Novel FABP4+C1q+ macrophages enhance antitumor immunity and associated with response to neoadjuvant pembrolizumab and chemotherapy in NSCLC via AMPK/JAK/STAT axis

Abstract

Immune checkpoint inhibitors (ICIs) immunotherapy facilitates new approaches to achieve precision cancer treatment. A growing number of patients with non-small cell lung cancer (NSCLC) have benefited from treatment with neoadjuvant ICIs combined with chemotherapy. However, the mechanisms and associations between the therapeutic efficacy of neoadjuvant pembrolizumab and chemotherapy (NAPC) and macrophage subsets are still unclear. We performed single-cell RNA sequencing (scRNA-seq) and identified a novel FABP4⁺C1q⁺ macrophage subtype, which exhibited stronger proinflammatory cytokine production and phagocytic ability. This subtype was found to be more abundant in tumor tissues and lymph nodes of major pathological response (MPR) patients compared to non-MPR patients, and was associated with a good efficacy of NAPC. Multiplex fluorescent immunohistochemical (mIHC) staining was subsequently used to verify our findings. Further mechanistic studies indicated that FABP4 and C1q regulate the expression of proinflammatory cytokines synergistically. In addition, FABP4 and C1q promote fatty acid synthesis, enhance anti-apoptosis ability and phagocytic ability of macrophage via the interaction of AMPK/JAK/STAT axis. This study provides novel insights into the underlying mechanisms and predictive biomarkers of NAPC. Our findings contribute to improving the prognosis of patients with NSCLC by potentially guiding more precise patient selection and treatment strategies. NOVELTY & IMPACT STATEMENTS: We identified a group of macrophages (FABP4⁺C1q⁺ macrophages) related to the therapeutic efficacy of neoadjuvant chemoimmunotherapy. FABP4⁺C1q⁺ macrophages highly expressed proinflammatory cytokines-related genes and had a strong cytokine production and phagocytic ability. We believe that our study provides a novel insight into the synergistic mechanism of neoadjuvant ICI combined with chemotherapy and may lead to improved clinical outcomes in patients with NSCLC in the future.

Fig. 1. Single-cell landscape of macrophages and its association with neoadjuvant pembrolizumab and chemotherapy (NAPC) in NSCLC patients.

A Uniform manifold approximation and projection (UMAP) of myeloid immune cells in 11 scRNA-sequenced samples. A total of 14 myeloid cell clusters were identified. B Cell-type annotation of myeloid immune cells. C Cell-type annotation markers, and the annotation process. MARCO, MSR1, and MRC1 were used as macrophage markers; CLEC9A, LAMP3, CD1C, and PLD4 were used as dendritic cell markers; CD14, CD33, S100A8, S100A9, S100A12, and VCAN were used as monocyte markers; MS4A2, CPA3 and TPSAB1 were used as mast cell markers; and NRGN, PPBP, PF4, and OST4 were used as markers of megakaryocytes. D Based on the acquired major pathological response, seven patients with NAPC were divided into two groups (MPR and non-MPR groups). E UMAP of myeloid immune cells in the treatment-naïve, MPR, and non-MPR groups. F Changes in the proportion of C0, C3, C11 macrophages in total number containing all tissues in treatment-naïve, MPR, and non-MPR groups.

Fig. 2. C0-macrophages, a novel gene signature macrophage subset: expression of FABP4 and C1q simultaneously with high expression of proinflammatory cytokines.

A Identification of novel marker genes in different clusters of myeloid immune cells after sorting the genes by log|Fold Change| and selecting the top ten marker genes. B UMAP of the expression of novel marker genes (FABP4, C1QA, C1QB, C1QC) of C0-macrophages. C Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of characteristic genes in FABP4⁺C1q⁺ macrophages. D The cytokines (IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-12) expression levels of FABP4⁺C1q⁺ macrophages and other macrophage subsets in our scRNA-seq profile (Blue: C0-macrophages; Red: other macrophages).

Fig. 3. Enrichment of FABP4⁺C1q⁺ macrophages in tumor tissues and lymph nodes indicated good therapeutic efficacy of NAPC and good prognosis in NSCLC.

A, B Comparison of FABP4 and C1QA expression levels in tumor tissues (T), lymph nodes (LN), distal normal tissues (D) of MPR and non-MPR groups. C Representative mIHC staining of NSCLC tumor tissues, lymph nodes, distal normal lung tissues showing CD68 (purple), C1q (green), and FABP4 (orange). Original magnification: ×200. Scale bar: 50 µm. D The number of FABP4⁺C1q⁺ macrophages infiltration in tumor tissues among naïve, MPR, non-MPR NSCLC patients. E Analysis of correlation between the level of tumor infiltrate FABP4⁺C1q⁺ macrophages (low and high) with OS and RFS in NSCLC, using Kaplan–Meier survival analysis. F Correlation between the number of tumor infiltrate FABP4⁺C1q⁺ macrophages and CD4⁺ T cells, CD20⁺ B cells, CD8⁺ T cells.

Fig. 4. FABP4⁺C1q⁺ macrophages have stronger proinflammatory cytokines secretion ability, phagocytic functions, and anti-apoptosis ability.

A, B The proportion of FABP4⁺C1q⁺ macrophages in CD14⁺ monocytes, M0, M1, and M2 macrophages from human PBMCs. C The production levels of inflammatory cytokines (TNF-α, IL-6, IL-1β) in FABP4⁺C1q⁺ and FABP4⁺C1q⁻macrophages (n = 3). D The phagocytic ability of negative control and C1q knockdown macrophages (n = 3). E Knockdown of FABP4 in PBMCs-derived macrophages increased cell apoptosis (n = 3).

Fig. 5. FABP4 and C1q regulate the expression of inflammatory cytokines in FABP4⁺C1q⁺macrophages synergistically.

A The efficiency of FABP4 knockdown in PBMCs-derived macrophages (n = 3). B The mRNA expression levels of IL-6 and IL-1β in siNC and siFABP4 macrophages. C The mRNA expression levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β) in FABP4⁺C1q⁺ and FABP4⁺C1q⁻ PBMCs-derived macrophages (n = 3). D The efficiency of C1q knockdown in macrophages detected by RT-qPCR and western blot (n = 3). E, F The proinflammatory cytokines (TNF-α, IL-6, IL-1β) expression and production ability in siNC and siC1q macrophages determined using RT-qPCR and flow cytometry (n = 3).

Fig. 6. AMPK phosphorylation level decreased in FABP4⁺C1q⁺macrophages.

A The mRNA expression levels of fatty acid synthase (FASN), and fatty acid lyase (CPT1, ACADM) in siNC and siFABP4 macrophages (n = 3). B The protein levels of AMPK, and pAMPK in FABP4⁺C1q⁺ and FABP4⁺C1q⁻ PBMCs-derived macrophages (n = 3). C The mRNA expression levels of C1QA, C1QB, C1QC of macrophages treated with AMPK activator. D The protein levels of C1q, AMPK, and pAMPK from PBMC-derived macrophages after treatment with AMPK activator (AICAR) (n = 3).

Fig. 7. FABP4⁺C1q⁺macrophage exert secretion of inflammatory cytokines and phagocytic function through the AMPK/JAK/STAT3 pathway.

A The protein levels of STAT3, and pSTAT3 in siNC and siFABP4 groups of macrophages (n = 3). B C1q subunit gene (C1QA, C1QB, C1QC) promoter region potential STAT3 binding sites, displaying the top four binding sites in the score.