Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy

Abstract

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA⁺) tumors compared to PRE MNA⁺ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA⁺ tumor cells were more immunogenic compared to PRE MNA⁺ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA⁺ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA⁺ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA⁺ neuroblastoma by targeting the immune microenvironment.

Keywords: MYCN amplification; Neuroblastoma; chemotherapy; macrophages.

Figure 1.

Immune cell infiltration in POST MNA+ neuroblastoma tumors a. Heatmap showing differentially expressed genes in PRE and POST MNA+ neuroblastoma tumors. The heatmap is based on (unsupervised) clustering of the genes that differed significantly in differential expression analyses. b. Top 20 biological processes upregulated in POST MNA+ neuroblastoma tumors. c. Expression of macrophage-related genes in POST MNA- and MNA+ neuroblastoma tumors compared to PRE MNA- and MNA+ neuroblastoma tumors, respectively. *Indicates significant differential expression, p-value. d. Representative IHC image for macrophages (CD68+ cells, here in a POST MNA+ neuroblastoma tumor). Scale bar = 100μm. e. Percentage of CD68+ cells in neuroblastoma tumors (1-7 tumor samples per patient) divided by treatment status and MYCN amplification. f. Mean percentage of CD68+ cells in patients’ PRE and POST tumors. Mean ± SD, Mann-Whitney test.

Figure 2.

Infiltration of tumor associated macrophages in POST MNA+ neuroblastoma tumors a. Representative image of high-resolution spatial proteomics on neuroblastoma tumors stained for immune cells (CD45+, green) and tumor cells (chromogranin A+, purple). Circles indicate regions of interest (ROI). Red blood cell autofluorescence can be seen between ROI. b. Volcano plot of immune cell related proteins. Unpaired t-tests, p-value <-2 or > 2. c. Heatmap and dendrogram showing unsupervised hierarchical clustering of multiple PRE and POST tumor samples from two patients with MNA+ neuroblastoma, for the proteins that had significantly different expression comparing PRE and POST tumor samples. d. Representative IHC images for TAMs (CD163+ cells in PRE and POST MNA+ tumors from the same patient). Black arrow indicate CD163+ cells. Scale bar = 100 µm.

Figure 3.

Monocyte/macrophage chemoattractant protein CCL2 increases in POST MNA+ tumors a. Genes related to cytokines and chemokines and their receptors in POST-treatment MNA- and MNA+ neuroblastoma tumors as compared to pre-treatment MNA- and MNA+ neuroblastoma tumors, respectively. *Indicates significant differential expression, p-value. b. Gene expression of CCL2 in PDX#3 tumors; untreated, collected during COJEC treatment or POST COJEC treatment. Mean ± SD, Mann-Whitney test. c. Representative IHC images for CCL2 in a PRE and POST MNA+ neuroblastoma tumor. Scale bar = 100μm. d. H-score of CCL2 protein levels in clinical MNA+ neuroblastomas. Mean ± SD, Mann-Whitney test. e. Data analysis of scRNA-seq sample from one treated MNA+ neuroblastoma tumor, visualized using a common UMAP embedding is tumor and stroma compartment. f. gene expression shown in UMAP plot.

Figure 4.

Macrophage infiltration and altered genetic landscape in POST-tumors a. Correlations between the percentage of CD68+ cells and the number of copy number aberrations (CNA) in clinical PRE and POST tumor samples. b. Number of CNA in clinical neuroblastoma tumor samples. Mean ± SD, Mann-Whitney test.

Figure 5.

Inhibition of macrophage recruitment prevent POST tumor regrowth a. Schematic illustration of experimental approach. b. Tumor growth kinetics of untreated (n = 4) and COJEC treated PDX#3 tumors (n = 12). The treatment continued for three weeks after which the tumors were left to regrow for an additional three weeks. Seven of the COJEC-treated mice were administrated with anti-CSF1R during the last three weeks. Mean ± SD. c. Tumor volume of POST treatment (n = 5) and POST treatment + anti-CSF1R treated tumors (n = 7) last day of experiment (day 42). Mean ± SD, Mann-Whitney test. d. Representative IHC images for mouse CD206 in untreated (i), POST (ii) and POST+anti-CSF1R (iii- CD206+ cells absent; iv- few dispersed CD206+ cells present) PDX#3 tumors. Black arrows indicate CD206+ cells. e. Immunohistochemistry statistics of d. using Mann-Whitney test. Mean ± SD. f. Representative IHC images for human CCL2 in untreated and POST PDX#3 tumors. Scale bar = 100 µm.

Figure 6.

Immunogenic tumor cells have a growth advantage after treatment due to their ability to recruit pro-tumor macrophages. Blocking the recruitment of macrophages after chemotherapy prevents the outgrowth of immunogenic tumor cells.