as defined by a BMI of 30 or greater) with patient VFI tertile revealed that a significantly higher proportion of obese cases were seen in bottom VFI tertile than in the top tertile (p=0.009) (Supplementary Figure S5B). This surprising result suggests that only 15% of the VFI high cases would be classified as obese using the prevailing method of identifying obese patients in retrospective studies. In addition, no association was found between standard BMI categories and tumor inflammation state (Supplementary Figure S5A). These results link visceral adiposity in lung cancer patients to potential immune dysfunction expected in the obese host, and they further highlight the markedly different relationships that exist between VFI and BMI and lung cancer biology.
有趣的是，研究传统肥胖状态（定义为 BMI 大于或等于 30）与患者 VFI 分位数之间的相关性，发现肥胖病例在底部 VFI 分位数中的比例明显高于顶部分位数（p=0.009）（附图 S5B）。这一令人惊讶的结果表明，仅有 15%的 VFI 高病例会被归类为肥胖，使用目前在回顾性研究中识别肥胖患者的方法。此外，标准 BMI 分类与肿瘤炎症状态之间没有发现关联（附图 S5A）。这些结果将肺癌患者的内脏脂肪与肥胖宿主中预期的免疫功能障碍联系起来，并进一步突出了 VFI 与 BMI 以及肺癌生物学之间存在的明显不同关系。

Obesity exacerbates lung cancer progression in mice while altering TME gene expression
肥胖加剧了小鼠肺癌的进展，同时改变了 TME 基因表达

In parallel, we observed the effects of obesity on tumor progression and anti-tumor immunity in widely used pre-clinical models of lung cancer. For this, the in vivo conditions present in overweight and obese lung cancer patients were recreated using a well-characterized approach for diet-induced obesity (DIO) in mice. Prolonged administration of high fat diet (e.g., one containing 60% calories from fat) to mice of an obesity-susceptible genetic background, such as C57BL/6, results in progressive weight gain and an accumulation of visceral fat⁴² compared to normal diet-fed control mice. Cohorts of obese and normal weight mice were injected subcutaneously (s.c.) with Lewis lung carcinoma (LLC) cells, and subsequent tumor development was monitored. As generally seen in implantable mouse tumor models^{14, 37}, obese mice supported more robust tumor growth compared to non-obese controls in this model (Figure 3A). In a complementary model of pulmonary metastatic disease, obese mice challenged intravenously (i.v.) with a modest number of luciferase-expressing LLC (LLC-Luc) cells developed markedly enhanced lung tumor burden compared to normal weight controls within 26 days post-injection (Figure 3B,​,C).C). These results demonstrate the decidedly pro-tumor effects associated with obesity in mouse models that align closely with the relationship between lung cancer outcomes and central obesity brought to light by the use of VFI in our clinical studies.
同时，我们观察了肥胖对肺癌肿瘤进展和抗肿瘤免疫的影响，使用广泛应用的肺癌临床前模型。为此，我们利用一种经过良好表征的饮食诱导肥胖（DIO）的方法，在小鼠中再现了超重和肥胖肺癌患者体内条件。将高脂饮食（例如，含有 60%脂肪热量的饮食）长期投与肥胖易感基因背景的小鼠（如 C57BL/6），导致逐渐体重增加和内脏脂肪积累，与正常饮食对照小鼠相比。将肥胖和正常体重的小鼠队列皮下注射 Lewis 肺癌细胞（LLC），并监测随后的肿瘤发展。如一般植入小鼠肿瘤模型中所见，相比于非肥胖对照组，肥胖小鼠在该模型中支持更强劲的肿瘤生长（图 3A）。在肺转移疾病的补充模型中，肥胖小鼠经静脉挑战（i.v.）。) 具有适量的荧光素酶表达的 LLC（LLC-Luc）细胞的小鼠，在注射后 26 天内与正常体重对照组相比，肺肿瘤负担明显增加（图 3B，C）。这些结果表明，肥胖与小鼠模型中明显的促肿瘤效应密切相关，与我们临床研究中使用 VFI 揭示的肺癌结果和中心性肥胖之间的关系密切相关。

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Figure 3. 图 3。

Obesity-mediated effects on progression of Lewis lung carcinoma tumors in mice.
肥胖对小鼠 Lewis 肺癌肿瘤进展的影响。

