Myeloid Clusters Are Associated with a Pro-Metastatic Environment and Poor Prognosis in Smoking-Related Early Stage Non-Small Cell Lung Cancer

Background: This study aimed to understand the role of myeloid cell clusters in uninvolved regional lymph nodes from early stage non-small cell lung cancer patients. Methods: Uninvolved regional lymph node sections from 67 patients with stage I–III resected non-small cell lung cancer were immunostained to detect myeloid clusters, STAT3 activity and occult metastasis. Anthracosis intensity, myeloid cluster infiltration associated with anthracosis and pSTAT3 level were scored and correlated with patient survival. Multivariate Cox regression analysis was performed with prognostic variables. Human macrophages were used for in vitro nicotine treatment. Results: $CD68^+$ myeloid clusters associated with anthracosis and with an immunosuppressive and metastasis-promoting phenotype and elevated overall STAT3 activity were observed in uninvolved lymph nodes. In patients with a smoking history, myeloid cluster score significantly correlated with anthracosis intensity and pSTAT3 level (P<0.01). Nicotine activated STAT3 in macrophages in long-term culture. $CD68^+$ myeloid clusters correlated and colocalized with occult metastasis. Myeloid cluster score was an independent prognostic factor (P = 0.049) and was associated with survival by Kaplan-Maier estimate in patients with a history of smoking (P = 0.055). The combination of myeloid cluster score with either lymph node stage or pSTAT3 level defined two populations with a significant difference in survival (P = 0.024 and P = 0.004, respectively). Conclusions: Myeloid clusters facilitate a pro-metastatic microenvironment in uninvolved regional lymph nodes and associate with occult metastasis in early stage non-small cell lung cancer. Myeloid cluster score is an independent prognostic factor for survival in patients with a history of smoking, and may present a novel method to inform therapy choices in the adjuvant setting. Further validation studies are warranted.Economic

Patient demographics and tumor characteristics.

<p>NA, not applicable.</p

Myeloid clusters and pSTAT3 predicts survival in NSCLC patients with a smoking history.

<p>Kaplan-Meier curves showing the survival of patients grouped by: (A) myeloid cluster infiltration associated with anthracosis, (B) overall pSTAT3 level, (C) the combination of nodal stage and myeloid clusters associated with anthracosis, (D) the combination of myeloid clusters associated with anthracosis and overall pSTAT3 level (n = 49).</p

Positive correlations between myeloid clusters and STAT3 activity in patients with a smoking history.

<p>(A) In patients with a smoking history, but not in those without a history of smoking, myeloid cluster infiltration associated with anthracosis correlates with anthracosis intensity and overall STAT3 activity; anthracosis correlates with STAT3 activity in myeloid clusters associated with anthracosis. The correlation was determined with nonparametric Spearman's rank correlation test. Shown are means ± SEM, ** P<0.01, *** P<0.001. (B) Nicotine activates STAT3 in human macrophages. Western blotting showing pSTAT3 and total STAT3 expression in human THP-1-derived macrophages and human primary monocyte-derived macrophages after nicotine treatment with or without AZD1480.</p

Scoring method.

<p>HE staining and IHC staining for CD68 or pSTAT3 showing representative images for the scoring of anthracosis intensity, anthracotic myeloid cluster infiltration, overall STAT3 activity and anthracotic myeloid cell STAT3 activity. The number on the upper-left corner of each image shows the score. (Scale bar, 200 µm)</p

Prominent myeloid clusters associated with anthracosis and elevated STAT3 activity in NSCLC uninvolved LNs.

<p>(A) HE staining of normal LNs from individuals without cancer (n = 4; left two panels) and uninvolved regional LNs from NSCLC patients (middle two panels) showing anthracotic carbon pigment (indicated with arrows; Scale bar, 20 µm) in patient LNs. Immunofluorescent staining for CD68 (right panel) demonstrating anthracotic pigment is within CD68⁺ cells (Note the black dots indicated with small arrows in CD68⁺ cells; Scale bar, 20 µm). (B) IHC staining showing prominent CD68⁺ myeloid clusters associated with anthracosis and highly activated STAT3 in uninvolved NSCLC regional LNs (right panels) compared to normal LNs (n = 4; left panels) (Scale bar, 200 µm).</p

Myeloid clusters in uninvolved LNs colocalize with occult metastasis and cFn.

<p>(A) IHC staining showing occult metastasis in “uninvolved” LNs (top two panels) and metastatic tumor cells in an involved LN as the positive control (lower left panel) (Scale bar, 50 µm). The bar graph shows the correlation between myeloid cluster infiltration associated with anthracosis and the presence of occult metastasis (OM). The P value was determined by nonparametric Spearman's rank correlation test. (B) Confocal images showing the colocalization of pan-CK⁺ NSCLC cells (arrows) and CD68⁺ myeloid clusters associated with anthracosis. (Scale bar, 50 µm) (C) Confocal images showing the colocalization of myeloid clusters associated with anthracosis (arrows) and cFn. (Scale bar, 50 µm)</p

Tumor-promoting phenotype of the myeloid clusters.

<p>IHC staining demonstrating the expression of CD163, IL-6, IL-10, VEGF-A, MMP-9, SDF-1 and Bcl-xL by the myeloid cells associated with anthracosis. For each protein, representative images showing positive and negative staining areas were selected from the same slide. The quantification was performed by analyzing random images of myeloid cluster areas associated with anthracosis and other areas (10 images for each group) from 10 patients. Two-tailed Student's <i>t</i>-test was used for statistical analysis. Shown are means ± SEM, *** P<0.001. (Scale bar, 200 µm for 100× and 400×; 25 µm for 1600×)</p