Spatial transcriptomics of macrophage infiltration in non-small cell lung cancer reveals determinants of sensitivity and resistance to anti-PD1/PD-L1 antibodies

Abstract

Background: Tumor-associated macrophages (TAMs) having immunosuppressive properties are one of the most abundant immune cells in the tumor microenvironment (TME). Preclinical studies have highlighted the potential role of TAMs in resistance to immune checkpoint blockers (ICBs). Here, we investigated the predictive value of TAM infiltration in patients with non-small cell lung cancer (NSCLC) treated with ICBs and characterized their transcriptomic profiles.

Methods: Tumor samples were collected from 152 patients with NSCLC before ICB treatment onset. After immunohistochemical staining and image analysis, the correlation between CD163+ cell infiltration and survival was analyzed. Spatial transcriptomic analyses were performed using the NanoString GeoMx Immune Pathways assay to compare the gene expression profile of tumors with high or low levels of CD163+ cell infiltration and to identify determinants of response to ICBs in tumors with high CD163+ infiltration.

Results: Low intratumoral CD163+ cell infiltration was associated with longer progression-free survival (PFS; HR 0.61, 95% CI 0.40 to 0.94, p=0.023) and overall survival (OS; HR 0.48, 95% CI 0.28 to 0.80, p=0.004) under ICB treatment. Spatial transcriptomic profiles of 16 tumors revealed the upregulation of ITGAM, CD27, and CCL5 in tumors with high CD163+ cell infiltration. Moreover, in tumors with high macrophage infiltration, the upregulation of genes associated with the interferon-γ signaling pathway and the M1 phenotype was associated with better responses under immunotherapy. Surprisingly, we found also a significantly higher expression of CSF1R in the tumors of responders. Analysis of three independent data sets confirmed that high CSF1R expression was associated with an increased durable clinical benefit rate (47% vs 6%, p=0.004), PFS (median 10.89 months vs 1.67 months, p=0.001), and OS (median 23.11 months vs 2.66 months, p<0.001) under ICB treatment.

Conclusions: Enrichment of TAMs in the TME of NSCLC is associated with resistance to immunotherapy regardless of the programmed death ligand 1 status and is driven by upregulation of CD27, ITGAM, and CCL5 gene expression within the tumor compartment. Our transcriptomic analyses identify new potential targets to alter TAM recruitment/polarization and highlight the complexity of the CSF1R pathway, which may not be a suitable target to improve ICB efficacy.

Keywords: immunotherapy; lung neoplasms; macrophages.

Figure 1

CD163+ cell tumor infiltration is correlated with poor clinical outcome. (A) Representative images of lung cancer sample stained with the multiplexed panel CD8/CD163/CK7—Objective 20× (B–C) Kaplan-Meier curves of progression-free survival (B) and overall survival (C) according to stroma CD8+ cell density. (D) Proportion of patients who experienced durable clinical benefit (DCB) or non-clinical benefit (NCB) according to their level of CD8+ stroma infiltration classified as high and low. P value was calculated using χ² test.(E–F) Kaplan-Meier curves of progression-free survival (E) and overall survival (F) according to tumor CD163 +cell density. (G) Proportion of patients who experienced DCB or NCB according to their level of CD163+ tumor infiltration classified as high and low. P value was calculated using χ² test.

Figure 2

Tumor expression of CCL5 induces CD163+ cell recruitment. (A) Tissue segmentation in tumor (red) and stroma (green) areas of illumination (AOI), performed on the GeoMX DSP platform. (B) Representation of CD45 (left) and PanCK (right) expression in the tumor and stroma AOIs. (C) Unsupervised clustering of tumor patient samples based on the averaged expression of the GeoMX Immune Pathways Panel probes in the tumor and stroma areas. Levels of CD163+ cell infiltration classified as high (red) and low (blue) are annotated. (D) Volcano plot representation of the gene differentially expressed by CD163+ high and low patients in their tumor areas. (E) Spearman correlation of the CD163+ cell density determined by IHC and the level of messenger RNA expression of indicated genes assessed by bulk RNA sequencing. DSP, Digital Spatial Profiler; IHC, immunohistochemistry.

Figure 3

M1-associated genes are enriched in immunotherapy-responsive patients with high level of CD163+ cell infiltration. (A) Unsupervised clustering of patient with tumor samples based on the averaged expression of the GeoMX Immune Pathways Panel probes in the tumor and stroma areas. The patient response classified as non-clinical benefit (NDB—blue) and durable clinical benefit (DCB—red) is annotated. (B) Volcano plot representation of the gene differentially expressed in the stroma areas of patients who experienced DCB and NCB. (C) tSNE visualization of 10× scRNA-seq of non-small cell lung cancer biopsy. Cells co-expressing CD68 or CD163 together with CSF1R are highlighted in blue and orange, respectively. (D) Representation of CSF1R expression in CD68+ and CD163+ cells, as assessed by scRNAseq. (E–F) Kaplan-Meier curves of progression-free survival (E) and overall survival (F) of patients according to the expression of CSF1R determined by RNAseq and classified as high or low. (G) Proportion of patients who experienced DCB or NCB according to their level of CSF1R expression determined by RNAseq and classified as high and low. P value was calculated using χ² test. IFN, interferon; RNAseq, RNA sequencing; scRNA -seq, single cell RNAseq. tSNE, t-distributed stochastic neighbor embedding.