A. C57BL/6 mice were fed a high-fat (to induce obesity) or normal diet for ~14–16 weeks before s.c. implantation of 10×10⁵ cells by s.c. injection into the shaved flanks. Mean tumor volumes were calculated using the formula: volume=0.5×lengthxwidth². (B,C) Obese and normal mice were challenged with intravenous tail vein injection of 0.25×10⁶ LLC-Luc cells. 26 days after injection, tumor burden (pulmonary metastases) were visualized by BLI after i.p. injection of d-luciferin (150mg/kg) using IVIS technology. P < 0.05 (*), < 0.02 (**), <0.001 (***) in standard t test. Error bars depict the SEM. Shown are a representative image (B) and mean tumor burden measurements (A and C) from 3–5 independent experiments.
C57BL/6 小鼠在皮下植入 10×10 ⁵ 细胞前，先饲喂高脂（诱导肥胖）或正常饮食约 14-16 周。平均肿瘤体积使用公式计算：体积=0.5×长度×宽度 ² 。肥胖和正常小鼠接受尾静脉注射 0.25×10 ⁶ LLC-Luc 细胞挑战。注射后 26 天，通过 i.p.注射 d-琥珀酸酯（150mg/kg）使用 IVIS 技术可视化肿瘤负担（肺转移）的 BLI。标准 t 检验中 P < 0.05（*），< 0.02（**），<0.001（***）。误差线表示 SEM。显示了 3-5 个独立实验的代表图像（B）和平均肿瘤负担测量（A 和 C）。

To gain insight into the potential mechanisms underlying the effects of obesity on tumor progression, we set out to document the transcriptomic changes in the TME associated with obesity. RNA was harvested from s.c. tumor sections (n=6 each) generated in the experiments described in Figure 3A, and RNASeq analysis was carried out. Expression of 187 and 217 genes was respectively up- and down-regulated by > 1.2x in tumors of obese compared to normal with adjusted Wald test p < 0.05 in analysis with DESeq2⁴³. Single-sample geneset enrichment analysis (GSEA) using the geneset variation analysis (GSVA) method⁴⁴ revealed significant divergences of multiple genesets related to metabolism and cancer biology (Bayes-moderated t test p < 0.05 with false discovery rate [FDR] < 0.20).
为了深入了解肥胖对肿瘤进展影响的潜在机制，我们着手记录与肥胖相关的肿瘤微环境的转录组变化。从实验中生成的皮下肿瘤切片（每组 n=6）中收集了 RNA，进行了 RNA 测序分析。与正常对照相比，肥胖肿瘤中分别有 187 和 217 个基因的表达上调和下调超过 1.2 倍，DESeq2 分析中调整的 Wald 检验 p < 0.05。使用基因集变异分析（GSVA）方法进行单样本基因集富集分析（GSEA）显示，与代谢和癌症生物学相关的多个基因集存在显著差异（Bayes 调节的 t 检验 p < 0.05，假发现率[FDR] < 0.20）。

Of the 18,025 genes identified as expressed in the tumors, expression of only 33 differed by ≥ 1.5-fold between obese and control mice at FDR < 0.05 (Supplementary Table S6). The five genes that were most up-regulated in obese tumors compared to controls were all found to either encode proteins involved in fat metabolism (i.e., pyruvate dehydrogenase kinase 4, lipase K, fatty acid binding protein 4) and lipid oxidation in particular⁴⁵ or to have been previously associated with adiposity (i.e., angiotension II receptor 2⁴⁶, bone morphogenetic protein 5⁴⁷). Examination of gene expression at the level of biological processes revealed a 1.2–1.3-fold enrichment of the gene sets involved in adipogenesis and oxidative phosphorylation in the tumors of obese mice. As might be expected given the robust tumor growth in the obese mice, genes involved in angiogenesis, epithelial-mesenchymal transition, hypoxia, and glycolysis were also upregulated in obese tumors. On the other hand, expression of genes associated with the immunologically relevant IL6-JAK-STAT3 signaling pathway were significantly reduced in the tumors of obese compared to control mice. Meanwhile expression of Stat4, a central driver of Th1 immunity and Areg, a gene known to be upregulated by Tregs in peripheral tissues (including lungs) were nominally down- and up-modulated in obese tumors, respectively (Supplementary Figure S5, Supplementary Table S6).
在被识别为在肿瘤中表达的 18,025 个基因中，仅有 33 个基因在肥胖和对照小鼠之间的表达差异≥1.5 倍，在 FDR < 0.05 时（附表 S6）。与对照组相比，在肥胖肿瘤中上调最多的五个基因都被发现要么编码参与脂肪代谢的蛋白质（即丙酮酸脱氢酶激酶 4，脂肪酶 K，脂肪酸结合蛋白 4）和特别是脂质氧化，要么与肥胖有关（即血管紧张素 II 受体 2，骨形态发生蛋白 5）。对生物过程水平的基因表达进行检查显示，在肥胖小鼠的肿瘤中，涉及脂肪生成和氧化磷酸化的基因集的富集程度为 1.2-1.3 倍。鉴于肥胖小鼠肿瘤的强劲生长，与血管生成、上皮间质转化、缺氧和糖酵解有关的基因也在肥胖肿瘤中上调。另一方面，与免疫相关的 IL6-JAK-STAT3 信号通路相关的基因在肥胖小鼠的肿瘤中表达显著降低，与对照小鼠相比。 与此同时，Stat4 的表达，作为 Th1 免疫的中心驱动因子，以及 Areg，一个已知在外周组织（包括肺部）中被 Tregs 上调的基因，在肥胖肿瘤中分别名义上被下调和上调调节（见附图 S5，附表 S6）。

These findings suggest that obesity’s still poorly understood impact on the TME and its potential fueling of tumor progression may be multi-faceted in nature, involving processes capable of aiding tumors directly and by opposing the activity of immune cells in the TME. Indications of reduced IL-6/STAT3 signaling were surprising, however, since elevated serum levels of IL-6 have been reported in the obese⁴⁸ and this cascade is generally thought to have multiple-pro-tumor effects in cancer cells themselves including expression of genes that promote cell proliferation, survival, angiogenesis, invasiveness, metastasis, and stemness⁴⁹. Interestingly though, an inverse relationship has been reported between STAT3 and commitment to oxidative metabolism/TCA cycle activity in other cancers (i.e., prostate cancer)⁵⁰. Also, given the known involvement of STAT3 signaling in glycolytic metabolism⁵¹ a reduced engagement of this cascade could also reflect a favoring of oxidative metabolism in the TME. Indeed, metabolic pathways that consume the available oxygen and enforce hypoxia in the TME were recently shown to be an obstacle for the mounting of effective immune cell activity⁵², and altered lipid transport and metabolism that can alter the availability of free fatty acids can also modify immune function in the TME³⁷. A relative upregulation of genes involved in TGF-beta-signaling suggests that along with these more recently described mechanisms obesity may enhance the notoriously anti-inflammatory cytokine contributing to a staunchly immune-suppressive TME under obese conditions.
这些发现表明，肥胖对肿瘤微环境的影响仍然不为人所了解，以及其对肿瘤进展的潜在推动可能是多方面的，涉及到能够直接帮助肿瘤并通过对抗肿瘤微环境中的免疫细胞活动的过程。然而，降低的 IL-6/STAT3 信号的迹象令人惊讶，因为已经报道了肥胖者血清 IL-6 水平升高，而这种级联通常被认为在癌细胞中具有多种促肿瘤效应，包括促进细胞增殖、存活、血管生成、侵袭、转移和干性基因的表达。然而，有趣的是，在其他癌症（如前列腺癌）中已经报道了 STAT3 与氧化代谢/TCA 循环活动承诺之间的反向关系。此外，鉴于已知 STAT3 信号在糖酵解代谢中的参与，这种级联的减少也可能反映了对肿瘤微环境中氧化代谢的偏爱。 事实上，最近显示，消耗可用氧气并在肿瘤微环境中引发缺氧的代谢途径成为有效免疫细胞活性的障碍 ⁵² ，而改变脂质运输和代谢也可以改变游离脂肪酸的可用性，从而在肿瘤微环境中修改免疫功能 ³⁷ 。参与 TGF-beta 信号通路的基因的相对上调表明，除了这些最近描述的机制外，肥胖可能增强臭名昭著的抗炎细胞因子，从而在肥胖条件下促进免疫抑制的肿瘤微环境。

Obesity attenuates anti-lung cancer immune responses in mice
肥胖减弱小鼠抗肺癌免疫反应

It is well appreciated that metabolic factors play a non-trivial part in regulating the phenotypic differentiation, fitness, and activity of immune cells^{53, 54}. Obesity is also known to have a profound effect on metabolism at an organismal or systemic level that is accompanied by immune dysfunction and smoldering inflammation⁵⁵. Yet, the precise effects of obesity on critical participants in the cellular response to lung cancer in mice and patients with lung cancer are just beginning to be understood. We therefore set out to dissect the impact of the obese state on the immune cell constituents of the TME in the mice described in Figure 3A using a multi-color flow cytometry-based approach. Our findings revealed multiple indications that obesity had a deleterious effect on the potency of the anti-tumor response.
代谢因素在调节免疫细胞的表型分化、适应性和活性中起着重要作用，这一点是被充分认可的。肥胖也被认为对有机体或系统水平的新陈代谢产生深远影响，伴随着免疫功能障碍和潜在炎症。然而，肥胖对小鼠和肺癌患者细胞反应中的关键参与者的确切影响才刚刚开始被理解。因此，我们着手解剖肥胖状态对图 3A 中描述的小鼠 TME 中免疫细胞成分的影响，采用基于多色流式细胞术的方法。我们的研究结果显示，肥胖对抗肿瘤反应的效力产生了不利影响。

Leukocyte infiltration of s.c. LLC tumors was markedly suppressed in obese (DIO) mice relative to normal weight (NORM) control tumors. This reduced intratumoral cellularity reflected a relative dearth of CD4+ and CD8+ T cells in the obese TME (Figure 4A). The potential tumoricidal CD8+ T cell compartment of obese mice were further observed to express considerably elevated levels of the immune checkpoint/exhaustion markers LAG3 and PD-1 on their surface relative to their normal weight counterparts (Figure 4B) in line with previous assessments of T cell phenotypes in obese tumor-bearing mice^{14, 56}. Similarly enhanced checkpoint expression levels were seen on conventional Thelper CD4+ T (Tconv) cells from DIO tumors (data not shown), and the CD8+ T cells recovered from obese mouse tumors stained more prominently with the dead cell-identifying dye LD Aqua (Figure 4C), indicative of an obesity-related defect in the fitness as well as the anti-tumor potential of intra-tumoral cells. Further suggesting an obesity-associated suppression of anti-tumor responses, the numbers of TILs capable of producing the tumoricidal Th1 cytokine IFNγ were much lower in the TILs recovered from DIO mice (Figure 4D).
肥胖（DIO）小鼠的皮下 LLC 肿瘤中白细胞浸润明显受抑制，相对于正常体重（NORM）对照肿瘤。这种降低的肿瘤内细胞密度反映了肥胖 TME 中 CD4+和 CD8+ T 细胞相对匮乏（图 4A）。观察到肥胖小鼠的潜在杀肿瘤 CD8+ T 细胞亚群在表面上表达的免疫检查点/疲劳标志物 LAG3 和 PD-1 的水平明显升高，相对于其正常体重对照（图 4B），与以往对肥胖肿瘤患小鼠 T 细胞表型的评估一致。类似地，DIO 肿瘤中的传统辅助 T 细胞 CD4+ T（Tconv）细胞上也观察到增强的检查点表达水平（未显示数据），并且从肥胖小鼠肿瘤中恢复的 CD8+ T 细胞在死细胞识别染料 LD Aqua 上染色更加明显（图 4C），表明肥胖相关缺陷影响了肿瘤内细胞的健康状态以及抗肿瘤潜力。 进一步表明肥胖相关的抑制抗肿瘤反应，从 DIO 小鼠中恢复的 TILs 中能够产生抗肿瘤 Th1 细胞因子 IFNγ的细胞数量要低得多（图 4D）。

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Figure 4. 图 4。

Obesity-mediated effects on the anti-tumor immune response to s.c. LLC tumors.
肥胖对皮下 LLC 肿瘤抗肿瘤免疫反应的影响。

C57BL/6 mice were fed a high-fat (to induce obesity) or normal diet for 16 weeks before implantation of 1×10⁵ cells by injection in shaved flanks (n=5–7/group). The density of tumor infiltrating leukocytes (TILs) were found, and flow cytometry analysis of tumor cell suspensions obtained in the experiment presented in Fig. 3 revealed the frequencies of CD4+ and CD8+ T cells amongst the tumor infiltrating leukocytes (TILs; A). The proportions of CD8+ T cells expressing the checkpoint molecules LAG3 and PD-1 and the viable fraction of CD8+ T cells were found and quantified (B, C). The levels of IFNγ producing TILs in obese and non-obese mice were also found (D). The relative proportions of Tregs among the TILs were found (E) as well as the levels of activated Treg markers. (F) and the frequencies of Foxp3+ Tregs expressing the checkpoint molecules LAG3 and PD-1 (F). P < 0.05 (*), < 0.02 (**), (****)<0.001 in standard t test. Error bars depict the SEM. Shown are representative flow plots and the mean quantification of replicates from one of 2–3 independent experiments.
C57BL/6 小鼠在植入 1×10 ⁵ 细胞前 16 周被喂高脂肪（诱导肥胖）或正常饮食（每组 n=5-7）注射在剃光的侧腹部。在图 3 中呈现的实验中获得的肿瘤细胞悬液的流式细胞术分析显示肿瘤浸润白细胞（TILs）的 CD4+和 CD8+ T 细胞的频率（A）。发现和量化了表达检查点分子 LAG3 和 PD-1 的 CD8+ T 细胞的比例以及 CD8+ T 细胞的存活分数（B，C）。还发现了肥胖和非肥胖小鼠中 IFNγ产生的 TILs 水平（D）。发现了 TILs 中 Tregs 的相对比例（E）以及激活的 Treg 标记物的水平（F）和表达检查点分子 LAG3 和 PD-1 的 Foxp3+ Tregs 的频率（F）。标准 t 检验中 P < 0.05（*），< 0.02（**），（****）<0.001。误差线表示 SEM。显示了代表性的流式细胞图和来自 2-3 个独立实验中的重复的平均量化。

In addition to effector cell deficits, enhanced suppressive cell phenotypes were also seen in the tumors of obese mice. Foxp3+ regulatory T (Treg) cells were modestly yet consistently enriched in the tumors of obese mice (Figure 4E). Moreover, these obese tumor-infiltrating Tregs displayed considerable up-regulated markers of an activated phenotype (CD44, ICOS) (Figure 4F). Since the activated or effector-like Treg phenotype is associated with both robust suppressive potency and a tendency to accumulate in murine and human tumors^{57, 58}, this observation suggests that obesity may bolster this subpopulation of suppressor cell known to be responsible for pathological immune suppression in the cancer setting. Concordantly, the levels of immune checkpoint molecules LAG3 and PD-1 expected to be expressed on the surface of intra-tumoral Tregs⁵⁷ were enhanced in DIO Tregs compared to those recovered from controls (Figure 4G). We also observed a marked elevation in the abundance of tumor CD11b+GR1(Ly6C/G)+ myeloid derived suppressor cells (MDSCs) in obese mice-an observation in agreement with previous studies¹³ and these cells displayed higher levels of PD-L1 on their surface in the obese setting compared to normal weight controls (Figure 5A, ​,B).B). Obesity also appeared to bolster proportions of potentially suppressive tumor-associated macrophages (CD11b+/F480+) as well as expression of the immune checkpoint molecule PDL-1 on these cells (Figure 5C, ​,D).D). In general, these differences in T and myeloid cell phenotypes were muted in other tissues surveyed including tumor-draining and distal lymph nodes and the spleens compared to the tumors of obese and control mice (data not shown) suggesting the immune cells of the TME may be particularly susceptible to obesity-related modulation. Interestingly, our findings suggest that, in contrast to the known ability of obesity to down-regulate Treg abundance in visceral fat⁵⁹ and blood, obesity can actually enhance Treg presence and activation in the TME and tumor-associated tissues. This previously unappreciated effect, potentially in concert with the potentiation of several other notoriously suppressive elements of the immune response (i.e., MDSCs, PD-L1:PD-1 signaling, exhausted CD8 T cells) may contribute to the enhanced tumor progression seen in obese mice.
除了效应细胞缺陷外，肥胖小鼠肿瘤中也观察到增强的抑制性细胞表型。Foxp3+ 调节性 T（Treg）细胞在肥胖小鼠肿瘤中略微但一贯地富集（图 4E）。此外，这些肥胖肿瘤浸润的 Treg 细胞显示出相当多的激活表型标记（CD44，ICOS）（图 4F）。由于激活或效应样 Treg 表型与强大的抑制能力和在小鼠和人类肿瘤中积累的倾向相关 ^{57, 58} ，这一观察表明肥胖可能增强这种已知负责癌症环境中病理免疫抑制的抑制细胞亚群。一致地，预计在肿瘤内 Treg 细胞表面表达的免疫检查点分子 LAG3 和 PD-1 的水平 ⁵⁷ 在 DIO Treg 细胞中比从对照组中恢复的细胞增强（图 4G）。 我们还观察到在肥胖小鼠中肿瘤 CD11b+GR1(Ly6C/G)+髓源抑制细胞(MDSCs)的丰度显著升高-这一观察结果与先前的研究一致 ¹³ ，并且与正常体重对照组相比，这些细胞在肥胖环境中表面的 PD-L1 水平更高(图 5A, B)。肥胖还似乎增强了潜在抑制性肿瘤相关巨噬细胞(CD11b+/F480+)的比例，以及这些细胞上 PD-L1 的表达(图 5C, D)。总的来说，在其他受调查的组织中，包括肿瘤引流和远端淋巴结以及脾脏，T 和髓细胞表型的差异相对较小，与肥胖和对照小鼠的肿瘤相比(未显示数据)，这表明 TME 的免疫细胞可能特别容易受到与肥胖相关的调节。有趣的是，我们的发现表明，与已知的肥胖降低内脏脂肪 ⁵⁹ 和血液中 Treg 丰度的能力相反，肥胖实际上可以增强 Treg 在 TME 和肿瘤相关组织中的存在和活化。 这种先前未被重视的效应，可能与免疫应答中几种其他臭名昭著的抑制元素的增强作用相结合（即 MDSCs、PD-L1:PD-1 信号通路、疲惫的 CD8 T 细胞），可能有助于肥胖小鼠中观察到的增强肿瘤进展。

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Figure 5. 图 5。

Obesity-mediated effects on myeloid derived suppressor cells in s.c. LLC tumors.
肥胖对皮下 LLC 肿瘤中髓源性抑制细胞的影响。

C57BL/6 mice were fed a high-fat (to induce obesity) or normal diet before implantation of 1×105 cells by injection in shaved flanks (n=5–7/group). Flow cytometry analysis of tumor cell suspensions obtained from Fig. 3. The frequencies of MDSC (GR1+/CD11b+) among the TILs were found by flow cytometry (A) as were levels of PD-L1 expression on these suppressor cells (B). Similarly, we determined the frequencies of tumor associated macrophages (CD11b+/F480+) and the surface levels of PDL1 on these cells (C,D). P < 0.05 (*), < 0.02 (**), in standard t test. Error bars depict the SEM. Shown are representative flow plots and the mean quantification of replicates from one of 2 independent experiments.
C57BL/6 小鼠在植入 1×105 个细胞之前被喂高脂肪（诱导肥胖）或正常饮食（每组 n=5-7）注射在剃光的侧腹部。从图 3 获得的肿瘤细胞悬液进行流式细胞术分析。通过流式细胞术发现 TILs 中 MDSC（GR1+/CD11b+）的频率（A），以及这些抑制细胞上 PD-L1 表达水平（B）。同样，我们确定了肿瘤相关巨噬细胞（CD11b+/F480+）的频率以及这些细胞上 PDL1 的表面水平（C，D）。在标准 t 检验中 P < 0.05（*），< 0.02（**）。误差线代表 SEM。显示了代表性的流式图和来自 2 个独立实验之一的重复的平均量化。

We thank the Genomics Shared Resource facility of RPCCC for performing the RNA sequencing experiments.
我们感谢 RPCCC 的基因组共享资源中心进行 RNA 测序实验。